Haem Flashcards

Question

WBC morphology - general

Answer 1

a. Critical to assess morphology – machine may miss blasts, cannot identify left shift b. Lymphocytes i. Reed-Sternberg cell = giant, multinucleated B lymphocyte ii. Smudge cell = lymphocyte damaged during preparation of blood smear indicating cell fragility, seen in CLL and other lymphoproliferative disorders c. Neutrophils i. Blasts  promyelocyte  myelocyte  metamyelocyte  band  mature neutrophil ii. Normally only mature neutrophils (2-4 lobed nucleus) and band neutrophils (immediate precursor with horseshoe-shaped nucleus) are found in circulation iii. Hyper segmented neutrophil = >5 lobes 1. Megaloblastic process – B12 or folate deficiency iv. Left shift 1. Increase in granulocyte precursors (bands, metamyelocytes, myelocytes, promyelocytes, blasts) in circulation 2. Acute infections, hypoxia, shock, CML v. Other 1. IT ratio – used in neonates (immature forms/total neutrophils) 2. Neutrophil toxic changes – bacterial infection

Answer 2

1. PRBC = RBC with plasma removed a. + anticoagulant + citrate b. + phosphate + dextrose 2. Indications a. Should be based on need to optimise tissue delivery b. O2 delivery = Hb x CO (HR x CV) x oxygen saturation x 1.34 c. Consider: i. Expected trajectory of Hb – haemolysis vs. blood loss ii. Ability to cope with tachycardia 3. General rules a. Hb <70g/L = although lower thresholds may be acceptable in patients without symptoms and where specific therapy (eg iron) is available. b. Transfusion may be indicated at higher thresholds for specific situations: i. Hb <70-100g/L during surgery associated with major blood loss or if evidence of impaired oxygen transport ii. Hb <80g/L; patients on a chronic transfusion regimen or during marrow suppressive therapy (for symptom control and appropriate growth) iii. Hb <100g/L; only for very select populations (eg. neonates) 4. Transfusion equation = 0.5 x weight x (target Hb – current Hb) • The increase in hemoglobin from 1 unit of RBCs will be approximately 1 g/dL; the increase in hematocrit will be approximately 3 percentage points

Answer 3

a. Leukodepletion (all products in Victoria automatically leukodepleted) i. Involves ‘filtering’ of blood product ii. Removes most WBC (by size) but not all iii. Reduces risk of non-haemolytic transfusion reactions, cytokine based reactions and infections such as CMV b. Washing i. Blood product washed in normal saline ii. Removes plasma proteins iii. Reduces risk of allergic reactions c. Irradiation i. Kills T-lymphocytes ii. Reduces risk of GVHD (AND risk of similar haplotypes reacting to each other  eg related donors) iii. Not required for acellular products d. CMV negative i. Requires CMV negative donors ii. For CMV -ve transplant patients

Answer 4

6. RBC typing a. Goal = to reduce risk of immune reactions b. Typing done for: i. ABO group ii. Rh group iii. Common antigens – Kell, Duffy 1. Kell = most common antibody implicated in haemolytic disease of the newborn, take special care in females pre child birth a. Antibodies to RBC antigens can occur to i. Natural exposure – carbohydrates that mimic blood antigens ii. Autoantibodies – against autologous blood group antigens iii. Allogeneic exposure – from transfusion/ pregnancy 1. IgG mediated can cross the placenta b. ABO system i. Oligosaccharide (CHO) antigen on RBC surface ii. Genes determining ABO type found on chromosome 9 iii. A and B alleles are codominant so both A and B antigens will be expressed on the RBC whenever either allele is present iv. Blood groups = A (AA, AO), B (BB, BO), AB, O v. H antigen – required to bind AB antigens to RBC surface, if not present then patient is O blood group despite genotype A, B, AB = Bombay phenotype vi. Production of antibodies 1. Natural production of antibodies against antigens not on cell surface a. E.g. A + blood group – then natural production of Anti-B 2. ABO incompatibility – IgM production to sensitization against Ag not present on own RBC a. IgM cannot cross the placenta 3. AB antibodies begin to appear 2-8months after birth and occur without external exposure c. Rhesus system i. Rh system comprises 61 antigens 1. D antigen = most immunogenic and important Rh antigen  autosomal dominant a. RH +ve refers to presence of Rh D protein (15% of the population lack D) 2. Others = C, E ii. Routine Rh typing of doors and patients only tests for the presence/absence of D iii. Antibody production 1. Development of antibodies to Rhesus only occurs due to prior sensitisation – NOT naturally occurring eg. prior transfusion, pregnancy 2. Results in the production of IgG antibodies (cf. ABO antibodies IgM) 3. IgG can cross the placenta iv. In a negative person exposed to +ve blood 1. Spontaneous reactions rarely occur - person needs to be exposed to lots of Rh antigen before agglutinins develop to cause a significant transfusion reaction 2. Usually causes a mild delayed transfusion reaction, which is increased on second exposure

Answer 5

G&H Also known as Group and Screen or Group and Hold. Group and Save is the sample processing that determines the patient blood group (ABO and RhD) and screens for any atypical antibodies. The process takes around 40 minutes and no blood is issued. If patient blood have atypical red cell antibodies, the laboratory will do additional tests to identify them. CROSSMATCH A crossmatch is the final step of pretransfusion compatibility testing, to request blood from the laboratory. Crossmatching involves physically mixing of patient’s blood with the donor’s blood, in order to see if any immune reaction occurs After ensuring that donnor blood is compatible, the donor blood is issued and can be transfused to the patient. This process takes around 40 minutes, in addition to the 40 minutes required to G&S the blood. It is not possible for the laboratory to provide crossmatched blood without having processed a G&S sample first.

Answer 6

a. In Australia, blood is tested for 5 transmissible disease: HIV, HBV, HCV, HTLV (human T-cell lymphotropic virus), syphilis b. Specifically: i. HBV surface Ag; HCV antibody; HIV-1 and HIV-2 antibody; HTLV-I and HTLV-II antibody; syphilis Ab ii. ALSO now test for HIV-1 and HCV RNA – looks for the actual presence of the virus, not just the immune response – this reduced the ‘window period’ (time between exposure to a virus and appearance of detectable antibodies) – see table below iii. Also test for malaria in donors who have resided in or travelled to a malarial area c. Donors are notified of abnormal result

Answer 7

a. Infection i. HIV = 1/2 million – last occurred in late 90s ii. HCV - 1/1.5-2 million iii. Bacterial contamination = 1/5 million – usually Yersinia and gram negatives iv. Transfusion related sepsis 1/17,000 b. Acute i. Volume overload ii. Non-haemolytic febrile transfusion reactions 1. Anti-HLA antibodies directed against contaminated leukocytes  cytokine release 2. Results in fever/chills/rigors 3. Reduced by giving leukocyte depleted blood (all blood in Aus) iii. Acute haemolytic transfusion reaction 1. Recipient with preformed antibodies against donor blood = destruction of donor RBC entering the circulation 2. Can be due to ABO/ Rh/ Kell / Duffy 3. Clinical features = chest/ flank pain, fever, tachycardia, hypotension, AKI 4. Rx = stop transfusion, give saline. Check Coombs/ haptoglobin, free Hb and repeat X match iv. Allergy 1. Urticaria (1%) 2. Anaphylaxis (beware IgA deficient patients with anti-IgA antibodies) v. Transfusion related acute lung injury (TRALI) 1. 1/5000 2. Donor plasma has anti-HLA / polymorph antibodies that bind to WBC  cytokine production 3. Cause pulmonary oedema/ARDS  fever, tachycardia, hypoxia 4. Within 2-8hrs of transfusion 5. V uncommon in kids (more common in elderly females) vi. Citrate toxicity (the anticoagulant in blood products) vii. Hyperkalaemia : RBC lysis,  in irradiated cells c. Delayed i. Refractory thrombocytopaenia after multiple transfusions (due to sepsis, DIC, antibodies against HLA types / human platelet antigens) ii. Delayed haemolytic transfusion reaction 1. Low level alloantibodies not picked up on cross match (often against Rh/ Kidd) 2. Bind to donor RBC, instigate extravascular haemolysis 3. 2-10 days post transfusion: fever, haemolysis, jaundice, positive Coomb’s test iii. Transfusion related GVHD = 8-10 days post transfusion 1. Results in rash, fever, abnormal LFTs, TENs 2. Reaction between HLA types = viable T cells in transfusion attack patient cells 3. 100% fatal 4. Does not occur in immunocompetent recipient as they have far greater WBC 5. Prevented by using irradiated products 6. Risk factors a. Any immunodeficiency b. Neonates c. HLA matched donor (i.e. the donor has 2 haplotypes that are the same, and the recipient has 1 of those haplotypes but not the other  recipient won’t mount a response as it recognizes the 2 haplotypes as same, but the graft will attack the host as it recognizes an abnormal haplotype) 7. Treatment = IVIG/immunosuppressants iv. Iron accumulation

Answer 8

1. Key points a. Kept at room temperature = highest risk of infection 2. Amount a. 5-20 ml/kg b. 5-10 ml/kg will raise platelet count by 50—100 x109/L 3. Methods of treating a. Washing b. Single donor apheresis (for adults, all pediatric packs single donor) c. HLA matched 4. Refractoriness a. Repeated transfusions/ pregnancy  allo-immunization b. Try apheresis/ HLA platelets 5. Risks a. Plts are stored at room temperature for 5 days only - HIGHEST risk of bacterial contamination!!! *** - EXAM Q • The platelet count increase from 5 to 6 units of whole blood-derived platelets or 1 unit of apheresis platelets (200-300mL) will be approximately 30,000/microL in an average sized adult

Answer 9

Platelets - separate card 1. Fresh frozen plasma a. Plasma is separated from whole blood  contains all components except for RBC, slightly diluted b. Contains i. All clotting factors ii. Some fibrinogen c. Indications i. Warfarin ii. Liver disease with abnormal coagulation iii. Acute DIC when there is bleeding or abnormal coagulation iv. Massive transfusion or cardiac bypass v. Deficiencies of clotting factors 2, 5, 10, 11 d. Neonate indications i. Massive transfusion ii. Haemorrhage due to vit K deficiency iii. DIC with bleeding e. Cross matching = needs to be plasma compatible 2. Cryoprecipitate a. FFP is frozen and thawed, the bit that thaws on top = cryo b. Contains i. Fibrinogen ii. vWF/ factor VIII/XIII c. Indications i. Dysfibrinogenemia ii. DIC iii. vWD if no other options available - haemophilia A d. Note much smaller volume required than with FFP 3. Cryo deplete plasma a. FFP with cryo removed b. Has less vWF/ factor 8 c. Good for TP if replacing ADAMST13 4. Prothrombinex a. 2, 9, 10, 7 (vit K dependent factors) b. For Warfarin reversal 5. Note - for adequate clotting, one needs: a. 25-30% normal clotting factors b. Fibrinogen 75-100 mg/dL c. No inhibitors

Answer 10

* Definition = RBC count, Hb level and total RBC volume all exceed the upper limits of normal * Postpubertal = total RBC mass >25% over the mean normal value (based on BSA) or Hb >185g/L (in males) or >165g/L (females) indicate absolute erythrocytosis * Haemoconcentration/relative polycythaemia = RBC mass is not increased and normalization of the plasma volume restores Hb to normal levels ie. decrease in plasma volume ie acute dehydration or burns Neonates: HCT >0.65 Children: Depends on age Adults: HCT >0.49 (men) >0.48 (women) Blood viscosity and hct have a linear relationship when the hct is <60 percent [6,9]. This relationship becomes exponential when the hct exceeds 65 percent, such that a small increase in hct is associated with a dramatic increase in viscosity.

Answer 11

= clonal/primary polycythaemia 1. Key points a. Rare in children b. Acquired clonal myeloproliferative disorder c. Primarily manifests as erythrocytosis +/- thrombocytosis, leukocytosis d. Risk factors = FHx, autoimmune disorder (eg. Crohn’s) 2. Genetics + pathogenesis a. Gain of function mutation of JAK2 (a cytoplasmic tyrosine kinase) is found in more than 90% of adult patients, but in <30% of children b. The erythropoietin receptor is normal, and serum erythropoietin levels are normal or low c. In vitro cultures do not require added erythropoietin to stimulate growth of erythroid precursors 3. Clinical manifestations a. Hepatosplenomegaly b. +/- hypertension c. +/- headache d. +/- SOB e. +/- neurologic symptoms f. Agranulocytosis – may cause diarrhea or pruritis from histamine release g. Thrombocytosis (+/- platelet dysfunction) may cause thrombosis or haemorrhage 4. Diagnostic criteria a. Major i. Hb >185g/L (men) or >165g/L (women) OR Hb/HCT>99th percentile or reference range for age, sex, altitude of resistant OR Hb >170 g/L (men) or Hb >150 g/L (women) if associated with a sustained increase of ≥2 g/dL from baseline that cannot be attributed to correction of iron deficiency OR elevated red cell mass >25% above mean normal predicted value ii. Presence of JAK2 or similar mutation b. Minor i. Bone marrow trilineage myeloproliferation ii. Subnormal serum erythropoietin level iii. Endogenous erythropoietin level iv. Endogenous erythroid colony growth c. Diagnosis i. Both major criteria and one minor criteria OR first major criteria and 2 minor criteria 5. Treatment a. Phlebotomy = alleviate symptoms of hyperviscosity and decrease risk of thrombosis b. Iron supplementation = prevent viscosity problems from iron-deficiency microcytosis or thrombocytosis c. Marked thrombocytosis = antiplatelet agents (ie aspirin) may reduce risk of thrombosis and bleeding d. Anti-proliferative treatments (hydroxyurea, anagrelide, interferon- α) i. If unsuccessful or progressive hepatosplenomegaly from above therapies e. JAk-2 inhibitors = active area of investigation 6. Prognosis a. Transformation of the disease into myelofibrosis or acute leukemia is rare in children b. Prolonged survival is not unusual

Answer 12

• Definition = when polycythaemia is caused by physiologic process that is not derived from a single cell 1. Congenital a. Lifelong or familial polycythaemia should trigger search for congenital b. May be transmitted as dominant or recessive disorders c. Aetiology i. Autosomal dominant 1. Hemoglobins that have increased oxygen affinity (P50 <20 mm Hg) 2. Erythropoietin receptor mutations resulting in an enhanced effect of erythropoietin 3. Mutations in the von Hippel–Lindau gene that result in altered intracellular oxygen sensing. 4. Hemoglobin M disease (autosomal dominant) causes methemoglobinemia and can lead to polycythemia. a. Cyanosis may occur in the presence of as little as 1.5 g/dL of methemoglobin ii. Autosomal recessive 1. 2,3-diphosphoglyceric acid deficiency (rare) – left shift of oxygen dissociation curve, increased oxygen affinity and consequence polycythaemia 2. Congenital methemoglobinemia – autosomal recessive deficiency of cytochrome b5 reductase may cause cyanosis and polycythemia d. Clinical manifestations i. Asymptomatic ii. Neurologic abnormalities - present in patients whose enzyme deficits are not limited to hematopoietic cells iii. Cyanosis in Haemoglobin M disease – uncommon in other hemoglobin variants unless hyperviscosity results in localized hypoxemia 2. Acquired a. Usually due to chronic arterial oxygen desaturation. b. Aetiology i. Hypoxic 1. Cardiovascular defects involving R-L shunts and pulmonary diseases interfering with proper oxygenation – most common cause hypoxic polycythemia 2. Living at high altitudes –Hb level increases 4% for each rise of 1,000 m in altitude ii. Partial obstruction of a renal artery rarely results in polycythemia. iii. Benign and malignant tumors that secrete erythropoietin. iv. Exogenous or endogenous excess of anabolic steroids also may cause polycythemia. v. A common spurious cause is decrease in plasma volume such as in mod-severe dehydration c. Clinical manifestations i. Cyanosis ii. Hyperemia of the sclera and mucous membranes iii. Clubbing iv. As the hematocrit rises to >65%, clinical manifestations of hyperviscosity, such as headache and hypertension, may require phlebotomy 3. Treatment a. Mild disease – observation b. Phlebotomy – prevent or treat symptoms of headache, dizziness, exertional dyspnoea i. When haematocrit >65-70% (Hb>23g/L), blood viscosity markedly increases ii. Apharesed blood should be replaced with plasma or saline to prevent hypovolaemia in patients accustomed with a chronically elevated blood volume c. Iron supplementation i. Increased demand for RBC production may cause iron deficiency ii. Iron deficient microcytic red cells are more rigid - increasing the risk of intracranial and other thrombosis iii. Periodic assessment of iron status, with treatment of iron deficiency should be performed

Answer 13

1. Key points a. Defined at haematocrit >65% during first week of life – venous sample b. Relationship between viscosity and Hct is linear below 60-65% but increases exponentially above this level c. Hyperviscosity is related to increased resistance to blood flow therefore increased risk circulatory impairment 2. Physiology a. Polycythemia results from increased red cell mass, with decreased, normal or increased plasma volume b. Haematocrit peaks at 4-6 hours of life, then drops slowly to value at birth and stays stable 3. Aetiology a. Chronic intrauterine hypoxia i. SGA ii. Post-dates iii. Maternal hypertension / smoking b. Excessive transfusion of blood i. Placental transfusion during cord clamping ii. Twin to twin transfusion syndrome iii. Maternofetal transfusion c. Endocrine i. Infants of diabetic mothers ii. Congenital adrenal hyperplasia iii. Neonatal thyrotoxicosis d. Syndromic i. Down’s syndrome ii. BWS 4. Clinical manifestations a. Lethargy/poor feeding b. Plethora i. Hyperbilirubinaemia c. Hyperviscosity i. Respiratory – pulmonary hypertension, RDS ii. CVS – tachypnea, cyanosis, tachycardia, cardiomegaly iii. CNS – apnea, irritability, lethargy, convulsions iv. GIT – NEC v. Renal – impaired renal function vi. Metabolic –thrombocytopenia, hypoglycaemia, hypocalcaemia 5. Treatment a. Goal – decrease Hct to 50-55% b. Partial exchange transfusion i. If symptomatic or Hct >75% ii. Volume exchanges (mL) = Observed Hct – desired Hct / observed Hct iii. Reverses physiological abnormality and decreases symptoms but has not been shown to improve long term outcomes

Answer 14

1. Definition a. Characterized by RBCs with irregular circumferential pointed projections 2. Aetiology a. Liver disease + vitamin E deficiency – due to alterations in cholesterol:phospholipid ratio b. Congenital Abetalipoproteinaemia or hypoprebetalipoproteinaemia c. Fat malabsorption d. Neuromuscular abnormalities e. Retinitis pigmentosa f. Normoproteinemic neuroacanthocytosis i. Chorea-acanthocytosis 1. Autosomal recessive 2. The production of acanthocytes in chorea-acanthocytosis appears related to altered Lyn kinase activity with increased tyrosine phosphorylation and altered linkage of band 3 to other RBC membrane proteins. ii. McLeod syndrome 1. X-linked recessive, rare 2. Absence of the KX (Kell) antigen, late-onset myopathy, peripheral neuropathy, chorea, splenomegaly, and hemolysis with acanthocytosis. 3. There is usually >3% acanthocytes on peripheral smear and caudate atrophy noted on MRI. iii. Pantothenate kinase-associated neurodegeneration 1. Autosomal recessive 2. Dystonia, rigidity, chorea, dysarthria, spasticity, retinopathy) iv. Huntington disease–like 2 1. Autosomal dominant

Answer 15

1. Anatomy a. Splenic precursor recognizable by 5 weeks gestation. b. Size i. Birth = 11g ii. Puberty = 135g iii. Diminishes in size during adulthood c. 15% have an accessory spleen d. The major splenic components i. Lymphoid compartment (white pulp) = periarterial lymphatic sheaths of T lymphocytes with embedded germinal centres containing B lymphocytes ii. Filtering system (red pulp) = skeleton of fixed reticular cells, mobile macrophages, partially collapsed endothelial passages (cords of Billroth), and splenic sinuses. iii. Marginal zone separates the red pulp from the white pulp = rich in dendritic APCs iv. Splenic capsule = smooth muscle and contracts in response to adrenaline e. Vascular i. 10% of the blood delivered to the spleen flows rapidly through a closed vascular network ii. 90% flows more slowly through an open system (the splenic cords), where it is filtered through 1-5 µm slits before entering the splenic sinuses 2. Function a. Haematopoiesis (fetal) i. Major splenic function at 3-6 mo of fetal life but then disappears. ii. Splenic haematopoiesis can be resumed in patients with myelofibrosis or severe haemolytic anaemia b. Reservoir i. Factor VIII and 1/3 of circulating platelet mass are sequestered in the spleen  can be released by stress or adrenaline injection. ii. Thrombocytosis and leucocytosis occur with loss of the splenic reservoir function c. Filtering i. Spleen receives 5-6% of the cardiac output, but normally contains only 25 mL of blood 1. Can retain much more when it enlarges  cytopenias ii. Filtering function facilitated by slow blood flow past macrophages and through small openings in the sinus walls iii. Removal of excess membrane on young RBCs 1. Loss of this function  target cells, poikilocytosis, decreased osmotic fragility iv. Main site for destruction of old RBCs – assumed by other reticuloendothelial cells post splenectomy v. Removes damaged/abnormal RBCs (ie spherocytes, Ab-coated RBCs) and damaged platelets vi. Intracytoplasmic inclusions may be removed from RBCs without cell lysis 1. Howell-Jolly bodies = nuclear remnants 2. Denatured Hb = Heinz bodies d. Host defence i. Largest lymphoid organ in the body ii. Contains nearly half of the body’s total Ig-producing B lymphocytes iii. Processes foreign material to stimulate production of opsonizing antibody iv. Produces properdin and tuftsin v. Phagocytosis to trap and destroy intracellular parasites vi. Minor role in antibody response to IM or s/cut injected antigens but is required for early Ab production after exposure to IV antigens vii. The spleen may be an important site of antibody production in ITP viii. Encapsulated bacteria

Answer 16

1. Key points a. Splenic edge >2 cm below the left costal margin  abnormal b. In most the spleen must be 2-3 x normal size before it is palpable c. Soft, thin spleen palpable 15% of neonates, 10% normal children, 5% adolescents. 2. Investigations a. FBE b. Peripheral smear c. US, CT, or technetium-99 sulfur colloid scan (also assesses splenic function) 4. Pseudosplenomgaly a. Abnormally long mesenteric connections may produce a wandering or ptotic spleen b. An enlarged left lobe of the liver c. LUQ mass d. Splenic hematoma e. Splenic cysts may contribute to splenomegaly or mimic i. Congenital (epidermoid) ii. Acquired (pseudocyst) after trauma or infarction iii. Usually asymptomatic, found on imaging f. Splenosis after splenic rupture – most not palpable g. Accessory spleen (present in 15% of normal individuals) – most not palpable h. Congenital polysplenism syndrome – cardiac defects, left-sided organ anomalies, bilobed lungs, biliary atresia, and pseudosplenomegaly

Answer 17

``` Anatomical • Cysts • Hamartomas • Polysplenia • Haemangioma ``` Haematological – hyperplasia • Acute and chronic haemolysis • Chronic iron deficiency • Extramedullary haematopoeisis Storage disease • Lipidosis, mucopolysaccharidoses, mucolipidoses, defects in CHO metabolism etc. Immunological/ inflammatory • All autoimmune conditions Infections • Almost all infections can cause splenomegaly Malignancies • Primary – leukaemia, lymphoma, angiosarcoma, Hodgkin • Metastasis Other • Heart failure • Portal hypertension

Answer 18

1. Key points a. Increased splenic function (sequestration or destruction of circulating cells)  peripheral blood cytopenias, increased bone marrow activity, splenomegaly b. Usually secondary to another disease c. Treatment i. Underlying condition ii. Splenectomy 2. Congestive splenomegaly (Banti syndrome) a. Secondary to obstruction in the hepatic, portal or splenic veins  hypersplenism b. Aetiology i. Hepatic inflammation, fibrosis and vascular obstruction secondary to Wilson disease, galactosemia, biliary atresia, and α1-antitrypsin deficiency ii. Congenital abnormalities (absence or hypoplasia) of the portal or splenic veins iii. Splenic venous flow may be obstructed by masses of sickled erythrocytes leading to an infarction iv. Obliteration of these vessels secondary to septic omphalitis or thrombophlebitis (spontaneous or as a result of umbilical veins catheterization in neonates) c. Management i. If spleen site of vascular obstruction splenectomy cures hypersplenism ii. Obstruction usually is in the hepatic or portal systems  portocaval shunting may be more helpful, because both portal hypertension and thrombocytopaenia contribute to variceal bleeding

Answer 19

1. Aetiology Congenital absence • Associations – complex cyanotic heart defects, dextrocardia, bilateral trilobed lungs, heterotropic abdominal organs (Ivemark syndrome) Sickle cell disease • May have splenic hypofunction as early as 6 months of age • Initially caused by vascular obstruction  reversed with RBC transfusion or hydroxyurea • The spleen eventually autoinfarcts and becomes fibrotic and permanently nonfunctional Functional hyposplenism • Normal neonates, especially if premature • Malaria • Post-radiation • Reticuloendothelial function overwhelmed – severe haemolytic anaemia, metabolic storage disease Other • Autoimmune disorders = SLE, RA, GN etc • Oncohaematological disorders • GI disorders • Hepatic disorders • Infectious • Iatrogenic = methyldopa, steroids, TPN, irradiation • Amyloidosis 2. Investigations a. Peripheral film i. RBC inclusions (Howell-Jolly bodies or Heinz bodies) ii. ‘pits’ on interference microscopy b. Poor uptake on technetium or other spleen scans c. +/- reduced IgM memory B cells may also be detected and is a risk factor for overwhelming sepsis - IgM memory B cells dependent on spleen for suvival, produced in marginal zone

Answer 20

1. Clinical manifestations a. Small splenic capsular tears -> abdominal or referred left shoulder pain (diaphragmatic irritation by blood) b. Larger tears with severe blood loss -> similar pain and signs of hypovolaemic shock 2. Risk factors a. Enlarged spleens (ie infectious mononucleosis) -> more likely to rupture with minor trauma b. Patients with splenomegaly should avoid contact sports/other activities that increase risk of splenic trauma 3. Investigations a. FBE – serial tests b. CT IV contrast – best imaging modality to assess splenic trauma 4. Treatment a. Small capsular injury i. Observation ii. PRBC if required iii. Restricted activities b. More marked abdominal bleeding i. Laparotomy +/- splenectomy if clinical instability/deterioration, if other organ damage suspected ii. Partial splenectomy if possible

Answer 21

1. Indications a. Splenic rupture b. Anatomic defects c. Severe transfusion dependent haemolytic anaemia d. Immune cytopaenias e. Metabolic storage disease f. Secondary hyposplenism 2. Complications a. Sudden, overwhelming post-splenectomy infections (sepsis or meningitis) b. Thromboembolic complications i. Not dependent on indication for splenectomy or platelet count iii. Proposed mechanisms – loss of filtering function of the spleen, allowing abnormal RBCs to remain in the circulation and activate the coagulation cascade. 3. Splenosis a. Splenosis refers to implants of splenic tissue resulting from spillage of cells following abdominal trauma or surgery. b. Up to 50% of children whose spleen is removed because of trauma have spontaneous splenosis c. Surgical splenosis (distributing small pieces of spleen throughout the abdomen) may decrease the risk of sepsis in patients whose splenectomy is necessitated by trauma d. The splenic tissue that regrows frequently has poor function

Answer 22

1. Overview a. Register with Spleen Australia b. Education c. Vaccination d. Antibiotic prophylaxis e. Rapid treatment of infections 2. Post-splenectomy sepsis a. Increased risk if children <5 years at the time of surgery i. Lifelong risk 5% - most occur within 2 year after splenectomy b. Risk impacted by indication i. Lower risk (2-4%) = trauma, RBC membrane defects, immune thrombocytopaenia ii. High risk (8-30%) = haemaglobinopathies, pre-existing immune deficiency (eg. Wiskott-Aldrich), reticuloendothelial blockade (eg. storage disease, severe haemolytic anaemia) c. Organisms i. Encapsulated bacteria = 80% (SHiNE SKiS = Strep pneumo, Haem influenzae, Neisseria, E. coli, Salmonella, Klebsiella, GBS) 1. Streptococcus pneumoniae >60% 2. Haemophilus influenzae 3. Neisseria meningitides ii. Protozoal infections – malaria and babesiosis iii. Capnocytophaga canimorsus or C. cynodegmi following animal bite d. Management i. Rapid treatment if febrile – emergency antibiotics at home ii. Broad spectrum cephalosporin (cefotaxime or ceftriaxone) iii. +/- vancomycin (to cover penicillin-resistant pneumococci)

Answer 23

a. Vaccination i. Timing 1. Elective = two weeks before scheduled operation 2. Emergency = >1 week after surgery ii. Pneumococcal 1. Prevenar 13 a. Primary course as per immunisation schedule b. One additional dose at >=12 months of age 2. Prevenar 23 a. One dose at 4-5 years of age b. Booster 5 years post initial dose iii. Meningococcal 1. Quadrivalent – MenA, C, W, Y135 2. Meningococcal B iv. Haemophilius 1. Primary course as per immunisation schedule 2. No booster required v. Annual influenza vaccine = influenza is a risk factor for secondary pneumococcal infections b. Prophylaxis i. Options 1. Oral amoxicillin 20 mg/kg daily 2. Oral phenoxymethylpenicillin (penicillin V) a. <5 years = 125 mg twice daily b. >5 years = 250 mg twice daily 3. Start prophylaxis in children with sickle cell as soon as diagnosed ii. Duration 1. Until 16 years of age 2. Minimum a. Up to the age of 5 years in children with Asplenia b. Up to the age of 5 years in patients who have hyposplenism due to sickle cell anaemia or other congenital haemaglobinopathy c. At least 3 years after splenectomy 3. Lifelong a. Severely immunosuppressed b. Splenectomy for haematological malignancy, particularly those with ongoing immunosuppression c. Episode of severe sepsis – particularly after 2nd c. Emergency antibiotics i. Augmentin 22.5 mg/kg orally twice daily

Answer 24

1. Definition = any cause of reduced haemoglobin below normal range for age and sex 2. Consequences a. ↓ O2 carrying capacity b. Tissue hypoxia c. Compensation i. Cardiac overactivity ii. Cardiorespiratory failure iii. Vasoconstriction with redistribution of blood flow to tissues with high oxygen dependence 3. Symptoms/signs suggestive of anaemia a. Pallor b. Pale conjunctivae c. Flow murmur d. Lethargy e. Poor growth f. Signs of cardiac failure g. Weakness h. Listlessness i. Shortness of breath 4. Classification a. Microcytic, normocytic or macrocytic b. Decreased production, increased destruction or blood loss i. Reticulocyte % or absolute number helpful in making distinction ii. Normal reticulocyte percentage of total RBCs= 1% 5. Investigations a. Every child i. FBE + Film ii. Reticulocyte count = indicates whether marrow is responding appropriately b. Further investigations dependent on MCV ``` Microcytosis - Iron deficiency - Thalassemia - Hereditary spherocytosis - Rare – sideroblastic anaemia (XLR, lead) ``` Normocytic, normochromic - Blood loss - Mixed nutritional deficiency - TEC - Anaemia chronic disease ``` Macrocytosis - B12/folate deficiency - Brisk reticulocytosis/ haemolysis - MDS - Fanconi’s/ Diamond Blackfan Anaemia - Congenital dyserythropoetic anaemia, osteoporosis ``` ``` Reduced production • Haematinic deficiency • Marrow failure • Marrow replacement • Anaemia of chronic disease • Ineffective erythropoiesis • Dyserythropoiesis ``` ``` Increased destruction • Immune o Autoimmune o Alloimmune • Non-immune o Inherited = Hb, membrane, enzyme o Other  Physical damage = MAHA, thermal, cardiac defects  Infectious agents = malaria ``` Increases losses • Bleeding – occult or massive

Answer 25

1. Bone marrow failure a. Congenital pure red cell aplasia i. Diamond-Blackfan anaemia ii. Aase syndrome b. Acquired pure red cell aplasia i. Autoimmune ii. Infections iii. Drugs iv. Transient erythroblastopaenia of childhood c. Malignancy d. Bone marrow failure syndromes i. Aplastic anaemia ii. Fanconi’s anaemia iii. Others e. Myelofibrosis i. Renal failure ii. Vitamin D deficiency iii. Hypoparathyroidism f. Bone disease = osteopetrosis 2. Impaired EPO production a. Hypothyroidism b. Starvation c. Chronic renal disease d. Anaemia of chronic disease 3. Disorders of erythroid maturation/ ineffective erythropoiesis a. Iron deficiency anaemia b. Sideroblastic anaemia c. Lead toxicity d. Megaloblastic anaemia e. Congenital dyserythropoetic anaemia

Answer 26

TAILS ``` Thalassaemia Anaemia of chronic disease Iron deficiency Lead poisoning Sideroblastic anaemia ```

Answer 27

1. Epidemiology a. Most common nutritional deficiency in children + most common cause of anaemia in children i. IDA caused by diet, most commonly occurs at 9-24 months in term infants b. Usually nutritional (insufficient red meat, fish, chicken, green vegetables, pulses; excessive cow's milk); rarely due to malabsorption or GI bleeding c. Can lead to reduced cognitive and psychomotor performance in the absence of anaemia 2. Physiology a. Most iron in neonates is in circulating Hb b. As the relatively high Hb concentration of the newborn infant falls during the first 2-3 months of life considerable iron is recycled – iron stores sufficient for blood formation in first 6-9 months of life c. Stores are depleted sooner in LBW infants, or infants with perinatal blood loss d. Delayed cord clamping (1-3 minutes) improves iron status and reduces risk of iron deficiency anaemia e. Iron absorption i. Absorbed in the duodenum in Fe 2+ (ferrous form), active transport across apical via DMT1 transporter (1-2 mg/day) ii. Once inside enterocyte, leaves via ferroportin transporter f. Regulation i. Enhanced by gastric acid, vitamin C, breast milk ii. Decreased by bovine milk proteins, egg whites, phytates, bran, calcium, zinc and lead iii. Absorption upregulated if liver stores are low – via hepcidin: if liver stores are full, hepcidin increases  binds to ferroportin and stops iron absorption g. Transport + storage i. Transported in blood bound to transferrin ii. Stored in liver and bone marrow (reticuloendothelial macrophages) iii. Body has minimal means of LOSING iron aside from blood loss, sloughed mucosal cells, menstruation iv. Iron stores last 5-6 months 3. Daily requirements a. Term infant body has 0.5 g of iron (compared to adult total 5g) b. Infants require 1 mg daily (daily intake of 8-10 mg as < 10% absorbed) c. Breast milk contains 1 mg/L of iron 4. Phases of iron deficiency a. Iron depletion = low ferritin, normal Hb and indices b. Iron deficiency = low ferritin and indices, Hb normal c. Iron deficiency anaemia

Answer 28

5. Clinical manifestations a. Most children are asymptomatic and are identified through screening b. Pallor most important clinical sign – only apparent when Hb falls to 70-80 g/L c. Irritability, anorexia, lethargy and flow murmurs – when Hb falls to <50 g/L d. Consequences i. Impaired neurocognitive function in infancy ii. Possible link with seizures, strokes, breath holding and exacerbation of RLS iii. Poor growth iv. Exercise intolerance v. Pica 6. Aetiology a. Low stores i. Prematurity b. Low intake i. Prolonged breast feeding without introduction of solids >6months ii. Cow’s milk in first year – increase intestinal blood loss, decrease iron absorption c. Blood loss i. Focal lesion – peptic ulcer, Meckel’s, haemangioma ii. GIT disease – IBD, Celiac, cow’s milk protein enteropathy iii. Hookworm infestation 7. Risk factors based on age a. Perinatal i. Maternal iron deficiency ii. Prematurity iii. Administrating of EPO for anaemia of prematurity iv. Perinatal haemorrhagic events (eg. TTTS or fetal-maternal haemorrhage, placenta praevia) b. Infancy i. Dietary factors 1. Lack of iron supplements for breastfed infants 2. Use of low iron infant formula 3. Feeding of unmodified (non-formula) cow’s milk, goat’s milk or soy milk 4. Insufficient iron rich complementary foods – should be introduced by 6 months ii. Other risk factors 1. Disorders with GI blood loss – eg. milk protein induced proctocolitis 2. Malabsorptive disease c. 1 to 12 years i. Dietary risk factors 1. Excessive intake of cow’s milk – no more than 500ml per day 2. Insufficient iron in foods ii. Other risk factors 1. Disorders with GI blood loss (eg. inflammatory bowel disease or chronic gastritis) 2. Malabsorptive disease eg. celiac, worms 3. Obesity

Answer 29

8. Investigations a. FBE = low MCV, elevated RDW, common to have thrombocytosis b. Film = ‘cigar cells’, target cells , tear dropping, anisocytosis c. Iron studies i. L ferritin -> L iron -> H transferrin -> L transferrin sat -> L Hb -> L MCV ii. Serum iron is biphasic and unreliable except to monitor compliance with replacement therapy iii. Ferritin 1. If low = deficient 2. If normal = can reflect acute phase reaction iv. Transferrin usually elevated in Fe deficiency v. Soluble transferrin receptors (present on erythroid cells) 1. Ratio of transferrin receptor: ferritin > 2 suggestive of iron deficiency anaemia 2. Useful In patients with chronic inflammation d. Bone marrow – iron stain can also be performed 9. DDx a. Lead poisoning i. Elevated whole blood lead ii. Film = basophilic stippling b. Alpha/beta trait i. Diagnosis of exclusion ii. If <6months – will NOT be iron deficiency or B thal (no B chains yet) so microcytic anaemia either A thal or blood loss iii. Thal vs IDA 1. RCC – normal or elevated in thalassaemia, low in IDA 2. MCV – low MCV out of proportion to degree of anaemia in thalassaemia 3. RDW – normal in thalassaemia, elevated in IDA (anisocytosis) c. Chronic disease i. Ferritin usually elevated ii. Serum transferrin receptor levels may be useful – not affected by inflammation, increased in iron deficiency (very sensitive), normal in anaemia of chronic disease

Answer 30

10. Management a. Iron supplementation i. Aim for iron supplementation at 2 - 6mg/kg/day of elemental iron ii. 2mg/kg/day is the preventative dose for iron deficiency and also effective in mild-moderate deficiency 1. The higher range doses are usually only necessary for severe deficiency, and iron studies should be monitored carefully to prevent overload 2. Higher doses should be divided (up to tds) to reduce gastric irritation iii. Supplementation usually = reticulocyte response in 48-72 hours iv. IV therapy = poor compliance, inability to tolerate due to GI issues v. Assessing response 1. 12-24 hours = subjective improvement 2. 72 hours = reticulocytosis 3. 7-10 days = Hb levels increase 4. 1-3 months = repletion of stores – always treat for 3 months vi. Cause of poor response 1. Non-compliance (++++) 2. Ongoing losses 3. Insufficient duration 4. High gastric pH 5. Inhibitors of absorption 6. Incorrect diagnosis – thalassaemia, chronic disease, sideroblastic anaemia b. Prevention i. Encourage breast feeding – then transition to additional source of iron (E.g. cereals) ii. In non-breast fed infants use iron-fortified formula iii. Encourage vitamin C intake iv. Introduce iron rich foods from 6 months (E.g. meats) v. Avoid unmodified cow’s milk until 12 months

Answer 31

1. Defect in iron absorption a. Iron refractory iron deficiency anaemia b. Defect in transmembrane proteins, autosomal recessive c. Unresponsive to oral iron, partially responsive to IV 2. Defects of iron recycling a. Aceruloplasminemia – iron cannot be appropriately transported from macrophages to plasma 3. Defects in mitochondrial iron utilization = sideroblastic anaemia

Answer 32

1. Definition = ring sideroblasts are present on the BMA stained for iron 2. Aetiology a. Hereditary i. XLR – most common form ALAS gene X chromosome 1. Presents during late childhood 2. Splenomegaly 3. Respond to B6 ii. Autosomal = dominant or recessive iii. Pearson Syndrome 1. Refractory sideroblastic anemia – MACROCYTIC not microcytic 2. Exocrine pancreatic dysfunction b. Acquired i. Myelodysplasia ii. Nutritional (copper, B6 deficiency) or toxins (lead, zinc) iii. Drugs (EtOH, isoniazid, chloramphenicol) iv. Hypothermia v. Uremia vi. Hyperthyroidism 3. Congenital sideroblastic anaemia a. Germline mutation in nuclear or mitochondrial genes b. 1/3 do not have an identifiable gene c. Genetics + pathogenesis ii. Impaired heme synthesis 1. Defective steps in heme synthesis in cytoplasm = porphyria 2. Defective steps of heme synthesis in mitochondrion = sideroblastic anaemia a. d-ALA or ferrochelatase  incorporation of iron into porphyrin ring b. Accumulation of iron in mitochondria of nucleated RBC (sideroblasts) d. Clinical manifestations i. Severe anaemia– infancy, milder forms – early adulthood ii. Pallor, icterus iii. Iron overload without transfusion history iv. Moderate splenomegaly and/or hepatomegaly e. Investigations i. FBE – hypochromic, microcytic, high RDW (microcytic RBC mixed with normal) ii. Film – ringed sideroblasts iii. Iron studies – ↑ serum iron ↑ ferritin ↑ transferrin saturation; ↓ transferrin f. Treatment i. Severity of anaemia varies – some require no treatment, some regular RBC transfusions ii. Stem cell transplant in transfusion dependent iii. Vitamin B6 – X linked form responsive iv. Management of Fe overload

Answer 33

``` • Interferes with heme synthesis including D-ALA and ferrochelatase -> microcytic hypochromic anaemia • Coarse basophilic stippling • Elevated Lead levels + protoporphyrins • Clinical features o Anaemia o Discolouration of gums o Abdominal pain o Peripheral neuropathy ```

Answer 34

1. Normal newborn 2. Massive reticulocytosis 3. Megaloblastic anaemia a. Nutritional b. IEM c. Drug effect eg. azathioprine 4. Liver disease 5. Hypothyroidism

Answer 35

1. Overview a. Megaloblastic anaemia= group of disorders cause by impaired DNA synthesis b. RBCs are larger than normal at every developmental stage and there is maturational asynchrony between the nucleus and cytoplasm c. Delayed nuclear development becomes increasingly evidence as cell divisions proceed d. Myeloid and platelet precursors are also affected 2. Investigations a. FBE: MCV >100fL, +/- thrombocytopaenia, leukopenia b. Film i. Large RBCs (often oval) ii. Hypersegmented neutrophils, many have >5 lobes c. Bone marrow: +/- metamyelocytes and neutrophil bands 3. Aetiology a. Majority – folic acid or vitamin B12 deficiency (cobalamin) – vitamins essential for DNA synthesis b. Rarely – inborn errors of metabolism

Answer 36

a. Source i. Found in animal products only (meat, eggs, fish and milk)– synthesized by microorganisms ii. B12 is a generic term encompassing all biologically active cobalamins iii. Methylcobalamin and adenosylcobalamin are the metabolically active derivatives b. Absorption i. Stomach 1. Chief cells  pepsinogen  pepsin  breaks down protein to release B12 2. B12 binds to R protein (also called haptocorrin = HC, salivary glycoprotein) ii. Duodenum 1. Lipase/amylase/protease degrade protein R 2. Gastric antrum parietal cells  intrinsic factor 3. IF + B12 iii. Ileum = absorption of B12 + IF via receptor mediated endocytosis c. Transport i. Transported in plasma bound to transcobalamin II (required to transport cobalamin into cells) d. Roles i. Serve as cofactors in 2 essential metabolic reactions 1. Methylation of homocysteine to methionine (via methionine synthase) 2. Conversion of methyl-malonyl-coenzyme A (CoA) to succinyl CoA (via L-methyl-malonyl-CoA mutase) ii. Product and byproducts of these enzymatic reactions are critical to DNA, RNA and protein synthesis iii. Deficiency = high homocysteine, high MMA  causes neurological regression e. Requirements + Stores i. Require 6-9 mcg / day ii. Older children and adults usually have 3-5 year stores of vitamin B12 iii. Young infants may manifest symptoms as early as 6- 18 months

Answer 37

``` 2. Aetiology by age Birth-6 months • Severe maternal deficiency • Metabolic causes 6 months- mid childhood • Dietary deficiency • Maternal deficiency (deficiency in BF infants) • Malabsorption Mid childhood onwards • Juvenile pernicious anaemia • Gastritis • Malabsorption • Medication ``` 3. Aetiology a. Reduced intake = breastmilk in vitamin B12 deficient mothers, vegan diet b. Impaired absorption i. Gastric abnormalities 1. Pernicious anaemia – Ab to IF or gastric parietal cells 2. Hereditary intrinsic factor deficiency 3. Gastrectomy/ bariatric surgery 4. Gastritis 5. Autoimmune atrophic gastritis ii. Small bowel disease 1. Malabsorption syndrome 2. Ileal resection or bypass 3. IBD eg. Crohn’s 4. Celiac disease 5. Bacterial overgrowth 6. Tapeworm iii. Drugs which block or impair absorption = neomycin, Biguanides (metformin), PPI + H2 antagonists iv. Pancreatic disease = insufficiency c. Impaired transport = inherited transcobalamin II deficiency d. Impaired utilisation i. Methylmalonic acidurais ii. Methylcobalamin deficiency

Answer 38

5. Investigations a. Film i. Hyper segmented neutrophils (>5 neutrophils with >5 lobes) ii. Oval macrocytes iii. Macrocytic anaemia +/- leukopenia +/- thrombocytopenia iv. +/- teardrop cells b. Active (holotranscobalamin) = measures TCII/B12 i. Most sensitive ii. Measurement of total B12 is NOT sensitive or specific c. If B12 deficiency is confirmed i. Urinary methylmalonic acid (MMA) and serum homocysteine 1. One or both are elevated in almost all patients with clinical deficiency, but decrease immediately after treatment 2. Homocysteine (ONLY) may also be elevated in folate deficiency d. Haemolysis screen – LDH often very high e. Iron studies + red cell folate – for coexistant deficiency f. Bone marrow aspirate usually not necessary i. If one shows hypercellular – left shift ii. Megaloblasts + giant metamyelocytes 6. Management a. For infants and those with neurological involvement – standard replacement is IM B12 (cyanocobalamin) b. Oral doses are poorly absorbed (0.5-4%) c. High doses can be effective for lower risk cases (ie. older children/adults WITHOUT evidence of tissue deficiency ie. no clinical features and normal homocysteine/MMA) d. Regimens vary i. Infants with clinical deficiency (macrocytic anaemia or neurological involvement) 1. 250 - 1000mcg intramuscular B12, on alternate days for 1-2 weeks, then 250mcg weekly 2. Short-term parenteral therapy is often sufficient, especially if maternal deficiency is proven 3. Switch to oral supplements once child is well, no diarrhoea, feeding improved, and maternal stores replaced ii. Older children with mild disease an alternative would be: 1000mcg oral daily iii. Subclinical, dietary deficiency 1. 50-200 mcg oral daily (generally as 100mcg tablets, also available as sublingual sprays/tablets). 2. Increase dietary intake iv. Supplement (not deficient, no dietary intake) 1. 50-100mcg daily or alternate daily v. Pernicious anaemia – oral B12 1000 ug/ day

Answer 39

• AR failure to absorb and transport B12 • Serum B12 levels are normal – need to check “active B12” o >80% of serum cobalmin is bound to haptocorrin (HC) • Manifests first week of life – FTT, diarrhoea, vomiting, glossitis, neurologic abnormalities + megaloblastic anaemia • Diagnosis o Severe megaloblastic anaemia o Normal serum B12 and folate levels o No evidence of any other inborn errors of metabolism • Treatment o Large parenteral doses of B12 – ‘overcomes’ transcobalamin deficiency o Death in infancy if untreated

Answer 40

1. Background a. Folates are essential for DNA replication and cellular proliferation b. Biologically active folates are derived from folic acid (pteroic acid conjugated to glutamic acid) and serve as donors and acceptors in biosynthetic pathways c. To form functional compounds – folates must be reduced to tetrahydrofolates (by enzyme – dihydrofolate reductase) 2. Physiology a. Source i. Widely available from food – 1/3 meat and fish, 1/3 cereals and bread, 1/3 fruit and vegetables (may be less in cow’s milk) ii. Folates are heat labile and water soluble – decreased amounts of vitamin with heating/boiling iii. NO folate in goat’s milk b. Folic acid  monoglutamated, naturally occurring folates  polyglutamated c. Absorption i. Polyglutamated form – less efficiently absorbed than monoglutamated ii. Dietary folate polyglutamates are hydrolysed to simple folates iii. Primarily absorbed in the proximal small intestine iv. Folates travel in blood stream and are taken into cells as unconjugated methyltetrahydrofolate v. Subsequently reconjugated/polyglutamated in the cell d. Function i. Folates act in numerous single carbon reactions 1. Synthesis of methionine from homocysteine 2. Purine and pyrimidine metabolism ii. Circulates in plasma as 5-methyl THF iii. Body folate stores limited to several weeks 1. Acute folate deficiency may develop in hospitalized patients

Answer 41

a. Inadequate folate intake i. Increased requirements 1. Pregnancy a. Supplementation recommended to prevent NTD b. Folate-deficient mothers generally do not give birth to infants with folate deficiency due to selective transfer of folate to the fetus via placental folate receptors 2. Accelerated growth after birth a. Increases demands for folic acid b. Premature, unwell infants and those with certain hemolytic disorders will have particularly high folate requirements. c. Dietary sources – goat’s milk inadequate 3. Haemolysis ii. Malnutrition 1. Most common cause in older children 2. Increased risk – patients with hemoglobinopathies, infections, and/or malabsorption 3. Body stores of folate are limited; deficiency can develop quickly in malnourished individuals a. On a folate-free diet, megaloblastic anaemia will occur after 2-3 months b. Decreased folate absorption i. Chronic diarrheal states – chronic infectious enteritis ii. Diffuse inflammatory disease iii. Coeliac disease iv. Enteroenteric fistulas v. Previous intestinal surgery vi. Certain anticonvulsant drugs – phenytoin, primidone, phenobarbital vii. Alcohol overuse c. Acquired and congenital disorders of folate metabolism or transport i. Hereditary folate malabsorption (HFM) b. Inability to absorb folic acid and derivatives ii. Enzyme deficiencies (extremely uncommon) 1. Functional methionine synthase deficiency 2. Dihydrofolate reductase deficiency 3. NOTE: methylenetetrahydrofolate (MTHFR) deficiency is the MOST common inborn error of folate metabolism iii. Drug induced 1. Methotrexate – prevents formation of active form of folate 2. Pyrimethamine, trimethoprim – folic acid deficiency

Answer 42

4. Incidence a. Megaloblastic anaemia as a consequence of folate deficiency is rare b. Peak incidence 4-7 months (earlier than iron deficiency) 5. Clinical manifestations a. Irritability (older – depression, dementia, psychosis) b. Chronic diarrhea c. Poor weight gain d. Advanced – haemorrhages from thrombocytopaenia 7. Treatment a. MUST exclude vitamin B12 deficiency before treating b. Folic acid – oral or parenteral for 3-4 weeks, 0.5-0.1mg/day c. Haematological response in 72 hours d. Maintenance therapy – multivitamin e. Preconception folate supplements for prevention NTD i. Fefol/FGF inadequate in pregnant women with increased folate requirements

Answer 43

a. FBE = macrocytic anaemia, low reticulocytes +/- neutropaenia +/- thrombocytopaenia i. Note: acute folate deficiency not macrocytic b. Film = nucleated RBC with megaloblastic morphology, variation in RBC size/shape, neutrophils large some with hypersegmented nuclei c. Bone marrow i. Hypercellular (due to erythroid hyperplasia) ii. Megaloblastic changes prominent iii. Large, abnormal neutrophilic forms (giant metamyelocytes) with cytoplasmic vacuolation d. Serum folic acids levels = < 3ng/ml (normal 5-20ng/ml) e. RBC folate= is a better indicator of chronic deficiency (normal 150-600ng/ml) f. Iron = normal/elevated g. Vitamin B12 = normal/elevated h. LDH (marker of ineffective erythropoiesis) = markedly elevated i. Homocysteine = elevated j. MMA = normal

Answer 44

a. Rare AR disorder of megaloblastic anaemia b. Presents in 1st yr of life c. Most common metabolic error in the de novo synthesis of pyrimidines and therefore affects nucleic acid synthesis d. Clinical manifestations i. Growth failure ii. Developmental retardation e. Investigations i. Severe megaloblastic anemia ii. Normal serum B12 and folate levels iii. No evidence of TC deficiency iv. Increased urinary orotic acid f. Treatment i. Responds promptly to administration of uridine (refractory to B12 or folic acid)

Answer 45

Thiamine-responsive megaloblastic anaemia a. Very rare autosomal recessive disorder b. Clinical manifestations i. Megaloblastic anemia ii. Sensorineural deafness iii. Diabetes mellitus iv. Thiamine-responsive megaloblastic anemia usually presents in infancy c. Investigations i. Bone marrow – megaloblastic changes, ringed sideroblasts. d. Treatment i. Thiamine supplementation = reverses the anemia and diabetes but not existing hearing defect

Answer 46

Inherited red cell aplasia Ribosomopathy 1. Key points a. Rare, congenital bone marrow failure syndrome b. Up to 50% of affected individuals have additional extrahematopoietic anomalies 2. Genetics + pathogenesis a. Predominantly AD d. Mutations in 1 of the 10 ribosomal protein genes (RP) identified in 50-70% of cases e. DBA is a ribosomopathy - mechanism by which RP haploinsufficiency leads to DBA is unclear Key features - anaemia (normochromic, macrocytic) - reticulocytopenia - insufficient/absent RBC precursors in BM - congenital anomalies

Answer 47

3. Clinical manifestations a. Usually becomes symptomatic in early infancy, >90% of cases recognized before 1 year of life b. Anaemia (normochromic and macrocytic) + reticulocytopaenia i. Profound anaemia evident by 2-6 months of age ii. 25% of patient’s anemic at birth and hydrops fetalis occurs rarely c. Congenital abnormalities = 50% i. Craniofacial (50%) – hypertelorism, snub nose, high arched palate ii. Skeletal anomalies (30%) – mostly upper limb and hand 1. Thumb abnormalities – flattening of thenar eminence and triphalangeal thumb 2. Radial pulse may be absent iii. Genitourinary (38%) = absent kidney, horseshoe kidney, hypospadias iv. Cardiac (30%) = VSD, ASD, CoA, complex cardiac disease v. Ophthalmologic vi. Musculoskeletal = growth retardation, syndactyly d. Short stature common – unclear if disease related vs treatment e. Malignancy i. Cancer predisposition syndrome ii. Increased risk of myelodysplastic syndrome, AML, colon carcinoma, osteogenic sarcoma, female genetic cancers 4. Investigations a. FBE = normochromic macrocytic anaemia i. +/- thrombocytosis, thrombocytopenia (rare) and occasionally neutropenia ii. Low reticulocyte percentages b. Blood film = red cell patterns similar to foetal population – increased fetal Hb and expression of ‘I’ antigen c. Bone marrow = erythrocyte precursors markedly reduced in most i. NORMAL myeloid/megakarocyte lines d. Serum iron levels = elevated e. HbF = often elevated f. Erythrocyte adenosine deaminase (ADA) activity = increased in most

Answer 48

a. Anaemia with low reticulocyte count  transient erythroblastopaenia of childhood (TEC) i. Relatively late onset, but occasionally onset <6mo of age ii. Don't typically see macrocytosis, congenital anomalies, fetal red cell characteristics, elevated erythrocyte ADA b. Macrocytic bone marrow failure syndromes = FA, SDS, DC, MDS c. Haemolytic disease of the newborn i. Can mimic features of DBA if protracted course and coupled with markedly reduced erythropoiesis ii. Anaemia usually resolves spontaneously 5-8 weeks of age d. Aplastic crisis in chronic haemolytic disease i. Reticulocytopaenia and decreased numbers of RBC precursors ii. Often occurs after first several mo of life iii. Often caused by parvovirus B19 in utero = pure RBC aplasia in infancy, hydrops fetalis at birth

Answer 49

6. Treatment a. Mainstay = corticosteroids i. 80% of patients initially respond ii. RBC precursors in BM within 1-3 weeks; followed by reticulocytosis iii. Hb can reach normal limits in 4-6 weeks iv. Adequate response = Hb >9 v. Aim lowest dose to maintain Hb vi. Side effects – reduced BMD, cataracts/glaucoma, avascular necrosis fem head vii. Other – Bactrim prophylaxis, PPI, stress steroid regime when unwell b. Transfusions i. For non-responders OR fail to tolerate side effect profile ii. Transfusions every 3-5 weeks c. Spontaneous remission has been reported d. Haematopoietic stem cell transplantation (HSCT) can be curative i. Best outcomes in HLA matched sibling donors < 9 years of age 7. Prognosis a. Actual survival – 75% at 40 years (87% maintained on steroids) and 57% for transfusion dependent patients b. Deaths i. Treatment related (67%) ii. Malignancy or severe aplastic anaemia (22%)

Answer 50

Diamond Blackfan Anaemia versus Transient erythroblastopenia of childhood ``` DBA Age: <1 year Antecedent history: None Physical anomalies: 1/3 MCV: Increased Haemaglobin F: Increased i-Antigen: Increased ``` ``` TEC Age: >1 year Antecedent history: Viral illness Physical anomalies: None MCV: Normal HbF: Normal i-Ag: Normal ```

Answer 51

1. Background a. Rare mitochondrial disorder which presents with hypoplastic anaemia 2. Genetics + pathogenesis a. Mitochondrial DNA deletion of variable size and location b. Variable clinical picture – heterogeneity in different tissues and patients c. Proportion of deleted mtDNA in BM correlated to severity of hematological picture 3. Clinical a. Marrow failure typically appears in neonatal period b. Multi-organ involvement i. FTT ii. Exocrine pancreas dysfunction iii. Liver and renal tubular defects iv. Malabsorption v. Myopathy 4. Investigations a. FBE = macrocytic anaemia, occasionally neutropaenia and thrombocytopaenia b. Bone marrow = vacuolated erythroblasts and myeloblasts, ringed sideroblasts (unique variant of congenital sideroblastic anaemia) c. Elevated haemoglobin F level 5. DDx a. Diamond Blackfan anaemia b. Transient erythroblastopaenia of childhood c. Kearns-Sayre syndrome – similar mitochondrial DNA deletion 6. Treatment a. Supportive b. Red cell transfusions to correct anaemia c. Granulocyte colony stimulating factor to reverse episodes of severe anaemia

Answer 52

1. Key points a. Mainly occurs previously healthy children between 6 mo-3 years b. More prevalent than congenital hypoplastic anaemia (Diamond-Blackfan) 2. Aetiology a. Suppression of erythropoiesis linked to IgG, IgM and cell mediated mechanism b. Familial cases reported – suggesting hereditary component c. Often follows viral illness (no specific virus implicated) d. Rarely caused by parvovirus induced RBC aplasia in children with hemolytic anaemia or congenital or acquired immunodeficiencies 3. Clinical manifestations a. Severe, transient hypoplastic anaemia b. Most older than 12mo at onset c. Anaemia develops slowly, significant symptoms only with severe anaemia 4. Investigations a. FBE i. Reticulocytopaenia, mod-severe normocytic anaemia (due to temporary suppression of erythropoiesis), MCV normal for age ii. Some degree of neutropaenia in 20% iii. Platelets normal or elevated - thrombocytosis presumably caused by increase erythropoietin, which has some homology with thrombopoietin b. HbF levels = normal before recovery phase c. RBC adenosine deaminase levels = normal (cf elevated in congenital hypoplastic anaemia) 5. Differential diagnosis a. Congenital hypoplastic anaemia i. Differences in age at onset, age-related MCV, HbF and adenosine deaminase b. Iron deficiency anaemia in infants receiving milk as main caloric source i. Peak occurrence at similar age ii. Differences in MCV 6. Prognosis a. Virtually all children recover within 1-2 months 7. Treatment a. RBC transfusions if severe anaemia

Answer 53

1. Key points a. Best-documented viral cause of RBC aplasia in patients with chronic haemolysis, immunocompromised and fetuses in utero b. Does NOT cause significant anaemia in immunocompetent individuals with normal red cell life spans 2. Pathogenesis a. Small, non-enveloped single-stranded virus is particularly infective and cytotoxic to marrow erythroid progenitor cells – binding to red cell P antigen 3. Clinical manifestations a. Causes erythema infectiosum (fifth disease) b. Usually transient with recovery occurring in <2 weeks c. Anaemia either not present or not appreciated in otherwise normal children whose peripheral RBC life span is 100-120 days 4. Investigations a. Parvovirus IgG/IgM + PCR b. Decreased/absent erythroid precursors c. Bone marrow i. Characteristic nuclear inclusions in erythroblasts ii. Giant pronormoblasts

Answer 54

a. Chronic haemolysis i. RBC lifespan is shorter in patients with haemolysis (eg. spherocytosis, immune haemolytic anaemia, sickle cell) ii. Brief cessation of erythropoiesis can cause severe anaemia – aplastic crisis iii. Parvovirus-induced aplastic crisis usually occurs only once in children with chronic haemolysis iv. Recovery from mod-severe anaemia usually spontaneous, heralded by a wave of nucleated RBCs and subsequent reticulocytosis in the peripheral blood v. Treatment = RBC transfusion if symptomatic from anaemia b. Immunodeficiency i. Chronic parvovirus infection in immunosuppressed children ii. Resultant pure RBC aplasia may be severe, and affected children may be thought to have TEC – no spontaneous recovery and >1 transfusion required iii. Note PCR usually required for diagnosis as serology impaired iv. Treatment = high dose IVIG – contains neutralizing Ab to parvovirus and is effective in short term c. Miscarriage and hydrops fetalis i. Parvovirus infection and destruction of erythroid precursors in utero ii. Associated with increased fetal wastage in the first and second trimesters iii. Born with hydrops fetalis and anaemia iv. Investigations 1. Immunologic tolerance to the virus can prevent usual development of specific Ab 2. Persistent congenital parvovirus – detected by PCR blood and/or bone marrow

Answer 55

1. Drugs = chloramphenicol a. Can inhibit erythropoiesis in a dose-dependent manner b. Reversible reticulocytopenia, erythroid hypoplasia, and vacuolated pronormoblasts in the bone marrow c. Distinct from the idiosyncratic and rare development of severe aplastic anemia in chloramphenicol recipients. 2. Chronic kidney disease a. Acquired Ab-mediated pure red cell aplasia rare complication in CKD patients treated with erythropoiesis-stimulating agents b. Treatment i. Discontinue EPO ii. RBC transfusion iii. Immunosuppression iv. Renal Tx

Answer 56

1. Key points a. Conditions where there is ongoing immune activation – infection, malignancy, autoimmunity, GVHD b. A similar anemia is associated with CKD c. Normocytic, normochromic, hypoproliferative anemia d. Associated ↓ serum iron and ↓ transferrin saturation 2. Pathogenesis a. Decreased red cell life span = cytokines (eg. IL-1) may increase macrophages ability to destroy RBC b. Impaired erythropoiesis = immune cell/cytokine driven EPO production + BM suppression c. Increased uptake of iron in the reticuloendothelial system i. Diversion of iron from the circulation into the reticuloendothelial system results in functional iron deficiency  impaired heme synthesis and iron-restricted erythropoiesis ii. Inflammation-associated excess synthesis of hepcidin (protein which controls intestinal absorption + tissue distribution); hepcidin synthesized by hepatocytes and expressed in other cells (monocytes) 1. Functions by binding to and initiating the degradation of the iron exporter, ferroportin.

Answer 57

3. Clinical manifestations a. Signs and symptoms associated with underlying disease b. Mild-mod anaemia can affect QoL 4. Investigations a. FBE = normochromic, normocytic anaemia (Hb 60-90) i. Modest hypochromia and microcytosis in some patients, particularly if concomitant iron deficiency ii. Low or normal absolute reticulocyte counts iii. Leukocytosis b. Iron studies i. Serum iron level = low 1. NO increase in serum transferrin that occurs in iron deficiency (low to normal) ii. Ferritin level = normal or increased c. Bone marrow = normal cellularity; RBC precursors decreased or adequate, marrow hemosiderin may be increased, and granulocytic hyperplasia may be present. 5. Treatment a. Treatment of underlying disorder = anemia will improve or resolve b. Transfusions rarely required c. Erythropoietic stimulating agents (ESAs) ie EPO i. Increase Hb level and improve activity ii. Concurrent treatment with iron usually necessary for optimal effect iii. Response variable d. Iron deficiency patients i. Difficult to identify iron deficiency in patients with an inflammatory disease ii. May be no response to iron therapy as persistent inflammation impairs iron absorption and utilization iii. IV iron may further increase hepcidin production.

Answer 58

1. Key points a. Usually normocytic, and the absolute reticulocyte count is normal or low b. Most common in ESRF c. Associated with incidence of LVH, impaired physical activity, reduced QoL + hospitalisation/ mortality 2. Pathogenesis a. Decreased EPO production = MAIN CAUSE b. Absolute or functional iron deficiency from chronic blood loss – blood sampling, surgery, dialysis c. Disturbances in the iron metabolic pathway d. Higher hepcidin levels i. Hepcidin is filtered by the glomerulus and excreted by the kidney ii. Serum concentrations are increased in patients with decreased GFR e. Inflammation f. Hyperparathyroidism and deficiencies of vitamin B12, folate, and carnitine

Answer 59

3. Investigations a. Anemia in children with CKD is defined by age i. Hemoglobin (Hb) 1. <110 g/L (0.5-5 yr) 2. <115 g/L (5-12 yr) 3. <120 g/L (12-15 yr) 4. <130 g/L (males older than 15 yr) 5. <120 g/L (females older than 15 yr). b. FBE i. Normocytic and normochromic (unless concomitant Fe deficiency or vitamin deficiency) ii. Absolute reticulocyte count – low iii. White cell and platelet counts – normal iv. Ferritin – low if iron deficiency, high if inflammation c. EPO = low 4. Treatment a. Oral iron therapy = all CKD patients with anaemia i. Oral iron at 3-6 mg of elemental iron/kg of target dry weight once daily for 3 mo ii. Consider IV iron if no improvement in transferrin saturation and/or ferritin b. IV iron therapy = consider in patients on dialysis c. ESAs = mainstay of therapy i. All children with CKD when Hb concentrations are at 90-100 g/L, with a goal of 110-120 g/L ii. Dosing varies with age and dialysis modality iii. Continue with Fe supplementation iv. Note: subset of patients is hyporesponsive to ESAs v. Complication = Ab mediated pure red cell aplasia 1. Must STOP ESA (antibodies can target endogeneous EPO as well)

Answer 60

1. Overview a. Heterogeneous class of inherited disorders resulting from abnormalities of late erythropoiesis. b. Rare conditions characterized by variable degrees of anemia, ineffective erythropoiesis, and secondary hemochromatosis c. Dyserythropoiesis is the major cause of anemia but a shortened half-life of circulating red cells may also contribute. d. The CDAs have historically been classified into 3 major types (I, II, and II) based upon distinctive bone marrow morphology and clinical features, although additional subgroups and variants have also been identified 2. Type 1 CDA i. AR ii. Causative gene (CDAN1) encodes codanin-1 i. Most cases recognized in childhood/adolescence – rarely diagnosed in utero i. Treatment primarily supportive – do not respond to erythropoietin 3. Type II aka HEMPAS a. Hereditary erythroblastic multinuclearity with a positive acidified serum test b. Most common form of CDA – families mostly from Europe + Middle East c. Erythroblast multinuclearity and circulating RBCs that are sensitive to lysis by acidified normal serum. i. AR i. Diagnosis is usually made later in life (cf CDA 1), milder anaemia and semiology g. Prognosis = usually normal life expectancy h. Ddx = hereditary spherocytosis 4. CDA III a. Extremely rare, ill-defined entity manifested by a mild-to-moderate macrocytic anemia. i. AD

Answer 61

1. Normal Hb a. Increases with gestational age i. Term Hb 170 g/L ii. VLBW Hb 150-160 g/L b. Physiologic ↓ in Hb i. Term 8-12/52 – 110 g/L ii. Preterm 6/52 - 70-100 g/L 2. Blood volume at birth a. 90mL/kg = 3.5kg infant = 300mL 3. Early vs late cord clamping a. Controversy b. Benefits delayed cord clamping – improved blood volume, reduced iron deficiency in childhood c. Disadvantages delayed cord clamping – increasing plethora/polycythemia, increasing hyperbilirubaemia d. Current recommendation clamp at 1 minute in uncompromised infants 4. Causes of neonatal anaemia a. Physiological b. Anaemia or prematurity c. Haemorrhage (external) i. APH ii. Fetomateral transfusion iii. Twin-twin transfusion d. Neonatal internal haemorrhage i. Traumatic – E.g. sub-galeal, umbilical cord snap ii. Coagulopathy iii. Thrombocytopenia iv. Haemorrhagic disease newborn e. Haemolysis i. Inherited ii. Acquired a. Congenital aplasia At birth 1. Haemorrhage – APH, FMH, TTTS 2. Traumatic haemorrhage – sub-galeal, umbilical cord snap First few days 1. Haemolysis 2. Coagulopathy / HDN Later 1. Physiological 2. Prematurity 3. Haemorrhagic disease newborn 4. Haemolysis 5. Congenital aplasia

Answer 62

1. Key points a. Full term infants have higher Hb + larger RBC than older children + adults b. Decline in Hb from first week of life to 6-8 weeks = physiologic anaemia of infancy c. Term infants – Hb reaches nadir of 11 g/dL at 8-12 weeks after birth (EPO prevents further decline) d. Preterm infants – more pronounced nadir, usually occurs at 3-12 weeks after birth in infants <32 weeks e. Onset inversely proportional to gestational age, resolves by 3-6 months 2. Pathogenesis a. Increase in blood oxygen content and delivery at birth i. Respiration = more O2 for binding to Hb  Hb–oxygen saturation increases from 50% to >95% ii. Fetal to adult Hb synthesis after birth  replacement of high-oxygen-affinity fetal Hb with lower-affinity adult Hb, capable of delivering more oxygen to tissues b. The increase in blood oxygen content and delivery  downregulation of EPO production  suppression of erythropoiesis  aged RBCs that are removed from the circulation are not replaced  Hb level decrease c. Hb concentration declines until tissue oxygen requirement > oxygen delivery – occurs at 8-12 weeks of age when the Hb concentration is about 110 g/L  EPO production increases and erythropoiesis resumes d. The supply of stored reticuloendothelial iron, derived from previously degraded RBCs, remains sufficient for this renewed Hb synthesis, even in the absence of dietary iron intake, until approximately 20 weeks of age 3. Investigations a. Normocytic and normochromic RBC b. Low reticulocyte count c. Serum EPO low 5. Triggers a. Some dietary factors, such as folic acid deficiency, can aggravate physiologic anemia b. Unless significant blood loss, iron stores should be sufficient to maintain erythropoiesis early on c. Vitamin E deficiency does NOT play a role in anemia of prematurity 6. Management a. Iron supplementation b. Monitoring c. Transfusion d. EPO = LIMITED efficacy in decreasing the number of blood donors to which infant exposed to; therefore not generally recommended e. Restrict phlebotomy

Answer 63

i. More extreme + rapid – nadir of 70-90 g/L reached by 3-6 weeks of age – lower in very small babies ii. Additional factors for premature neonates 1. Blood loss from repeated phlebotomies 2. Suboptimal erythropoietic response a. Increased demand of erythropoiesis  shortened RBC life span (40-60 days) and accelerated expansion of RBC mass that accompanies rapid rate of growth b. Plasma EPO levels are lower than would be expected for the degree of anemia i. Switch from liver (normal fetal source of EPO) to kidney synthesis is not accelerated in prematurity  reliance on the liver as the primary site for synthesis (less sensitive than kidney)  reduced responsiveness to anemia iii. Treatment 1. Transfusions – optimal Hb not established a. Note decline in Hb in VLBW infants associated with abnormal clinical signs NOT benign and requires transfusions b. Beneficial effect NOT documented when administered for poor weight gain, respiratory difficulties and abnormal HR c. Negative associations i. Neurodevelopmental outcomes poorer in liberally transfused ii. NEC (late exposure to PRBC) iii. IVH (early transfusions) iv. Donor exposure d. In early preterm infants’ half-life of transfused RBCs is about 30 days 2. Recombinant human EPO if symptomatic (? Increased risk of ROP)

Answer 64

1. Definition a. Reduction below normal values of all 3 peripheral lineages – leucocytes, platelets and erythrocytes b. Pancytopaenia requires microscopic examination of a bone marrow biopsy specimen and a marrow aspirate to assess overall cellularity and morphology 2. Aetiology a. Hypocellular marrow i. Inherited (‘constitutional’) marrow failure syndromes ii. Acquired aplastic anaemia of various aetiologies iii. Hypoplastic variant of myelodysplastic syndrome (MDS) iv. Some cases of paroxysmal nocturnal haemoglobinuria with pancytopaenia b. Cellular marrow i. Primary bone disease ie acute leukaemia and MDS ii. Secondary to systemic disease, such as autoimmune disorders (SLE), vitamin B12 or folate deficiency and storage disease (Gaucher and Niemann-Pick diseases), overwhelming infection, sarcoidosis, hypersplenism c. Bone marrow infiltration i. Metastatic solid tumours ii. Myelofibrosis iii. Haemophagocytic lymphiohistiocytosis iv. Osteoporosis

Answer 65

Inherited/constitutional pancytopenia 1. Key points a. Aplastic anaemia = pancytopaenia + hypocellular bone marrow b. 30% of causes of paediatric marrow failure – Fanconi most common 2. Aetiology a. Inherited i. Fanconi anaemia ii. Schwachman-Diamond syndrome iii. Dyskeratosis congenital iv. Congenital amegakaryocytic thrombocytopenia v. Reticular dysgenesis vi. Unclassified inherited bone marrow failure syndromes vii. Other genetic syndromes = Down syndrome, Dubowitz syndrome, Seckel syndrome, Schimke immunoosseous dysplasia, Cartilage-hair hypoplasia, Noonan syndrome b. Acquired i. Drugs, chemicals, radiation ii. Viral infection, immune disorder iii. MDS iv. PNH

Answer 66

a. AR – one uncommon form X-linked b. Sibling discordance in clinical + haematological findings (even monozygotic twins) c. Mutation in FA (FANC) gene – resulting in chromosomal fragility i. Wild-type FANC gene product recognise and repair DNA ii. Mutant gene proteins lead to genomic instability and chromosome fragility. iii. Corrective effect (complementation) – cell fusion of FA cells with normal cells/cells from unrelated patients with FA produces a corrective effect on chromosomal fragility – allows subtyping iv. Multiple different subtypes (complementation groups) – A-P - genes prefixed with FANC (FANCA, FANCB); NOTE FANCD1 is identical to the breast cancer susceptibility gene, BRCA2 d. Consequences i. Inability to remove oxygen free-radicals  oxidative damage ii. Leukocyte telomerase length reduced + telomerase activity increased  high proliferative rate of marrow progenitors that ultimately leads to their premature senescence iii. Increased marrow cell apoptosis occurs and is mediated by Fas

Answer 67

a. Presentation i. 75% of patients are 3-14 years at diagnosis ii. At presentation, patients with FA may have 1. Typical physical anomalies and abnormal hematologic findings (majority of the patients) 2. Normal physical features but abnormal hematologic findings (about one-third of patients) 3. Physical anomalies and normal hematologic findings (unknown percentage) b. Haematological i. Marrow failure usually in the first decade of life ii. Initially – thrombocytopaenia, RBC macrocytosis iii. Subsequent onset of granulocytopenia, anaemia iv. Months to years – severe aplasia c. Skin (most common, 55%) i. Hyperpigmentation of the trunk, neck, and intertriginous areas ii. Café-au-lait spots and vitiligo, alone or in combination d. Short stature (51%) – may be aggravated by abnormal GH secretion or hypothyroidism e. Skeletal (upper limb 43%, lower limb 10%) i. Absence of radii ii. Thumbs that are hypoplastic, supernumerary, bifid, or absent are common iii. Anomalies of the feet iv. Congenital hip dislocation v. Leg abnormalities vi. The “r” radial pulse may be weak or absent f. Genitourinary (35%, mostly male) i. +/- underdeveloped penis; undescended, atrophic, or absence of the testes; hypospadias or phimosis ii. Malformations of the vagina, uterus, and ovary g. Facies i. Microcephaly ii. Small eyes, epicanthal folds iii. Abnormal shape, size, or positioning of the ears, deafness (9%) h. Renal = ectopic, pelvic, or horseshoe kidneys (21%) i. Cardiovascular and gastrointestinal malformations also occur (11%) j. Malignancy i. Solid tumours = SCC of the head, neck, upper oesophagus, vulva and/or anus, cervix, lower oesophagus; HPV suspected in pathogenesis ii. Therapy related oral cancer post BMT iii. Androgen therapy = benign and malignant liver tumors occur (adenomas, hepatomas) 1. Peliosis hepatis (blood-filled hepatic sinusoids) – reversible when androgen therapy is discontinued, and tumors may regress. iv. Clonal marrow cytogenetic abnormalities – advanced MDS and AML (15% leukaemia by age 35)

Answer 68

a. FBE i. Marrow failure usually in the first decade of life ii. Initially – thrombocytopaenia, RBC macrocytosis iii. Subsequent onset of granulocytopenia, anaemia iv. Months to years – severe aplasia b. AFP = stable elevation c. BMA = hypocellular, fatty d. Chromosome fragility = fragility (spontaneous chromatid breaks etc) i. Lymphocyte chromosomal breakage study using DEB or mitomycin C = unique to Fanconi Anaemia ii. Skin fibroblasts = testing of skin fibroblasts instead of lymphocytes confirms diagnosis 1. 10-15% have somatic mosaicism – therefore lymphocytes may not show chromosomal fragility due to mixed population e. Prenatal diagnosis = abnormal chromosome breakage analysis and genetic testing can be performed in amniotic fluid cells or in tissue from a chorionic villus biopsy f. Genetic testing i. Large number of FANC genes - genetic diagnosis has traditionally with complementation testing. ii. Determining whether cellular hypersensitivity to crosslinking agents (e.g., mitomycin C or radiation) or immunoblotting for FANCD2 is restored after generating hybridoma of the patient cells with known genetic complementation cells or after transducing the cells with a known FANC gene iii. The mutant gene or the complementation group is deduced when a specific wild-type FANC gene corrects the abnormal chromosome fragility

Answer 69

4. Treatment a. Surveillance i. Endocrinology 1. Monitor growth velocity 2. Screening for glucose intolerance + hyperinsulinism – annually or biannually ii. Cancer 1. FBE 1-3 monthly 2. BMA + biopsy annually to surveil for leukaemia and MDS 3. Annual assessment for solid tumours including gynaecological cancers in pubertal females b. HSCT i. Only curative therapy ii. Patients <10 years with FA – undergo transplantation with HLA-identical sibling donor >80% survival 1. Survival rates lower for patients >10 yr old iii. May do MUD for children without HLA-matched sibling donor – 50% survival c. Androgens i. Oral oxymetholone daily ii. Response in 50% of patients 1. Reticulocytosis and a rise in Hb within 1-2 months  WBC  platelets iii. May take months to achieve the max response – then taper androgen dose but not ceased iv. ? Add prednisolone - may counter androgen-induced growth acceleration and prevent thrombocytopenic bleeding by promoting vascular stability v. Usually becomes refractory as the disease progresses vi. AE = masculinization, elevated hepatic enzymes, cholestasis, peliosis hepatis, and liver tumors. 1. Require screening d. GCSF + EPO i. Increases ANC +/- platelets and Hb levels ii. Possible increased risk of marrow cells with clonal cytogenetic abnormalities such as monosomy 7 iii. Combined with EPO to boost Hb iv. Short-term treatment – patients lose response after 1 year due to marrow failure e. Future = gene therapy 5. Prognosis a. Cases reported in the 1990s, the projected median survival was >30 yr of age b. Careful surveillance has improved survival

Answer 70

1. Genetics + pathogenesis a. AR b. Mutant gene SBDS (chromosome 7q11) in 90% i. The wild-type gene protein product is involved in ribosomal biogenesis ii. Found in 80-90% of patients iii. Results in ribosomal dysfunction in 90-95% of patients c. Pancreatic insufficiency is a result of failure of pancreatic acinar development – fatty replacement d. BM failure - dysfunctional stem cells, accelerated apoptosis of marrow progenitors and a defective marrow microenvironment that does not support and maintain normal hematopoiesis 4. Differential diagnosis a. Fanconi Anaemia i. Similarities – marrow dysfunction, growth failure ii. Differences – SDS has pancreatic insufficiency, lacks characteristic skeletal abnormalities, normal chromosomal breakage study with DEB. b. Pearson syndrome i. Refractory sideroblastic anemia, cytoplasmic vacuolization of bone marrow precursors, lactic acidosis, exocrine pancreatic insufficiency, and a diagnostic mitochondrial DNA mutation is similar ii. Differ in clinical course, morphologic features of the bone marrow, and gene mutation iii. Severe anemia requiring transfusion (cf neutropenia) is present from birth to 1 yr of age.

Answer 71

a. Pancreatic insufficiency i. 2nd most common cause of pancreatic insufficiency in children ii. Extensive lipomatous changes in the pancreas iii. Variable abnormality of enzyme secretion iv. 50% of patients show an age-related improvement (4 years) in pancreatic function v. Despite adequate pancreatic replacement and correction of malabsorption, poor growth commonly continues b. Haematological i. Neutropenia (may be cyclic, neutrophil chemotaxis defects) occurs in at least 85-100% 1. Common cause of congenital neutropenia 2. Neutrophils may have a defect in mobility, migration, and chemotaxis owing to alterations in neutrophil cytoskeletal or microtubular function 3. Recurrent pyogenic infections (otitis media, pneumonia, OM, dermatitis, sepsis) = frequent and are common cause of death 4. Can have neonatal sepsis and early death ii. Thrombocytopenia found in 25% of patients iii. Anaemia in 50% iv. Pancytopenia in 10-25% v. Myelodysplastic syndromes in 30% - 10% acute myeloid and other leukaemias c. Skeletal defects i. Often not apparent until patient >2 years ii. Delayed bone maturation iii. Metaphyseal dysostosis/chondrodysplasia iv. Flared ribs v. Thoracic dystrophy d. Failure to thrive + short stature i. Birth weight low and by 6/12 are typically below the 5th centile for weight and height ii. Independent of nutrition e. Oher i. +/- hepatomegaly and elevation of liver enzymes ii. +/- neurocognitive problems and poor social skills iii. Dental disease

Answer 72

3. Investigations a. FBE = neutropenia, thrombocytopenia, anaemia, pancytopenia i. There is no increased chromosomal breakage after DEB testing of SDS lymphocyte b. Pancreatic function tests i. Impaired enzyme secretion, but with preservation of ductal function ii. Serum trypsinogen and isoamylase levels are reduced iii. 72 hour stool collection – fat malabsorption c. Immunoglobulins i. +/- B cell defects with 1 or more of the following: low IgG or IgG subclasses, low % of circulating B lymphocytes, decreased in vitro B-cell proliferation, and lack of specific antibody production ii. +/- low percentage of circulating T cells, subsets, or NK cells, and decreased in vitro T-cell proliferation d. Bone marrow = varying degrees of marrow hypoplasia and fat infiltration e. CT/US = fatty replacement of pancreatic tissue 5. Treatment a. Surveillance i. Regular FBE, 3 monthly ii. For malignant myeloid transformation 1. Serial bone marrow aspirations for smears and cytogenetics and marrow biopsy. 2. One recommendation is to perform marrow testing every 1-2 yr b. Treatment of pancreatic insufficiency = replacement as for CF c. G-CSF = for profound neutropenia ? predisposition to MDS/acute leukaemia d. Transfusion = for management of severe anemia or thrombocytopenia e. Allogeneic HSCT = only curative option i. Traditional myeloblastic HSCT resulted in treatment-related mortality in 35-50% of the patients ii. HSCT for severe marrow failure has produced 50-70% survival rate iii. Fludarabine-based protocols using reduced-intensity conditioning - safer and effective for SDS HSCT f. +/- androgens + steroids (some evidence that blood counts have improved) 6. Prognosis a. Median age of survival 35 years b. With pancytopenia age of survival reduced to 24 years c. 50% of patients experience spontaneous conversion from pancreatic insufficiency to pancreatic sufficiency as a result of improvement in pancreatic enzyme secretion

Answer 73

1. Genetics + pathogenesis a. Variable – most patients male consistent with X-linked recessive; others AD or AR b. Mutations in genes that encode components critical for telomere maintenance c. Impaired telomere maintenance -> short telomere = KEY FEATURE in peripheral blood of ALL individuals d. Progressive depletion of hematopoietic stem cells because of premature senescence (cessation of cell division) -> pancytopenia 3. Laboratory a. FBE = thrombocytopaenia and/or anaemia -> pancytopaenia + aplastic anaemia +/- macrocytosis b. Fetal hemoglobin = elevated initially c. Bone marrow = initially hypercellular -> symmetric depletion of all cell lines d. Immune tests = + reduced or elevated Ig values, decreased B- and/or T-lymphocyte count, and reduction of or absence of lymphocyte proliferative responses to phytohemagglutinin. e. Primary skin fibroblast culture = abnormal morphologic features and doubling rate and show unbalanced chromosome rearrangements, such as dicentrics, tricentrics, and translocations, in the absence of DEB

Answer 74

a. Cutaneous findings = diagnostic mucocutaneous (ectodermal) triad i. Lacy reticulate skin pigmentation of the upper body = can involve entire body, progressive ii. Mucosal leukoplakia = usually involves the oral mucosa (78%), especially the tongue but may also be seen in the conjunctiva and the anal, urethral, or genital mucosa; usually seen later in life iii. Nail dystrophy (88%) = involves hands and feet, usually starts with longitudinal ridging, splitting, or pterygium formation and may progress to complete nail loss; usually develops in first 10 years iv. Other features = hyperhidrosis of palms and soles, hair loss b. Bone marrow failure (90%) i. Severe aplastic anaemia in 50% - usually second decade c. Malignancy i. Develops in approximately 10-15%, usually in the 3rd and 4th decades of life ii. Predisposed to MDS as well as to solid tumors iii. 40% of cancers are squamous cell carcinomas of the head and neck (tongue, mouth, pharynx) d. Eye abnormalities (50%) i. Excessive tearing (epiphora) secondary to nasolacrimal duct obstruction common. ii. Other = conjunctivitis, blepharitis, loss of eyelashes, strabismus, cataracts, and optic atrophy e. Dental decay and early loss of teeth f. Skeletal abnormalities (20%) = osteoporosis, AVN, scoliosis + mandibular hypoplasia g. Genitourinary abnormalities = hypoplastic testes, hypospadias, phimosis, urethral stenosis, horseshoe kidney h. Gastrointestinal (10%) = esophageal strictures, vascular lesions causing bleeding, hepatomegaly, and fibrosis i. Respiratory = reduced diffusion capacity and/or a restrictive defect +/- pulmonary fibrosis

Answer 75

5. Treatment a. Androgens = can induce marrow function in 50% b. Cytokine therapy with granulocyte-macrophage colony-stimulating factor or with G-CSF alone or combined with EPO = improvement in neutrophils c. Allogenic HSCT = to correct marrow failure – survival 50% i. Vascular lesions and fibrosis involving various organs not prevented by HSCT ii. Up to 40% of patients with DC experience fatal pulmonary complications after transplantation 6. Prognosis a. Mean age of death for patients with DC who are diagnosed in childhood is approximately 30 years b. Cause of death – BM failure, complications of HSCT, cancer, fetal pulmonary problems and GIT bleeding

Answer 76

1. Key points a. Rarest of the 4 major inherited pancytopenias 2. Genetics + pathogenesis a. AR – carriers have normal haematology b. Mutations in MPL – gene for receptor of thrombopoietin i. Growth factor which promotes HSC survival – therefore results in evolution to aplastic anaemia ii. Stimulates megakaryocyte proliferation and maturation c. Genotype–phenotype correlations predict disease course and prognosis d. Biologically active plasma thrombopoietin is consistently elevated in all patients with CAMT 3. Classification a. CAMT type I i. Nonsense mutations = complete loss of function of the thrombopoietin receptor ii. Persistently low platelet counts (absent megakaryocytes) + rapid progression to pancytopaenia iii. Mortality close to 100% b. CAMPT type II i. Missense mutations ii. Milder course, a transient increase in platelets during the 1st yr of life iii. Delayed onset, if any, of pancytopenia, indicating residual receptor function iv. Lower mortality; can have serious complications

Answer 77

a. Haematological i. Thrombocytopaenia – petechial rash, bruising or bleeding 1. Usually presents in first year of life ii. Subsequent pancytopaenia caused by aplastic anaemia b. Malignancy i. Clonal marrow cell cytogenetic abnormalities (eg. monosomy 7 and trisomy 8); risk of MDS and acute leukaemia c. Physical abnormalities = 20% i. MOST have normal physical and imaging features ii. Cerebellar and cerebral atrophy are frequent iii. Developmental delay is a prominent feature iv. Congenital heart disease – ASD defects, VSD, PDA, tetralogy of Fallot, and coarctation of the aorta. v. Other anomalies include abnormal hips or feet, kidney malformations, eye anomalies, and cleft or high-arched palate. vi. Some patients have microcephaly and an abnormal facies

Answer 78

5. Lab a. FBE = thrombocytopaenia, subsequent aplastic anaemia +/- macrocytosis b. +/- elevated hemoglobin F c. +/- increased expression of i antigen. d. Bone marrow i. Initially normal cellularity with marked reduction or absence of megakaryocytes. ii. Aplastic anemia develops - marrow cellularity is decreased, with fatty replacement; erythropoietic and granulopoietic lineages are also symmetrically reduced e. Mutational analysis – confirms diagnosis f. Do NOT show increased chromosomal breakage when exposed to DEB 6. Treatment a. Corticosteroids NOT effective b. Platelet transfusions i. Single-donor filtered platelets are preferred to minimize sensitization ii. Leukodepleted platelet units might be adequate, CMV free if considering HSCT c. HSCT = curative, ideally HLA-matched sibling donor d. Other i. +/- Androgens for aplastic anaemia – may result in partial improvement ii. +/- Interleukin-3 iii. +/- Thrombomimetic agents – the induction of fibrosis by these agents and the risk of MDS/leukemia in CAMT render HSCT the preferred treatment for patients with severe cytopenia

Answer 79

1. Key points a. Hallmark of aplastic anemia i. Peripheral pancytopenia ii. Hypoplastic or aplastic bone marrow b. Classified as moderate or severe depending on cell count c. Pancytopenia results in increased risks of cardiac failure, infection, bleeding, and fatigue 2. Aetiology a. Radiation, drugs, and chemicals i. Idiosyncratic = chloramphenicol, antiepileptics, gold; ecstasy b. Viruses = CMV, EBV, hepatitis B, hepatitis C, hepatitis (seronegative hepatitis), HIV i. Parvovirus B19 is classically associated with isolated red blood cell aplasia, but in patients with sickle cell disease or immunodeficiency can result in transient pancytopenia c. Immune diseases – eosinophilic fasciitis, hypoimmunoglobulinemia, thymoma d. Pregnancy e. Paroxysmal nocturnal hemoglobinuria f. Marrow replacement – leukemia, myelodysplasia, myelofibrosis g. Autoimmune h. Other – cryptic DC (no physical stigmata), telomerase reverse transcriptase haploinsufficiency

Answer 80

3. Complications of pancytopaenia a. Life-threatening bleeding from prolonged thrombocytopenia b. Infection secondary to protracted neutropenia – serious bacterial infections, invasive mycoses c. Regular PRBC i. Developing alloantibodies to red cell antigens ii. Iron overload requiring chelation 4. Investigations a. FBE = pancytopaenia, reticulocyte count assesses erythropoietic activity b. Film +/- red blood cell, leukocyte, and platelet morphologic features c. Chromosomal breakage analysis = to exclude Fanconi anemia d. Fetal Hb – suggests congenital pancytopenia but is not diagnostic e. To assess for PNH – flow cytometric analysis of erythrocytes for CD55 and CD59 f. BM – morphologic features, cellularity, and cytogenetic abnormalities

Answer 81

5. Treatment a. Allogeneic HSCT i. 90% chance of long-term survival in HLA–identical family member donor ii. Only 1 in 5 patients has a HLA–matched sibling donor  not an option for the majority of patients iii. MUD and T-cell depleted haploidentical related transplant – response rate 90% b. Immunosuppression i. Agents = ATG, cyclosporine, tacrolimus 1. Response rate of 70-80%, median time to response is 6 months 2. Relapse in 30% following discontinuation of treatment – some require longterm cyclosporine a. Of those who relapse 50% respond to a second course of ATG and cyclosporine 3. Risks = clonal bone marrow disease (leukaemia), MDS, PNH after immunosuppression 4. GCSF sometimes used 5. Higher reticulocytes = higher probability of response to immunosuppression + survival ii. High dose cyclophosphamide 1. Previously used in patients with newly diagnosed aplastic anaemia + refractory cases 2. Leads to prolonged severe pancytopenia = risk of life-threatening infection, especially fungal. c. Other i. Eltrombopag (oral thrombopoietin mimetic agent) = haematological response ii. Other therapies with inconsistent results – androgens, corticosteroids, and plasmapheresis 6. Prognosis a. Spontaneous recovery rarely occurs b. Untreated - overall mortality rate of approximately 50% within 6 mo of diagnosis and of >75% overall i. Infection and hemorrhage being the major causes of morbidity and mortality c. Majority of children with acquired severe aplastic anemia respond to SCT or immunosuppression

Answer 82

a. Very rare in children, but when it occurs clinical course more aggressive b. Classification i. Refractory cytopenia of childhood (peripheral blasts <2% and marrow blasts <5%) ii. Refractory anaemia with excess blasts (peripheral blasts 2-19% and/or marrow blasts 5-19%) iii. Refractory anaemia with excess blasts in transformation (peripheral and/or marrow blasts 20-29%) iv. Disease in children with >30% blasts is usually defined as acute myelocytic leukemia c. Associations i. Down syndrome ii. Severe congenital neutropenia iii. Noonan syndrome iv. Fanconi anaemia v. Trisomy 8 mosaicism vi. Neurofibromatosis vii. Schwachman syndrome d. Genetics i. Significant clonal abnormalities in 50% of patients with MDS ii. Monosomy 7 and being most common but prognostically neutral iii. Those with a structurally complex karyotype have a very poor outcome e. Progression i. The transition time from paediatric MDS to acute leukemia is relatively short (14-26 mo)  aggressive treatment, such as HSCT, must be considered shortly after diagnosis f. Treatment i. Allogeneic HSCT – survival rate is approximately 60% ii. Exception – MDS and AML in children with DS 1. Highly responsive to conventional chemotherapy 2. Survival >80% iii. DNA hypomethylating agents azacitidine and decitabine - used in treating MDS without a known molecular target and have some effect

Answer 83

1. Pathogenesis a. Hemolysis = premature destruction of RBCs b. When rate of destruction exceeds capacity of BM to produce RBCs  anaemia c. Normal RBC survival time – 110-120 days (half-life: 55-60 days) i. 0.85% of the most senescent RBCs are removed and replaced each day d. During hemolysis i. RBC survival is shortened, RBC count falls ii. EPO is increased iii. Stimulation of marrow activity  heightened RBC production, reflected in increased reticulocyte % e. Bone marrow response i. Marrow ↑ output 2-3–fold acutely - maximum of 6-8 fold in long-standing hemolysis ii. The reticulocyte % can be corrected to measure the magnitude of marrow production in response to hemolysis as follows: 1. Reticulocyte index is essentially a measure of RBC production per day 2. Reticulocyte index= reticulocyte % x (observed haematocrit/normal haematocrit) x (1/μ) a. µ = maturation factor of 1-3 related to the severity of the anemia 3. Normal reticulocyte index is 1.0; therefore, the index measures the fold increase in erythropoiesis (e.g., 2-fold, 3-fold). 4. As anemia becomes more severe, the EPO increases and reticulocytes are released from the marrow earlier; they are identifiable as reticulocytes in the blood that last for >1 day 3. Pathogenesis of RBC destruction a. Three heme-binding proteins in the plasma are altered during hemolysis i. Hemoglobin binds to haptoglobin and haemopexin  reduced plasma concentration ii. Oxidized heme binds to albumin to form methaemalbumin  increased in plasma b. If capacity of binding molecules is exceeded  free Hb appears in the plasma (sign of intravascular haemolysis) i. Free Hb dissociates into dimers and is filtered by the kidneys ii. When the tubular reabsorptive capacity of the kidneys for Hb is exceeded  free Hb in urine c. Iron loss in urine  iron deficiency if chronic intravascular haemolysis d. When hemoglobin is degraded, an α-methene bridge is broken in the cyclic tetrapyrrole of the heme moiety, with release of carbon monoxide (CO) i. Amount of CO in the blood or expired air  dynamic measure of the hemolytic rate ii. End-tidal CO is not available in most clinical laboratories to measure hemolysis e. Haematocrit depends on severity + haemolysis + erythropoietic response f. Aplastic crisis i. The shortened RBC life span and heightened RBC production  marked susceptibility to aplastic or hypoplastic crises ii. Characterized by 1. Erythroid marrow failure 2. Reticulocytopenia 3. Rapid reduction in hemoglobin and hematocrit to extremely low levels a. Life-threatening decline in hematocrit that usually lasts 10-14 days iii. Transient erythroid marrow failure usually only has a mild effect on normal RBC lifespan BUT has a greater effect if RBC lifespan shortened by haemolysis iv. Parvovirus B19 (erythrocytotropic) most common cause of aplastic crisis

Answer 84

4. Clinical manifestations a. Pallor and anaemia b. Jaundice c. Dark urine d. Splenomegaly 2. Complications a. Erythroid hyperplasia i. Secondary to chronic hemolytic anemia in children (especially thalassemia) ii. May be extensive – medullary spaces expand at the expense of the cortical bone. iii. A propensity of long bones # iv. Diagnosis – clinical examination or XR of skull and long bones b. Gallstones = composed of calcium bilirubinate may be formed in children with chronic hemolysis as young as 4

Answer 85

a. FBE + Film b. Reticulocyte count = elevated c. Serum markers of haemolysis i. Unconjugated hyperbilirubinaemia ii. Elevated LDH iii. Elevated free plasma Hb iv. Low or absent haptoglobin d. Urinary and faecal urobilinogen – due to increased biliary excretion of heme pigment derivatives e. Measure half-life i. Radioisotope Na251CrO4 = normal half-life 25-35 days ii. RBC biotin labelling f. Blood group g. Coombs test i. Direct (DAT) – detects antibodies/complement on surface of RBC  USED FOR AIHA 1. Anti-IgG (Coombs reagent) + patient RBC 2. Uses a. Autoimmune haemolysis – warm, cold b. Drug induced haemolysis c. Alloimmune haemolysis ii. Indirect – Detects antibodies in the serum 1. Anti-IgG + synthetic RBC + patient plasma 2. Uses a. Pre transfusion testing ie crossmatching b. Prenatal screening of pregnant woman do Rh antigen h. Definitive i. Flow cytometry for eosin-5 malemide staining ii. Hb electrophoresis iii. RBC enzyme assays – G6PD, pyruvate kinase

Answer 86

Haemolysis Acute blood loss Anaemia therapy (iron, B12) short term

Answer 87

``` Intrinsic/Cellular • Resulting from intrinsic abnormalities of the membrane, enzymes, or hemoglobin • Majority inherited (note: paroxysmal nocturnal hemoglobinuria is acquired) 1. Membrane a. Hereditary spherocytosis b. Hereditary elliptocytosis c. Hereditary pyropoikilocytosis d. Hereditary stomatocytosis e. PNH (‘combined mechanism’) 2. Enzyme a. Pyruvate kinase deficiency b. G6PD deficiency 3. Haemaglobinopathies a. Sickle cell disease b. Thalassaemias c. Unstable Hb ``` ``` Extracellular • Resulting from antibodies, mechanical factors, or plasma factor • Most acquired (note: abetalipoproteinemia with acanthocytosis is inherited) 1. Autoimmune a. ‘Warm’ antibody b. ‘Cold’ antibody 2. Fragmentation haemolysis a. DIC, TTP, HUS, aHUS b. ECMO c. Prosthetic heart valve d. Burns/thermal injury e. Hypersplenism 3. Plasma factors a. Liver disease b. Abetalipoproteinaemia c. Infection d. Wilson’s disease ```

Answer 88

1. Key points a. 1 in 5,000 – most common inherited abnormality 2. Genetics + pathogenesis a. AD most common (75%), less common AR (25%) b. 25% have no previous family history – new mutations, autosomal recessive or non-paternity c. 5 proteins – all involved in cell shape i. Ankyrin-1 – most common (dominant and recessive defects) ii. β -spectrin – most common (dominant) iii. Band 3 (dominant) iv. α-spectrin (recessive) v. Protein 4.2 (recessive) d. Deficiency results in uncoupling in the “vertical” interactions of the lipid bilayer skeleton and subsequent release of membrane microvesicles  loss of surface area without loss of volume  sphere e. Associated increase in cation permeability, cation transport, adenosine triphosphate use, and glycolysis f. The decreased deformability of the spherocytic RBCs impairs cell passage from the splenic cords to the splenic sinuses  spherocytic RBCs destroyed prematurely in the spleen 4. Complications a. Splenic sequestration crisis b. Gout c. Cardiomyopathy d. Priapism e. Leg ulcers f. Spinocerebellar degeneration

Answer 89

3. Clinical manifestations a. Can become symptomatic at any time – some asymptomatic until adulthood b. Neonatal period i. Anemia and hyperbilirubinemia 1. Severe anemia = pallor, jaundice, fatigue, exercise intolerance etc 2. BM hyperplasia  expansion of the diploe of skull (lesser extent than thal major) ii. Hemolysis may be more prominent in the newborn because HbF binds 2,3-BPG poorly  increased level of free 2,3-BPG  destabilizes interactions among spectrin, actin, and protein 4.1 iii. Infants do not mount an adequate reticulocyte response until several months after birth 1. The need for exchange transfusion at birth or transfusions in infancy is not indicative of more severe disease later in life c. After infancy i. Splenomegaly common ii. Bilirubin gallstone formation – can occur as early as 4-5 years, occur in almost all adults iii. Crises 1. Aplastic crises – primarily as a result of parvovirus B19 infection 2. Hypoplastic crises – associated with various other infections iv. High RBC turnover in the setting of erythroid marrow failure can result in profound anemia (hematocrit <10%), high-output heart failure, cardiovascular collapse, and death v. WBC and platelets – can also fall 5. Investigations a. FBE i. Hb 60-100g/L, MCV normal ii. Reticulocytosis, reticulocyte % increased to 6-20% (mean 10%) iii. MCHC often increased b. Film i. Polychromatophilic reticulocytes and spherocytes (smaller and hyperchromic) ii. Spherocytes normally >15% of cells c. Haemolysis = unconjugated hyperbilirubinaemia, reduced haptoglobin d. Flow cytometric EMA (eosin-5-maleimide) binding test e. Osmotic fragility test

Answer 90

6. Diagnosis a. Positive family history AND presence of typical clinical and laboratory features i. Splenomegaly + spherocytes on the blood smear + reticulocytosis + elevated MCHC ii. If present no additional testing is required to confirm diagnosis b. If diagnosis is less certain i. High predictive value 1. Flow cytometric EMA (eosin-5-maleimide) binding test 7. DDx diagnosis a. Isoimmune haemolysis ie haemolytic disease of the newborn b. Autoimmune haemolysis - characterized by spherocytes, may be evidence of previously normal values for hemoglobin, hematocrit, and reticulocyte count c. Rare causes of spherocytes – Wilson disease , DIC/ HUS, infx, severe burns

Answer 91

a. General Supportive Care i. Phototherapy +/- exchange transfusion = close monitoring of newborns ii. Transfusion 1. Minority of infants transfusion dependent, uncommon >6-12 months 2. Aplastic crisis secondary to parvovirus 3. Hypoplastic crises with other infections iii. Folic acid = recommended (moderate and severe HS) due to increased erythropoiesis b. Splenectomy i. Spherocytes destroyed in spleen  splenectomy eliminates most haemolysis ii. Splenectomy  reduction in anaemia, hyperbilirubinaemia, gallstones iii. Indications = severe HS (after age 6 due to risk of post-splenectomy sepsis in young children) 1. Frequent transfusions 2. Poor growth 3. Massive splenomegaly with risk of rupture iv. Considered = moderate HS and frequent hypoplastic or aplastic crises, poor growth, or cardiomegaly v. NOT recommended for mild HS vi. Usual post-splenectomy prophylaxis (penicillin) + vaccinations (pneumocococcus, Hib, meningococcus) vii. Complications 1. Lifelong increased risk for sepsis, particularly pneumococcal species. 2. Immediate surgical morbidity 3. Postsplenectomy thrombocytosis – common, requires no treatment and resolves spontaneously c. Cholecystectomy = sometimes required for gallstones

Answer 92

1. Key points a. Elongated, oval, or elliptically shaped RBCs b. Hemolytic anemia in these disorders ranges from absent to life-threatening c. Less common than spherocytosis d. Rare in Western populations, more common among West Africans [spectrin mutations = malaria resistance] 2. Genetics + pathogenesis a. Dominant disorder – if 2 abnormal alleles (HPP) severe haemolytic anaemia b. Abnormalities of α- and β-spectrin and defective spectrin heterodimer self-association (other less common) c. Defects in horizontal protein interactions result in gross membrane fragmentation  weakening of cytoskeletal interactions  membrane instability  fragmentation, hemolysis, microcytic or spherocytic RBCs d. Transient augmented fragmentation and hemolysis in the newborn i. Results from the presence of HbF that binds poorly to the glycolytic intermediate 2,3-DPG ii. The increased 2,3-DPG destabilizes the spectrin–actin–protein 4.1 complex  membrane instability iii. Excess 2,3-DPG does NOT affect RBC shape/survival in infants without HE 3. Clinical manifestations a. Varies markedly in severity b. Mild hereditary elliptocytosis produces no symptoms; can be an incidental finding c. Neonatal poikilocytosis (shape variation) and hemolysis i. Neonatal jaundice, even though characteristic elliptocytosis might not be evident at that time. ii. Film – bizarre poikilocytes and pyknocytes d. Chronic haemolytic anaemia = anaemia, jaundice, splenomegaly, osseous changes, cholelithiasis 5. Investigations a. FBE = reticulocyte % increased (reflects severity of hemolysis) b. Blood film = various degrees of elongation, can be rod shaped +/- other abnormal RBC shapes depending on the severity of the haemolysis ie microcytes, spherocytes, and other poikilocytes c. +/- erythroid hyperplasia +/- indirect hyperbilirubinemia d. Protein separation and analysis techniques (to establish the specific protein abnormality) e. Eosin-5-maleimide binding test - detects binding to protein band 3 by flow cytometry, may be useful in conjunction with the MCV in diagnosing hereditary elliptocytosis and hereditary spherocytosis 6. Treatment a. Morphologic abnormality on the blood film without evident hemolysis – no treatment is necessary b. Chronic hemolysis i. Folic acid = 1 mg daily to prevent secondary folic acid deficiency. ii. Splenectomy = consider if Hb <100 g/L and reticulocyte count >10% (ie chronic haemolysis) iii. Note: The RBCs on the blood film may be more abnormal after splenectomy, even though hemoglobin increases and reticulocytes decrease

Answer 93

1. Key points a. AD group of disorders b. RBCs are cup-shaped, creating a mouth shaped area (stoma) of central pallor instead of the usual circular area of central pallor c. Increased red cell cation permeability 2. Classification a. Hydrocytosis (Overhydrated) i. Most severe form ii. Pathophysiology 1. Excess intracellular Na and water content and decreased intracellular potassium content -> cells subsequently develop increased cation content and water, and thus swell resulting in osmotic fragility iii. Clinical features 1. Moderate-severe haemolysis 2. Jaundice 3. Splenomegaly 4. Cholelithiasis iv. Investigations 1. FBE – macrocytosis 2. Film – large number of stomatocytes b. Xerocytosis (Underhydrated) i. More common form of hereditary stomatocytosis and usually results in a milder anemia ii. Pathophysiology 1. The underlying cation defect is a net loss of RBC potassium that is not accompanied by an increase in sodium -> dehydration of erythrocyte iii. Clinical features 1. Mild compensated macrocytic hemolytic anemia 2. Jaundice and splenomegaly a. Recurrent episodes of abdominal pain b. Budd-Chiari syndrome (hepatic veins) c. Splenomegaly (splenic vein) iv. Investigations 1. FBE – increased MCHC (2° cellular dehydration) 2. Blood film – variable numbers of stomatocytes and/or target cell d. Acquired stomatocytosis i. Liver disease, alcoholism, malignancy, and cardiovascular disease. ii. Stomatocytes can be seen on the blood smears of normal patients as a result of drying artifact. 3. Treatment a. Severe – RBC transfusion b. Splenectomy NOT recommended for cation-leaky hereditary stomatocytosis i. Not effective ii. Severe complications 1. Pulmonary hypertension 2. Thromboembolic events

Answer 94

1. Key points a. Rare disorder in children 2. Pathogenesis a. Abnormality of marrow stem cells affecting each blood cell lineage b. An acquired somatic mutation that results in a defect in cell membrane proteins -> RBCs and other cells susceptible to damage by normal plasma complement proteins 3. Clinical manifestations a. Marrow failure (60%) b. Intermittent or chronic anaemia – often with intravascular haemolysis c. Thrombosis and thromboembolic phenomena (>5%) – unknown mechanism i. Abdominal venous thrombosis 1. Recurrent episodes of abdominal pain 2. Budd-Chiari syndrome (hepatic veins) 3. Splenomegaly (splenic vein) d. Abdominal pain, recurrent (>10%) e. Back and head pain f. Note: Hemoglobinuria – rarely seen in children i. Nocturnal and morning hemoglobinuria is a classic finding in adults, hemolysis is worse during sleep 4. Investigations a. FBE i. Elevated reticulocyte percentage, thrombocytopaenia, leucopaenia ii. Initially normocytic anaemia, but might become microcytic (if iron deficiency develops) b. Chronic intravascular haemolysis – hemosiderinuria, elevated reticulocyte percentage, low serum haptoglobin, increased LDH c. Markedly reduced levels of RBC acetylcholinesterase activity and decay-accelerating factor d. Flow cytometry – diagnostic test of choice – identify glucolipid-bound membrane proteins

Answer 95

5. Prognosis a. The mortality in PNH is related primarily to the development of aplastic anemia or thrombotic complications. b. The predicted survival rate for children before the development of eculizumab (see Treatment below) was 80% at 5 yr, 60% at 10 yr, and 28% at 20 yr. 6. Complications a. Marrow failure and severe thrombosis are the most serious complications in children i. Hypoplastic or aplastic pancytopenia can precede or follow the diagnosis of PNH b. Rarely may progress to acute myelogenous leukemia 7. Treatment a. Acute haemolytic episode = steroids b. Marrow failure = androgens (e.g., fluoxymesterone [Halotestin]), antithymocyte globulin, cyclosporine, and growth factors (e.g., erythropoietin and granulocyte colony-stimulating factor) c. Bone marrow transplantation (if a suitable donor exists) d. Eculizumab i. MAb against complement component C5 ii. Interrupts the excessive complement destruction of RBC and activation of platelets iii. It decreases the rate of hemolysis, stabilizes hemoglobin levels, reduces the number of transfusions, and reduces the risk of thrombosis. iv. Medication does not improve the hematopoietic clonal expansion or prevent marrow failure v. Meningococcal vaccine before beginning therapy e. +/- prolonged anticoagulation (heparin or LMWH) therapy may be of benefit when thrombosis occur f. +/- iron therapy – chronic urinary loss of iron as hemosiderin

Answer 96

• Pathways affected in enzyme defects o Hexose monophosphate shunt and glutathione pathways (eg. G6PD, glutathione synthase) o Glycolytic pathways (eg. pyruvate kinase, hexokinase, glucose phosphate isomerase) • Key features o Most AR o Most mild and uncommon (exception – G6PD) o Classified as ‘non-spherocytic’ as spherocytic RBC are usually absent and osmotic fragility testing is normal • Pathogenesis o Deficiencies of glycolytic enzymes = compromise ATP generation  inadequate energy  shortened RBC lifespan [Embden Meyerhof pathway] o Deficiencies of glutathione pathway = inadequate levels of glutathione  impairs ability to handle oxidative stress  HB denatures and precipitates into Heinz bodies  damage RBC membrane  intravascular and extravascular haemolysis

Answer 97

1. Key points a. Cause of chronic haemolysis b. Second most common RBC enzyme defect c. Results in extravascular haemolysis d. Common in Northern Europe 2. Genetics a. Autosomal recessive b. 2 mammalian PK genes -> only the PKLR gene is expressed in red cells (chromosome 1q21) c. >180 different gene mutations 3. Pathogenesis a. PK enzyme functions in the energy-producing glycolytic pathway – metabolises glucose to ATP b. PK catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate by removal of a phosphate group c. The phosphate group from PEP is transferred to ADP to create one molecule of ATP d. Impaired PK activity -> low levels of ATP, pyruvate, and the oxidized form of nicotinamide adenine dinucleotide (NAD+) -> cannot maintain water + potassium content -> rigid + reduced lifespan (+ other upstream effects e.g. that shift Hb dissociation curve) 4. Clinical manifestations a. Variable – neonatal jaundice to adult diagnosis of HA b. Commonly mild jaundice in adulthood with pallor, jaundice + splenomegaly c. A severe form of the disease has a relatively high incidence among the Amish of the midwestern United States 5. Investigations a. FBE – Increased reticulocytes, mild macrocytosis (reflect the elevated reticulocyte count) b. Film i. Polychromatophilia (more staining with some dyes 2° to too many immature RBCs) ii. Spherocytes are uncommon, but a few spiculated pyknocytes may be found c. RBC PK activity or increase in Km for substrate phosphoenolpyruvate (high Km variant) d. PKLR gene mutation 6. Treatment a. Treatment of neonatal jaundice if required – exchange transfusion b. Folic acid supplementation c. PRBC – severe anaemia or aplastic crisis i. +/- iron chelation d. Splenectomy – children >5-6 years to increase Hb levels i. Not curative ii. May be followed by higher hemoglobin levels and by strikingly high (30-60%) reticulocyte counts.

Answer 98

1. Key points a. Most common enzyme disorder b. Responsible for 2 clinical syndromes = episodic HA + chronic non-spherocytic haemolytic anaemia 2. Genetics + pathogenesis a. X-linked b. Normal variants include G6PDB+ and G6PDA+ c. G6PD catalyses the conversion of glucose-6-phosphate to 6-phosphogluconic acid i. The reaction produces NADPH, which maintains glutathione (GSH) in a reduced, functional state ii. Reduced GSH provides protection against oxidative threats from certain drugs and infections that would otherwise cause precipitation of haemoglobin (Heinz bodies) or damage the RBC membrane d. Mutations most often single base changes -> amino acid substitution + destabilization of enzyme i. Milder disease = mutations near the amino terminus of the G6PD molecule ii. Chronic nonspherocytic hemolytic anemia = mutations clustered near the carboxyl terminus e. Heterozygous females have intermediate enzymatic activity 3. Epidemiology a. More than 400 million people worldwide – overall 4.9% global prevalence b. Global distribution parallels malaria – ‘balanced polymorphism’ = advantage of resistance to falciparum malaria in heterozygous females outweighs the small negative effect on affected hemizygous males c. Mutations i. G6PD A – African ii. G6PD B – (G6PD Mediterranean) iii. G6PD canton – 5% of Chinese population

Answer 99

4. Clinical manifestations a. Most asymptomatic in absence of triggering factors (infection, drugs, fava beans) b. Haemolysis 24-48 hours after a patient has ingested a substance with oxidative properties c. Degree of haemolysis depends on inciting agent, amount ingested, severity of enzyme deficiency d. Severe i. Symptomatic anaemia ii. Haemoglobinuria iii. Jaundice e. Neonate i. G6PD deficiency can produce haemolysis in the neonatal period ii. Severe neonatal jaundice if G6PD deficiency AND mutation in promoter of UGT1A (eg. Gilberts) iii. Oxidant drugs in pregnancy  HA and jaundice may be apparent at birth 5. Triggers a. Medications i. Antibacterials = sulfonamides, dapsone, trimethoprim-sulfamethoxazole, nalidixic acid, chloramphenicol, nitrofurantoin ii. Antimalarials = primaquine, pamaquine, chloroquine, quinacrine iii. Antihelminths = Beta-Naphthol, stibophen, niridazole iv. Others = Acetanilide, Vit K analogues, methylene blue, toluidine blue, probenecid, dimercaprol, acetylsalicyclic acid, phenazopyridine, rasburicase b. Chemicals = phenylhydrazine, benzene, naphthalene (moth balls), 2,4,6-Trinitrotoluene c. Illness = diabetes acidosis, hepatitis, sepsis d. Favism i. In some individuals, ingestion of fava beans causes an acute, severe hemolytic syndrome ii. Fava beans contain divicine, isouramil, convicine  leads to production of hydrogen peroxide and other reactive oxygen products iii. More frequently associated with G6PD B-

Answer 100

6. Investigations a. FBE = anaemia + decreased haematocrit b. Film i. Unstained or supravital preparations  Heinz bodies (precipitated Hb) 1. Not visible on Wright-stained blood film 2. Heinz bodies seen only within the first 3-4 days of illness (rapidly cleared from blood) ii. Anisopoikilocytosis – ‘bite cells’ 1. Anisopoikilocytosis = abnormality in the shape or size of erythrocytes iii. Polychromasia (bluish, larger RBCs) representing reticulocytosis c. Haemaglobinuria = if episode is severe  haptoglobin saturated  free Hb in plasma and subsequently urine d. G6PD activity = reduced ≤10% of normal (note: cannot test if recent transfusion) 7. Treatment a. Prevention i. Males from ethnic groups with high incidence should be tested prior to giving oxidative drugs ii. Infants with severe neonatal jaundice who belong to these ethnic groups also require testing for G6PD deficiency because of their heightened risk for this defect b. Cease oxidant agent – generally results in recovery c. Severe haemolysis – supportive care ie. blood transfusions

Answer 101

1. Autoimmune haemolytic anaemia a. Most common b. Type 2 hypersensitivity c. Classification i. Warm-reactive AIHA ii. Cold agglutinin disease iii. Paroxysmal cold haemaglobinuria 2. Drug induced haemolysis a. Penicillins, cephalosporins, tetracycline, erythromycin b. Probenecid c. Paracetamol and ibuprofen 3. Alloimmune haemolysis a. Haemolytic disease of newborn b. Alloimmune haemolytic transfusion reaction

Answer 102

1. Key points a. Autoantibodies bind patient’s own RBC resulting in premature destruction b. When haemolysis > bone marrow replacement = anaemia c. Positive direct antiglobulin (Coombs) test 2. Pathogenesis a. Autoantibodies directed against RBC membrane antigens b. As for other autoimmune diseases exact mechanism of antibody production unknown c. The antibodies usually react to epitopes (antigens) that are ‘public’ or common to all RBCs ie Rh proteins 5. Clinical manifestations a. Non-specific and common to other types of HA b. Anaemia = weakness, fatigue, SOB, dizziness, pallor c. Haemolysis = jaundice, icterus, painless dark urine 6. Investigations a. FBE i. Anaemia – often severe (<7 /dL) ii. WBC and platelets normal – if leukopenia or thrombocytopaenia suggests bone marrow failure, MAHA or BM involvement iii. Elevated reticulocyte count iv. Thrombocytopaenia/leucopaenia – suggest bone marrow failure, microangiopathic haemolytic anaemia (HUS, TTP), BM involvement b. Film i. Warm AIHA = numerous small spherocytes predominate ii. Cold-reactive AIHA = clumped or agglutinated cells iii. Polychromasia = reflects reticulocytosis iv. Howell-Jolly bodies and nucleated RBC = reflects accelerated erythropoiesis and/or prior splenectomy c. Coombs test = DAT +ve d. Cold agglutinins titre = highest dilution of serum sample at which agglutination of thee RBC is usually seen e. Urinalysis = intravascular haemolysis -> haemaglobin -> haemaglobinuria f. Serum markers of haemolysis = NOT specific for AIHA i. Unconjugated hyperbilirubinaemia ii. Elevated LDH and AST iii. Low haptoglobin

Answer 103

a. Warm i. Most common – 60-90% of cases ii. Usually IgG preferentially binds at 37 degrees (“warm” antibodies) iii. Mechanism of haemolysis 1. Extravascular – mainly in the spleen a. Results in anaemia, conjugated hyperbilirubinaemia, high LDH, low haptoglobin b. Occasionally splenomegaly 2. Occasionally IgG present to fix complement causing intravascular haemolysis iv. Spherocytes on blood film b. Cold i. Relatively uncommon un children – 10% of cases ii. Most commonly occurs after Mycoplasma or EBV infection iii. IgM autoantibodies bind RBC at temperatures <37°C iv. Fix complement – results in anaemia due to two mechanisms 1. C’ mediated intravascular haemolysis – predominant mechanism 2. Immune-mediated extravascular clearance – mainly hepatic macrophages v. NOTE: only C’ is detected on RBCs because the IgM is removed during the washing steps of the DAT 4. Primary vs secondary a. Primary = 40-50% of cases, mostly warm-reactive b. Secondary i. Autoimmune ii. Immunodeficiency iii. Evans syndrome iv. Malignancy v. Infection vi. Transplantation vii. Drugs

Answer 104

1. Aetiology a. Primary b. Secondary – underlying disease ie lymphoproliferative disorder, SLE, Evans syndrome, or immunodeficiency 2. Clinical a. Acute transient type = 70-80% i. Lasting 3-6 months and occurring predominantly in children ages 2-12 years ii. Frequently preceded by an infection, usually respiratory iii. Onset may be acute, with collapse, pallor, jaundice, fever and hemoglobinuria OR more gradual, with primarily fatigue and pallor iv. The spleen is usually enlarged and is the primary site of destruction of IgG-coated RBCs v. Underlying systemic disorders are unusual vi. Prognosis = consistent response to glucocorticoids, low mortality rate + full recovery are characteristic b. Prolonged and chronic course i. More frequent in infants and children > 12 years old ii. Hemolysis may continue for many months to years iii. Response to glucocorticoids is variable and inconsistent iv. Prognosis = mortality rate 10%, death often attributable to an underlying systemic disease 3. Investigations a. FBE = anaemia (profound <60 g/L), leukocytosis, platelets normal i. Note can have low reticulocytes early in episode ii. Concomitant ITP sometimes occurs -> Evans syndrome b. Film = spherocytes, polychromasia (reflects reticulocytes) nucleated RBC c. Direct antiglobulin test = strongly positive i. Free antibody can sometimes be demonstrated in the serum (indirect Coombs) ii. Antibodies are active at 35-40°C (“warm” antibodies) and most often belong to the IgG class iii. They do not require complement for activity and are usually incomplete antibodies that do not produce agglutination in vitro

Answer 105

4. Treatment a. Mild disease and compensated hemolysis may not require treatment b. Folic acid supplementation c. PRBC = if haemodynamic instability prior to definitive treatment i. Test for underlying allo-antibody which can cause rapid hemolysis of transfused cells 1. Alloantibodies unlikely if not previously pregnant or previously transfused d. Glucocorticoids = for severe disease i. Mechanism – decrease the rate of hemolysis by blocking macrophage function by down regulating Fcγ receptor expression, decreasing the production of autoantibody and perhaps enhancing the elution of antibody from the RBCs ii. Continue until rate of hemolysis decreases and then wean iii. Disease tends to remit spontaneous within a few weeks or months iv. Coombs test may remain positive even after the Hb level returns to normal v. Usually safe to discontinue once direct Coombs test result becomes negative e. IVIG = if refractory to glucocorticoid f. Rituximab = Mab against CD20+ B cells i. Can be useful in chronic cases refractory to conventional therapy g. Plasmapheresis = has been used in refectory cases but generally not helpful h. Splenectomy = may be beneficial 5. Prognosis a. Acute idiopathic autoimmune hemolytic disease in childhood varies in severity but is self-limited b. Death from untreatable anaemia is rare c. 30% of patients have chronic haemolysis, often associated with underlying disease (eg. SLE, lymphoma) d. Presence of antiphospholipid antibodies in adult patients with immune hemolysis predisposes to thrombosis

Answer 106

1. Key points a. Less common in children b. May result in spurious macrocytosis (as RBC clumped together) 2. Aetiology a. Primary or idiopathic b. Secondary to infections such as those from Mycoplasma pneumoniae and Epstein-Barr virus c. Secondary to lymphoproliferative disorders. 3. Pathogenesis a. Cold antibodies usually have specificity for the oligosaccharide antigens of the I/i system b. M. pneumoniae infection  increased titres of anti-I Ab c. EBV infection  anti-i Ab – less haemolysis in adults as fewer i antigens on RBC d. Each IgM molecule has the potential to activate a C1 molecule  large amounts of C’ are found on the RBCs i. C’ mediated intravascular lysis ii. Destruction in liver 4. Direct antiglobulin test a. Cold antibodies agglutinate RBCs at temperatures <37°C b. They are primarily the IgM class and require complement for hemolytic activity c. The highest temperature at which RBC agglutination occurs is called the thermal amplitude d. A higher thermal aptitude antibody –one that can bind to RBCs at temperatures achievable in the body – results in hemolysis with exposure to a cold environment. e. High antibody titres are associated with a high thermal amplitude

Answer 107

5. Clinical manifestations a. 1-2 weeks post febrile illness b. Dark coloured urine, often after exposure to cold c. Anaemia i. Usually results in acute, self-limited episode of haemolysis ii. Severity related to amplitude of Ab and thermal amplitude of Ab (depends on IgM titre) iii. Agglutination triggered by exposure to cold (external temperature OR ingestion of foods) d. Acrocyanosis e. Fatigue f. Weakness or dyspnoea on exertion 6. Treatment a. Folic acid supplementation b. Indications for treatment i. Symptomatic anaemia ii. Cold dependence iii. Disabling circulatory symptoms c. Treatment options i. Avoidance of cold ii. Rituximab – most commonly used iii. Bortezomib – proteasome inhibitor iv. Cytotoxic agents d. Treatment of underlying disease e. Note glucocorticoids less effective + splenectomy NOT useful as predominantly intravascular haemolysis

Answer 108

Hapten’ mechanism (immune but not autoimmune) a. Example = penicillin or sometimes cephalosprorins b. Drugs bind tightly to the RBC membrane c. Antibodies to the drug, either newly or previously formed, bind to the drug molecules on RBCs, mediating their destruction in the spleen d. Direct antiglobulin test: Positive (anti-IgG) e. Haemolysis – extravascular 2. Ternary (immune) complex a. Example = quinine and quinidine b. Drugs do not bind to RBCs but, rather, form part of a ‘ternary’ complex, consisting of the drug, a RBC membrane antigen, and an antibody that recognizes both c. Direct antiglobulin test: Positive (anti-C3) d. Haemolysis – intravascular 3. Autoantibody induction a. Example = Methyldopa and sometimes cephalosporins b. By unknown mechanism incite true autoantibodies to RBC membrane antigens, so that the presence of the drug is not required to cause haemolysis c. Direct antiglobulin test: Positive (anti-IgG) d. Haemolysis – extravascular i. Cephalosporins are the most common cause of drug immune hemolytic anaemia

Answer 109

1. Pathogenesis a. Destruction of fetal RBC by maternal IgG antibodies due to the presence of Ag not present in maternal blood b. Ab produced when fetal RBC produce Ag not expressed by the mother c. Requires maternal-fetal haemorrhage or exposure with all antigens except ABO (pre-existing Ab in serum) 2. Clinical manifestations a. Highly variable – depends on type of HDFN i. ABO incompatibility – usually minor ii. Rhesus or minor blood group incompatibilities – more often severe b. Mild-moderate i. Self-limited haemolytic disease ii. Hyperbilirubinaemia in first 24 hours c. Severe i. Skin edema, pleural or pericardial effusion or ascites ii. May present at delivery with shock or near shock

Answer 110

a. Rh hemolytic disease i. Most common Alloimmune HDN (despite introduction of anti-D) ii. Maternal sensitization = either through transfusion or pregnancy with Rh +ve offspring 1. In the absence of transfusions Rh HDFN generally does NOT occur in 1st pregnancy iii. Rh positive blood from fetus into mother  mother develops Anti-D antibody 1. Initially IgM  IgG (only IgG can cross placenta & cause disease) iv. Highest risk if ABO match + Rh mismatch - when mother and fetus are also incompatible with ABO, mother is partially protected against sensitization by rapid removal of Rh-positive cells from circulation by pre-existing Anti-A or anti-B which are IgM & do not cross placenta b. ABO incompatibility i. A, B, AB and O ii. 3-6 months of age, start making A or B antibodies (found in food and bacteria) that they do not posses iii. ABO Alloimmune HDN can occur in 1st pregnancy 1. Group O – anti-A and anti-B Ab – IgG and able to cross placenta 2. Occurs almost exclusively in mothers who are BG O with A, B, AB + babies 3. Haemolysis is more common with Anti-A than with Anti-B 4. Individuals with BG A or B have antibodies against A or B – IgM usually therefore cannot cross placenta iv. Occurs in 15% of all pregnancy, only cause HDN in 4% v. Less severe than Rh - low antigenicity of ABO factors in fetus and newborn infant may account for low incidence of hemolytic disease c. Minor blood group i. Kell, Duffy, E, MNS ii. Ranges from mild to severe including hydrops fetalis iii. Kell HDN can be severe and require intrauterine intervention 1. Overall rare 0.1% women 2. Much more likely to haemolyse and have severe anaemia if mismatch 3. Less likely to have high bilirubin

Answer 111

a. Prevention i. Maternal blood group and antibody screening, serial antibody titres if positive or at risk – indirect Coomb’s test ii. Anti-D x2 throughout pregnancy iii. If high antibody titers, measure serial fetal MCA velocities – increased MCA velocity correlates with fetal anaemia b. Fetal management i. Intrauterine – if high risk and MCA dopplers abnormal, consider fetal cord sampling to determine Hb +/- fetal transfusion ii. Post-natal 1. Delivery at tertiary centre 2. Immediate FBE, DCT, BG, SBR on cord sample 3. Serial SBR 4. Phototherapy 5. Exchange transfusion if severe (removes maternal Ig) 6. PRBC transfusion in severe anaemia (match to mothers blood)

Answer 112

a. Aetiology i. ECMO ii. Prosthetic heart valve iii. Burns/thermal injury iv. Hypersplenism v. MAHA 1. TTP 2. HUS 3. TBI 4. Post Tx 5. Malignancy 6. Drugs a. Calcineurin inhibitors = cyclosporin, tacrolimus b. Sirolimus c. Mitomycin C d. Clopidogrel b. Pathogenesis i. Microvascular damage 1. RBCs sheared by fibrin in the capillaries during intravascular coagulation 2. Renovascular disease accompanies the HUS or TTP ii. Larger vessel damage 1. Kasabach-Merritt syndrome (giant hemangioma and thrombocytopenia) 2. Replacement heart valve is poorly epithelialized c. Investigations i. Film – “schistocytes,” or fragmented cells, as well as polychromatophilia ii. Secondary IDA due to urinary Hb and hemosiderin iron loss d. Treatment = variable depending on underlying problem; PRBC often not helpful as are destroyed

Answer 113

1. Haematopoiesis a. <6 weeks gestation = yolk sac b. 6-40weeks = stem cells migrate to bone marrow, liver and spleen – LIVER predominant site in fetal life c. Infancy = red bone marrow main site of RBC production 2. Haemaglobin a. Haemoglobin occupies 33% of the RBC cytoplasm b. Tetrameric protein c. 4x heme groups attach to 4 x globin chains, with each haeme group carrying one O2 molecule d. Two pairs of globin polypeptide chains: i. One pair of alpha-globin chains ii. One pair of non-alpha-globin chains (beta-, gamma-, or delta-globin) 3. Genetics a. Two Hb gene clusters located at the end of the short arms of chromosomes 16 and 11 i. Chromosome 16: alpha (α) gene cluster (Zeta, alpha 1/2) ii. On chromosome 11: beta (β) gene cluster (epsilon, gamma, delta, beta) 4. Types - Embryonic: epsilon+zeta (+gamma/alpha) - first 8wks - Foetal: alpha+gamma - Adult: alpha+beta - Adult2: alpha+delta 5. Normal Hb types a. From 6 months of age i. ≥95% HbA ii. ≤3.5% HbA2 iii. <2.5% HbF b. No substitute for alpha-globin in the formation of any of the normal hemoglobins following birth c. Absence of any alpha-globin  generally incompatible with extrauterine life 6. Haemaglobinopathies a. Either produce abnormal Hb  sickle cell b. Reduced amount of normal globin  thalassemia c. Investigations i. HEP, HPLC or isoelectric focusing used to identify variant Hbs ii. Separates variant Hb’s based on difference in charge iii. Sickle solubility testing detects only HbS, positive in sickle cell trait

Answer 114

1. Epidemiology = 90% African-American, 10% Hispanic 2. Classification a. Sickle cell anaemia = homozygous HbSS i. Both β-globin alleles have the sickle cell mutation (βs) ii. HbS is commonly as high as 90% of the total Hb b. Sickle cell disease = HbS >50% of all Hb i. Sickle cell anaemia ii. Compound heterozygotes – one β-globin allele includes the sickle cell mutation and the second β-globin allele includes a gene mutation other than the sickle cell mutation ie. HbC, β-thalassemia 3. Genetics a. AR b. Single base-pair change c. Thymine for adenine (GAG to GTG) at the sixth codon of the β-globin gene  valine instead of glutamine 4. Pathogenesis a. Hb molecules do not usually interact with each other; HbS results in a conformational change in Hb tetramer b. In the deoxygenated state, HbS molecules interact with each other forming rigid polymers  “sickled” shape c. Sickled erythrocytes are rigid and obstruct small blood vessels (VOC)  adhesion of leucocytes  local inflammation  disturbance in vasomotor tone (NO) d. Lung is only organ capable of reversing the polymers – therefore compromises the degree of reversibility e. Viscosity vaso-occlusion (erythrocyte sickling) i. Haemoglobin level ii. Vaso-occlusive pain crisis iii. Acute chest syndrome iv. Osteonecrosis f. Haemolysis-endothelial dysfunction (proliferative vasculopathy) i. Serum LDH ii. Reticulocyte count iii. Plasma Hb and arginase iv. Pulmonary HTN, priapism, leg ulcers, ? stroke 5. Investigations a. FBE = Hb 65-85, MCV 80-100, reticulocytes 5-15% b. Film = sickle cells, Polychromasia (reticulocytosis), Howell-Jolly bodies c. Hb electrophoresis = HbS d. Genetic testing

Answer 115

a. Overview i. Acute PRBC – anaemia, complicated VOC episode, ACS, aplastic crisis, splenic sequestration ii. Chronic PRBC – growth failure, frequent hospital admissions, stroke (secondary prevention) or primary prevention (elevated TCD) iii. Hydroxyurea iv. Folic acid v. Health maintenance – immunizations, nutrition, social supports, education b. Transfusions ii. Special requirements for sickle cell patients (at risk of stroke) iii. Indications 1. Acute intermittent a. Treatment of acute complication (eg. ACS, aplastic crisis, sequestration, CVA) b. To prevent surgery-related ACS in patient with abnormal TCD or MRI 2. Chronic transfusions a. Increase amount of HBA, reduce % of sickle cells; keep Hb < 100 / not increase by more than 30 as this increases risk of vaso-occlusive crises b. Indications i. Growth failure ii. Frequent hospital admissions iii. Stroke/ stroke prevention + Previous splenic sequestration iv. Risk of alloantibodies to less common RBC surface antigens – more extensive matching done to identify donor units C-, E- and Kell-Ag negative v. Blood transfusion methods 1. Automated/manual red cell exchange = removed patients blood, replace with donor blood 3. Simple transfusion = least preferable due to high net positive iron balance vi. Risks 1. TTI 2. Iron overload 3. Alloimmunisation (donor exposure, ethnic mismatch, inflam state) 4. Acute and delayed HTR 5. Hyperhaemolysis – specific to sickle cell disease, Ab attack transfused PRBC, lower Hb 6. Hyperviscosity c. Pharmacological i. Hydroxyurea 1. Only drug proven effective in reducing the frequency of painful episodes, ACS, dactylitis 2. Multiple mechanisms of action (Increases HbF, Improves NO metabolism, Reduces interaction between RBC and endothelium) 3. Indications a. Renal protection, prevention of proteinuria b. Silent cerebral ischaemia/preservation neurocognitive functioning c. Splenic complications d. Cardiopulmonary function/improved exercise tolerance e. Use in SC/Sbeta+ 4. Contraindications = pregnant or breastfeeding 5. Monitoring a. FBE, LFT and UECs – every 4 weeks during dose escalation then 3/12 b. HbS and HbF % every 3 months d. Good response = increased Hb, induction of HbF, increased MCV, decreased neut e. Discontinue if neutrophil or platelet count drops 6. Failure of therapy = non-adherence inadequate attempt MTD 7. Adverse effects a. Bone marrow suppression b. Leukaemia, leg ulcers no risk ii. Fe chelators (see thalassaemia notes) iii. Prophylactic penicillin = until age 5 at least iv. Routine immunization, annual flu vaccine + additional pneumococcal/ meningococcal d. Curative i. HSCT ii. Indications = ACS, stroke, abnormal TCD

Answer 116

a. Complications of blood transfusions (infections, iron overload, if require chelation – annual audiogram and organ toxicity monitoring (LFTs and pituitary function testing) because of iron deposition) b. Spleen palpation i. Splenomegaly common complication, splenic sequestration can be life threatening c. Transcranial Doppler ultrasound i. Primary stroke prevention d. Pulmonary and asthma screening i. Asthma associated with increased sickle cell disease morbidity and mortality ii. Annual screening iii. If snoring and daytime somnolence – consider OSA and referral to sleep specialist e. Retinopathy i. Annual screening from age 10 with ophthalmologist f. Renal i. Sickle-cell associated renal disease starts in infancy and may not become clinically manifested until adulthood ii. Screening for early signs 1. Annual urinalysis – proteinuria, albumin: creatinine ratio iii. Education re enuresis and priapism g. Echo i. Screening tool for pulmonary artery hypertension iii. Recommended patients with severe cardiopulmonary symptoms to be referred to cardiologist for formal evaluation

Answer 117

* Anaemia/haemolysis/ reduced RBC survival -> anaemia, aplastic crisis (parvovirus), jaundice, gallstones * Acute VOC -> painful VOC, ACS, stroke, splenic sequestration, priapism * Asplenia –> invasive infection * Chronic organ damage –> spleen, kidneys, lung, brain, eyes, hips (AVN), cardiac (iron toxicity)

Answer 118

a. Acquired immunodeficiency due to splenic infarction – by 5 years most children functional asplenia b. Pathogens i. At risk of encapsulated organisms (Streptococcus pneumoniae, Hib, and Neisseria meningitidis) ii. Salmonella osteomyelitis c. Site of infection i. Rate of bacteraemia <1% ii. Increased risk of osteomyelitis – often diaphysis of long bone cf. metaphyseal region d. Treatment i. Inpatient or outpatient management depending on how unwell the child is ii. Note risk of immune haemolysis with ceftriaxone iii. Management of Asplenia – vaccinations, prophylaxis, early treatment of infection 1. Generally prophylaxis until age 5

Answer 119

a. Parvovirus B19 results in temporary red cell aplasia and profound anaemia b. Usually only occurs once c. Consider in children with fever and reticulocytopaenia d. Treatment = PRBC e. Complications = pain, splenic sequestration, ACS, GN, stroke

Answer 120

a. Reduced incidence due to early diagnosis + education b. Can occur as early as 5 weeks of age – usually 6 months to 2 years c. May be triggered by fever, bacteraemia, or viral infections d. Clinical manifestations i. Rapid spleen enlargement causing left-sided abdominal pain ii. Hypovolemia as a result of the trapping of blood in the spleen iii. Signs of profound anaemia e. Investigations i. Decline in Hb of at least 20 g/L from the patient’s baseline, total Hb may fall below 30 g/L ii. +/- reticulocytosis iii. +/- thrombocytopaenia f. Treatment i. Supportive – fluid or blood 1. PRBC aborts RBC sickling in the spleen, allowing release of the patient’s sequestered blood cells, often raising the Hb above baseline ii. Typically recommend only 5 mL/kg of red blood cells because the goal is to prevent hypovolemia g. Complications i. Autotransfusion – the blood sequestered in the spleen is released and dramatically increases the Hb concentration, putting the patient at risk for hyperviscosity syndrome ii. Recurrent episodes 1. Occur in 2/3 of patients – usually within 6 months of previous episode 2. Prophylactic splenectomy only effective treatment 3. Blood transfusion does not reduce the risk of recurrent splenic sequestration

Answer 121

a. Most common cause for hospital admission + high mortality b. Acute chest syndrome i. Acute chest syndrome (ACS) = life-threatening pulmonary complication of sickle cell ii. Leading cause of ICU admission/mortality iii. Peak age 2-4 years iv. The most frequent event preceding ACS is a painful episode requiring systemic opioid treatment v. Aetiology 1. Infection = common, only 30% of ACS episodes will have positive sputum or BAL culture a. Most common pathogens – S. pneumoniae, Mycoplasma pneumoniae, and Chlamydia sp. 2. Fat emboli arising from infarcted bone marrow – consider if rapid onset resp distress and altered mental status 3. Pulmonary infarction 4. Atelectasis 5. Asthma, bronchospasm 6. Chronic hypoxia (OSA) vi. Pathogenesis 1. Hypoxia  sickling  vaso-occlusion ischaemia  endothelial injury 2. Pain  hypoventilation  hypoxia  sickling  pain vii. Clinical 1. Fever, respiratory distress, hypoxia, cough, or chest pain 2. +/- petechial rash viii. Diagnostic criteria 1. New radiodensity on CXR = single lobe involvement (predominantly left lower lobe), multiple lobes (most often both lower lobes) and/or pleural effusions, either unilateral or bilateral. 2. Plus any 2 of the following: fever, respiratory distress, hypoxia, cough, or chest pain ix. Risks = HbSS, low HbF, high WCC, hypoventilation x. Investigations 1. Blood cultures 2. NPA 3. Daily FBE 4. Continuous pulse oximetry 5. CXR xi. Treatment 1. Oxygen if drop by 4% over baseline, or values < 90% 2. Empirical antibiotics (clinical overlap between pneumonia and ACS) 3. Blood transfusion - simple or exchange (manual or automated) a. Only method to abort a rapidly progressing episode of ACS 4. Optimum pain control and fluid management 5. Chest physiotherapy xii. Prevention 1. Incentive spirometry and period ambulation 2. Observation 3. Cautious use of IV fluids 4. Hydroxyurea 5. Treatment of infections 6. Respiratory surveillance c. Pulmonary hypertension i. Major RF for death in adults with sickle cell anemia ii. The natural history is unknown

Answer 122

a. Dactylitis = hand-foot syndrome i. Often first manifestation of pain in infants and young children with sickle cell anemia 1. 50% of children by 2 years ii. Dactylitis often manifests with symmetric or unilateral swelling of the hands and/or feet iii. Difficult to distinguish from osteomyelitis b. Acute vasoactive pain i. Usually one episode per year requiring treatment ii. Clinical manifestations 1. Severe pain – most common chest, abdomen, extremities a. Skeletal pain may indicate bone or bone marrow infarction 2. Often abrupt iii. Aetiology = unknown, likely due to sickle cells obstructing blood flow resulting in ischaemia iv. Trigger = stress, infection, dehydration, hypoxia, acidosis, cold, swimming v. Treatment = analgesia, hydroxyurea (preventative)

Answer 123

a. Unwanted painful erection, usually between 3-9am b. Mean age of first episode is 15 years (90% by 20 years); reported in children as young as 3 years c. Prolonged (>4 hours) or stuttering (brief episodes that may occur in clusters) d. Both types occur from early childhood to adulthood e. Pathogenesis = low flow state due to venous stasis from sickling of RBC in corpora cavernosa f. Complication = recurrent prolonged episodes of priapism are associated with impotence. g. Treatment i. Supportive (hot shower, exercise, analgesia, urination) ii. A prolonged episode lasting >4 hr  urology referral for aspiration of blood from the corpora cavernosa + irrigation with dilute adrenaline iii. +/- simple blood transfusion and exchange transfusion – limited evidence h. Prevention = hydroxyurea

Answer 124

a. Primary toxic effect of blood transfusions b. Complications – organ damage and death c. Develop after 100ml/kg of red cell transfusion or about 10 transfusions d. Investigations i. Serum ferritin levels – most common measure 1. Note poor correlation with excessive iron in specific organs after 2 years of regular blood transfusion therapy ii. MRI – iron stores in heart and liver iii. Liver biopsy – standard for iron assessment, but does NOT accurately assess total body iron 1. Iron in liver is NOT equivalent to cardiac tissues e. Treatment i. Chelation – deferoxamine (S/c), deferasirox (effervescent tablet), deferiprone (oral) ii. Deferiprone – weekly FBE to monitor for neutropaenia

Answer 125

a. Renal disease i. Renal disease is a major comorbid condition that can lead to premature death. ii. Seven sickle cell disease nephropathies have been identified (1) gross haematuria (2) papillary necrosis, (3) nephrotic syndrome (4) renal infarction (5) hyposthenuria, (6) pyelonephritis, and (7) renal medullary carcinoma. iii. Clinical manifestations = haematuria, proteinuria, renal insufficiency, concentrating defects, hypertension b. Nocturnal enuresis i. High frequency of enuresis beyond early childhood – 9% of adolescents between 18-20 years of age ii. OSA is associated with an increased prevalence of enuresis (same as general population) iii. Most children with nocturnal enuresis do not have an aetiology iv. Biggest issue in clinical practice – important for long case

Answer 126

a. Include i. Stroke 1. Overt stroke = neurologic deficit lasting for >24 hr and/or abnormal neuroimaging 2. Silent cerebral infarct = no focal neurological deficits, diagnosed on imaging a. Commonly white matter between ACA and MCA b. Risk = male, lower Hb, higher BP, previous seizures, recurrent headache 3. Most at risk – HbSS and HbSβ-thalassemia zero 4. Ischaemic or haemorrhagic - ischaemic more common ii. Headaches iii. Seizures iv. Cerebral venous thrombosis – can mimic stroke v. PRES b. Investigations i. CTB/ MRIB with diffusion-weighted imaging - ischemic infarcts vs PRES. ii. Magnetic resonance (MR) venography –? cerebral venous thrombosis c. Treatment i. Supportive, aim Sp >96% ii. Transfuse within 1 hour to increase Hb to maximum of 100 g/L 1. Most efficient strategy to dramatically increase oxygen content of the blood if SaO2> 96% 2. If Hb threshold is exceeded oxygen delivery to the brain is limited due to hyperviscosity iii. Exchange transfusion at the time of acute stroke is associated with a decreased risk of second stroke when compared to simple transfusion alone d. Stroke prevention i. Transcranial Doppler ultrasonography (TCD) = from age 2 to 16 years, every 2 years 1. Assess blood velocity in terminal portion of ICA and proximal portion of MCA 2. Elevated time-averaged mean maximum (TAMM) blood-flow velocity >200 cm/sec increased risk for CVA ii. Blood transfusion therapy 1. If elevated start chronic transfusion to maintain HbS <30% to reduce risk of stroke a. Results in an 85% reduction in the rate of overt strokes b. Must continue indefinitely – cessation associated with increase in silent infarcts 2. Despite regular blood transfusion therapy, around 20% of patients will have a second stroke and 30% of this group will have a third stroke iii. Hydroxyurea iv. Red cell exchange v. HSCT

Answer 127

a. Chronic haemolysis results in formation of pigmented (bilirubin) gallstones b. Occur in 1/3 sickle cell patients by adulthood c. Symptoms – abdominal pain, nausea, vomiting d. Laparoscopic cholecystectomy if symptomatic

Answer 128

1. Key points a. Benign carrier condition b. NONE of the symptoms of sickle cell anaemia or other sickle cell diseases c. Normal lifespan 2. Epidemiology a. Varies throughout the world b. 7-10% of African Americans in the US 3. Genetics + pathogenesis a. One allele of the beta globin gene carries the sickle mutation and the other allele is normal, producing haemaglobin AS (HbAS) b. Production of HbS is influenced by the number of alpha-thalassemia genes present, and the amount of HbS c. Individuals with sickle cell trait – HbS level <50% 4. Investigations a. FBE – within normal range b. Hb analysis – diagnostic, revealing predominance of HbA, typically >50% and HbS<50% 5. Clinical manifestations a. Life expectancy = normal b. Urological + renal disease i. Haematuria ii. UTI iii. CKD iv. Renal papillary necrosis v. Renal medullary carcinoma – occurs predominantly in young adults and children vi. Microalbuminuria (adults) c. Vaso-occlusive phenomena i. Splenic infarction + splenic sequestration ii. Priapism iii. Hyphema (a pooling or collection of blood inside the anterior chamber of the eye (the space between the cornea and the iris)) d. Venous thrombo-embolism i. DVT e. Stroke f. Rhabdomyolysis + SCD i. Exercise-associated sudden death – likely second genetic factor and/or environmental factor ii. Exertional rhabdomyolysis g. Other i. Hyposthenuria (inability to appropriately concentrate urine) ii. Protection against falciparum malaria 6. Treatment a. Do not require exercise limitations b. Should receive maximum hydration and appropriate rest

Answer 129

1. Definition a. Group of genetic disorders of globin chain production in which there is an imbalance between the alpha-globin and beta-globin chain production b. The primary pathology in the thalassemia syndromes stems from the quantity of globin produced i. cf. sickle cell disease – quality of the β-globin gene produced 2. Key features a. Haemolytic anaemia b. Impaired iron handling 3. Epidemiology a. >200 mutations resulting in absent or decreased globin production b. 3% of the world’s population carry alleles for β -thalassemia c. SE Asia 5-10% of the population carry alleles for α-thalassemia (Review table in notes) - all generally microcytic +/- hypochromic/nucleated anaemias 4. Haemaglobinopathy screen a. Haemaglobin electrophoresis i. Haemaglobin A2 = α2δ2 ii. Haemaglobin F = α2γ2 iii. Haemaglobin Barts = γγγγ b. HPLC c. DNA analysis

Answer 130

1. Overview a. β-thalassemia syndromes usually require a β thalassemia mutation in both β-globin genes b. Carriers with a single β-globin mutation are generally asymptomatic, except for microcytosis and mild anaemia c. Becomes evident at 6 months of age when HbA usually becomes predominant (HbF prominent Hb prior to this) d. Nomenclature i. β0-thalassemia = no production of β-globin (QUANTITATIVE defect) 1. Homozygous for the β-thalassemia gene – cannot make any normal β chains (HbA) 2. More severe than β+-Thalassemia ii. β+ -thalassemia = decreased amounts of normal β-globin, but is still present (HbA) 2. Classification a. β-thalassemia major i. Severe β -Thalassemia patient who requires early transfusion therapy ii. Often homozygous for β0 mutations b. β-thalassemia intermedia i. Clinical diagnosis of a patient with less-severe clinical phenotype that usually does not require transfusion therapy in childhood ii. Many of these patients have at least 1 β+-Thalassemia mutation c. Other β-thalassemia syndromes i. Some β-thalassemia mutations have structural mutations such as HbE ii. Others, such as δβ-thalassemia or HPFH, are variants of β-thalassemia that have decreased production of β-globin gene with increased compensation of HbF iii. Concomitant alpha/ beta trait 1. AMELIORATES severity of beta thalassemia 2. Reduction in alpha globin synthesis reduces burden of alpha globin inclusions 3. Genetics a. AR b. 200-300 β-thalassemia alleles have been characterized 4. Pathogenesis a. Impaired production of beta chains + relative excess of alpha chains leads to an unstable Hb which precipitates i. Elevated HbA2 (α4 globin tetramers )  interact with RBC membrane and shorten cell production ii. Elevated HbF 5. Management a. Folate supplementation b. Blood transfusion = maintain Hb > 100 c. Manage Fe overload = generally start chelation before ferritin 2000 d. Splenectomy 6. Genetic counselling a. Both parents carrier – β Thalassaemia minor (may be silent, microcytosis only or confused with iron deficiency) b. Compound heterozygosity with other haemoglobinopathies eg Hb E/β, Hb S/β, Hb C/β thal also possible c. Prenatal diagnosis – fetal blood sampling/ CVS  requires SEQUENCING

Answer 131

1. Key points a. Symptomatic in late infancy (6-12mo) when HbA predominates b. If untreated – progressive HA, with profound weakness and cardiac decompensation from 6 months of life 2. Genetics + pathogenesis a. Homozygous β0β0= NO beta chains b. Inadequate β-globin gene production leading to decreased levels of normal Hb (HbA) i. Alpha-chains combine with γ-chains  HbF (α2γ2) dominant hemoglobin ii. δ-chain synthesis is not affected  increase in HbA2 production (α2δ2) c. Unbalanced α and β-globin chain production i. α -globin chains in excess  α globin tetramers (α4)  RBC inclusions ii. Free α -globin chains and inclusions are unstable  precipitate and damage RBC membrane shortening RBC lifespan d. Overall ineffective erythropoiesis results in erythroid hyperactivity and massive marrow expansion e. Coinheritances with alpha-thalassemia mutation is common + decreases severity of the β-thalassemia disease (reduce burden excess chains)

Answer 132

a. Haematological i. Anaemia ii. Jaundice + pigment gallstones iii. Hepatosplenomegaly 1. Due to chronic haemolysis 2. Exacerbated by extramedullary haemopoiesis 3. Typically develops in the first few years of life - splenectomy may be required b. Skeletal changes i. Thalassaemia facies = maxilla hyperplasia, flat nasal bridge, frontal bossing ii. Pathological bone fractures c. Iron overload i. Liver cirrhosis, carcinoma ii. Diabetes, hypothyroidism, hypogonadism, GH deficiency, hypoparathyroidism, DM , CCF iii. Osteopenia, osteomalacia iv. ↑ risk of Listeria, Yersinia, Salmonella d. Growth impairment i. Chronic anaemia ii. Hypermetabolic state due to ineffective erythropoiesis iii. Nutrient deficiencies iv. Toxicities of iron chelation v. Endocrinopathies e. Endocrine + metabolic abnormalities i. Hypogonadism, hypothyroidism, insulin resistance + growth impairment; partly due to iron deposition f. Other i. HF and arrythmias – associated with hepatic iron ii. Pulmonary abnormalities + pulmonary hypertension iii. Thrombosis iv. Leg ulcers

Answer 133

a. Bloods i. FBE 1. Progressive anaemia after the newborn period, falls to <60g/L unless transfused 2. Microcytosis, hypochromia (low MCH or MCHC) 3. Inappropriately low reticulocyte count (<8%) ii. Film – nucleated RBC, targeting, marked anisopoikilocytosis iii. Unconjugated bilirubin – elevated iv. Iron studies – elevated ferritin and transferrin saturation (even if not transfused) b. CXR = bone marrow hyperplasia c. Hb electrophoresis i. HbF (α2γ2) = elevated ii. HbA2 production (α2δ2) = elevated d. Endocrinological i. Bone age (XR of wrist and hand) ii. Thyroid function (TSH and FT4) treatment iii. Hypothalamic-pituitary-gonadal function: Gonadotrophin-Releasing-Hormone (GnRH) iv. Stimulation test for LH and FSH v. Sex steroids (serum testosterone, serum 17-, Estradiol) vi. Pelvic ultrasound to assess ovarian and uterine size vii. Transglutaminase antibodies viii. In selected cases 1. GH stimulation test 2. Insulin growth factor-I (IGF-I) 3. Insulin growth factor binding protein -3 (IGFBP-3), plasma zinc

Answer 134

a. Chronic transfusions i. Improves QOL and reduces complication ii. Usually necessary from 2 months to 2 years – rarely later iii. Transfusion indicated when signs of ineffective erythropoiesis 1. Growth failure, bone deformities secondary to marrow expansion, hepatosplenomegaly iv. Guidelines 1. Extended matching, leucocyte deplete 3. CMV-negative units for SCT candidates 4. Goal to maintain a pre-transfusion Hb of 95-105 g/L b. Management of transfusion-induced haemosiderosis i. Iron deposition 1. Liver = 1st site, occurs after 1 year 2. Endocrine system = 2nd site of deposition 3. Cardiac dysfunction = occurs after 8-10 years of transfusion ii. Monitoring 1. Serial serum ferritin levels – screening, but may inaccurately calculate quantitative iron stores 2. Non-invasive measurement of quantitative organ injury a. Quantitative liver MRI – best indicator of total body stores b. T2 MRI to determine quantitative cardiac iron (T2 MRI) should be obtained after 7 years of transfusion therapy iii. Chelation therapy 1. Usually commences after 1 year of transfusion therapy and correlates with the serum ferritin >1,000 ng/mL and/or a liver iron concentration of >2,500 µg/g dry weight 2. Goal to prevent hemosiderosis-induced tissue injury and avoid chelation toxicity a. Chelation toxicity increases as iron stores decrease 3. 3 available iron chelators a. Desferrioxamine – s/cut or IV b. Deferasirox – oral c. Deferiprone c. Hydroxyurea i. DNA antimetabolite ii. Increases stress erythropoiesis -> increased HbF production iii. Used in SCD, some patients with β-thalassemia intermedia iv. Studies in β-thalassemia major are limited d. HSCT e. Splenectomy i. May be required in patients who develop hypersplenism 1. Have a falling steady state Hb and/or a rising transfusion requirement ii. In thalassemia intermedia -> increased risk of venous thrombosis, pulmonary hypertension, leg ulcers, and silent cerebral infarction

Answer 135

a. Cardiac i. Cardiac failure and/or arrythmia 1. Major cause of death in thalassemia 2. Monitoring = serial echo, cardiac MRI 3. Treatment = intensive combination chelation therapy ii. Pulmonary hypertension 1. Frequently occurs in non-transfused thalassemia patients 2. May be an indication for transfusion therapy b. Endocrine i. Endocrine function progressively declines with age ii. Secondary to haemosiderosis and nutritional deficiencies iii. Iron deposition in the pituitary and endocrine glands can result in multiple endocrinopatheis 1. Hypothyroidism, hypogonadotrophic gonadism, growth hormone deficiency, delayed puberty, hypoparathyroidism, diabetes, osteoporosis and adrenal insufficiency iv. Monitoring starts at age 5 OR after at least 3 years of chronic transfusions 1. Height, weight, sitting height (truncal shortening is a cx of rx: desferrioxamine) 2. Nutritional assessments 3. Most patients need vitamin D, calcium, vitamin B, vitamin C, zinc replacement 4. Fertility is a concern

Answer 136

``` Significant cause of morbidity - siderophilic (iron loving - necessary for growth) Chelation (desferrioxamine) makes iron MORE available to Yersinia Transmission largely food borne (pork) Presentation - diarrhoea, abdo pain, fever - pharyngitis - can be confused with appendicitis - erythema nodosum, reactive arthritis ```

Answer 137

1. Key points a. Not chronically transfused but may sporadically require transfusions 2. Genetics + Pathogenesis a. Genetics variable = β0β+ or β+β+ b. Coinheritance of α-thalassemia trait or polymorphisms of globin promoters such as BCL11 i. May convert thal major to  nontransfusion-dependent thalassemia intermedia c. HbEβ-thalassemia (50%) - common cause of both transfusion-dependent and nontransfusion-dependent thalassemia 3. Clinical manifestations a. May be clinically silent. The typical age of presentation is two to four years of age. b. May have some complications of thal major – severity depends on degree of ineffective erythropoiesis c. Extramedullary haematopoeisis can occur in the vertebral canal, compressing the spinal cord and causing neurological symptoms emergency requiring immediate local radiation therapy to halt erythropoiesis 4. Investigations a. FBE i. Microcytic anaemia ii. Hb 70g/L (range 60-100 d/L) iii. Persistently normal RDW b. Electrophoresis i. HbF = elevated ii. HbA2 = elevated

Answer 138

• One defective beta globin gene o Complete absence of the beta globin protein β0 o Reduced synthesis of the beta globin protein β+ • Normal Hb, ↑HbA2 (>3.5%), ↑ HbF • Hb 95-120 • MCV 65-75

Answer 139

1. Key points a. Most common in SE Asia 2. Pathogenesis a. 2 genes with 2 maternal and 2 paternal alleles control α-globin production -> 4 α-globin gene alleles b. Absence or reduction in α-globulin production - the more genes affected, the more severe the disease c. Nomenclature i. αo mutation = no α-chains produced from the gene ii. α+ mutations = decreased amount of α-globin chain 3. Genetics a. Type of mutation i. Deletion mutations common b. An excess of β- and γ-globin chains are produced i. Form Bart hemoglobin (γ4) in fetal life and HbH (β4) after birth ii. Abnormal tetramers 1. Non-functional with high oxygen affinity 2. Do not transport oxygen 3. Result in extravascular haemolysis 4. Investigations a. Hb electrophoresis = may be useful if Barts Hb detected i. May be normal in carrier / trait if normal Hb produced ii. Haemoglobin Barts elevated in all newborns with alpha thalassemia mutations (carriers/trait and HbH) b. Genetic testing = PCR/ gene sequencing i. Targeted mutational analysis by PCR ii. Full gene sequencing c. Prenatal diagnosis can be performed via CVS/ amniocentesis/ molecular testing 5. Genetic counseling a. At risk population – African American, other – SEA, middle Eastern, Indian b. Known family history c. Suspicion of red cell indices eg microcytosis d. Haemoglobin electrophoresis may be unhelpful for carrier and trait as no abnormal Hb produced in adults e. Genetic testing and partner screening = PCR, gene sequencing f. Prenatal diagnosis – CVS/amniocentesis – molecular testing g. Cis vs trans has important implications for future pregnancies 6. Management (general) a. Folate supplementation b. Consider splenectomy c. Intermittent transfusion during severe anaemia for nondeletional HbH disease d. Avoid exposure to oxidative medications

Answer 140

a. Deletion of 1 α-globin allele b. Common in African-Americans c. Clinically silent d. Investigations i. FBE = normal MCV and MCH ii. Normal electrophoresis (isoelectric focus – IEF, haemaglobin electrophoresis – HEF) iii. During newborn period, <3% Hb Bart observed iv. DNA analysis – required for diagnosis

Answer 141

a. Deletion of 2 α-globin gene alleles b. The α-globin alleles can be lost - trans (different chromosome) or cis (same chromosome) i. African descent – trans configuration more common ii. Asia, Mediterranean region – cis deletions most common c. Investigations i. FBE 1. Mild microcytosis MCV 65-75 2. Mild anaemia Hb 10.5-12 g/L 3. Low MCH ii. Hb analysis normal (except during newborn period where Hb Bart <8% but >3%)

Answer 142

a. Deletion of 3 α-globin alleles = Haemoglobin H disease b. Classification i. Deletional HbH 1. Excess β chains form a tetramer (β4)= HbH – unstable and results in haemolysis ii. Non-deletional HbH 1. More severe form of HbH disease 2. Nondeletional α-globin mutations with 2 allele deletions c. Investigations i. FBE – microcytic anaemia (Hb 7-10, MCV 55-65), film Heinz bodies i. HbH 1. Newborn period – excess in γ-tetramers are present and Hb Bart is commonly >25% 2. Later in childhood – excess of β-globin chain tetramers that results in HbH ii. DNA mutation analysis d. Clinical manifestations i. Chronic anaemia ii. Mild splenomegaly iii. +/- scleral icterus or cholelithiasis e. Treatment i. Ongoing monitoring of growth and organ dysfunction ii. Dietary supplement with folate and multivitamins iii. Chronic transfusion not commonly required as Hb range 70-110 g/L – may require intermittent iv. May need transfusions with illness/pregnancy, 25% need some transfusion support v. Splenectomy – occasionally indicated 1. High risk of post splenectomy thrombosis  aspirin/ anticoagulant therapy considered f. Complications i. Haemosiderosis = secondary to gastrointestinal iron absorption and/or transfusion exposure, ii. Because HbH is an unstable Hb sensitive to oxidative injury avoid oxidative medications

Answer 143

a. Deletion of all 4 α-globin gene alleles b. Clinical manifestations i. Profound anemia during fetal life -> hydrops fetalis 1. The ζ-globin gene must be present for fetal survival 2. There are no normal hemoglobins present at birth (primarily Hb Bart, with Hb Gower 1, Gower 2, and Portland) ii. Massive hepatomegaly due to extramedullary haematopoeisis iii. Significant morbidity in mother during pregnancy iv. Often incompatible with life c. Investigations i. FBE = Hb 4-10 g/L d. Management i. Intrauterine transfusion – can improve fetal survival ii. If the fetus survives, immediate exchange transfusion is indicated iii. Transfusion dependent iv. HSCT only cure

Answer 144

1. Include a. Hb E/β Thalassaemia (50%) i. Hemoglobin E (HbE), a mutation of the beta globin chain, is associated with reduced expression (ie, it is a hemoglobin mutation which also expresses a thalassemic blood picture) and is mildly unstable to oxidative damage. b. Haemoglobin H disease (deletional/non deletional forms) 2. Pathogenesis a. Anaemia b. Haemolysis c. Ineffective erythropoiesis 3. Treatment a. Surveillance i. Growth and development ii. Complications of haemolysis/iron overload iii. Thrombosis b. Episodic transfusions i. Surgery, infection, pregnancy c. Role of splenectomy 4. Transfusions a. Regular lifelong transfusions not required, intermittent transfusion necessary during specific time periods (growth/pregnancy) or clinical situations (infection/surgery/illness) 5. Iron overload in NTDT a. Ineffective erythropoiesis (reduced hepcidin (NTDT) i. Increased GI absorption (ferroportin) ii. Transfusion iii. Increased recycling from RES

Answer 145

1. Key points a. Children have higher transfusion requirement per kg body weight common in early years, may influence rate of iron loading b. Impact of splenomegaly on transfusion requirement c. Evidence of iron loading present in young children – eg. beta thal major liver siderosis reported <3.5 years, cardiac pancreatic and pituitary iron loading, observed < 10 years 2. Pathophysiology a. One blood transfusion = 200mg iron i. Transfusion-dependent patients receive 0.1-0.8 mg/kg of iron each day (140mg/kg each year) ii. There is no physiological mechanism to excrete excess iron iii. 0.4-0.5mg/kg daily iron excretion is required in order to achieve iron balance in a transfusion-dependent individual b. Highly toxic labile iron  free radical  lipid peroxidation, DNA modifications, protein damage i. TGF-β1  collagen synthesis  fibrosis ii. Organelle damage  cell death iii. Resultant organ damage 3. Organ systems affected (generally liver, heart, endo) a. Pituitary – hypogonadotropic, hypogonadism b. Thyroid – hypothyroidism c. Parathyroid – hypoparathyroidism d. Heart – cardiomegaly e. Liver – cirrhosis, carcinoma f. Pancreas – diabetes g. Gonads – hypogonadotropic hypogonadism h. Bones – osteomalacia, osteoporosis

Answer 146

5. Investigations a. Serum ferritin i. Multiple cofounders ii. Correlates with cardiac overload and survival – rare for cardiac Fe overload with ferritin <1500 ug/L iii. Poor correlation with hepatic iron iv. Frequent monitoring, trends important v. Previous concern re chelation with ferritin <500ug/L (++SE) chelation holiday or dose reduction 1. Risks/SE of unbound iron chelators if no LPI b. Liver biopsy – rarely done (MRI) c. Echo d. Endocrine investigations e. Noninvasive measures – Liver/ cardiac MRI 6. Recommendation a. 3/12 serum ferritin – trend for individual utilized b. Liver iron – MRI to measure LIC (concentration) annually, and LFTs c. Cardiac T2 - begin 8-10 years, then 2nd yearly d. Echo – EF or GBPS (annual) e. Other – assessment for endocrinopathies f. Abdo U/S liver biopsy – HCC, cirrhosis, fibrosis

Answer 147

a. Chelation therapy can prevent (+/- reverse) complications caused by excess iron b. Key points i. Timely initiation ii. Close monitoring + continuous adjustment iii. Factor in higher transfusion requirement iv. Aim to maintain adequate Hb without toxicity for growth and development c. Indications for starting i. Children = >12-18 months transfusions, ferritin 1500-2000 ug/L (previously? wait until school age) ii. Adults = 10-20 units transfused, ferritin 1000-2500 ug/L d. Monitoring i. Liver/renal function ii. Audiology iii. Ophthalmology iv. Urine – Ca/Cr ratio v. Bone health – Vit D, Ca, ALP e. Goal i. Maintain LIC in normal/low range (2-7 mg/d dry weight) ii. Maintain serum ferritin 500-1500ug/L iii. If lower serum ferritin, concern chelation will decrease iron stores  higher toxicity from chelators iv. Normal T2*, plus echo v. Normal growth and puberty f. Agents iii. Generally 1. < 4 years: deferasirox (licensed > 6 years or if other CI or ineffective) 2. > 4 years: desferrioxamine or deferasirox

Answer 148

1. Key points a. Mutations are associated with a decrease in the production of either or both β- and δ-globins b. Imbalance in the α: non-α synthetic ratio characteristic of thalassemia c. More than 20 variants of HPFH have been described 2. δβ0 forms a. Deletions of the entire δ- and β-globin gene sequences b. The most common form in the United States is the Black (HPFH 1) variant. c. δ and β gene deletions  production only of γ-globin and formation of HbF (α2γ2). d. In the homozygous form, no manifestations of thalassemia are present i. There is only HbF with very mild anemia and slight microcytosis 3. Associations a. When inherited with other variant Hb  HbF is elevated into the 20-30% range b. When inherited with HbS  amelioration of sickle cell disease with fewer complications

Answer 149

a. Mutation for HbC is at the same site as HbS with substitution of lysine for glutamine (cf valine) b. Pathogenesis i. HbC crystallises, disrupting the RBC membrane ii. No sickling! c. Phenotypes i. Haemoglobin C trait (HbAC) = asymptomatic ii. Haemoglobin C disease (HbCC) 1. Mild anaemia 2. Splenomegaly 3. Cholelithiasis 4. Rare – spontaneous splenic rupture d. Diagnosis = usually newborn screening programs (USA) i. Film - HbC crystals may be visible on peripheral smear

Answer 150

a. Abnormal Hb resulting from qualitative mutation in the beta-globin gene b. Second most common globin mutation worldwide c. Phenotypes i. HbE/ beta thalassemia 1. Common in South East Asians 2. HbE = abnormal Hb with single point mutation in B chain 3. Similar presentation to moderately severe thalassemia ii. HbE/E homozygous: mild haemolytic anaemia with splenomegaly iii. HbE/ sickle – mild haemolytic anaemia with splenomegaly iv. HbE heterozygous – no effect, may have low MCV + target cells

Answer 151

1. Key points a. Clinical syndrome caused by increased serum concentration of methaemoglobin b. In normal individuals, oxidation of Hb to MetHb occurs at a slow rate, 0.5-3% i. Countered by MetHb reduction to maintain a steady state of 1% MetHb 2. Pathogenesis a. Methemoglobin = altered state of Hb in which the ferrous (Fe++) ions of heme are oxidized to the ferric (Fe+++) state i. Unable to reversibly bind oxygen ii. Oxygen affinity of any remaining globins ferrous haemes in the Hb tetramer are increased b. Net effect i. "left-shifted"  the remainder of the haemes in the hemoglobin tetramer have an INCREASED affinity for oxygen ii. Functional anaemia (ie, the amount of functional hemoglobin is less than the measured level of total hemoglobin) iii. Impaired oxygen delivery to tissues c. Consequences = compensatory polycythemia/erythrocytosis d. Reduction of MetHb i. Physiological/ predominant pathway = NADH-dependent reaction catalysed by cytochrome B5 reductase ii. Alternative pathway = utilizes NADPH generated by G6PD in the hexose monophosphate shunt and requires an extrinsic electron acceptor to be activated (eg. methylene blue, ascorbic acid, riboflavin) e. Infants = vulnerable to Hb oxidation as i. Erythrocytes have half the amount of cytochrome b5 reductase seen in adults ii. Fetal hemoglobin is more susceptible to oxidation than HbA iii. The more alkaline infant GIT promotes the growth of nitrite-producing Gram-negative bacteria 3. Aetiology a. Congenital forms = autosomal recessive i. Type I (functional deficiency of cytochrome B5 reductase in RBC only) 1. Cyanosis and asymptomatic ii. Types II (deficiency of cytochrome b5 in all cells) 1. Cyanosis, developmentally delayed, most affected infants die in 1st year of life b. Acquired forms i. Drugs – dapsone, topical anaesthetics, inhaled NO, aniline ii. Medical conditions – sepsis, drug overdose, sickle cell disease iii. Miscellaneous – fume inhalation, industrial chemicals, pesticides

Answer 152

4. Clinical manifestations a. When MetHb levels are >1.5 g/24 hr, cyanosis is visible (15-20% MetHb) i. A level of 70% MetHb is lethal b. Methaemoglobin can color the blood brown c. Asymptomatic initially  headache, fatigue, dyspnea, lethargy  respiratory depression, altered conscious state, seizures  death 5. Diagnosis a. Suspected by presence of clinical “cyanosis” in presence of normal paO2 on ABG i. Hypoxia does NOT improve with increased administration of oxygen b. Pulse oximetry may be inaccurate c. Lab test 6. Treatment a. Congenital i. Avoid causative drugs – nitrites, aniline derivatives ii. Treatment of cyanosis in individuals with type I and II cytochrome b5 reductase deficiency is cosmetic only iii. Ascorbic acid 1. Daily oral treatment, 200-500 mg/day in divided doses 2. Gradually reduces the MetHb to approximately 10% of the total pigment and alleviates the cyanosis as long as therapy is continued 3. AE = hyperoxaluria and renal stone formation iv. Riboflavin = 400 mg daily v. Methylene blue 1. IV (1-2 mg/kg initially), is used to treat toxic methemoglobinemia 2. An oral dose can be administered (100-300 mg PO per day) as maintenance therapy 3. Should not be used in patients with G6PD deficiency – results in haemolysis b. Acquired i. Stop the drug/ treat underlying conditions ii. If symptomatic – supportive care, blood transfusion, methylene blue and ascorbic acid iii. Hyperbaric oxygen

Answer 153

a. Vascular spasm i. Smooth muscle in vessel wall contracts, reducing blood loss ii. Enhanced by thromboxane A2 released by platelets b. Platelet plug formation i. Endothelial damage exposes collagen, vWF bridges collagen + platelets via Gp1b receptors ii. ACTIVATION = platelets swell, protrude pseudopods and contractile proteins contract, releasing granules (ADP + thromboxane) iii. AGGREGATION = activated platelets express Gp2b/3a – binds fibrinogen onto activated platelets, mediates clot retraction iv. SECRETION 1. ADP + 5HT = recruit additional platelets 2. TxA2 = platelet recruitment and vasoconstriction c. Clot formation i. Occurs within 15 seconds to 2 minutes ii. Formation of fibrin clot via coagulation cascade d. Fibrinolysis i. Plasminogen is converted to plasmin by kallikrein (activated by factor VII) ii. Plasmin contains trypsin, which digests fibrin, fibrinogen, factors II/V/VII/VIII iii. Plasminogen is present in clots, but does not become activated until tissue plasminogen activator released (often a few days after clot formation)

Answer 154

a. Extrinsic – activated by tissue factor i. Damage to vessel wall exposes/releases tissue factor ii. Tissue factor activates factor VII iii. TF +VIIa leads to activation of Factor X (start of common pathway) b. Intrinsic – triggered by contact activation with platelet receptor 2b3a i. Activation of Factor XII (as contacts collagen) ii. Factor XII activates Factor XI (requires high MW kinogen, prekallikrein) iii. Factor XI activates Factor IX iv. Factor IXa + FVIII + phospholipids  activates factor X c. Common pathway i. Activation of Factor X ii. Xa + Ca+ phospholipid + V = prothrombinase complex = cleaves prothrombin  thrombin 1. Factor V accelerates this activity iii. Thrombin cleaves fibrinogen to fibrin 1. Fibrinogen formed in the liver 2. Thrombin removes a LMW peptide from fibrinogen to create a fibrin monomer 3. Fibrin monomers can polymerize with each other to form long fibrin factors iv. Factor 13 a crosslinks fibrin to stabilize the clot v. Thrombin also provides positive feedback 1. Converts prothrombin into more thrombin 2. Accelerates actions of factors FVIII-XII d. “Clotting factor complexes” form on surface of platelets i. Prothrombinase complex = factor Xa+Va+ prothrombin, with calcium + phospholipid ii. Tenase complex (IXa + VIII + X)

Answer 155

a. Intact endothelial wall i. ‘smooth surface’ stops platelets adhering ii. Glycocalyx (mucopolysaccharide) repels clotting factors b. Protein C and Protein S i. Activated protein C degrades factors Va and VIIIa (protein S = cofactor) ii. Protein C activated by thrombin-thrombomodulin complex c. Antithrombin 3 i. Combines with loose thrombin not adsorbed to fibrin fibres ii. Reduces the amount of clot iii. Mainly regulates thrombin and factor Xa iv. To a lesser extent, modulates 9,11 and 12 a d. Thrombomodulin i. Sits in endothelial membrane ii. Binds thrombin

Answer 156

• Normal newborn has reduced levels of most procoagulants and anticoagulants – more marked in preterm infant • Coagulation factors do NOT cross the placenta o Synthesised dependently by neonatal liver o During gestation – progressive maturation and increase in clotting factor production • Extremely premature infant o Prolonged PT and APTT values o Reduction in anticoagulant proteins (protein C, protein S, ATIII) • The vitamin K dependent factors – 2, 7, 9, 10 and contact factors 11, 12 are reduced by approximatley 50% adult values • Vitamin K facilitates the post-transcriptional carboxlylation of factors 2, 7, 9, 10 • Adults vs newobrn o Factors V, VIII, XIII and fibrinogen are all similar to adult values o Plasma concentrations of anticoagulant proteins – antithrombin, protein C, protein S are signfiicantly lower at birth than during adult years o Plasminogen is reduced by approximaly 50% o Platelet counts are normal o vWF concentrations are increased in neonates * Protein C and protein S are physiologically reduced - normal factors V and VIII are not balanced with their regulatory proteins * Physiologic deficiency of vitamin K dependent procoagulant proteins (factor II, VII, IX, and X) is partially balanced by the physiologic reduction of AT-III * The net effect is that newborns (especially premature infants) are at increased risk of complications of bleeding, clotting or both

Answer 157

``` COAGULATION (e.g. haemophilia) Skin – Petechiae Not usually seen Ecchymoses Common – Large one or more Soft tissue hematoma Characteristic Joint hemorrhages Characteristic – Hallmark of the disease Delayed bleeding Common Bleeding from superficial skin abrasions Uncommon Family history of bleeding Common Sex of the patient Predominantly male ``` ``` PLATELETS / blood vessels (e.g. VWD) Skin – Petechiae: Characteristic Ecchymoses: Characteristic (small, scattered) Soft tissue hematoma: Rare Joint hemorrhages: Not usually seen Delayed bleeding: Rare Bleeding from superficial skin abrasions: Common and persistent Family history of bleeding: Rare Sex of the patient: Predominantly female ```

Answer 158

i. PFA-100 measures platelet adhesion-aggregation in whole blood at high shear when exposed to either collagen –epinephrine or collagen-ADP 1. Expressed as the closure time measured in seconds 2. Sensitive for severe forms of vWD and platelet dysfunction 3. Variable specificity ii. Affected by medications including NSAID, valproic acid

Answer 159

a. APTT = addition of surface activator, assesses INTRINSIC pathway i. Presence of fibrin detected via electromechanical method ii. Assesses intrinsic pathway – VIII, IX, X, XI, XII iii. Prolonged 1. Heparin 2. Lupus anticoagulant 3. Factor deficiency – haemophilia A and B b. PT = addition of tissue factor, assesses EXTRINSIC pathway i. Assesses common pathway and factor FVII ii. INR = standardization of PT to account for differences in lab reagents iii. Prolonged 1. Factor VII deficiency / inhibitor 2. Vitamin K deficiency (mild) or early liver disease 3. Warfarin c. Fibrinogen i. If quantity > 1, not cause of bleeding (unless DIC) ii. Is also an acute phase reactant iii. If low  use cryoprecipitate iv. If normal  FFP a. Thrombin time i. Time taken to convert fibrinogen to fibrin ii. Often used to determine if heparinized

Answer 160

i. Measure of fibrinolysis – formed by plasmin degradation of cross-linked fibrin ii. Elevated in DIC/ DVT

Answer 161

Extrinsic pathway * Factor VII deficiency / inhibitor * Mild vit K deficiency * Warfarin therapy * Early liver disease * Lupus anticoagulant

Answer 162

Intrinsic pathway * Factor VIII/IX/XI/XII deficiency or inhibition * vWF disease * Heparin * Lupus anticoagulant

Answer 163

* Prothrombin/ fibrinogen/ factor V/X def/inhibitor * DIC (D-dimer elevated, cf liver disease which D dimer is normal) * Liver disease * Severe vitamin K deficiency

Answer 164

* Thrombocytopenia * Platelet function disorder * Factor XIII def (produced in endothelial cells rather than liver - normal in liver disease) * vWF disease

Answer 165

Fibrinogen

Answer 166

Prothrombin

Answer 167

``` Inheritance X-linked Factor deficiency Factor VIII Bleeding sites Muscle, joint, surgical Ix PT N APTT Prolonged Bleeding time N Factor VIII coagulant activity ↓ Von Willebrand factor activity N Factor IX N Platelet aggregation N Ristocetin-induced platelet agglutination N Treatment DDAVP Recombinant factor VIII ```

Answer 168

``` X-linked Deficiency: Factor IX Bleeding sites: Muscle, joint, surgical Ix PT: N APTT: Prolonged Bleeding time: N Factor VIII coagulant activity: N Von Willebrand factor activity: N Factor IX: reduced Platelet aggregation: N Ristocetin-induced platelet agglutination: N Treat: Recombinant factor IX ```

Answer 169

``` Autosomal dominant Deficiency: vWF and VIIIC Bleeding sites: Mucous membranes, skin, surgical, menstrual PT: N APTT: Prolonged or normal Bleeding time: Prolonged or normal Factor 7 activity: ↓ or N vWF activity: ↓ Factor 9 activity: N Platelet aggregation: N Ristocetin-induced platelet agglutination: Normal, low or increased Treat: DDAVP or vWF concentrate ```

Answer 170

A: VIII B: IX C: XI

Answer 171

1. Epidemiology a. 1:5000 males - 85% factor VIII deficiency and 10-15% factor IX deficiency b. No racial predilection, appears in all ethnic groups 2. Pathogenesis a. Factors VIII and IX participate to form ‘tenase’ or factor-X activating complex (with phospholipid + calcium) b. In vitro = factor X is activated by either the complex of factors VIII and IX or the complex TF (tissue factor) and factor VII c. In vivo = complex of factor VIIa and TF activate factor IX to initiate clotting d. In the lab = prothrombin (PT) measures the activation of factor X by factor VII and is therefore normal in patients with factor VIII or factor IX deficiency e. Consequences i. Clot formation is delayed ii. Failure to tightly cross-link fibrin iii. Re-bleeding occurs during the physiologic lysis of clots or with minimal new trauma 3. Genetics a. X-linked – the genes for factors VIII and IX are carried near the terminus of the long arm of the X chromosome i. Majority have reduced production d. Some female carriers of haemophilia A or B have mild bleeding disorders (lyonization of the X chromosome) 4. Classification a. Severity = classified on the basis of the patients baseline level of factor VIII or factor IX i. By definition, 1IU of each factor is defined as that amount in 1ml of normal plasma ii. Therefore 100ml of normal plasma has 100IU/dL (100% activity) of each factor iii. % activity  refers to the percentage found in normal plasma (100% activity) b. Classification i. Severe hemophilia = <1% activity = spontaneous bleeding ii. Moderate hemophilia = 1-5% activity = mild trauma to induce bleeding iii. Mild hemophilia = >5% activity = significant trauma to induce bleeding

Answer 172

5. Clinical manifestations a. Most commonly present when child begins to mobilise if not diagnosed as a neonate b. Newborn i. FVIII and FIX do not cross placenta – therefore bleeding may occur in fetus/ newborn ii. Intracranial hemorrhage – 2% of neonates iii. Bleeding with circumcision – 30% of males c. Bleeding from minor trauma d. Haematomas e. Haemarthrosis = characteristic of haemophilia i. May be induced by minor trauma, however many spontaneous ii. Ankle most common early joint, knees and elbows in older children iii. Warm, tingling sensation in the joint as the first sign of an early joint haemorrhage iv. Repeated bleeding episodes into the same joint  may become a target joint v. Spontaneous bleeding may occur due to underlying joint damage f. Muscle haemorrhages i. Iliopsoas muscle haemorrhage = can result in large amount of blood loss resulting in shock 1. Present with vague referred pain in groin, hip held in flexed internally rotated position 2. Diagnosis clinically and confirmed with imaging g. Life-threatening bleeding i. CNS or upper airway ii. Exsanguination – external trauma, GI, iliopsoas 6. Investigations a. Prolonged APTT = if severe 2-3x of normal b. Normal PT/INR c. Normalisation of PTT with mixing studies i. If does not correct – inhibitor present ii. 25-35% of patients with haemophilia who receive infusions of factor VIII/IX inhibitor present iii. Quantitative Bethesda assay for inhibitors should be performed to measure antibody titre d. Specific assay for factors VIII and IX – confirm the diagnosis i. Note difficulty in newborn diagnosis 1. FVIII falsely elevated as acute phase reactant 2. FIX physiologically low in newborn ii. Haemostatic level 1. FVIII >30-40% 2. FIX >25-30% iii. Normal >=50% e. Ratio of FVIII: vWF – sometimes used to diagnose carrier i. Factor VIII is carried in plasma by von Willebrand factor f. Genetic testing

Answer 173

a. Lifestyle - prevention i. Avoid trauma (but should encourage exercise/sport/activity) ii. Anticipatory guidance (car seats, seatbelts, bike helmets, avoiding high risk behaviour) iii. Avoid aspirin and NSAIDS that affect platelet function b. Desmopressin – factor VIII only i. Indicated for mild FVIII deficiency (haemophilia A) ii. Desmopressin stimulates release of endogenously produced FVIII – peaks after 30-60 minutes iii. Concentrated intranasal form available c. Recombinant factor – factor VIII and factor IX i. Acute therapy 1. Mild to moderate bleeding = aim 35-50% 2. Life-threatening or major hemorrhages = aim 100 IU/dL or 100% 3. Calculation for recombinant factor a. Dose of rFVIII(U)=% desired (rise in rFVIII) x body weight (kg) x 0.5 b. Dose of rFIX(U)=% desired (rise in rFIX) x body weight (kg) x 1.4 4. Benefits a. Avoid the infectious risk of plasma-derived transfusion-transmitted disease ii. Prophylaxis 1. Superior to episodic treatment in preventing debilitating joint disease 2. Recommended for severe hemophilia, usually initiated with the 1st or 2nd joint haemorrhage 3. May require CVC insertion 4. Treatment every 2-3 days to maintain measurable plasma level of clotting factor (1-2%) when assayed just before the next infusion (trough level) a. Often done as home therapy 5. If moderate arthropathy develops, prevention of future bleeding will require higher plasma levels of clotting factors 6. If target joints develop, ‘secondary’ prophylaxis is often initiated

Answer 174

a. Chronic arthropathy i. Occurs due to inflammatory response following heamarthrosis 1. Synovium thickens and projects into the joint - susceptible to being pinched and may induce further haemorrhage 2. Cartilaginous surface becomes eroded - joint susceptible to articular fusion ii. Bleeding into target joint results in severe pain as no space to accommodate blood iii. If target joint develops – short or long-term prophylaxis indicated b. Development of inhibitor to either factor VIII or factor IX i. Usually occurs shortly after factor replacement therapy has been initiated ii. The inhibitors are primarily IgG antibodies iii. More common in haemophilia A than B (and more common if severe as less endogenous factor) 1. 30% of patients with severe haemophilia A 2. 5% of patients with severe haemophilia B a. Many patients with haemophilia B make an inactive dysfunctional protein therefore less likely to make an Ab to replacement iv. Manifestations 1. First sign usually failure of bleeding episode to respond to replacement 2. HOWEVER – does NOT lead to marked increase in frequency of bleeding 3. Results in more difficult to control bleeds, and number of bleeding episodes may increase over time due to development of musculoskeletal complications v. Classification 1. Degree of response in Bethesda units classify patients with factor VIII or IX inhibitors 2. High responders = titres above 5 Bethesda units at any time a. Increase in Ab titre after each exposure; begins within 2-3 days, peaks at 7-21 days, and may persist for years b. High inhibitor levels render treatment with FVIII preparations ineffective 3. Low responders = titres <5 Bethesda units a. Primarily occur early in treatment in young children b. Much less common in patients with moderate and mild haemophilia A vi. Risk factors 1. High purified factor IX or recombinant factor IX 2. Some anti-factor IX inhibitors induce anaphylaxis vii. Management 1. Many patients with an inhibitor lose it with continued regular infusions 2. Inhibitor eradiation = immune tolerance 3. Rituximab = used off label as an alternative therapy with high titres in whom immune tolerance programs have failed 4. Some centres have trialed prednisolone +/- cyclosporine 5. Bypassing products = activated FVIIa (rFVIIa – NovoVII) or activated prothrombin complex concentrates (factor VIII inhibitor bypassing activity) a. Activated FVII can directly activated factor X – bypasses need for FVIII and FIX b. Available products i. Recombinant activated FVII – rFVIIa (NovoSeven) ii. Activated prothrombinex complex (aPCCs) 6. Recombinant porcine (pig) FVIII – also available c. Transfusion-transmitted infection i. Uncommon with modern screening techniques d. Complications of central access i. Thrombosis ii. Infection e. Obesity i. Complicated venous access ii. Clotting factor usage iii. Exacerbation of joint disease

Answer 175

Factor XI deficiency 1. Key points a. Autosomal deficiency associated with mild-moderate bleeding symptoms b. Frequently encountered in Ashkenazi Jewish people c. Bleeding NOT correlated with the amount of factor XI 2. Clinical manifestations a. Bleeding tendency not as severe as in factor VIII or factor IX deficiency b. Bleeding with surgery – particularly in oral cavity (sites of high fibrinolytic activity) c. Chronic joint bleeding is rarely a problem and for most patients 3. Pathogenesis a. Factor XI augments thrombin generation and leads to activation of the fibrinolytic inhibitor thrombin –activatable fibrinolysis inhibitor 4. Investigations a. Prolonged PTT i. Homozygous deficiency of factor XI PTT is often longer than it is with patients with either severe factor VIII or factor IX deficiency ii. The paradox of fewer clinical symptoms in combination with longer PTT is surprising, but it occurs because factor VIIa can activate factor IX in vivo. b. Genetic studies 5. Treatment a. Replacement pre-operatively with FFP i. Plasma infusions of 1 IU/kg usually increase the plasma concentration by 2%. ii. Infusion of plasma at 10-15 mL/kg will result in a plasma level of 20-30%, which is usually sufficient to control moderate hemorrhage iii. Frequent infusions of plasma would be necessary to achieve higher levels of factor XI. iv. Because the half-life of factor XI is usually ≥48 hr, maintaining adequate levels of factor XI commonly is not difficult b. Fibrinolytic inhibitors like aminocaproic acid = particularly with oral surgery

Answer 176

Can have deficiencies in any other (not VIII, IX, XI, VWF) factor Less common - essentially all autosomal genes -> less frequent and often heterozygous with less impairment Symptoms vary Treatment depends on cause and symptoms - usually replacement of factor (e.g. FFP)

Answer 177

Von Willebrand disease

Answer 178

Epidemiology a. Most common inherited bleeding disorder b. Estimated prevalence at 1:100 to 1:10 000, M=W Type 1 = 80% = mild-mod quantitative defect Type 2 = 20% = qualitative Type 3 = rare = complete deficiency 2. Genetics a. Most AD - short arm of Chromosome 12 codes for vWF b. Genotype does not correlate with phenotype – genetic modifiers 3. vWF Ag a. Key points i. Large glycoprotein ii. Synthesized in megakaryocytes + endothelial cells iii. Stored in platelet alpha granules + endothelial cell Weibel-Palade bodies iv. Circulates as different sized multimers, high MW the most active form v. Cleaved by circulating ADAMTS13 to various multimer sizes b. Functions i. Adheres to subendothelial matrix after vascular damage ii. Adheres to platelets via glycoprotein Ib receptor, with resultant activation of platelets 1. Shear stress induces a conformational change in VWF that facilitates its ability to bind platelets through a binding site on platelet glycoprotein 1b (GP1b) iii. Acts as the carrier protein of factor VII

Answer 179

4. Clinical manifestations a. Mucosal bleeding (similar to other platelet defects) – including epistaxis b. Easy bruising c. Menorrhagia d. Surgical bleeding – particularly with dental extractions and T&As e. Joint bleeds – severe type 3 f. Most patients have a FHx of bleeding 5. Classification Type 1 (partial quantitative deficiency) • Most common – 75% • Variable bleeding severity; mild to severe • AD inheritance • VWF activity and RIPA decreased • RIPA may be normal in mild disease • Multimer electrophoresis: all multimers present and uniformly decreased Type 3 (severe quantitative deficiency) • Rare • Clinically similar to hemophilia A with joint and soft tissue bleeding • Severe mucosal bleeding • AR inheritance • No detectable vWF • Factor VIII levels low (1 to 10%) • Multimer electrophoresis: Undetectable or too faint to visualize Type 2 (qualitative variant) - multiple types (A (most common), B, M, N) - decreased VWF activity - generally reduced large multimers - Factor VII levels normal

Answer 180

a. FBE = anaemia +/- thrombocytopaenia (type 2B, platelet type pseudo-vWD) b. Bleeding time = prolonged c. Coagulation studies = APTT may be prolonged (if FVIII low), but normal in mild type I d. Mixing study = if APTT prolonged, will be corrected with mixing study e. vWF panel i. VWF antigen (VWF : Ag) = measures the total amount of VWF protein present 1. VWF : Ag <20-30 IU/dL are most likely to have a genetic defect in VWF 2. VWF : Ag between 30 and 50 IU/dL are said to have “low VWF” 3. Neonates – reach adult levels by 6 months ii. VWF activity = typically performed using the ristocetin cofactor activity assay (VWF : RCo) and provides a measure of the amount of functional VWF 1. Ristocetin initiates binding vWF to platelet GpIb iii. FVIII = VWF protects factor VIII from proteolysis, therefore, decreased plasma VWF or a mutation in the VWF binding site for factor VIII can lead to ↓ factor VIII concentrations 1. If decreased sufficiently results in prolonged APTT iv. VWF multimer distribution 1. Blood group = blood group O associated with reduced vWF antigen (group: AB > B > A > O) f. Desmopressin challenge i. 30 minute infusion at 0.3 mcg /kg - vWF antigen measured at 30 minutes, 1 hours and 4 hours ii. Done in case of need for surgery iii. Contraindicated in children < 2 years of age – case of hyponatraemic seizures iv. 80% respond g. NOTE i. Increase VWF levels – stress, exercise, and pregnancy ii. Decrease VWF levels –hypothyroidism, and medications, such as valproic acid, blood group O

Answer 181

a. “Significant mucocutaneous bleeding” i. Nose bleeding > 2 episodes without trauma not stopped by a short compression of < 10 minutes or > 1 episode requiring blood transfusion ii. Cutaneous bruising requiring medical presentation with no hx of trauma iii. Prolonged bleeding from a wound > 15 minutes/ recurring during 7 days after wounding iv. Spont GI bleeding v. Heavy prolonged/ recurrent bleeding after tooth extraction/ tonsillectomy or adenoidectomy vi. Menorrhagia b. Family history (clinical/biochemical) c. Laboratory criteria i. Levels of both VWF: Ag + RICOF < 2 SD below population mean and ABO adjusted mean ii. Occurs on at least 2 occasions

Answer 182

a. Non-transfusional i. Desmopressin – increases the amount of circulating VWF by release from endothelial cells 1. May increase level of VWF + Factor VIII by 3-5 x over 8-10 hours 4. Side effects – vasodilation – facial flushing, headache, nausea and tingling 5. Can be given intranasally but results in tachyphylaxis (rapidly diminishing response to successive doses of a drug) after repeated administration ii. Tranexamic acid 1. Antifibrinolytic agent 2. Prevents dissolution of haemostatic plug 3. Can be given o/IV  good for menorrhagia, epistaxis b. Tarnsfusional i. Plasma-derived FVIII – contains vWF (recombinant FVII contains NO vWF) = biostate 1. Ratio of FVIII: vWF is 1:2 c. Treatment by type i. Type 1 = desmopressin ii. Type 2 + 3 = VWF containing concentrates d. Monitoring i. VWF and FVIII levels to tailor treatment for surgeries and major trauma 1. Plasma FVIII most important in surgical or soft tissue bleeding 2. Functional measures of VWF most important for mucosal bleeding e. Adjunct i. Hormonal therapy – menorrhagia ii. Local treatment ie nasal cautery or packing- epistaxis iii. Iron therapy – if iron deficient iv. Avoid NSAIDs

Answer 183

1. Key points a. Rare in term infants b. More common in premature infants 2. Pathogenesis a. Transient deficiency in vitamin K dependent clotting factors - due to lack of free vitamin K from mother and absence of the bacterial intestinal flora which normally synthesize vitamin K b. Consequence i. Moderate decrease in FII, VII, IX an X occurs in all newborn infants by 48-72 hours after birth ii. Gradually return to normal by 7-10 days of age c. Accentuated and prolonged deficiency between 2nd and 7th day of life results in prolonged bleeding d. Note breastmilk is a poor source of vitamin K 4. Investigations a. PT = prolonged b. APTT = prolonged c. Prothrombin, FVII, IX and X = decreased 5. Prevention a. IM 1 mg vitamin K – prevents decrease in vitamin K dependent clotting factors in full term infants i. Not uniformly effective in preventing HDN – particularly premature and breastfed infants 6. Treatment a. FFP b. PRBC

Answer 184

3. Clinical manifestations a. Prodromal or warning signs (mild bleeding) may occur before serious ICH EARLY-ONSET DISEASE Age 0-24 hr Site of hemorrhage: Cephalohaematoma, Subgaleal, Intracranial, Gastrointestinal, Umbilicus, Intraabdominal Etiology/risks: Maternal drugs (phenobarbital, phenytoin, warfarin, rifampin, isoniazid) that interfere with vitamin K, Inherited coagulopathy Prevention: Possibly, administrations of vitamin K to infant at birth or to mother (20 mg) before birth Avoid high-risk medications Incidence: Very rare CLASSIC DISEASE 2-7 days Bleeding: Gastrointestinal, Ear-nose-throat-mucosal, Intracranial, Circumcision, Cutaneous, Injection sites Aetiology: Vitamin K deficiency, Breastfeeding Prevention: Prevented by IM vitamin K at birth Oral vitamin K regimens require repeated dosing over time Incidence: ≈ 2% if infant not given vitamin K LATE ONSET DISEASE 1-6 mo Bleeding: Intracranial, Gastrointestinal, Cutaneous, Ear-nose-throat-mucosal, Injection sites, Thoracic Aetiology: Cholestasis—malabsorption of vitamin K (biliary atresia, cystic fibrosis, hepatitis), Abetalipoprotein deficiency, Idiopathic in Asian breastfed infants, Warfarin ingestion Prevention: Prevented by IM and high-dose oral vitamin K during periods of malabsorption or cholestasis Incidence: Dependent on primary disease

Answer 185

1. Key points a. DIC is a thrombotic microangiopathy b. Features i. Consumption of clotting factors, platelets and anticoagulant proteins  haemorrhagic state ii. Widespread intravascular deposition of fibrin leading to tissue ischaemia and necrosis iii. Haemolytic anaemia 2. Aetiology a. Any life threatening systemic disease associated with hypoxia, acidosis, tissue necrosis, shock and/or endothelial damage may trigger DIC: i. Sepsis – meningococcal, bacterial, viral, malaria, fungal ii. Shock/asphyxia iii. Trauma/injury iv. Snake/insect bites b. Malignancy – haematological c. Microangiopathic disorders – TTE/HUS, giant haemangioma (Kasabach-Merritt) d. GIT – hepatitis, IBD, pancreatitis e. Thrombotic disorders = antithrombin 3 deficiency, homozygous protein C deficiency 3. Pathogenesis a. Endothelial tissue damage (due to hypoxia/acidosis/tissue necrosis) b. Widespread activation of coag cascade c. Coag activation + platelet aggregation = ↓ plt, ↓ fibrinogen, ↓ APTT, ↓ fibrin degradation products d. Consumptive coagulopathy + platelets = bleeding e. Haemolysis due to mechanical obstruction by clot in small vessels f. Shock

Answer 186

4. Clinical manifestations a. Accompanies a severe systemic disease process, usually with shock b. Bleeding frequently first occurs from sites of venepuncture or surgical incision c. Petechiae, ecchymoses d. Tissue necrosis – infarction of large areas of skin, subcutaneous tissue, kidneys e. Anaemia secondary to haemolysis may develop rapidly owing to microangiopathic anaemia 5. Investigations a. FBE = thrombocytopenia, anaemia b. Film = fragmented, burr and helmet shaped RBC (schistiocytes) c. Clotting studies i. Coagulation factors (II, V, VIII and fibrinogen) = reduced ii. APTT, PT-INR, thrombin times = prolonged d. ↑ D-dimer (fibrin breakdown products) 6. Treatment a. First two steps most critical i. Treat the trigger that caused DIC (eg. infection) ii. Restore normal homeostasis by correcting shock, acidosis, hypoxia b. Blood component replacement therapy i. Platelet infusion – for thrombocytopenia ii. Cryoprecipitate – for hypofibrinogenaemia iii. FFP – for replacement of other coagulation factors and natural inhibitors c. Heparin = limited to patients with vascular thrombosis in association with DIC who require prophylaxis as they are at high risk of venous thromboembolism

Answer 187

1. Epidemiology a. Infants <1 year old account for the largest proportion of pediatric VTEs, with second peak in adolescence 2. Risk factors a. CVC = most important RF – 90% neonatal VTE 60% of childhood VTE i. CVCs may damage endothelial lining and/or cause blood flow disruption b. Inherited thrombophilia i. Factor V Leiden ii. Prothrombin G20210A mutation iii. Antithrombin deficiency iv. Protein C deficiency v. Protein S deficiency c. Anatomic i. May-Thurner syndrome = compression of the left iliac vein by the overlying right iliac artery) 1. Consider if present spontaneously with left iliofemoral thrombosis ii. Thoracic outlet obstruction (Paget-Schroetter synd) = effort related axillary-subclavian thrombosis d. Other conditions i. Infection ii. Malignancy iii. Congenital heart disease iv. Trauma v. Nephrotic syndrome

Answer 188

a. Extremity DVT/ central line associated i. Clinical manifestations 1. Extremity pain, swelling, discolouration 2. Often more subtle and chronic – CVC occlusion or sepsis, prominent venous collaterals on chest, face, neck ii. History of current of recent CVC in that extremity very suggestive b. Pulmonary embolism i. Clinical manifestations 1. SOB, pleuritic chest pain, cough, haemoptysis, fever 2. Massive PE – hypotension, right-heart failure c. Cerebral sinovenous thrombosis i. Clinical manifestations 1. Neonates – seizures 2. Older children – headache, vomiting, seizures, focal signs (papilledema, abducens palsy) ii. Some patients may have concurrent sinusitis or mastoiditis d. Renal vein thrombosis i. Most common spontaneous TE in neonates - 25% of cases are bilateral ii. Clinical manifestations = 1) haematuria 2) abdominal mass 3) thrombocytopaenia (consumptive) iii. Increased risk – IDM e. Portal vein thrombosis i. Risk factors = liver transplantation, infections, splenectomy, sickle cell disease, chemotherapy ii. Clinical manifestations = acute abdomen, chronic hypertension (splenomegaly, varices) f. Peripheral arterial thrombosis i. With the exception of stroke, the majority of arterial TEs in children are secondary to catheters 1. Neonates (umbilical artery line), older children cardiac cath ii. Clinical manifestations = cold, pale, blue extremity with poor/absent pulses g. Stroke i. Clinical manifestations = hemiparesis, LOC, seizures ii. Secondary intracranial disease – sickle cell disease, vasculopathy, traumatic arterial dissection iii. Venous thrombi that embolise to arterial circulation – placental thrombi, CHD, PFO h. Rapidly progressive thrombosis/ thrombotic storm i. Rapid progression of multi-focal thrombosis is a rare complication of APS, heparin-induced thrombocytopenia with thrombosis or TTP while on appropriate antithrombotic therapy ii. Multiple organ dysfunction develops due to small vessel occlusion and elevated d-dimer levels iii. Recurrences may occur as well as post-thrombotic syndrome (PTS) iv. Treatment = anticoagulation (short term thrombin inhibitors or fondaparinux, long term warfarin)

Answer 189

4. Investigations a. FBE b. Coagulation studies - baseline PT and APTT – to assess coagulation status c. +/- LFT and UEC d. +/- d-dimer e. +/- APS work up (lupus anticoagulant antibody, anticardiolipin antibody, anti β2 glycoprotein antibodies) f. +/- thrombophilia screen = influences duration of treatment g. Imaging studies i. Doppler ultrasound = most commonly used imaging for upper and lower VTE ii. Spiral CT = PE iii. CT =may be useful in proximal thrombosis iv. MR venography =may be useful in proximal thrombosis v. MRI + venography or diffusion weighting =for cerebral sinovenous thrombosis or acute ischaemic stroke 5. Treatment b. Options i. Anticoagulation = 6 weeks to 3 months 1. Inherited thrombophilia, recurrent thrombosis, APS may require indefinite treatment 2. LMWH preferred to UFH ii. Thrombolysis iii. Surgery – life or limb-threatening thrombosis if contraindication to thrombolysis iv. Observation 6. Complications a. Recurrent thrombosis – local or distant b. Post-thrombotic syndrome i. Thrombosed vessel may partially or fully recanalyze or may remain occluded ii. Over time an occluded deep vein may cause venous hypertension, resulting in blood flow being directed from the deep system into the superficial veins iii. Symptoms = pain, swelling, oedema, discolouration, ulceration iv. Highest risk in first 2 years, but continues to increase over time

Answer 190

1. Key points a. Absolute risk of thrombosis in children is low – 0.07/ 100 000 b. Thrombophilia testing controversial for provoked VTE (eg. catheter associated) c. Challenging to interpret thrombophilia studies in children i. Neonates have decreased concentrations of protein C, protein S and AT – these increase rapidly over the first 6 months of life ii. Protein C concentration remains below adult levels throughout much of childhood iii. Non genetics factors which may influence results of inherited thrombophilia testing 1. Acute thrombosis, infection, inflammation, hepatic dysfunction, nephrotic syndrome, medication and Vitamin K deficiency d. Elevated levels of factor VIII and homocysteine are associated with thrombosis, but these are less well characterized and not necessarily genetically determined 2. Aetiology a. Deficiency/ abnormalities of coagulation inhibitors: i. Antithrombin deficiency ii. Protein C/ S deficiency iii. Activated protein C resistance (Factor V Leiden) iv. Thrombomodulin deficiency b. Metabolic defects i. Hyperhomocysteinemia c. Abnormality of coagulation enzyme/ cofactor i. Prothrombin mutation ii. Elevated factor 7/9/10/11 iii. Factor V Leiden

Answer 191

APC resistance a. Most common inherited RF for thrombosis b. 5% of white population heterozygous for mutation, less prevalent in other ethnic groups c. Risk of thrombosis i. Heterozygotes – 5-7 fold increase in risk of venous thrombosis ii. Homozygotes – relative risk of 80-100 d. Genetics i. Factor V Leiden = single point mutation in the factor V gene (G to A at nucleotide 1691) resulting in replacement of arginine with glutamine at amino acid 506  prevents binding of FV to aPC e. Pathogenesis i. Factor V is a procoagulant clotting factor that amplifies the production of thrombin (Factor IIa) – the central enzyme that converts fibrinogen (Factor I) to fibrin (Factor Ia) resulting in clot formation ii. Thrombin slows down its own creation by creating a negative feedback loop via activation of protein C which produces activated protein C (aPC) iii. aPC degrades activated factor Va – reducing thrombin formation f. Investigations i. APC resistance assay – APTT undertaken in presence and absence of exogenous APC and results expressed as ratio (APC should prolong APTT; in FVL doesn’t happen) ii. DNA analysis for F5 – FISH g. Management i. Anticoagulation may be considered long-term in those with recurrent VTE, multiple thrombophilic disorders, or other RF (E.g. pregnancy, HRT, travel), in homozygotes ii. Not recommended for asymptomatic heterozygotes but advice given about increased risk of pregnancy loss, and discourage use of OCP

Answer 192

a. Second most common inherited thrombophilia b. Results in prothrombin level 30% higher than normal individuals c. Genetics + pathogenesis i. Prothrombin is the precursor of thrombin – end-product of coagulation cascade ii. G20210A mutation results from substitution of A for G at position 20210 – non-coding region of the prothrombin gene (3’ UTR corresponding to the messenger RNA responsible for polyadenylation) iii. Mutation is considered a gain-of-function mutation as it causes increased prothrombin function

Answer 193

a. Rarer than above genetic mutations (Factor V Leiden, Prothrombin 20210), but stronger risk of thrombosis b. Clinical manifestations i. Heterozygous deficiencies – do not normally present in childhood ii. Homozygous defects may result in significant symptoms in infancy 1. Neonates with homozygous deficiencies in protein C, protein S and AT may present with purpura fulminans = rapidly spreading purpuric skin lesions resulting from thromboses of the small dermal vessels followed by bleeding into the skin 2. Risk of cerebral thrombosis, ophthalmic thrombosis, DIC, large vessel thrombosis c. Management i. Purpuric skin lesions – initial replacement with FFP ii. Protein C and AT concentrates are also available

Answer 194

a. Inborn error of metabolism caused by deficiency of cystathione-β-synthase b. Very rare c. Increases risk of venous and arterial thrombosis - common in young patients with homocystinuria d. Plasma levels of homocysteine exceed 100μmol/L e. Much more common are mild-moderate elevations of homocysteine i. May be acquired or associated with a polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene ii. Testing for polymorphisms for MTHFR not indicated – as polymorphisms are not associated with venous thromboembolism

Answer 195

1. Key points a. Inhibitors = antibodies that react or cross react with clotting factors or components used in coagulation screening tests (phospholipids), thereby prolonging screening tests such as PT and PTT i. Some are autoantibodies that react with phospholipid interfering with clotting in vitro but not in vivo b. Antiphospholipid syndrome most common (= lupus anticoagulant) i. Found in patients with SLE, collagen vascular disease and HIV ii. In otherwise healthy children lupus-like anticoagulant can develop following viral infection – NOT associated with bleeding or thrombosis c. Antibodies to FVIII are most common 2. Investigations a. Depending on the target of the antibody = PT, PTT, or both may be prolonged b. Mixing studies = patient plasma containing inhibitor + normal plasma (1:1) i. Antibody directed against the active site of clotting factor (FVIII or factor IX) will NOT correct on 1:1 mixing with normal plasma ii. Antibody that lead to increased clearance of the factor (prothrombin) WILL correct c. Specific factor assays – used to determine which factor is involved 3. Treatment a. Recombinant factor VIIa or activated prothrombin complex concentrate b. Immunosuppressive agents c. Inhibitors seen with an underlying disease eg. SLE disappear when treated

Answer 196

1. Key points a. Occurs as primary condition OR associated with SLE b. Antiphospholipid antibodies i. Heterogenous group of antibodies directed against phospholipid binding protein ii. Include = anticardiolipin Ab, anti-beta-2 glycoprotein antibody, lupus anticoagulant c. When to suspect i. One or more otherwise unexplained venous or arterial thrombotic events ii. Fetal death after 10 weeks gestation, premature birth due to severe pre-eclampsia or placental insufficiency, or multiple embryonic losses 2. Clinical manifestations a. Fetal loss b. Venous and arterial thrombosis i. More often associated with predisposition to thrombosis than bleeding symptoms ii. However, bleeding symptoms may be caused by thrombocytopaenia (as a manifestation of APS or lupus itself) or rarely, by a coexistent specific autoantibody against prothrombin (factor II) 3. Investigations a. Lupus anticoagulant antibody, anticardiolipin antibody, anti β2 glycoprotein antibodies 4. Treatment a. Long term anticoagulation (high risk of recurrence)

Answer 197

1. Overview a. Non-nucleated cellular fragments produced by megakaryocytes within the bone marrow and other tissues b. Megakaryocytes are large polyploidy cells c. When the megakaryocyte approaches maturity, budding of the cytoplasm occurs and large numbers of platelets are liberated d. Lifespan – 10-14 days 2. Thrombopoietin (TPO) a. Primary growth factor that controls platelet production b. Levels of TPO correlate inversely with platelet number and megakaryocyte mass c. Levels of TPO are higher in the thrombocytopenic states associated with decreased marrow megakaryopoiesis and may be variable in states of increased platelet production 3. Surface receptors a. Adhesive proteins – vWF, fibrinogen b. Receptors for agonist that triggers platelet aggregation – thrombin, collagen adenosine diphosphate (ADP) 4. Mechanism of haemostatic plug formation a. Injury = exposure of adhesive and pro-coagulant proteins  vWF binds sub-endothelial collagen b. Platelet adhesion = vWF undergoes conformational change that induces binding of the platelet to glycoprotein Ib (GPIb) c. Platelet activation i. Generate thromboxane A2 (via arachidonic acid + COX pathway) ii. Platelets release agonists (ADP, ATP, Ca2+, serotonin, coagulation factors) d. Hemostatic plug formation i. Binding of vWF to the GPIb complex triggers a complex signaling cascade that results in activation of the fibrinogen receptor, the major platelet integrin glycoprotein αIIb-β3 (GPIIb-IIIa) ii. Circulating fibrinogen binds to its receptor on the activated platelets aggregating platelets iii. Results in formation of a hemostatic plug at the site of vascular injury e. Other mediators i. Serotonin and histamine that are liberated during activation increase local vasoconstriction ii. Platelet provides a catalytic surface on which coagulation factors assemble and eventually generate thrombin through a sequential series of enzymatic cleavages

Answer 198

1. Definition a. Normal platelet count is 150-450 x 109/L b. Thrombocytopaenia - <150 x 109/L i. <75 – risk of bleeding after major surgery, trauma ii. <50 – spontaneous bleeding, mostly skin – petechiae, purpura iii. <20 – noticeable hemorrhage – epistaxis, gingival iv. <10 – possible life threatening haemorrhage (mucosa, CNS) 2. Classification schema a. Platelet size – large, normal and small b. Mode of acquisition – congenital or acquired c. Mechanism – destruction or production 4. General management a. DDAVP (desmopressin) i. ↑ vWF from endothelium, increases aggregation ii. Rapid action b. Oestrogens – may be useful for uraemia, VWD c. Tranexamic acid d. Platelet transfusion e. Activated factor VII

Answer 199

a. DESTRUCTIVE i. Immune-mediated 1. ITP 2. Drug-induced 3. SLE 4. Autoimmune lymphoproliferative syndrome 5. Neonatal Alloimmune thrombocytopaenia 6. Post-transplant thrombocytopaenia ii. Non-immune 1. HUS, TTP + DIC 2. Major surgery or trauma 3. Kasabach-Merit syndrome 4. ECMO 5. Hypersplenism 6. Hypothermia b. DECREASED PRODUCTION i. Infection = EBV, CMV, parvovirus, varicella, sepsis ii. Nutritional = B12, folate, iron iii. Acquired bone marrow failure 1. Aplastic anaemia 2. MDS 3. Chemotherapy + radiotherapy iv. Infiltrative bone marrow disorders v. Genetic causes of impaired thrombopoesis 1. Wiskott-Aldrich 2. Inherited bone marrow failure syndromes a. Fanconi anaemia b. Dyskeratosis congenital c. Scwachmann-Diamond syndrome d. Congenital amekagaryocyic thrombocytopaenia 3. TAR 4. Amegakaryocytic thrombocytopaenia with radioulnar synostosis 5. Familial platelet disorder with predisposition to haematological malignancy 6. Bernard-Soulier syndrome 7. MYH9 related disorders

Answer 200

Immune thrombocytopenic purpura 1. Key points a. Previously idiopathic thrombocytopenic purpura b. Most common cause of acute thrombocytopaenia in an otherwise well child c. 1 in 20 000 d. Time course i. Newly diagnosed = within 3 months ii. Persistent ITP = 3-12 months iii. Chronic ITP = >12 months 2. Epidemiology a. Acute ITP i. 1-8years (peak 3-4years) ii. M = F iii. Increased prevalence in spring b. Chronic ITP i. Adolescents and adults ii. F > M iii. No seasonal variation 3. Classification a. Primary = absence of other causes b. Secondary = immune-mediated thrombocytopaenia with other underlying cause (eg. SLE, HIV) 4. Pathogenesis a. Autoantibody (usually IgG) directed against platelet surface  thrombocytopaenia b. Ag target unknown – commonly GP IIb/IIIa complex c. Ab coated platelets short half-life as they undergo clearance by tissue macrophages – mainly spleen d. Most common viruses have been described in association with ITP – EBV, HIV i. EBV-related ITP = short duration and follows the course of infectious mononucleosis ii. HIV associated ITP = chronic iii. Following H pylori or rarely post vaccines

Answer 201

5. Clinical manifestations a. Sudden onset generalised petechiae and purpura b. Preceding viral illness 1-4 weeks prior (50-65% of cases) or vaccination c. Bleeding = gums or mucous membranes most common i. Serious haemorrhage in 3%, ICH in 0.5% d. Lack of systemic signs or symptoms e. Splenomegaly, lymphadenopathy, bone pain and pallor are rare -> consider DDx 6. Investigations a. FBE = thrombocytopaenia (<20 x 109/L common), normal platelet size i. Note may have mild anaemia if significant bleeding – uncommon b. Coag = normal (not always required) c. Bone marrow = NOT usually done, only if >1 cell line affected or abnormal film i. Megakaryocytes = normal or increased number, may appear immature d. HIV test = in at-risk populations, especially sexually active teens e. DAT = if unexplained anemia 7. DDx a. Systemic signs and symptoms (inc hepatosplenomegaly) = malignancy, SLE or other systemic illness b. Splenomegaly = liver disease or portal venous thrombosis c. Anaemia = Evans syndrome, HUS, TTP d. Excessive bleeding = vWD e. Longstanding thrombocytopaenia = congenital thrombocytopaenia f. Wiskott-Aldrich syndrome = young males with small platelets, Phx – eczema, recurrent infection

Answer 202

8. Treatment a. Education important b. Monitoring of platelets c. Pharmacological i. Corticosteroids 1. No clear criteria for steroids over IVIG – generally considered if active mucosal bleeding 2. 1mg/kg for 5 days = potential regimen 3. Proposed action a. Improve vascular integrity b. ↓ Antibody affinity for PLT membrane c. ↓ Antibody production d. ↓ Phagocytosis of antibody coated PLT ii. IVIG = chronic ITP 1. Binds Fc receptor, results in prolonged survival of antibody coated platelets 2. ↑ platelets within 24 hours, usually persists for 3-4 weeks 3. More rapid than steroids (superior first line when rapid response required/desired) iii. IV anti-D therapy 1. For Rh positive patients ONLY 2. Causes a rise in platelet count >20 × 109/L within 48-72 hr – associated mild anaemia 3. RBC-antibody complexes bind to macrophage Fc receptors + interfere with platelet destruction d. Splenectomy i. Older child with severe chronic ITP ii. Life-threatening haemorrhage complicating acute ITP 9. Outcome a. Severe bleeding is rare, <1% of patients develop intracranial haemorrhage b. In 70-80% of children who present with acute ITP – spontaneous resolution occurs within 6 months i. More likely to resolve in younger children c. Chronic ITP (20%)

Answer 203

>12 months 1. Key points a. 20% of patients who present with acute ITP go on to have chronic ITP b. Incidence 50% in adolescents c. Must carefully reconsider diagnosis d. More likely to have underlying autoimmune disorder 2. Investigations a. Viral tests b. Autoimmune screen c. Immunodeficiency screen d. BMA 3. Treatment a. Indication for treatment – platelet count <20 b. IVIG c. Rituximab – has been used in chronic ITP d. Splenectomy –complete remission in 64-88% of children with chronic ITP i. Indications 1. Persistent bleeding or v low PLT 2. Emergency splenectomy  life threatening bleeding ii. Risks 1. Post-surgical bleeding 2. Overwhelming sepsis = higher risk if <5 years iii. Efficacy 1. ↑ PLT  60-90% 2. Refractory chronic ITP – 25-30%  ongoing bleeding problem post splenectomy 3. Assess for accessory spleen

Answer 204

1. Mechanisms a. Immune process b. Megakaryocyte injury 2. Drugs a. Valproic acid b. Phenytoin c. Carbamazepine d. Sulfonamides e. Vancomycin f. Bactrim g. Heparin-induced thrombocytopaenia i. Seldom seen in paediatrics ii. Mechanism – antibody directed against the heparin–platelet factor 4 complex iii. Recommended treatment in adults – argatroban, lepirudin, or danaparoid Drugs affecting platelet FUNCTION • Aspirin: irreversibly inhibit COX  inhibits platelet aggregation • NSAIDs: reversibly inhibit COX • Clopidogrel: inhibits platelet aggregation

Answer 205

1. Key points a. Clinically similar to HUS b. More common in adults occasionally seen in children 2. Pathogenesis a. ADAMTS-13 protease deficiency – responsible for cleaving VWF multimers b. Without this cleavage ultra-large VWF multimers circulate stimulating microvascular platelet thrombi c. Results in microangiopathic haemolytic anaemia and thrombocytopaenia 3. Classification a. Congenital i. Autosomal recessive ii. Inherited deficiency in ADAMTS-13 iii. Results in TTP in young children b. Acquired i. Acquired Ab mediated destruction of ADAMTS-13 4. Clinical manifestations a. Initial manifestations are often nonspecific (weakness, pain, emesis) b. Clinically similar to HUS – renal involvement more prominent in HUS, CNS changes more prominent in TTP c. Classic pentad i. Fever ii. Microangiopathic haemolytic anaemia iii. Thrombocytopaenia iv. Abnormal renal function v. CNS changes 5. Investigations a. FBE = microangiopathic haemolytic anaemia, elevated reticulocytes, thrombocytopaenia b. Film = morphologically abnormal RBCs, with schistocytes, spherocytes, helmet cells c. Coagulation studies – non diagnostic d. Metalloproteinase – low e. +/- elevated BUN f. +/- elevated creatinine 6. Treatment a. Plasmapheresis i. Effective in 80-95% of cases ii. Only used if acquired ie. antibody mediated b. ADAMTS13 replacement (FFP) = if congenital deficiency c. Refractory cases i. Rituximab ii. Corticosteroids iii. Splenectomy

Answer 206

1. Key points a. One of the main causes of AKI in children b. Triggered by gastroenteritis (E. coli) 2. Classification a. Primary causes = atypical HUS i. Complement gene mutations ii. Antibodies to C’ factor H iii. Inborn errors of cobalamin C metabolism iv. DGKE gene mutation b. Secondary i. Infection 1. Shiga-toxin producing E coli (STEC) = 90% 2. Streptococcus pneumoniae 3. Human immunodeficiency viral infection ii. Drug toxicity iii. Rare in those with autoimmune conditions 3. Clinical manifestations a. Haemolytic anaemia b. Thrombocytopenia c. Acute renal failure 4. Investigations a. Film – helmet cells, spherocytes, schistocytes, burr cells and other distorted forms b. Elevated D dimer but normal other coagulation screen c. Normal megakaryocytes on bone marrow d. Urine – protein, RBCs, casts 5. Treatment a. Fluid management b. Dialysis c. Plasmapheresis performed if neurological involvement

Answer 207

1. Key points a. Life-threatening complication i. 70% of Kaposiform haemangioendothelioma ii. 10% of tufted angiomas b. Profound thrombocytopaenia + consumptive coagulopathy 2. Clinical manifestations a. Haemangioma – may be internal 3. Pathogenesis a. Platelet trapping + activation inside haemangioma  consumption of coagulation factors and fibrinogen b. Arteriovenous malformation within the lesions can cause heart failure 4. Investigations a. FBE = thrombocytopaenia b. Film = microangiopathic changes c. Coagulation factors i. Prolonged INR, APTT ii. Low fibrinogen iii. Elevated D-dimer d. Imaging = MRI 5. Treatment a. Various treatments = propranolol, surgical excision (if possible), laser photocoagulation, high-dose corticosteroids, local radiation therapy, antiangiogenic agents, such as interferon-α2, and vincristine b. Antifibrinolytic therapy with ε-aminocaproic acid (Amicar)

Answer 208

Key points a. Relatively rare 2. Aetiology a. Increased destruction or consumption of platelets i. Immune thrombocytopaenia 1. Autoimmune 2. Alloimmune 3. Drug-induced ii. Peripheral consumptions 1. Hypersplenism 2. Kasabach-Merritt syndrome 3. DIC 4. Thrombosis 5. Type 2B VWD b. Deceased production i. Congenital thrombocytopaenia ii. Infiltrative BM disorders iii. Infection-associated BM suppression 1. Congenital infections – rubella, CMV, toxo, syphilis 2. Perinatal bacterial infections – particularly GNB iv. Pre-eclampsia c. Miscellaneous i. Asphyxia ii. Dilution Aetiology by age: FETAL Alloimmune thrombocytopaenia Congenital infection – CMV, toxo, rubella, HIV Aneuploidy – T18, T13, T21 or triploidy Autoimmune – ITP, SLE Severe Rh haemolytic disease Congenital/inherited – Wiskott Aldrich syndrome ``` EARLY ONSET <72 HOURS Placental insufficiency – PET, IUGR, diabetes Perinatal asphyxia Perinatal infection – E.coli, GBE, HSV DIC Alloimmune thrombocytopaenia Autoimmune condition – ITP, SLE Congenital/inherited – TAR, CAMT ``` LATE ONSET >72 HOURS Late onset sepsis NEC Congenital/inherited – TAR, CAMT

Answer 209

1. Pathogenesis a. Ab-mediated thrombocytopenia in the newborn (equivalent of RhD of newborn) b. Transplacental transfer of maternal antibodies directed against antigens present on fetal platelets (but not mothers) c. Antibodies present from early first trimester d. Note that first pregnancies can be affected – subsequent more severely affected i. The most common cause is incompatibility for the platelet alloantigen HPA-1a 1. 98% of Caucasians are HPA1  most NAIT occurs with non-HPA1a mother + HPA1a foetus 2. 2% of women are hPA1a negative (1b homozygous), 10% have antibodies 2. Clinical manifestations a. Apparently well neonate  generalised petechiae and purpura within first few days b. Intracranial haemorrhage – up to 30% (prenatal and perinatally) 3. Investigations a. Platelets – moderate to severe thrombocytopaenia b. Maternal platelets – normal c. Maternal alloantibody – maternal alloantibodies directed against the father’s platelets i. Note titre not predictive of degree of fetal thrombocytopaenia d. Platelet Ag typing e. CNS imaging – CrUSS + MRI 4. Differential diagnosis a. Transplacental transfer of maternal antiplatelet autoantibodies (maternal ITP) b. Viral or bacterial infection – more common 5. Treatment a. IVIG prenatally to the mother = 2nd trimester onwards i. Fetal platelet count can be monitored by percutaneous umbilical blood sampling b. Deliver by LUSCS c. Platelet transfusion postnatally = ideally washed maternal platelets (share maternal alloantigens) i. Cause a rise in platelet counts to provide effective haemostasis d. IVIG +/- methylpred 6. Prognosis – platelets rise by 4 weeks as antibody levels fall, resolves over 2-4 months a. 1st pregnancies can be affected, but subsequent pregnancies are earlier onset and more severe b. Previous ICH confers higher risk

Answer 210

1. Key points a. Occurs in 10-15% pregnancies complicated by ITP b. Tends to be more severe with lower maternal platelet count c. Risk factors for severe disease – maternal splenectomy, thrombocytopenia in preceding sibling, severe thrombocytopenia in pregnancy 2. Pathogenesis a. Maternal antibodies against both maternal + fetal platelets b. IgG crosses placenta and cause fetal disease c. Initial ↓ in PLT count on delivery as infant acquires splenic function, destroys antibody sensitized platelets 3. Clinical manifestations a. Well babies – may have petechiae/ bruising/ bleeding b. Lower risk of haemorrhage than those with NATP (<1%) 4. Treatment a. Nil antenatal management required b. Postnatally – monitor platelets for first 3 days i. Platelet transfusion (less effective as antibodies attack platelets), +/- IVIG +/- steroids to infant 5. Prognosis - Usually resolves within 2-4 months of delivery

Answer 211

Thrombocytopenia-absent radius syndrome 1. Clinical manifestation a. Severe thrombocytopaenia i. At birth or in the first week b. Bilateral absent radii – thumbs ALWAYs present c. Congenital heart disease in 1/3 of patients d. Intolerance to cow’s milk formula (present in 50%) 2. Natural history = thrombocytopaenia frequently remits over the first few years of life 3. Investigations = thrombocytopenia (absence or hypoplasia of megakaryocytes) 4. Differential diagnosis a. Amegakaryocytic thrombocytopenia with radioulnar synostosis i. Caused by a mutation in the HOXA11 gene ii. Different from TAR syndrome, this mutation causes marrow aplasia

Answer 212

1. Thrombocytopaenia with small platelets 2. Immunodeficiency 3. Malignancy risk 4. Eczema

Answer 213

• A diverse number of hereditary thrombocytopenia syndromes - Sebastian, Epstein, May-Hegglin, and Fechtner syndromes • Key features o AD o Macrothrombocytopenia, usually mild and not progressive o Neutrophil inclusion bodies o Variety of physical anomalies  Sensorineural deafness  Renal disease  Eye disease – cataracts • Pathophysiology – mutations in the MYH9 gene (non-muscle myosin-IIa heavy chain)

Answer 214

1. Normal platelet function a. Platelet adherence i. Initiated by exposure of the vascular subendothelium following injury to endothelial surface ii. Platelet exposed to collagen, fibronectin, vWF, fibrinogen and thrombospondin – interact via glycoprotein (GP) receptor on platelet surface (GpIa/IIa) b. Platelet activation i. Receptor-ligand interaction results in platelet activation -> degranulation 1. Platelet alpha granules = VWF, platelet factor 4, thrombospondin, fibrinogen, beta-thromboglobulin, platelet-derived growth factor 2. Platelet dense granules = ADP, serotonin c. Platelet aggregation i. GPIIb/IIIa (αIIbβ3) conformational change allows platelet aggregation ii. Fibrinogen binds to the conformationally altered integrin receptor on two or more adjacent platelets – resulting in aggregation d. Interaction with coagulation factors i. Interact with circulating coagulation factors – provide a scaffold for activation of phospholipid dependent coagulation factors 2. Assessing platelet function a. FBE + peripheral smear i. Large platelets = accelerated platelet turnover ii. Very large platelets + thrombocytopaenia (macrothrombocytopaenia) = Bernard-Soulier and other giant platelet syndromes iii. Small platelet = characteristic of Wiskott-Aldrich syndrome b. Platelet aggregation assays i. Agonists = ADP, arachidonic acid, collagen, adrenaline, thrombin, ristocetin c. Automated platelet function screening tests i. Platelet function analysed (PFA-100) - measures platelet adhesion and aggregation in whole blood at high shear when the blood is exposed to either collagen-epinephrine or collagen-ADP d. Bleeding time i. Previously used as screening test, now rarely used as affected by plt count and function 3. Treatment a. Successful treatment depends on the severity of both the diagnosis and the haemorrhagic event. b. Desmopressin i. 0.3 µg/kg IV ii. Stimulates increase levels of vWF and FVIII iii. Corrects bleeding time + augments haemostasis in patients with mild to moderate platelet function defects

Answer 215

1. Systemic illnesses associated with platelet dysfunction a. Include – liver disease, kidney disease (uremia) and disorders that trigger increased amounts of fibrin degradation products b. These disorders cause prolonged bleeding time and are often associated with other abnormalities of the coagulation mechanism c. Treatment i. Treatment of primary illness most important ii. Desmopressin – augmenting haemostasis and correcting bleeding time iii. +/- platelets iv. +/- cryoprecipitate 2. Drugs a. Include i. Aspirin, valproic acid, high dose penicillin ii. Specific agents to inhibit plt function – clopidogrel (block platelet ADP receptor) and αIIbβ3 receptor antagonists b. Aspirin i. Aspirin irreversibly aceylates the enzyme COX, which is critical to the formation of thromboxane A2 ii. Usually causes moderate platelet dysfunction that becomes more prominent if there is another abnormality of the hemostatic mechanism

Answer 216

1. Severe congenital platelet dysfunction a. Key points i. Present with petechiae and purpura shortly after birth, especially after vaginal delivery ii. Defects in the platelet GPIb complex (the VWF receptor) or the αIIb-β3 complex (the fibrinogen receptor) cause severe congenital platelet dysfunction b. Bernard-Soulier syndrome i. Severe congenital platelet function disorder ii. Genetics 1. AR 2. Genes forming the GPIb complex of glycoproteins Ibα, Ibβ, V, and IX iii. Pathogenesis 1. Absence or severe deficiency of the VWF receptor (GPIb complex) on the platelet membrane 2. The GPIb complex interacts with the platelet cytoskeleton; a defect in this interaction is believed to be the cause of the large platelet size iv. Investigations 1. FBE = thrombocytopenia, with GIANT platelets 2. Markedly prolonged bleeding time (>20 min) or PFA-100 closure time 3. Platelet aggregation tests a. Absent ristocetin-induced platelet aggregation - ristocetin induces the binding of VWF to platelets and agglutinates platelets b. Normal aggregation to all other agonists 4. VWF studies = normal 5. Flow cytometry of platelet glycoproteins = confirm diagnosis v. Treatment 1. Platelet transfusion – sometimes develop Ab 2. Recombinant FVIIa 3. HSCT c. Glanzmann thrombasthenia i. Severe platelet dysfunction that yields prolonged bleeding time and a normal platelet count. ii. Genetics + pathogenesis 1. AR – mutation in gene for αIIb or β3 2. Caused by deficiency of the platelet fibrinogen receptor αIIb-β3 a. Major integrin complex on the platelet surface that undergoes conformational changes by inside out signalling when platelets are activated b. Fibrinogen binds to this complex when the platelet is activated and causes platelets to aggregate iii. Investigations 1. FBE = platelets normal morphological size 2. PFA-100 closure time + bleeding time = markedly prolonged 3. Aggregation = abnormal or absent aggregation with all agonists used except ristocetin, because ristocetin agglutinates platelets and does not require a metabolically active platelet 4. Flow cytometry of platelet glycoproteins = confirm diagnosis iv. Treatment 1. Platelet transfusion – sometimes develop Ab 2. Recombinant FVIIa 3. HSCT 2. Hereditary deficiency of platelet storage granules a. Dense body deficiency b. Gray platelet syndrome 3. Other hereditary disorders of platelet function a. Defects in platelet signalling/activation and release of granules b. Defects in COX pathway

Answer 217

1. Overview a. Disorders of the vessel walls or supporting structures mimic the findings of a bleeding disorder, although the coagulation studies are usually normal b. Findings of petechiae and purpuric lesions are often attributable to an underling vasculitis, vasculopathy c. Skin biopsy can be particularly helpful in elucidating the type of vascular pathology 2. Disorders a. Henoch-Schonlein Purpura b. Ehlers-Danlos Syndrome c. Other acquired disorders i. Scurvy, chronic corticosteroid therapy, severe malnutrition ii. Associated with ‘weakening’ of the collagen matrix that supports the blood vessels iii. These factors are associated with easy bruising, bleeding gums and loosening of teeth iv. Skin lesions hat initially appear to be petechiae and purpura may be seen in vasculitic syndromes, such as SLE

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