1
Q
Apt-Downey test
A
To differentiate fetal blood from swallowed maternal blood in the evaluation of bloody stools. Mix specimen with 3-5 ml of tap water and centrifuge. Supernatant must have a pink color to proceed. To 5 parts of supernatant, add 1 part of 0.25 N (1%) NaOH. A pink color persisting over 2 minutes indicates fetal hemoglobin. Adult hemoglobin gives a pink color that becomes yellowish brown in 2 minutes or less indicating denaturation of the adult hemoglobin.
2
Q
Mercury poisoning mimics what?
A
Pheochromocytoma
3
Q
Arsenic poisoning is associated with what smell?
A
Garlic
4
Q
D-xylose test for enteric vs. pancreatic causes of malabsorption.
A
oral dose of d-xylose after an overnight fast, and then urinary excretion of this molecule is monitored. Results: a) ↑ urine d-xylose = normal. b) ↓ urine d-xylose = enteric cause. (Regardless of pancreatic disease.) Make sure there’s no chronic renal failure. Normal result: high blood and urine levels of xylose – indicates good xylose absorption by the intestines; suggests sx 2/2 pancreatic insufficiency, bile insufficiency High blood levels but low urine levels: kidney dysfunction Low blood and urine levels: poor xylose absorption, 2/2 bacterial overgrowth in the intestines, parasitic infections, a shortened bowel, celiac disease
5
Q
Fecal elastate test
A
1) Stool test for extreme pancreatic insufficiency. (100% sensitive, 93% specific). 2) Stool needs to be well formed. (Diluted stool → false positive). 3) Is non-invasive, therefore, most often used for children with cystic fibrosis.
6
Q
Light blue top tube – contents and purpose
A
3.2% sodium citrate – anticoagulant (binds Ca++) Used for coag studies
7
Q
Red/gold top tube – contents and purpose
A
Serum tube, +/- clot activator (glass/silica particles) or gel (separates serum from cellular components of blood) Used for chemistry, serology, immunology
8
Q
Green top tube – contents and purpose
A
Sodium lithium or heparin, +/- gel – anticoagulant (inhibits thrombin and thromboplastin) Used for stat and routine chemistry
9
Q
Lavender/pink top tube – contents and purpose
A
Potassium EDTA – anticoagulant (binds Ca++) Used for hematology, blood bank
10
Q
Gray top tube – contents and purpose
A
Fluoride + oxalate – Fl inhibits glycolysis, oxalate acts as an anticoagulant (binds Ca++) Used for glucose msrmt (esp when testing will be delayed), blood alcohol testing, lactate testing
11
Q
Biotin interference: – effect on sandwich immunoassays – effect on competitive assays
A
Sandwich: prone to false decreases Competitive: prone to false increases
12
Q
Friedewald Equation for LDL-C
A
LDL-C = total cholesterol - HDL cholesterol - (trigylcerides/5) Tri/5 = estimation of VLDL, but not always accurate
13
Q
Jaffe method used to measure which analyte? Interferences?
A
Creatinine (alkaline picrate determination) Subject to positive interference by many analytes, most notably ketones (i.e. beta-hydroxybutyrate), glucose, cephalosporins, A1c, proteins
14
Q
Hereditary angioedema:
- inheritance pattern and age of onset?
- mutations and relative prevalence of Type I, II, and III
- C1 protein inhibitor concentration and activity in each type
- what class of meds are contraindicated in these pts?
- mechanism of acquired angioedema?
A
Autosomal dominant; presents at puberty
Type I (80-85%)
– mutations in SERPING1 gene that results in decreased production of C1 inhibitor protein, resulting in overstimulated complement system. Both C1 protein inhibitor concentration and activity are reduced.
Type II (15-20%)
– also SERPING1 mutations, but resulting in a normal levels of a dysfunctional protein that’s incapable of suppressing complement activation. C1 protein inhibitor concentration will be normal or increased, but activity will be reduced.
Type III (<1%)
– mutations in factor XII (12) gene that increases bradykinin levels and produces swelling.
–
ACEis are contraindicated.
–
Acquired angioedema
– 2/2 auto-Ab against C1 inhibitor protein (same as in type I and type II)
– both C1 protein inhibitor concentration and activity are reduced.
15
Q
Thyroid hormones in pregnancy:
- Thyroid binding globulin
– does it increase or decrease?
- Total T3 and T4
– increase or decrease?
- Free T4 ____ in first trimester, then ___ in second and third trimesters - TSH ___ in first trimester, then ____ in second and third trimesters
A
- TBG increases (increased hepatic synthesis, decreased metabolism)
- Total T3 and T4 increase (same reasons as TBG) - Free T4 INCREASES in first trimester, then DECREASES in second and third trimesters
- TSH DECREASES in first trimester, then INCREASES in second and third trimesters -> hCG and TSH share the same alpha subunit. Thus the thyroid gets stimulated by alpha-hCG to produce more T3 and T4 and free T4 in the first trimester. These hormone products thus enact negative feedback and decrease TSH levels in the first trimester.
16
Q
Autoimmune lymphoproliferative syndrome (ALPS) - phenotype of T-cells? - inheritance pattern? - affected genes?
A
- T cells are CD4-, CD8- (double -) but CD3+ –> generalized LAD, autoimmune disease phenotype - autosomal dominant - FAS, FASLG, CASP10
17
Q
Explain possible lab consequences of the following dietary habits: 1. prolonged fasting 2. carb-restricted diets 3. intake of fish, meat, dietary iron, or horseradish 4. diet high in serotonin-rich foods like bananas, avocados, pineapples
A
- hyperbilirubinemia 2. increased urine ketones 3. positive fecal occult blood 4. increased urinary 5-HIAA (5-hydroxyindolacetic acid)
18
Q
Compare/contrast Type I vs. Type II Crigler-Najjar Syndrome in terms of: - age of onset - inheritance method - Typical unconjugated/indirect bili range - UDP glucuronosyltransferase 1-A1 (UGTA1) enzyme activity - effectiveness of phenobarbital - risk of kernicterus
A
Type I: - onset at birth (jaundiced baby; persistent jaundice) - autosomal recessive - very high unconjugated bili (17-50 mg/dL – normal <1 mg/dL) - no UGT activity –> means phenobarbital won’t work - risk of kernicterus very high Type II (Arias syndrome) - onset in late childhood/puberty - autosomal recessive - moderately high unconjugated bili (5-20 mg/dL) - reduced UGT activity – thus bile is still pigmented, but mostly just monoconjugated – thus phenobarbital can help reduce serum bili by at least 25% - normal liver enzymes - risk of kernicterus low
19
Q
Gilbert’s Syndrome - defective gene/enzyme - inheritance pattern - how is the Dx made? - age of Dx - Rx
A
- UDP glucuronosyltransferase 1-A1 (UGTA1) gene/enzyme – same as in Crigler-Najjar, but results in decreased activity - AR or AD, depending on the mutation - Dx made on basis of higher levels of unconjugated bili in the blood without evidence other liver problems/red cell breakdown - presents in late childhood to early adulthood, but some may be asymptomatic lifelong. Classically jaundice/icterus during times of stress. - Usually don’t need Rx, but can give phenobarbital to decrease jaundice if needed
20
Q
Dubin-Johnson syndrome vs Rotor Syndrome - gene defect and inheritance pattern - pathophysiology - color of the liver - Dx - Rx
A
DJ - AR loss of function mutations in the ABCC2 gene leading to a defective canalicular multiple drug resistance protein 2 (MRP2), which impairs excretion of conjugated bilirubin from the liver - polymerized epinephrine metabolites (NOT bilirubin) lead to a darkly pigmented liver Dx: - Oral cholecystogram study –> gallbladder not visible - urine coproporphyrin content normal; >80% being isomer 1 (where normal urine contains more isomer 3 than 1) Rx: - generally not needed. OCPs and pregnancy can lead to overt jaundice. Rotor - AR defect in SLCO1B1 or SLCO1B3 gene –> make organic anion transporting polypeptide 1B1 (OATP1B1) and organic anion transporting polypeptide 1B3 (OATP1B3) that are responsible for transporting bili and other compounds from the blood into the liver for clearance. Defects in these genes produce defective proteins that lead to less efficient uptake of bili by the liver and less effective clearance –> jaundice. - liver is normal-colored Dx: -increased conjugated hyperbilirubinemia (usually 2-5 mg/dL; maybe as high as 20 mg/dL) in the setting of normal liver enzymes and alk phos - urine coproporphyrin content HIGH; <70% being isomer 1 - Rx: phenobarbital to reduce jaundice
21
Q
Cryoglobulinemia:
I
II
III
Dz assoc w/ each?
Relative prevalences?
Common clinical features of dz?
A
I (10-15%): - monoclonal IgG, IgM, IgA, or kappa/lambda light chains - associated w/ heme dz: MGUS/plasma cell myeloma or Waldenstrom, CLL
II (50-60%): - polyclonal IgG (or rarely IgA) + monoclonal IgM w/ rheum factor activity (bind polyclonal Igs, activate complement, form tissue deposits, may cause small vessel vasculitis) - associated w/ infectious dz, particularly hep C, HIV; heme dzs esp B cell disorders; autoimmune dz
III (25-30%) - polyclonal IgM w/ rheum factor activity + polyclonal IgG or IgA - associated w/ autoimmune dz (Sjogren > SLE, RA); infectious dz esp hep C Clinical pres: 1) Increase blood viscosity (HA, confusion, blurry vision, hearing loss, epistaxi) 2) deposit in small arteries and capillaries -> infarction and necrosis, esp of ears, distal extremities, and kidneys 3) in type II and III, deposit on blood vessel epithelium and activate complement -> cryoglobulinemic vasculitis
22
Q
Minimum Hgb to be an RBC donor? (Autologous donor, how long before surgery?)
A
12.5 g/dL (11 g/dL, 72 hours minimum before surgery)
23
Q
How often can you donate platelets after: - an apheresis donation? - a double/triple apheresis donation? - in one week? - in one year?
A
- 2 days - 7 days - 2 times/week - 24 times/year
24
Q
Smallpox vaccine and blood donation deferral periods
A
- 3 weeks OR after the scab falls off naturally, whichever is later OR - 2 months, if the scab is manually picked off
25
West Nile Virus blood donation deferral period
120 days/4 months
26
Allogeneic organ/skin/bone marrow transplantation -- blood donation deferral period
12 months (But if the BMT was for leukemia/lymphoma, indefinite deferral)
27
Blood donation deferral period for: - receiving any unlicensed vaccine - receiving the Hep B immunoglobulin vaccine - receiving a non-prophylactic rabies vaccine
12 months
28
Blood donation deferral periods for: - traveling to a malaria-endemic region? - living in a malaria-endemic region? (how is this defined?) - having completed Rx for malarial infection?
Travel: 12 months Living (for \>5 consecutive years): 3 years Completed Rx for malaria: 3 years
29
Blood donation questionnaire -- do the following ?s pertain to risk of bovine spongiform encephalopathy (BSE, aka Mad Cow dz) or CJD/vCJD? 1. Have you lived in the UK for \>3 mo between 1980-1996? 2. Have you lived in France for \>5 years between 1980-present? 3. Have you ever received a dura mater transplant, pituitary GH injections, or bovine insulin injections?
1. BSE 2. BSE 3. vCJD
30
Describe these plasma exchange adverse rxns: - anaphylaxis (in what pt popn?) - air embolus (and how is it treated?)
Anaphalaxis: seen in patients on ACEis who took meds 24-30 hours before plasma exchange, and when 5% albumin was the replacement fluid Air embolus: 5 mL/kg entering venous system -\> blockage of pulmonary outflow tract -\> severe cardiopulmonary complications Rx: place patient in Trendelenburg or lateral decubitus position (head up or down); may be able to remove embolus with percutaneous needle drainage.
31
Name and describe category I plasma exchange indications: - neurological - hematological - renal - metabolic
Neuro: - acute Guillain-Barre syndrome - chronic inflammatory demyelinating neuropathy - myasthenia gravis - polyneuropathy assoc w/ paraproteinemias - PANDAS -- pediatric autoimmune neuropsychiatric disorders associated w/ Strep infections (including Tourette syndrome, OCD) Heme: - TTP (replace w/ FFP/cryo supernatant) - Atypical HUS (autoAb to factor H) - Hyperviscosity syndromes (paraproteinemias) - Severe/symptomatic cryoglobulinemias - ticlopidine-associated thrombotic microangiopathy (TMA) -- because there are ADAMST13-autoantibodies Renal: - Goodpasture's syndrome (anti-GBM Abs) - ANCA-associated rapidly progressive glomerulonephritis - recurrent focal segmental glomerular sclerosis (to decrease levels of "permeability factor") - Ab-mediated renal transplant rejection Metabolic: - homozygous familial hypercholesterolemia - fulminant Wilson's dz
32
How much of an offending substance is remaining after 1 round of plasma exchange?
~37% remaining // 63% removed Calculate using y=y0\*e^(Lambda-#of plasma exchanges) Lambda = Euler's # = 2.718 y=remaining substance conc y0=initial substance conc Solving for y = y0\*0.37
33
What are some category II indications for therapeutic plasma exchange? - neurological - hematological - immunological - metabolic
Neuro: - LEMS (Lambert Eaton myasthenic syndrome) - Acute MS exacerbation - Chronic focal encephalitis - Neuromyelitis optica Heme: - ABO incompatible BMT - Pure red cell aplasia - life-threatening cold agglutinin dz (use warmed 5% albumin to prevent cryoprec) - clopidogrel-associated thrombotic microangiopathy (TMA) Immune: - catastrophic antiphospholipid syndrome - cerebral SLE Metabolic: - Refsum's dz - heterozygous familial hypercholesterolemia
34
What are some category I indications for: 1. leukacytapharesis (WBC removal) 2. red cell exchange (removing RBCs and replacing them with donor RBCs) 3. erythrocytapharesis (removing RBCs and replacing them with saline or albumin) 4. extracorporeal photopheresis (ECP) - exposing WBCs to 8-methoxypsoralen + UVA irradiation before returning them to the patient 5. What are category II recommendations for ECP?
1. hyperleukocytosis w/ leukostasis 2. acute stroke in the setting of sickle cell disease; severe babesiosis 3. hereditary hemochromatosis 4. erythrodermic cutaneous T-cell lymphoma (Sezary syndrome) 5. Cellular or recurrent rejection of (or prophylaxis against) a cardiac transplant; skin involvement in acute or chronic GVHD; bronchiolitis obliterans syndrome in lung allograft rejection
35
What are the three most important HLA alleles for transplant? What two others are also important, but not associated with survival diffs?
HLA-A, HLA-B, and HLA-DRB -- most highly expressed HLA-DP, HLA-DQ
36
First line Rx of ITP (3 things, incl one specific to a blood group)?
Second line Rx?
Second/third line Rx if there's still substantial bleeding risk? molecular mechanism and side effects?
First line: steroids, IVIg, anti-D in Rh+ patients with intact spleens
Uncontrolled diabetes mellitus is a contraindication to using steroids. In these patients, use IVIg + RhIg.
Second line: rituximab, dapsone, splenectomy (but not in kids).
Third line: TPO mimetics like romiplastin and eltromogbopag. These bind to MPL (the TPO receptor) and stimulate platelet production. However, they do have serious long-term side effects incl thromboembolism, hepatic toxicity, bone marrow reticulin deposition, increased blast count, and cataracts. Don't use in MDS.
37
Thrombotic thrombocytopenic purpura:
- classic pentad?
- congenital vs. idiopathic vs. drug induced? How does Rx differ in each?
- fever, mental status changes, MAHA, thrombocytopenia, renal failure
- congenital: inherited ADAMST13 enzyme deficiency -\> inability to cleave ultralarge vWF multimers -\> platelet thrombi; shear-stress induced hemolysis. Treat w/ plasma infusion
- idiopathic: autoAb against ADAMST13 -\> ultralarge vWF multimers -\> platelet thrombi + shear stress induced hemolysis. Treat w/ plasmapharesis using FFP as the replacement fluid -- this removes the offending Abs and replenishes ADAMST13. If plasmapharesis isn't immediately available, transfuse FFP until plasmapharesis can be performed. Can also use immunosuppresants (usually steroids) to inhibit Ab production.
- Drug-induced: caused by cyclosporine, tacrolimus, clopidogrel, mitomycin C, gemcitabiine, and others; rx: stop the medication. Plasmapharesis commonly used but may not be helpful.
38
Rhogam dosing:
- during pregnancy and after birth in Rh- moms
- for bleeds (including Rosette test and Kleihauer-Betke test and calculation)
- how much fetal bleeding into maternal circulation can lead to alloimmunization?
Regular dosing:
- 1500 IU/300 micrograms, IV/IM, @ 28-30 weeks gestation
- 1500 IU/300 micrograms, IV/IM, within first 72 hrs postpartum
Bleeds:
- Trauma before 12 weeks: mini-dose of 150 ug -\> suppressees 2.5 mL Rh+ RBCs
- Trauma after 12 weeks: regular 1500 IU/300 ug dose, which generally covers 15 mL fetal RBCs or 30 mL fetal whole blood involved
- Massive trauma: do Kleihauer-Betke test (threshold 5 mL fetal blood in maternal circulation). Exposes RBCs to NaOH, which denatures HgA from mom but doesn't affect HbF from fetus, then stain w/ Shephard's method, and count 2000 cells to determine fetal cell %.
- If you're not sure whether massive trauma occured, do Rosette test -- incubate Rh- maternal venous whole blood w/ anti-Rho(D) immune globulin. Only fetal cells will bind anti-Rho(D). Then enzyme-digest the sample to isolate fetal cells -\> erythrocyte rosetting pattern which can be assessed by microscopy.
To determine Rhogam dosing:
of vials of 300 ug RhIG required = volume of fetal blood/30
OR
of vials required = (( % fetal cells from KB test)\*50 )/30 mL
-- round up if \>0.5, round down if \<0.5, and then +1 regardless
- as little as 10-30 uL of fetal bleeding can alloimmunize a pregnant patient
39
Two most common antigens implicated in NAIT?
HPA1a (approx 98% of people are HPA1a/HPA1a or HPA1a/HPA1b and won't form auto-Abs) HPA5b (rare)
40
Minimum specifications for the following blood products: 1. Platelets, apheresis collection 2. Platelets, non-apheresis collection 3. Residual leukocytes in a leukoreduced RBC unit 4. Residual leukocytes in a single leukoreduced platelet unit 5. Granulocytes, apheresis granulocytes 6. RBCs, granulocyte unit derived from whole blood
1. 3x10^11/300mL 2. 5.5x10^10/50 mL 3. \<5 \*10^6 4. \<8.3\*10^5 5. 1\* 10^10 6. \<2 mL RBCs
41
AABB standards for each unit of cryo: - must contain \>\_\_ of fibrinogen - must contain \>\_\_ of factor VIII Overall cryo contains which 5 components? Which has the longest t 1/2?
- must contain \>150 mg of fibrinogen - must contain \>80 IU of factor VIII Cryo contains: - fibrinogen - vWF - fibronectin - factor VIII - factor XIII (longest t 1/2: 5-10 days)
42
Paroxysmal Cold Hemoglobinuria (PCH) - caused by cold-reactive ___ complement-binding antibody that causes C3 to bind irreversibly to red cells at cold temps, most frequently w/ _ antigen specificity - commonly seen in kids after a ___ infection; presents w/ fever, jaundice, abdominal and back pain after cold exposure - the hemolysin binds at ___ temps and lyses at ___ temps and can be queried with the \_\_\_-\_\_\_\_ test.
Paroxysmal Cold Hemoglobinuria (PCH) - caused by cold-reactive IgG complement-binding antibody that causes C3 to bind irreversibly to red cells at cold temps, most frequently w/ P antigen specificity - commonly seen in kids after a viral infection; presents w/ fever, jaundice, abdominal and back pain after cold exposure - the hemolysin binds at COLD temps and lyses at WARM temps and can be queried with the Donath-Landsteiner test: -- pre-incubate at 4 C, then incubate at 37 C --\> lysis -- negative control is incubated at 37 C and should not lyse.
43
In one unit of pRBCs: - what's the volume? - what's the iron content?
- 500 mL - 200 mg
44
Platelet transfusion thresholds: \<80: \<50: \<30: \<20: \<10:
\<80: - CNS surgery - intracerebral hemorrhage \<50: - undergoing lumbar puncture or other major non-neurosurgical procedure \<30: - outpatients \<20: - undergoing minor surgical procedure \<10: - any hospitalized patient
45
IgG classes of: 1. anti-Rh antibodies 2. anti-complement antibodies 3. anti-hemophilic factor antibodies
1. IgG1 2. IgG3 3. IgG4
46
Compare/contrast: - bone marrow-derived stem cell transplants - peripherally-derived stem cell transplants - umbilical cord-derived stem cell transplants in terms of: - risk of chronic GVHD - speed of engraftment - risk of graft failure - invasiveness of collection process
Chronic GVHD: - umbilical \< bone marrow \< peripheral Engraftment: - peripheral is faster (~2 weeks for plts and neutrophils, vs. ~3 weeks in BM-derived) Risk of graft failure: - peripheral is lowest risk Invasive: - bone marrow collection is most invasive
47
Stem cells needed for transplant in:
- bone marrow derived allogenic
- bone marrow derived autologous
- peripherally derived allogenic
- peripherally derived autologous
How many apheresis procedures are needed to get that many cells in peripherally derived collections?
BM allogenic - bone marrow derived allogenic: - 3\*10^8/kg CD34+ cells - bone marrow derived autologous - 2\*10^8/kg - peripherally derived allogenic - 2-5 \*10^6/kg (2 minimum; 5 ideal; this means 140-350\*10^6 cells are harvested for a 70 kg patient) - peripherally derived autologous - 2\*10^6/kg Usually achieved in 1-2 rounds of apheresis. Usually enough HSCs are collected in donation for 2 transplants.
48
Pres of DMSO toxicity during a stem cell infusion?
How to prevent it?
Histamine release side effects -- coughing, flushing, rashes, wheezing, nausea, voming, CV instability; garlic taste in mouth Prevention: wash cells; give antihistamines
49
Rank the following blood groups in order of decreasing immunogenicity:
D
Fya
Kell
Kidd
Jka
Jkb
MNS
D
Kell
Jka
Fya
Jkb
Kidd
MNS
50
Formula for corrected count increment (CCI) to assess for platelet refractoriness. If CCI is \< ___ for \>\_ sequential plt transfusions, this suggests platelet refractoriness.
CCI = ((platelet increment/uL)\*(body surface area in squared meters))/(# of plts transfused\*10^11) If CCI is \< 7500 for \> 2 sequential plt transfusions, this suggests platelet refractoriness.
51
There are 3 categories of blood banks:
- FDA-registered transfusion services
- FDA-registered and licensed blood banks
- FDA-registered distribution centers
Define each.
FDA-registered transfusion services - perform only basic preparation activities, like preparing RBCs from whole blood, pooling components, performing bedside leukocyte reduction. They only collect blood in emergencies. FDA-registered and licensed blood banks - perform routine blood collection, including autologous donation. - perform irradiation, washing, leukocyte reduction in the blood bank; freezing, deglycerolization - perform ID testing on blood products FDA-registered distribution centers - act as depots for forwarding blood products
52
Do allogenic tissue suppliers have to register with the FDA? The American Association of Tissue Banks (AATB)?
FDA - Yes AATB - No; voluntary
53
How often must delivery dose verification be performed for blood irradiation, if the radiation source is: - cobalt 60 - cesium 137 - an alternate source
Cobalt 60: every 6 months Cesium 137: every 12 months Alternate: per manufacturer recs Any: anytime the instrument is installed, undergoes major repair, or relocated
54
Blood donation hypocalcemia:
- cause?
- presentation?
- rx?
- caused by the combo of: -- pretreatment of donor w/ a calcium resorption inhibiting drug, like furosemide or another loop diuretic -- exposure of citrate thru pheresis - presents w/ lightheadedness, nausea, extremity tingling - rx: IV calcium gluconate
55
Parvovirus B19 enters cells thru the _ antigen receptor and induces arrest of erythroid maturation at the ___ stage. Plasmodium vivax and knowlesi enter cells thru the _ antigen receptor. (think: evolutionary advantage turned transfusion nightmare) Shiga toxin and Streptococcus suis enter cells thru the __ antigen receptor.
Parvovirus B19 enters cells thru the P antigen receptor and induces arrest of erythroid maturation at the pronormoblast stage. Plasmodium vivax and knowlesi enter cells thru the Duffy antigen receptor. (why 70% of African-Ameicans and 100% of Gambians are Fy(a-b-) -- they lack Duffy on RBCs but express it on their other tissues) Shiga toxin and Streptococcus suis enter cells thru the PK antigen receptor.
56
A sample collected for pre-transfusion testing on Monday morning will expire at....
11:59 PM Thursday (midnight 3 days later)
57
Compare/contrast the direct antiglobulin test (DAT) with the indirect antiglobulin test (IAT)
DAT: - mix washed recipient RBCs with anti-human globulin - if no agglutination is observed, induce agglutination with check cells - false -s can result if: -- Abs are floating -- complement binds AHG instead of RBCs IAT: - mix serum/plasma with recipient RBCs, then wash to remove unbound globulins, then add AHG
58
In RBC antibody testing: - why add LISS (low ionic strength solution) or PEG? - what does pretreatment with enzymes like ficin and papain do? Which antigens are affected? (Hint: mnemonic)
LISS (6x lower ionic strength than normal saline) and PEG can promote antibody binding to RBCs Ficin and papain cleave negatively charged sialic acid molecules from RBC polysaccharides, thus decreasing surface charge and promoting agglutination. But it also destroys some antigens and enhances others: - Destroyed by enzyme digest: M, N, S, s, Fya, Fyb, Xga, InB - Enhanced by enzyme digest: P, Lewis, Rh, Kidd (Lewis is a Rhotten Peeing Kidd)
59
DTT (or BME) used during RBC antibody identification will help with differentiating __ and ___ class antibodies while also destroying certain red cell antigen classes, like \_\_\_\_.
differentiate IgM and IgG (will break apart IgM pentamers, unmasking IgGs underneath) destroy antigens from the Kell, Lutheran, Dombrock, Cromer, Indian, Cartrwright, LW, and Knops systems
60
Acquired hemophilia A: - presentation, incl differences between this and congenital hemophilia A? - at what FVIII levels can they experience severe bleeding? - how to treat mixing studies? - Rx?
- presentation, incl differences between this and congenital hemophilia A? Severe bleeding, usually of skin, deep MSK, retroperitoneal. Do NOT have hemarthrosis seen in congenital d. - at what FVIII levels can they experience severe bleeding? \>2-5% - how to treat mixing studies? incubate for 1-2 hours beforehand - Rx? PCCs such as FEIBA (factor 8 inhibitor bypass agent) or recombinant activated factor 7 preparation (i.e. Hemlibra) for active bleeding
61
Factor XIII (13) Deficiency
Normal function?
Congenital deficiency?
Acquired deficiency?
Rx?
Normally cross-links fibrin thru peptide bonds.
Congenital def: rare, AR. Assoc w/ delayed umbilical stump bleeding, bleeding after circumcision, hematomas, soft tissue bleeding, and recurrent spontaneous abortions.
Acquired def: Seen in Crohn's dz, ulcerative colitis, Henoch Schonlein purpura, liver cirrhosis, sepsis
Rx: cryo, factor XIII concentartions (Europe)
62
What are the 3 components of Vichrow's triad?
Endothelial injury Stasis (immobility) Hypercoagulability: - acquired \> genetic - acquired = pregnancy, cancer antiphospholipid syndrome - genetic more likely in a patient w/ a + FH, unprovoked clots, clotting while on anticoagulant, clotting at a young age, and/or clotting at unusual sites
63
Outline lab testing for a patient being worked up for a genetic hypercoagulability disorder.
Standard coag testing:
- PT
- PTT
Specialized coag testing:
- Antithrombin deficiency
- Protein C & S deficiency
For these you can measure free protein S by ELISA (to quantify biologically active protein S), total protein S by ELISA (to quantify total protein S, including C4b-bound), antigen activity test. This will help tease apart whether you may have a production deficiency vs. a functional deficiency.
Genetic testing:
- Factor V Leiden genotyping (clasically R506E; but screen first w/ the activated protein C resistance test. Factor V Liverpool I359T also leads to increased thrombosis. Factor V Cambridge R306T shows activated protein C resistance but doesn't raise thrombosis risk).
- MTHFR genotyping (faster and cheaper method: homocysteine level)
- Prothrombin gene mutations (G20210A in 3' UTR of factor 2 gene in Caucasians, or C20290T in African Americans)
64
Regarding protein C and S:
- how do they change in pregnancy?
- how can they change in patients on warfarin?
- how can they change in patients with factor V Leiden deficiency?
- how do they change in pregnancy?
Prot S is decreased
- how can they change in patients on warfarin?
Prot S can be decreased
- how can they change in patients with factor V Leiden deficiency?
Both Prot C and S levels decrease.
65
Factor V Leiden: - what % of Caucasians are affected? - what is the most common mutation? How does it lead to a hypercoagulable state? - what lab test is used to screen for this condition? - what is the risk for thrombosis in a homozygote? heterozygote?
- what % of Caucasians are affected? 5% (homo + hetero) - what is the most common mutation? How does it lead to a hypercoagulable state? Arg506Glu (R506E) --\> resistance to cleavage of factor V by activated protein C - what lab test is used to screen for this condition? APC resistance assay - what is the risk for thrombosis in a homozygote? heterozygote? 3-8x risk hetero 10-80x risk homo
66
Protein S deficiency:
- inheritance pattern?
- frequency of mild deficiency?
Compare/contrast total protein S, free protein S, and protein S function in:
- type I (quant def)
- type IIa (qual def)
- type IIb (qual def)
How much prot S is normally free?
Why not just do genetic testing?
autosomal dominant
mild 1:500; severe phenotype rare
Total prot S:
- decreased in type I
- normal in types IIa and IIb
Free protein S:
- decreased in types I and IIa
- normal in IIb
Prot S function - decreased in all
Approx 30-40% of prot S is free in plasma. Rest is bound to C4b.
Molecular testing isn't required for Dx -- it's complicated b/c there are two genes incl a pseudogene.
67
What is the most common inherited disorder of primary hemostasis?
Popn prev?
von Willebrand dz
1%
68
Expected von Willebrand multimer patterns in:
- type I vWD
- type 2A vWD
- type 2B vWD
- type 3 vWD
See image
69
In type I vWD:
1. is it common? is it severe?
2. inheritance pattern?
3. describe the deficiency pattern
4. under which circumstances might patients w/ type I vWD have normnal test resutls?
1. most common - 70% of all vWD cases. Seen in about 1% of people -- the most common inherited bleeding disorder. Least severe.
2. autosomal **dominant**
3. partial quantitative def. results in vWF and factor VIII being \<30% of normal
4. OCP use, stress, hemorrage, acute phase rxn
70
Type 2 vWD represent various qualitative deficiencies.
- inheritance pattern for all?
Differentiate:
2A
2B (what happens when you give DDAVP?)
2M
2N(ormandy) -- what factor deficiency does it mimic?
Platelet type vWF
**My cleavage sites changed, so now I'm small (2A)**
**I stick so close to vWF, so I can't reach high on the wall (2B)**
**Now I can't touch vWF using GPIIb, but I'm mostly fine (2M)**
**But I took after hemophilia A, and now to factor VIII I can't bind (2N)**
**All are autosomal dominant.**
**2A:**
VWF activity \< VWF antigen, but normal platelet count. Loss of HMW and IMQ vWF multimers on gel.
Mutation in the vWF protease cleavage site -\> increased enzymatic cleavage + lack of high and intermediate MW multimers
**2B:**
gain of function mutation leading to **increased** affinity for platelet binding to vWF ("stickier vWF"). Results in decreased platelet count.
vWF antigen and activity ratio is disproprotionately low (activity \< antigen). On gel, HMW multimers are absent -- looks very similar to platelet type vWF.
Giving DDAVP may paradoxically cause thrombocytopenia, though this is transient and most often not assoc. w/ bleeding or thrombosis.
**2M:**
loss of function mutation in GPIIb/V/IX binding site of vWF -\> decreased binding of vWF to platelets
decreased activity \< antigen, but normal (or very slightly normal) multimers
**2N:**
mutation in the factor VIII binding site of vWF.
Mimics hemophilia A because factor VIII is disproportionately decreased since vWF isn't able to properly bind to it, thus destabilzing it.
**Platelet type:**
- caused by gain-of-function mutation in GPIIb-alpha that leads to increased binding to vWF
- resembles 2B in lab testing.
71
Type 3 vWD
- inheritance pattern?
- characteristics?
- autosomal **recessive** (all other vWD is AD)
- complete/absolute quantitative deficiency of vWD -\> resembles hemophilia A
- most severe form; also the rarest
72
What molecules do platelets use to adhere to each other? What disease is this process absent/deficient in?
What molecules do platelets use to adhere to collagen? What disease is this process absent/deficient in? (Smock's mnemonic)
GPIIb/IIIa + fibrinogen to adhere to each other. Absent/def in Glanzmann's thrombasthenia
GPIb + vWF to adhere to collagen (activation of vWF thru shear force or ristocetin exposes platelet bidning and collagen binding domains on vWF). Absent/def in Bernard Soulier Syndrome ("GPIb has only 1 'B'")
73
Describe platelet aggregation study findings in the following diseases:
- Bernard Soulier syndrome
- Glanzmann's thrombosthenia
- vWD
- Acquired platelet storage pool deficiency
- Hermansky-Pudlak disorder
- Bernard Soulier syndrome (GPIb defect)
normal aggregation w/ ADP, epinephrine, collagen
absent response to ristocetin
- Glanzmann's thrombosthenia (GPIIb/IIIa defect)
normal aggregation with ristocetin
absent response to ADP, epinephrine, and collagen
- vWD
normal platelet agg response to ADP, epinephrine, and collagen
absent/decreased response to ristocetin (except for 2B, which has enhanced response to ristocetin)
- Acquired platelet storage pool deficiency (often associated with hematologic disorders like MDS)
abnormal response to all agonists
- Hermansky-Pudlak disorder (absence of dense granules)
normal 1st wave of aggregation, but absent 2nd wave
luminometry: no release of ADP
74
If platelets from the following disorders were analyzed by flow cytometry, which markers would be absent?
- Bernard Soulier syndrome
- Glanzmann's thrombastenia
- Bernard Soulier syndrome
GPIb = CD42b/CD61
- Glanzmann's thrombastenia
GPIIb/IIIa = CD41 (GPIIb) and GPIIIa (CD61) -- encoded by the ITGA2B and ITGB3 proteins, respectively
75
For coag testing:
- what color tube/tube additive is used?
- why is the tube inverted 3-6x?
- what temp is the sample sent to the lab at? Why?
- how is platelet poor plasma produced and defined?
- what color tube/tube additive is used?
Light blue top - sodium citrate
- why is the tube inverted 3-6x?
To mix thoroughly w/ sodium citrate and prevent fibrin clot formation
- what temp is the sample sent to the lab at? Why?
Room temp. Cold temps can activate factor 7 and produce erroneous results; may also precip vWF and artificially decrease its activity. Cold temps can also lead to plt membrane disruption and potentially increase the sample's phospholipid content.
- how is platelet poor plasma produced and defined?
Centrifugation. Want \<10K platelets/uL.
76
HIT:
Higher risk with UFH or LMWH?
Management of patients who present w/o thrombosis?
What is the 4T score for heparin-induced thrombocytopenia?
Describe each T and its scoring algorithm, and the overall scoring algorithm.
2-3x more likely w/ UFH
All HIT pts need to be on rapid-acting alternate anticoagulations b/c the risk of thrombotic complications w/i 30 days is 50% for those who don't have thrombosis @ presentation
**Thrombocytopenia:**
+2 if plts fall \>50%
+1 if plts fall 30-50%
+0 if plts fall \<30%
**Timing**
+2 if thombocytopenia appears 5-14 days after heparin exposure
+1 if thrombocytopenia appears \>14 days after heparin exposure
+0 if there's no hx of heparin exposure
**Thrombosis (or other sequelae)**
+2 for new thrombosis OR skin necrosis OR acute systemic rxn after IV heparin bolus
+1 for progressive or recurrent thrombosis, non-necrotizing skin lesions
+0 for no thrombosis
**oTher causes for thrombocytopenia:**
+2 if none are apparent
+1 if it's possible
+0 if there's definitely another cause
**Scoring:**
\<3: low probability of HIT (\<1-5%)
4-5: intermediate prob. of HIT (~14%)
6-8: high prob of HIT (~64%)
77
Name the members of the Mycobacterium tuberculosis complex.
*PaPa’s ABCCs of MTB*
M. pinnipedii, africanum, bovis, canetti, caprae, microti, tuberculosis, BCG (bovis Calmette Guerin)
78
Name the non-MTB complex Runyon I mycobacteria: the photochromogens
## Footnote
*Cold Kansas Asian Sea Monkeys need to be in the sun (turn yellow when exposed to UV light)*
M. szulgai (@ 24 C), kansasii, asiatum, marinum, simiae
79
Name the non-MTB complex Runyon II mycobacteria: the scotochromogens
## Footnote
*Flash Gordon’s Hot Alien Scrotum*
M. gordonae, szulgai (@ 37 C), xenopii, scrofula
80
Name the non-MTB complex Runyon III mycobacteria: the non-chromogens
MAC complex
Mycobacterium avium/intracellulare
81
Name the non-MTB complex Runyon IV mycobacteria: the fast growers (colonies w/i 7 days)
## Footnote
*“Dick Cheney is obsessed with making a quick fortune, while heat-resistant smeagol can spit his mucus quickly.”*
M. chelonae, abscessus, fortuitum, thermoresistibile, smegmatis, mucogenicum
82
Name the mycobacteria that produce niacin and reduce nitrate reduction.
"Nice sea monkey"
M. simiae and M. marinum produce niacin and do NOT reduce nitrate
M. tuberculosis is the only mycobacterium to both produce niacin AND reduce nitrate to nitrite
83
Name the DNA viruses.
HeHe PoPa ParAde
He=Herpes
He=HepaDNA (HBV; carries special RT)
Po=Pox (smallpox, molluscum contagiosum)
Pa=Papilloma, polyoma (JC, PML, demyelinating encephalopathy)
Par=parvovirus (ssDNA)
Ade=Adenovirus
84
Apixiban (Eliquis, 2012)
- route of administration?
- MOA?
- Antidote/reversal?
- Lab test to monitor efficacy?
Oral
Reversible direct inhibitor of factor Xa (both free and clot-bound)
Andexanet Alfa reversal for major life-threatening bleeding; otherwise PCCs
Anti-Xa assay
85
Rivaroxaban (Xarelto, 2011)
- route of administration?
- MOA?
- Antidote/reversal?
- Lab test to monitor efficacy?
Oral
Reversible direct inhibitor of factor Xa (both free and clot-bound)
Andexanet Alfa for major life-threatning bleeding; otherwise PCCs
Anti-Xa assay
86
Dabigatran (Pradaxa, 2010)
- route of administration?
- MOA?
- Antidote/reversal?
- Lab test to monitor efficacy?
Oral
Reversible thrombin inhibitor
Idarucizimab (Praxbind)
Thrombin time
87
Unfractionated heparin (5000-40,000 Dalton MW range)
- route of administration? (hint: 2)
- MOA?
- Antidote/reversal?
- Lab test to monitor efficacy?
IV or SQ -- but if given SQ isn't absorbed the gut and has a short half life (1-2 hrs)
Binds antithrombin III -\> induces confirmational change to activate antithrombin -\> inactivation of thrombin, factor Xa, and other proteases
Protamine reversal
Monitor using PTT, anti-Xa; therapeutic goal is 0.3-0.7 u/mL
88
LMWH (enoxaparin/Lovenox, other -parins; MW \<8000 Da)
- route of administration?
- MOA?
- Antidote/reversal?
- Lab test to monitor efficacy?
SQ or IV
Activates antithrombin III -\> inhibits factor Xa preferentially, not factor IIa
Protamine reversal, though this isn't as effective as protamine to reverse unfractionated heparin
Anti-Xa assay
89
Warfarin
- route of administration?
- MOA?
- Antidote/reversal, depending on different scenarios?
- Lab test to monitor efficacy?
Oral
Inhibits vitamin K-dependent clotting factors: 2, 7, 9, 10, C, and S
Reversal:
- if bleeding:
ABO-matched FFP @ 15 mL/kg - will repelenish factors 2, 7, and 10, but doesn't have enough factor 9.
Other options (controversial/used differentially in diff countries): recombinant factor 7a (quickly activates factor X to Xa and allows thrombi formation) -- but expensive and short t 1/2. FEIBA (activated PCC containing factor 7a + small amounts of 2a, 9a, 10a.
- if INR \>10 and patient is stable: d/c warfarin and give 2.5-5 mg vitamin K orally (only give IV vit K in life-threatning situations since it can cause anaphylaxis)
- INR \<10 and patient is stable/has very minor bleeding: withhold warfarin and wait; coagulapathy should start correcting w/i 24-36 hrs with full correction in 3-5 days
Lab monitoring: PT/INR
90
Eptifibatide (Integrellin) - antiplatelet drug
- Route
- MOA
- indication
- t 1/2
IV
reversible GPIIb/IIIa inhibitor; derived from the venom of a southeastern pygmy rattlesnake
acute coronary syndrome (unstable angina, NSTEMI -- reduces mortality and prevents non-fatal MI) and percutaneous coronary intervention (reduces ischemic events during stenting)
2-3 hours
91
Abciximab (Reopro; antiplatelet drug)
- Route
- MOA
- indication
- t 1/2
IV
Fab Ab fragment of a chimeric monoclonal Ab that reversibly binds platelet IIb/IIIa receptors -\> steric hindrance -\> prevents binding of fibrinogen, vWF, and other aggregation-promoting molecules during clot formation. Secondary MOA: binds Mac-1 integrin receptor on activated monocytes.
- Used in myocardial ischemia, PCI
10-30 min in plasma, but 4-5 days when platelet-bound
92
Clopidogrel (Plavix, anti-platelet)
- route?
- MOA?
- which other anti-platelet drugs share its target?
- which CYP enzymes are important in its metabolism?
- Indications?
- t 1/2?
Oral
Irreversible inhibitor of the platelet P2Y12 adenosine diphosphate receptor. Inhibition of this receptor prevents the downstream activation of the glycoprotein IIb/IIIa receptor complex which leads to reduced platelet aggregation
- other P2Y12 inhibitors: ticlopidine, prasugrel, cangrelor
Since clopidogrel is an inactive prodrug, it needs to be activated in two steps. CYP2C19 and CYP3A4 are needed. Patients w/ LOF of at least one CYP2C19 allele won't effectively metabolize the drug:
CYP2C19 \*1/\*1 - extensive metabolizers
CYP2C19 \*1/\*17 or \*17/\*17 - ultrarapid metabolizers
\*1/\*2, \*3, \*4, or \*8 - intermediate metabolizers
Any combo of 2, 3, 4, 8 - poor metabolizers - more common in East Asians
t 1/2 is 8 hours, but since it's an irreversible inhibitor, any bound clopidogrel will inhibit platelets for their entire lifespan, 7-10 days. Thus the drug shoudl be d/ced 5 days before surgery.
93
Salicylic Acid (Aspirin)
- Route
- MOA (3 targets)
- Indications
Oral (less commonly IV or suppository)
Derived from willow tree bark (salicin is the Latin word for willow)
Irreversible COX1 inhibitor; COX2 modifier; irreversible platelet thromboxane A2 blocker preventing platelet activation. Due to COX blocking, arachidonic acids are shuttled into the lipooxygenase pathway, producing anti-inflammatory lipoxins.
Angina (active/prophylactic), CV risk reduction, CRC, ischemic stroke (active/prophylactic), OA, revascularization procedures (prophylactic), RA, SLE, pain relief
94
Afibrinogenemia
- inheritance pattern?
- what genes can be mutated? Most common?
- Presentation?
- Rx?
AR
FGA (alpha), FGB (beta), FGG (gamma) genes -\> FGA most common
Presents as fibrinogen \<0.1 g/L and mild/severe bleeding abnormalities. Bleeding from the umbilical cord stump at birth is often the first sign.
Rx: fibrinogen replacement from plasma-derived fibrinogen concentrates (preferred); or cryoprecipitate; or plasma
95
What do the following parameters in thromboelastography (TEG) denote, and represent in terms of in vivo clotting ability? Which intervention should be given for each abnormal paramter?
R time
K and alpha angle
Max amplitude
Ly30, Ly60
What etiology underlies a TEG graph showing decreased R time and increaed max amplitude?
R time = initial clot generation -- assesses function of coagulation factors. If increased, give plasma.
K and alpha angle = speed of clot formation. Alpha angle correlates with fibrin production.
Max amplitude = clot strength. Assesses platelet function.
Ly30, Ly60 = % decrease in amplitude 30 or 60 minutes after the maximal amplitude, aka fibrinolysis. If a clot dissolves faster than expected, this represents excess fibrinolysis, and can be treated withh antifibrinolytics, like tranexamic acid, aminocaproic acid, or aprotinin.
96
Warfarin and pharmacogenomics:
- role of CYP2C9 genotype?
- role of VKORC1 genotype? (what protein does this encode?)
Patients who carry at least one copy of such a variant allele (such as CYP2C9\*2 and CYP2C9\*3) have reduced metabolism leading to higher warfarin concentrations. On average, they require a lower daily warfarin dose than patients who are homozygous for the wild-type CYP2C9\*1 allele, and may require more time (\>2-4 weeks) to achieve maximum INR effect.
The VKORC1 gene encodes the vitamin K epoxide reductase enzyme, the target of warfarin. Patients who carry the -1639G\>A polymorphism in the promoter region of the VKORC1 gene are more sensitive to warfarin and require lower doses.
97
Alpha and beta thalassemia
General mechanism of gene defect?
Location of each gene?
Alpha: deletions of HBA1 and HBA2 genes on chrom 16
Beta: missense mutations of HBB on chrom 11
98
In iron deficiency anemia, describe typical lab findings regarding:
- serum iron
- storage iron (ferritin)
- transferrin level
- transferrin saturation
- RDW
- MCHC
- bone marrow storage iron
- bone marrow sideroblasts
- bone marrow # of erythroid precursors
- serum iron: decreased
- storage iron (ferritin): increased
- transferrin level: decreased
- transferrin saturation: decreased
- RDW: normal or slightly elevated
- MCHC: normal
- bone marrow storage iron: increased
- bone marrow sideroblasts: decreased
- bone marrow # of erythroid precursors: normal
99
X-linked sideroblastic anemia
- caused by mutation in which gene?
- finding on BMBx?
Delta-aminolevulinic acid synthetase (ALA synthetase), an early step in heme biosynthesis
Characterized by numerous ringed sideroblasts on iron stain
100
In transfusion medicine, what precaution needs to be taken for people with IgA deficiency?
Relative frequency?
wash RBCs. A small % will develop anti-IgA in response to transfusion; a smaller % are at risk for anaphylaxis w/ transfusion
1:500
101
After transfusion of 1 U of pRBCs:
- how much is Hct expected to rise?
- how much is Hgb expected to rise?
Hct: increase by 3%
Hgb: increase by 1 g/dL
102
What are three benefits obtained by leukoreducing blood products?
1. decrease CMV transmission
2. decrease febrile nonhemolytic transfusion reactions
3. decrease HLA immunization for platelet units
103
True or false: anti-A1 Abs can be produced by all non-A1 blood types.
False. Only people with A2 or A2B blood type develop anti-A1 antibodies. People with type O and B blood develop a generic anti-A antibody.
104
Kell blood group:
- antigens?
- relative antigen prevalences in white people? black people?
- antigen type? CD?
- what protein is it linked to in the membrane, and clinical significance?
- what treatments destroy Kell antigens?
- what is the predominant IgG class involved in anti-Kell antibodies?
- what is the most common way to acquire an anti-K Ab?
K and k (chilano) -- differ by one amino acid (193 - Met in K, Thr in k). Also rare k alleles and K0 (K null; these individuals can produce anti-Ku/anti-KEL5 antigens)
91% of whites are K-/k+, 8% are K+/k+, and 0.2% are K+/k-. 98% of blacks are K-/k+.
CD238, type II membrane glycoprotein
Linked via disulfide bond to Xk protein; when Xk (the only allele of the Kx blood group) is absent, there's reduced expression of Kell and the McLeod phenotype. Xk is encoded on the X chromosome, and McLeod syndrome develops almost exclusively in males, manifesting as acanthocytosis and late onsent musuclar, neurological, and psych sx. Can cooccur w/ CGD b/c both the XK gene and the CYBB gene are on Xp21.1. Men w/ CGD + McLeod shouldn't be transfused b/c they'll develop allo-Abs that make all other blood products incompatible to them.
Antigens destroyed by trypsin/chymotrypsin mixture, or DTT, AET, or EDTA glycine. Resistant to other enzymes, including trypsin and chymotrypsin alone.
IgG1
Transfusion, not pregnancy...so some countries given girls and women of childbearing age only K- blood.
105
Name some conditions in which the following findings can be seen:
1. Burr cells
2. Basophilic stippling (and what's staining?)
3. Sickle cells
1. Artifact - delay of \>8 hrs between blood draw and smear prep (most common). Transfusion (reversible -- cells return to normal w/i 48 hours). Uremia.
2. Conditions that lead to defective or accelerated heme synthesis: Lead intoxication. Thalassemias. Hemoglobinopathies. Myelodysplasia. Megaloblastic anemia.
Basophilic stippling is the result fof granules of ribonucleoproteins and mitochondrial remnants.
106
Describe the levels of alpha thalassemia:
- silent carrier?
- trait - presumptively diagnostic tests?
- Hemoglobin H disease?
- Alpha thal major (aka hemoglobin Barts disease)?
Silent carrier: 1/4 genes defective. Clinically and hematologically silent; may show mild microcytosis and hypochromia.
Trait: 2/4 genes defective (in cis or trans). Hematologically mild (mild anemia). Presumptively dxed w/ elevated RBC, low MCV, low MCHC, but **normal** hemoglobin electrophoresis and iron studies
Hemoglobin H disease: 3/4 genes defective; usually chronic hemolyzers and transfusion-dependent; patients have low/normal Hb and severe microcytosis and hypochromia. Hb H refers to a hemoglobin tetratmer composed entirely of B-globin proteins. this is a fast-migrating Hb found at the edge of gels run at pH 8.2. H bodies in RBCs form when Hb H is oxidized.
Alpha thal major: 4/4 genes defective; usually embryonic lethal
107
Beta thalassemia:
- diff between ß+ and ß0?
- CBC and hemoglobin electrophoreis result for beta thal trait?
- B thal intermieda vs B thal major?
B+: gene producing decreased amounts of globin
B0: gene that does not produce B globin
Beta thal trait pts will have high RBCs, low MCV, low MCH, and an elevated hemoglobin A2 fraction on electrophoresis
B thal intermedia: B+/B+ genotype; not transfusion dependent except during times of stress
B thal major: B+/B0 or B0/B0 - transfusion dependent. Aka Cooley's disease
108
On which chromosome are the beta-globin like genes located?
What is the arranagement of this locus, from 5' to 3'? (hint: same order as in embryonic expression)
How can this arrangement result in delta beta thalassemias? Phenotype?
What about delta-beta fusions?
22
5' - epsilon – gamma-G – gamma-A – delta – beta - 3'.
A deletion can wipe out both delta and beta genes:
- (gamma-beta)0 trait: generally clinically silent, but hematologically resembles beta-thal, with normal A2 but elevated F on electrophoresis
- Homozygotes for (gamma-beta)0 or double hets for (gamma-beta)0 + beta thal usually present as beta thal intermedia; almost all hemoglobin is Hb F.
Alternately, the 5' portion of delta can fuse w/ the 3' portion of beta in a crossover event, leading to hemoglobin Lepore, a thalassemia b/c the delta promoter isn't efficient in making enough delta-beta fusion product.
- Lepore/normal hets present like beta-thalassemia minor.
- Lepore/Lepore has a phenotype between beta thal intermedia and major.
- Lepore runs @ S position on alkaline cellulose acetate, and between A and A2 on cation exchange chromatography.
109
Hemoglobin Barts
- composition?
- when is it seen?
- tetramer of gamma globulins
- moderately insoluble -- accumulates in tissue
- extremely high O2 affinity -- cannot release oxygen to tissues
- found mostly in hemoglobin Barts disease
110
Hemoglobin E:
- molecular cause?
- patient population?
- hemoglobin electrophoresis pattern?
- significance of hem E trait? hem E homozygosity?
- clinically significance w/ other Hb abnormalities?
- point mutation in Beta globulin gene: Glu26Lys (E26K). Leads to gain of +2 charge and leads to an alternate splice site that causes an unstable mRNA -\> both structural consequences and thalassemic red cell parameters.
- Southeast Asian
- Elutes in A2 region on electrophoresis. C-position on alkaline electrophoresis; A-position on acid electrophoresis
- Trait: normal to mild anemia; generally asymptoamtic.
- Homozygosity: clinically insignificant.
- HbE + Hb S = sickling disorder
- HbE + B-thal = thalassemia major
111
What is hemoglobin Gower-1? Gower-2?
Hemoglobin Gower 1 (also referred to as ζ2ε2 or HbE Gower-1) is a form of hemoglobin existing only during embryonic life, and is the primary embryonic hemoglobin. It is composed of two zeta chains and two epsilon chains, and is relatively unstable, breaking down easily.
Hemoglobin Gower 2 (also referred to as α2ε2 or HbE Gower-2) is a form of hemoglobin existing at low levels during embryonic and fetal life. It is composed of two alpha chains and two epsilon chains, and is somewhat unstable, though not as much as hemoglobin Gower 1
112
Hemoglobin-oxygen dissociation curve:
- x axis?
- y axis?
- what is the p50 value for hemoglobin?
- of the following, which conditions shift the curve to the left (decreasing O2 delivery to tissus) vs. to the right (increasing O2 delivery to tissues)?
1. fever vs. hypothermia
2. acidosis vs. alkalosis
3. increased 2,3-DPG vs. decreased 2,3-DPG (= 2,3-biphosphoglyceric acid)
4. CO2 binding to Hb
5. High affinity Hb like HbF, HbBarts, Hb Chesapeake
X: oxygen tension
Y: O2 sat
p50 (oxygen tension associated w/ 50% O2 sat) for Hb: 27 mm Hg
1. fever - right shift; hypothermia - left shift
2. acidosis - right shift; alkalosis - left shift
3. increased 2,3,-DPG - right shift; decreased 2,3-DPG - left shift (this is a glycolysis offshoot that binds and stabilized deoxygenated Hb. Elevated in people who live at high altitudes; pregnant women; airway obstruction; CHF)
4. Right shift
5. Left shift
113
Osmotic fragility test:
- readout of test?
- at what NaCl conc will normal RBCs be 100% lysed?
- in what conditions is osmotic fragility increased (more lysis at NaCl concentrations closer to normal serum osmalarity?)
- in what conditions is osmotic fragility decreased?
- measuring Hb in supernatant by spectrophotometry @ 540 nm
- 0.3%
- increased osmotic fragility: anything that decreases surface area:volume ratio: hereditary spherocytosis, hereditary elliptocytosis; hereditary pyropoikylocytosis (a subtype of HE); stomatocytes; warm autoimmune hemolytic anemia (2/2 to spherocyte production)
- decreased osmotic fragility: sickle cells, target cells, xerocytes, iron deficiency anemia, alpha or beta thalassemia, hyponatremia; chronic liver disease; hemoglobin C (target cells)
114
Rouleaux:
- stacks of how many red cells?
- changes to CBC?
- pathophysiology?
4 or more
Hgb unaffected, RBC falsely decreased, MCH falsely increased (b/c MCH = Hgb/RBC)
Caused by elevated levels of gamma-globulins (things that migrate in the gamma region of SPEPs, most notably Igs) or acute phase proteins like fibrinogens. Associated w/ lymphoprolif disorders (i.e., MM), chronic liver disease, chronic inflammatory disorders.
115
What is the function of ferrochetalase, and what is the consequence of enzyme defects?
What is zinc protoporphyrin (ZPP), and why is it significant?
What are some assay interferences when measuring ZPP?
Heme is formed when iron is inserted into protoporphyrin IX, catalzed by the mitochondrial homodimer enzyme ferrochetalase (the last step of heme synthesis).
Deficiencies of ferrochetalase (encoded by the FECH gene on chrom 18), can lead to erythropoietic protoporphyria (EPP). Features include photosensitivity, lichenification, hypo/hyper skin pigmentation, abdominal pain, and accumulation of protoporphyrin in the liver in 5-20% of pts leading to liver failure.
In the absence/deficiency of iron, some protoporhyrin binds zinc instead of iron, forming ZPP.
ZPP serum levels are increased in iron deficiency anemia and anemia of chronic disease. They are normal in thalassemias.
ZPP is measured via fluorescence, so it's subject to false elvations in the cases of hyperbilirubinemia and increased riboflavin (absorb at similar wavelengths).
116
Describe the reference method for measuring Hb, incl:
- reagent
- absorbance
- type of Hb not detected/measured in this method
- sources of error
Cyanmethemoglobin colorimetric method:
- RBCs lysed
- Drabkin's reagent (ferricyanide + KCN)
- Ferricyanide converts Hb to methemoglobin by oxidizing Fe2+ (ferric iron) to Fe3+ (ferrous iron).
- KCN converts methemoglobin to cyanmethemoglobin, which can be stably measured @ 540 nm
- measures all Hb forms except for sulfhemoflobin
- rxn takes about 10 minutes; reagants stable (but also potentially lethal)
- errors: incomplete conversion; lipemia; hyperproteinemia; pipetting
117
Hemoglobin C:
- molecular cause?
- patient population?
- hemoglobin electrophoresis pattern?
- sickle solubility test? CBC findings?
- significance of hem C trait? hem C homozygosity?
- clinically significance w/ other Hb abnormalities?
- beta chain missense: Glu6Lys (E6K) (vs. E26K in HbE, and E6V in HbS)
- found in 2-3% of African-Americans
- net +2 charge for a Hb tetratmer --\> slower migration than HbA and HbS on an alkaline gel; migrates in C position on acidic and alkaline gels and accounts for 35-45% of all Hb
- hem C trait: asymptomatic; smear maybe normal or maybe hypochromia/target cells. **Sickle solubility test is negative**.
- hem C + hem S (each inherited from a diff parent, since same a.a. is affected) = Hemoglobin SC disease, a clinically significant sickling disorder. Smear shows taco cells, boat cells and target cells.
118
Methemoglobinemia:
- which iron state?
- oxygen affinity?
- how does our metabolism normally keep this in check?
- which hemoglobin variant can result in methemoglobinemia?
- causes of acquired methemoglobinemia?
- rx?
- ferrous (Fe3+ iron)
- high oxygen affiity relative to normal Hb -\> cyanosis since tissues can't get oxygen
- NADH methemoglobin reductase (cytochrome b5 reductase) (major) and NADPH methemoglobin reductase, ascorbic acid, and the glutathione pathway, reduce Fe3+ to Fe2+
- Hemoglobin M
- acquired: certain antibiotics (dapsone), local anasthetics (benzocaine), nitrites
- initial rx: oxygen therapy, methylene blue (but don't give methylene blue to G6PD def patients since NADPH required for it to work). Life threatning cases: consider red cell exchange.
119
ID this species
Aspergillus flavus
120
ID this species
Aspergillus fumigatus
121
ID this species
Aspergillus niger
122
ID this species
Aspergillus terreus
123
Hepcidin
- class of molecule?
- where is it produced?
- when is it produced?
- function?
- if elevated, what tissue stain correlates?
- what are hepcidin levels in hemochromatosis patients?
25-amino acid peptide hormone
mostly liver
regulated by iron status; also an acute phase reactant; also upregulated by IL-6 and other cytokines
degrades the iron channel ferroprotein on macropages and gut enterocytes, thus trapping iron inside macrophages
Prussian blue iron stain will show increased iron in macrophages
Surprisingly hepcidin levels are LOW in pts w/ hematochromatosis (mechanism unknown)
124
Acanthocytes (spur cells) vs. echninocytes (burr cells)?
What causes acanthocytes?
Clinical scenarios where you'd see \>10% acanthocytes?
Other scenarios when you'd see acanthocytes, but @ \<10%?
Acanthocytes have irregularly distributed spicules of varying shapes and sizes; in burr cells spicules are uniformly sized and distributed.
Acanathocytes are due to changes in the lipid content of the red cell membrane.
Clinical scenarios w/ \>10% acanthocytes: McLeod blood group (absence of Kx protein of the Kell blood group); abetalipoproteinenamia (absence of apo B, preventing TGs from getting absorbed thru the GI tract); homozygous hypobetaproteinemias; advanced liver disease
\<10% acanthocytes: Lu blood gorup phenotype; post-splenectomy; MAHA (DIC, TTP, HUS); artifact
125
What are the components of HbA2?
Does it have increased or decreased O2 affinity compared to HbA?
In hemoglobin electrophoresis:
1) when is it normal?
2) when is it increased? what can cause a "false" increase?
3) when is it decreased?
Two alpha and two delta globulins
Similar O2 affinity as HbA
1) normal in alpha thalassemia trait (2/4 functional copies) -- combined w/ normal iron studies and microcytosis, it's diagnositc of alpha thal
2) increased in beta thal (most common); can also been seen in B12 and folate deficiency, hyperthyroidism, and antiretroviral therapy. Can be "falsely" increased in pts w/ HbE, since they elute at the same place.
3) decreased in alpha thal (1/4 functional copies); iron deficiency, lead poisoning, siderblastic anemia, hypothyroidism, delta chain variants
126
What is pictured on this Wright-Giemsa smear?
What is it composed of?
In what clinical scenarios might it be seen?
Howell-Jolly body - small, round basophilic inclusions
DNA. Forms when erythrocytes don't extrude all their DNA as they mature.
Asplenia or hyposplenia. Splenectomy, sickle cell disease (autosplenectomy), splenic irradiation.
127
Paroxysmal noctural hemoglobinuria
Defect?
What is a good screening test for PNH?
Which flow markers aid in the diagnosis of PNH?
Prior to flow, how was PNH diagnosed?
Will DAT be positive?
Rx?
Defect in assembly of glyosyl phosphatidylinositols (GPIs), most commonly due to a defect in the enzyme phosphatidlyinositol glycan A (PIGA) on the X chromosome. Proteins anchored via GPIs that are disrupted in PNH include decay accelerating factor (DAF/CD55, disrupts the formation of C3-convertase), and protectin (CD59, preents bidning of the MAC to RBCs).
Screening test: sucrose lysis test. Sucrose has low ionic strength and facilitates complement binding to RBCs.
Flow markers: CD55 and/or CD59; nowadays FLAER (binds selectively to GPI anchor)
Old gold standard: Acidified serum test (Ham's test), which involved mixing treated patient and control blood to prove that patient's hemolysis was caused by an increased susc to activated complement.
DAT negative, since hemolysis isn't antibody-mediated.
Rx: Eculizumab - binds C5 late in the complement cascade, inhibiting its cleavage by C5 converstase to C5a and C5b.
128
Bernard Soulier (BS) Syndrome
- inheritance?
- defect?
- presentation?
- CBC and platelet aggregation study findings?
- AR; extremely rare (only ~1000 estimated cases)
- GPIb/IX/V complex genes (GPIBA, GPIBB, or GP9 -- no cases involvoing GPV have been described) such that plts don't adhere to vWF
- thrombocytopenia, giant platelets, lack of ristocetin-induced platelet aggregation, and a decreased response to thrombin-induced aggregation
- mild to severe bleeding -- almost all pts will require transfusion at some point, but isoantibodes against the missing antigens may block the function of transfused platelets.
129
Reticulocytes:
Which supravital stains highlight them?
Which fluorescent stains highlight them?
How do you calculate reticulocyte index (RI)?
Reticulocyte production index (RPI)?
New methylene blue (NMB), brilliant cresyl blue
Thiazole orange, auramine O, ethidium bromide -- often used in automated analyzers
RI = (Reticulocytes\*Hct)/(normal Hct), where normal Hct is approx = 45
RPI = (reticulocytes\*Hct)/(normal Hct\*correction factor)
The correction factor ranges from 1-2.5 depending on Hct; I'm not memorizing these.
130
Sickle solubulity:
- SOP?
- Causes for true +s?
- Causes for false +s?
- Causes for false -s?
Prepare hemolysate by adding saponin to RBCs, then add phosphate solution containing sodium hydrosulfite, which will reduce HbS into liquid hemoglobin crystals called tactoids, making the solution cloudy.
Hold tubes against a white background w/ black lines. If lines can be read clearly behind the tube -\> negative. If lines can't be read -\> positive.
True +: hemoglobin S trait, hemoglobin SS, SC, SD, SG, SE, SO, S/B0, etc.
False +: anything that produces increased turbidity. High protein levels, hypergammaglobinemia (MM), cryoglobinemia, hyperlipedimia, polycythemia vera.
False -: severe anemia, elevated HbF (such as seen in newborns with Hb SS; this test isn't suitable for those \<6 mo old)
131
Platelet granules:
- In general, what type of organelle are these?
- composition and appearance of alpha granules? (Hint: PPPFFT)
- How are alpha granules associated w/ gray platelet syndrome?
- composition and appearance of dense bodies aka delta granules (Hint: CAN)?
- lysosome-related; this is why deficiencies in them also affect similar organelles in other heme cell lines, i.e. megs, melanosomes, lytic granules
Alpha granules:
- round, numerous, less electron dense. Bigger, like alpha males, and contain lots of protein.
- contain:
- P-selectin
- platelet factor 4 (PF4)
- Platelet-derived growth factor (PDGF)
- Fibronectin
- Fibrinogen and other factors (V, VIII, vWF)
- Transforming group factor beta (TGF-B)
- gray platelet syndrome = normally formed but leaky alpha granules, such that platelets don't appear granular. However, P-selectin still remains in the defective alpha granules.
Dense bodies aka delta granules:
- more electron dense (hence the name)
- contain:
- Cations (Mg++, Ca++)
- Amines (serotonin, histamine)
- Nucleotides (ATP, ADP, pyrophosphate)
132
List some disorders of malfunctioning/non-functioning platelet **granules,** specifying whether they affect alpha granules or dense (delta) granules.
Common features on aggregometry studies?
_Dense granule disorders:_
Idiopathic dense-granule disorder (delta-storage pool disease)
Idiopathic dense-granule deficiency
Hermansky-Pudlak syndrome
Chediak-Higashi syndrome
_Alpha granule disorders:_
Paris-Trosseau/Jacobsen syndrome (11q23 deletion including the FLI1 gene)
Gray platelet syndrome
Quebec platelet syndrome
Athrogryposis-renal dysfunction-cholestasis
_Common features:_
- absence of secodnary wave of aggegation in response to epinephrine
- delayed and reduced response ot collage
- impaired aggregation to low concentration of agonists
- high concentrations of ADP eliciting full, irreversible aggregation
- reduction in both the ocntent and ratio of ADP:ATP, or absence of release of ATP
133
Chediak-Higashi syndrome:
- inheritance
- gene defect
- skin and eye feature (what other platelet granule disorder is this seen in?)
- other notable presenting features
- CBC findings that help distinguish it from the other plt disorder w/ eye features
- AR; extremely rare (\<1000 cases; most die before age 10)
- LYST gene
- oculocutaneous albinism (shared w/ Hermansky Pudlak syndrome)
- infectious, lymphoproliferatoive accelerated phase
- presence of very large peroxidase + cytoplasmic granules in both heme and non-heme cells (i.e., neutrophils) -- absent in Hermansky-Pudlak
134
What do the following disorders have in common?
MYH9 (myosin heavy chain) disorders (May-Hegglin anomaly, Sebastian syndrome, Fechter syndrome, Epstein syndrome)
-- inheritance of MYH9 disorders?
-- what is a characteristic blood smear finding in MYH0 disorders?
-- clinical ftrs in MYH9 disorders?
Congenital amegakaryocytic thrombocytopenia (defect in MPL gene, which is the TPO receptor, and also commonly mutated in myeloid malignancies)
Amegakaryocytic thrombocytopenia w/ radioulnar synostosis
Thrombocytopenia absent radius syndrome
X-linked thrombocytopenia w/ dyserythropoesis
Disorders of abnormal platelet **number.**
MYH9 disorders are **mostly autosomal dominant**. All have giant platelets, may have Dohle-like inclusions in neutrophils (as depicted in this image) but most don't cause clinically significant bleeding problems.
Sx include hearing loss, nephritis, and cataracts.
135
Hermansky-Pudlak syndrome
- type of disorder?
- inheritance? associated country of origin?
- gene defect?
- features?
platelet storage pool defect / storage of abnormal fat-protein compound (lysosomal accumulation of ceroid lipofuscin)
- AR; rare but affects as high as 1:1800 Puerto Ricans; also seen in Ashkenazi Jews
- mutation in HPS1, 3, 4, 5, 6, or 7, or AP3B1
- oculocutaneous albinism + bleeding disorder + cellular storage disease (waxy ceroid accumulates in various tissues incl lungs and kidneys). Granulomatous colitis. Pulmonary fibrosis in 40s-50s is most common COD.
136
Immune thrombocytic purpura (ITP)
- true or false: most common autoimmune disorder during pregnancy
- lab abnormalities?
- what are the auto-Abs directed against?
- can they cross the placenta?
- true
- only low platelets
- AutoAbs against GPIIb/IIIa or GP1b-IX-V platelet proteins
Yes, they are IgGs. They can cause fetal or neonatal ITP.
137
Name the ligands that activate the following platelet receptors. Which is the most potent binding event?
- GPIIb/IIIa
- GPVI
- P2X1
- P2Y12 and P2Y1
Protease-activated receptor 1 and 4 (PAR-1, PAR4)
- GPIIb/IIIa: vWF, collagen
- GPVI: collagen
- P2X1: ATP -\> triggers intracellular Ca++ relaese
- P2Y12 and P2Y1: ADP -\> triggers activation
Protease-activated receptor 1 and 4 (PAR-1, PAR4): Thrombin. Thrombin-mediated cleavage of the PAR-1 protein is the most potent platelet activation event.
138
What are Weibel-Palade bodies, and what do they store?
Secretory organelles in endothelial cells
Contain:
- vWF (for primary hemostasis - binds and stabilizes platelets and factor VIII)
- P-selectin (for leukocyte adhesion)
- IL-8
- endothelin
- tissue plasminogen activator
139
Describe the following parameters/trends in all types of vWD:
- ristocetin cofactor activity (vWF:RCo in IU/dL), aka vWF activity
- vWF antigen (vWF:Ag in IU/dL)
- factor VIII coagulant activity in IU/dL
- aPTT
- vWF:RCO/VWF:Ag ratio
- RIPA (ristocetin-induced platelet aggregometry)
- vWF multimers
- should desmopressin be given?
See image.
140
Factor X deficiencies:
- which malignancies/medical conditions can lead to acquired factor X deficiencies?
- rx?
- systemic amyloidosis -\> amyloid factors thought to bind directly to factor X
- resporatory infections
- thymoma
- renal carcinoma, adrenal adenocarcinoma, AML)
- vitamin K def or liver disease -- but these lead to multi-factor deficiences
- rx: FFP, PCCs
141
Relate the following components of thrombosis:
- Factor Va
- Factor VIIIa
- Protein S
- Protein C
- APC
Protein C - a vitamin K-dependent zymogen. Activated with the help of thrombin (aka factor II) and protein S to APC (aka activated protein C).
APC inactivates factor Va and VIIIa. Its inactivation of factor Va involves cleaving at three different arginine residues. Protein S tags along and helps with one of these cleavage events.
In factor V Leiden, one of these three arginines is changed to a glutamine, basically making the mutant factor V "APC resistant."
Protein C deficiency is also possible, both congenital and acquired. Heterozygous protein C deficiency is found in 0.1-0.5% of the general population and increases risk of VTE 7x. Homozygous protein C deficiency can present as life-threatening purpura fulminans and DIC in newborns.
142
Which procoagulant/anticoagulant factors are synthesized BOTH inside and outside of the liver?
Which are synthesized ONLY in the liver?
Both hepatically and extra-hepatically: factor V, VIII, PAI-1, AT, protein C, protein S, tissue factor pathway inhibitor (TFPI)
Only in the liver: fibrinogen, plasminogen
CP boards
flashcards
Decks in class (2)
# Cards
