APS: Lab Criteria
- LA present in plasma on 2 or more occasions at least 12 weeks apart 2. aCL ab of IgG and/or IgM isotype in serum or plasma (titer >99th %ile by ELISA) on 2 or more occasions at least 12 weeks apart 3. anti-beta2 glycoprotein I ab of IgG and/or IgM in serum or plasma (titer >99th %ile by ELISA) on 2 or more occasions at least 12 weeks apart
APS Diagnosis
At least one clinical and one lab criteria are met
APS patients may have which false positive test?
-Serologic test of syphillis (ie positive VDRL or RPR, but neg treponemal assays) -Syphilis ag is used in these tests is cardiolipin mixed with cephaline and cholesterol
Antiphospholipid antibodies
-Abs directed against the proteins bound to phospholipids, and include lupus anticoagulant, anticardiolipin ab, and anti-beta2 glycoproetin I ab -increased with age, characteristically assd with thrombosis in elderly pts -10-20% of cases are seen in pts with SLE (secondary APS); 50% of patients with LA have SLE
Criteria for lupus anticoagulant
- Prolongation of phospholipid dependent clotting assay (aPTT) -prolonged clotting times are seen in screening assays containing low amount of phospholipid (eg dilute aPTT or aPTT-LA, dilute RVVT screening test); if LA is present, it will bind all phospholipid so that there is none available for clotting (more sensitive tests) -dRVVT test (which activates FX directly) is more specific than aPTT test for detection of LA b/c not influenced by deficiencies or inhibitors of clotting factors VIII, IX, XI 2. Evidence of an inhibitor demonstrated by mixing studies -the prolonged aPTT is NOT corrected by a 1:1 mix with normal platelet free plasma 3. Confirmation of the phospholipid-dependent nature of inhibitor -RVVT confirmatory test: RVVT corrects in this test b/c it contains high amount of phospholipid (bind all the LA in specimen, still excess phospholipid for clotting) 4. Lack of specific inhibition of any one coagulation factor
Anticardiolipin ab ELISA
IgG abs confer greater risk of thrombosis than do IgM or IgA
Anti-beta2glycoprotein-I ELISA
beta2glycoprotein-I is a naturally occurring inhibitor of coagulation and platelet aggregation
Most common cause of hereditary thrombophilia
Factor V Leiden mutation
Factor V Leiden mutation
-responsible for 90-95% of activated protein C resistance -inherited as AD -5% Caucasians have mutation (almost never in asains or AA) -G–>A substitution resulting in AA missense mutation that eliminates one of the 3 APC cleavage sites, resulting in factor V protein that is resistant to proteolytic cleavage by APC -heterozygotes have a 5-10 fold increased risk of venous thrombosis
Factor V Leiden mutation screening test
- APC added to pt plamsa + phospholipid + Ca2+ -APC degrades factors Va and VIIIa in samples from normal patients, prolonging the aPTT
- aPTT test with and w/o added APC are performed in parallel and a ratio of the two is calculated (aPTT with APC/aPTT)
- normal pts ratio >2.0
- Factor V Leiden heterozygotes ratio less than 2.0 and homozygotes ratio less than 1.5
Factor V Leiden mutation confirmatory assay
(DNA test) -used for pts with pos screen, pts on anticoag tx, or pts with coexisting lupus anticoagulant -RFLP PCR with WT yielding 2 PCR products and mutant yielding 1 PCR product (loss of MnII restriction endonuclease cleavage site)
Bethesda Assay
-performed for detection of inhibitor to FVIII -can also be used to quantify inhibitors to factors V, IX, X, XI, XII -serial dilution of pt plasma with PRP containing known amount of FVIII -incubated 2hrs at 37 degrees -residual FVIII activity is determined by FVIII assay and compared to normal control (which uses PNP + imidazole buffer and is processes similarly to pt sample)
Bethesda Assay Interpretation
-inhibitor strength measured in Bethesda units (1 Bethesda unit= amount of inhibitor that neutralizes 50% of the FVIII in normal plasma after 2 hours at 37 degrees) -calculation of the Bethesda titer is obtained from a log-log graph (x-axis: dilution of pts plasma; y-axis: % residual FVIII after incubation)
Low and High Inhibitor Titers
low inhibitor titer: 0.5-5 BU high inhibitor titer: >5 BU
Low and High Responders
low responder: pts with low inhibitor titer that does not rise on re-exposure to FVIII (treated initially with high dose FVIII in attempt to neutralize ab) high responder: pts with an inhibitor titer that rises sharply on re-exposure
Treatment for high responders and low responder who do not respond to FVIII infusion
give concentrates that bypass need for FVIII (activated PCC, activated FVII)
Detection of cryoglobulins
store serum at 4 deg Celcius for 3 days, centrifuge, electrophoresis on the precipitate that forms (cryoprecipitate)
Type I Cryoglobulinemia
monoclonal immunoglobulins assd with myeloma or WM
Type II Cryoglobulinemia
-most common type -mixture of polyclonal IgG and monoclonal IgM directed against IgG (rheumatoid factor)
Type III Cryoglobulinemia
Mixture of two polyclonal immunoglobulins
Mixed Cryoglobulinemia
-types II-III -usually in HCV infection -remaining cases in autoimmune dz (SLE), lymphoproliferative d/o, chronic infections -decreased complement levels -distinct clinical syndrome -tx with corticosteroids, plasapheresis, alpha IFN (HCV)
Mixed Cryoglobulinemia Clinical Syndrome
-palpable purpura (leukocytoclastic vasculitis) -arthralgia -hepatosplenomegaly -LAD -anemia -sensorineural deficits -glomerulonephritis
DIC labs
-prolonged PT, aPTT, TT -increased D-dimer, PF1.2, fibrinopeptide A -decreased fibrinogen, plts, ATIII -intravascular hemolysis (increased LDH and bili, schistocytes)
DIC pathogenesis
-circulating substance behaves like tissue factor causing widespread formation of microvascular thrombi -attempts to break down newly formed fibrin by fibrinolytic system -consumption of clotting factors -bleeding diathesis -primarily tx thrombosis b/c it causes more of the morbidity (use subQ low dose heparin or ATIII)
