Coag Flashcards
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)
Direct thrombin inhibitors
-include argatroban, bivalirudin, lepirudin -used to tx pts with HIT and thrombosis -mechanism: DTIs bind active site of thrombin and inhibit proteolytic activity toward fibrinogen
Direct thrombin inhibitors lab testing
-DTIs prolong aPTT and, to lesser extend, PT -PT and aPTT do not correct with a 1:1 mixing study (consistent with inhibitor) -TT is markedly prolonged
Factor XI Deficiency
-AKA hemophilia C -due to mutation in FXI gene -13% Ashkenazi Jews; rare in all other ethnicities -often milder bleeding than predicted by factor level ****isolated prolongation of the aPTT**** -treatment with FFP (no factor concentrate available) -dx/monitoring using factor XI assay
APS: Clinical Criteria
- Vascular thrombosis 2. Pregnancy morbidity: - unexplained fetal loss after 10 weeks gestation - premature birth (less than 34 weeks) due to preeclampsia, eclampsia, or placental insufficiency 3. At least 3 fetal losses prior to 10 weeks
Factor XIII Deficiency
-factor XIIIa forms cross-links with fibrin clot -deficiency causes poor wound healing, traumatic bleeding, postsurgical bleeding (delayed bleeding), umbilical stump bleeding -FXIII activity cannot be assessed by PT, aPTT, or TT as clotting endpoint detected in those assays occurs prior to clot stabilization or cross-linking
Screen for Factor XIII Deficiency
-pt plasma is re-calcified and allowed to clot -clot then suspended in 5M urea or 2% acetic acid, which disrupts hydrogen bonds but does not degrade FXIII-dependent covalent cross links -if FXIII is decreased (less 5%), there are no covalent bonds in fibrin clot and it will dissolve -if sufficient FXIII, clot will not dissolve in 5M urea or 2% acetic acid -test cannot identify heterozygous FXIII deficiency or homozygous pt with mild dificiency
Fibrinogen Assay
Clauss Fibrinogen Assay: -most commonly performed fibrinogen assay, modified thrombin time test -measures rate of clot formation after adding high concentration of throbin to citrated plasma -fibrinogen activity of sample is derived from a standard curve relating clotting time to plasma standards of known fibrinogen activity -normal: 170-410 mg/dL
Utility of fibrinogen assay
-dx of hypofibrinogenemia/dysfibrinogenemia -dx of DIC, liver failure, fibrinolysis -guide transfusion tx with cryoppt *in newborns, fibrinogen level may be slightly lower due to presence of fetal fibrinogen which is not assayed by the functional assay *therapeutic heparin levels and DTI drugs do not affect the test results b/c thrombin is present in excess
Other functional fibrinogen assays
-turbidimetric method: lower amnt thrombin is added to pt plasma, change in turbidity measured spectrophotometer -PT based assay: TF and phospholipids added to pt plasma to generate endogenous thrombin, turbidity/light scatter measured -immunologic method: pt plasma added to agar with anti-fibrinogen abs; zones of ppt are measured
Gray Platelet Syndrome (alpha granule deficiency)
- rare inherited d/o characterized by thrombocytopenia and large, abnormal plts (ghost like appearance)
- absence of alpha granules and their contents
- proteins normally stored in these organelles are spontaneously released from megas in the BM which leads to marrow fibrosis
- heterogeneity in both bleeding severity and response to plt function testing (in some pts, thrombin and/or collagen induced plt agg is markedly modified)
Pseudo-Gray Platelet Syndrome
- plts may appear markedly pale in pts without GPS due to extensive plt activation from EDTA exposure
- normal sized plts, normal plt agg studies, no thrombocytopenia
- recollection of specimen in citrate or hepain will demonstrate normal plt morphology
Hemophilia A
- most common congenital coag d/o
- X-linked recesssive
- bleeding (hemarthroses) due to Factor VIII def
- Mild: greater 5%
- Moderate: 1-5%
- Severe: less 1% (spont hemorrhage)
- prolonged PTT, corrects w/mixing studies
- give recombinant FVIII (half life=12 hrs)
- DDAVP in mild cases
- ~25% pts develop inhibitors (detect on 1:1 mixing study)
Hemophilia B
- X-linked recessive
- clinically identifical to Hemophilia A
- factor IX deficiency
- prolonged PTT, corrects with mixing studies
- Factor IX replacement differs from FVIII b/c only about 30% recovery and half life=8 hours (therefore give double calculated dose of recombinant FIX)
- 1-2% develop inhibitors
HIT type I
- 10-20% pts receiving unfractionated heparin
- plt count drops to ~100,000 (less than 50% decline from baseline)
- occurs within 2 days of heparinization and spontaneously resolves
- not immune mediated
- of no clinical consequence
- NOT a contraindicated for additional heparin use
HIT type II General
- 1% of pts receiving unfractionated heparin (LMW heparin much lower incidence)
- plt count drops to ~60,000
- usually occurs 4-10 days after starting heparin
HIT type II mechanism
- administration of unfractionated heparin induces the formation of IgG abs against heparin platelet factor 4 (hepartin-PF4) complex
- this complex binds to platelet surface leading to platelet activation, aggregation, premature removal from circulation
HIT Type II clinical
- pts at increased risk for arterial/venous thrombosis
- bleeding not major concern b/c plt count usually greater than 20, 000
- d/c all heparin
- use a DTI
HIT Type II diagnosis
- by ELISA using microtiter plates with heparin-PF4 complexes
- assay is very sensitive (negative result r/o HIT) but it is not specific
- strength of a positive test is measured by the optical density; OD>1.0 is associated with platelet-activating antibodies
PFA general
- whole blood (sodium citrate tube) aspirated through 150 µm aperature in a membrane that is coated with either collagen and epi (CEPI) or collagen and adenosine diphosphate (CADP)
- blood is passed through membrane at high shear rate to simulate the in vivo hemodynamics in the small capillaries
- the formation of adherent plt aggregates blocks the aperature and stops a timer- referred to as closure time and is reported in seconds
- combined measure of plt adhesion and aggregation (primary hemostasis)
PFA applications
- evaluation of bleeding pt
- determining presence of drug-inducced plt dysfunction
- determining pt compliance with ASA and other antiplt drugs
- determining plt functionality in high risk pregnancy
- evaluation of pts with suspected inherited or acquired plt d/o such as vWD
- monitoring DDAVP tx in pts with Type I vWD
PFA interpretation
- result is dependent on plt function, plasma vWF level, plt number, and to some extent hct
- normal PFA: CEPI less than 180s (excludes the presence of a sig plt function defect)
- ASA effect: CEPI greater than 180s(prolonged) and CADP less than 116s
- abnormal plt fx (other than ASA): CEPI greater than 180s (prolonged) and CADP greater than 116s (prolonged)
- the finding that both CEPI and CADP are prolonged suggests that plt fx is abnormal; however thrombocytopenia (less than 100) and anemia (hct less than 30%) should first be excluded as that can cause false positive PFA results
Platelets normal survival time in circulation
7-10 days
Alpha Granules
Contain large proteins:
- fibrinogen
- fibronectin
- PDGF
- P-selectin (adhesion molecule)
- PF4
- TGF-beta
- factors V and VIII
Dense Granules
Contain small molecules:
-ATP
- ADP
- serotonin
- calcium
- histamine
- epinephrine
Platelet Surface Antigens
- GP Ib/V/IX (vWF receptor, CD42)
- GP IIb/IIIa (fibrinogen receptor, CD41)
*GP IIIa= CD61*
- GP Ia/IIa (collagen receptor)
- GP Ic/IIa (fibronectin receptor)
- RBC ag (ABO, P, I, i, Le) but no Rh ag
- Class I MHC
Acquired Platelet Function Defects
- NSAIDS
- ASA
- PCN
- Uremia
- DIC
- MPNs
