Laboratory Investigation of Challenging Cases Laura A. Worfolk, Ph.D Scientific Director, Coagulation
Coagulation Cascade XII XIIa XI HMWK/Prekallikrein XIa VII Injury IX X IXa VIII Xa X TF TF/VIIa Prothrombin V Thrombin XIII Fibrinogen XIIIa Fibrin (soluble) Fibrin (insoluble) Confidential Do not copy or distribute 2
Cascade and Laboratory Testing Intrinsic & Contact Pathway XIIa XIa Injury Extrinsic Pathway IXa/VIIIa VIIa/TF aptt PT Common Pathway Xa /Va Prothrombin Thrombin Fibrinogen Fibrin Clot Confidential Do not copy or distribute 3
Case #1 52 year female, asymptomatic, pre-op testing reveals prolonged aptt and normal PT Possibilities: Lupus Anticoagulant (prolongs aptt, generally not PT) Factor deficiency (VIII, IX, XI, XII) Von Willebrand Disease (not likely) Pre-analytic factor (but PT is normal & prolongation is reproducible) Heparin (prolongs aptt, but not PT) Confidential Do not copy or distribute 4
Case #1 Test Result Reference PTT 81 sec 1:1 Mix 30 sec 1:1 Incubated Mix 31 sec Thrombin Time 20 sec Prothrombin Time 11.2 sec 22-34 sec 16-23 sec 9 11.5 sec Abnormal aptt corrected when mixed with normal pool plasma suggesting factor deficiency.. Confidential Do not copy or distribute 5
Case #1: Factor Testing Test Result Reference Factor VIII 66% 50-180% Factor IX 151% 60-160% Factor XI 126% 65-150% Factor XII 136% 50-150% Inhibitor pattern was not detected with any of the factor testing.. Confidential Do not copy or distribute 6
Inhibitors & Factor Testing Multiple dilutions of patient sample are tested to ensure an inhibitor is not affecting results; examples below: FVIII Dilution Normal Heparin FVIII Inhib. Lupus Anticoagulant 1:10 105% 15% 5% 85% 1:20 99% 45% 10% 127% 1:40 108% 63% 16% 154% 1:80 88% Final 104%?????? Confidential Do not copy or distribute 7
Case #1 Continued: Additional Testing Von Willebrand Factor testing all normal Lupus Anticoagulant testing negative What else? Prekallikrein: 1% (60-187%) Confidential Do not copy or distribute 8
Prekallikrein (Fletcher Factor) Deficiency PK: 609 aa protein w/ 58% homology to FXI 75% circulates bound to HK Deficiency first identified by Hathaway and colleagues in 1965, autosomal-recessive trait* Deficiency is not associated with a bleeding disorder, nor does it appear to protect against thrombotic disease Extremely rare (<100 case reports in the literature) and does not require treatment *Blood 1965;26:521 Confidential Do not copy or distribute 9
Contact Pathway & the APTT HK-PK α-kallikrein Prekallikrein FXII APTT Activator FXIIa APTT Contact Activators: Silica, Ellagic Acid, Kaolin FXI +CaCl2 FXIa Thrombin Generation HK=High Molecular Weight Kininogen Clot formation
Contact Pathway & the APTT HK-PK α-kallikrein Prekallikrein FXII APTT Activator FXIIa APTT Contact Activators: Silica, Ellagic Acid, Kaolin FXI +CaCl2 FXIa Thrombin Generation HK=High Molecular Weight Kininogen Prolonged Clotting Time
PK Deficiency and Modified APTT APTT Incubation Time Sample 5 min 10 min Normal Control 26.3 sec 24.1 sec Abnormal Control 40.5 sec 38.0 sec Patient 50.4 sec 38.8 sec Patient PK level was 15% Case Studies in Hematology & Coagulation. ASCP CaseSet, Gulati et al editors, 2012. Confidential Do not copy or distribute 12
Case #2 79 year old female: unknown history, sample received for Lupus Anticoagulant workup Lupus Anticoagulants (LA): One type of antiphospholipid antibody (APA) chx by prolongation of in-vitro phospholipid dependent clotting reactions Protein targets: B2GPI and Prothrombin LA s more closely associated with thrombotic events than other APAs Confidential Do not copy or distribute 13
How to test for LA s? Demonstration of phospholipid dependence using 2 different methods Evidence of inhibition by mixing studies Blood collection: before anticoagulant therapy or after discontinuation, platelet poor LA can not be conclusively determined if the TT is significantly prolonged. Lack of specific inhibition by coagulation factor/other circulating inhibitors J. Thromb Haemost 2009; 7:1737-40. Confidential Do not copy or distribute 14
Screening Tests: low phospholipid Dilute Russell Viper Venom (DRVVT): Snake venom used to directly activate FX Insensitive to deficiencies/inhibitors of XII, XI, VIII, IX & VII Sensitive to deficiencies/inhibitors of Factors X, V, II (i.e. Coumadin therapy) Sensitive aptt Sensitive to deficiencies/inhibitors of Factors VIII, IX, XI, XII Sensitive to anticoagulant therapies (heparin, thrombin inhibitors, coumadin) Confidential Do not copy or distribute 15
Confirmatory Tests DRVVT Confirm: High concentration of phospholipid (PL) neutralizes LA effect Results expressed as ratio: DRVVT Screen/Confirm Staclot LA (APTT based test): Hexagonal phase PL neutralizes LA effect Incorporates mixing study Confidential Do not copy or distribute 16
How do we test? Pathway #1 PTT-LA Normal Prolonged Hexagonal Phase Confirm: Pos or Neg Stop Pathway 1 or 2 positive: LA Detected Pathway #2 DRVVT Screen Normal Stop Prolonged DRVVT Confirm Negative Stop Positive Prolonged LA Pos DRVVT Mix Study Normal LA Neg Confidential Do not copy or distribute 17
LA Test Interpretation Sample Type PTT-LA Hexagonal Phase DRVVT Screen DRVVT Ratio DRVVT Mix Normal Normal Not Indicated Normal Not indicated LA Pos High Pos High Pos High (not corrected) FVIII Inhib High Normal or Pos Normal Not Indicated Heparin High Neg Normal Not Indicated Coumadin Normal- High Neg High Normal- Pos Normal (corrected) Confidential Do not copy or distribute 18
Case #2 continued: LA Work-up DRVVT Screen (<45 s) DRVVT Confirm & Mix 36 s PTT-LA (<40 s) NI Hexagonal Phase Confirm 95 s Positive Overall interpretation: LA detected by one pathway a bleeding history requires other coagulopathies be excluded.. Clinical History No past personal/family history of bleeding, inpatient for minor procedure but currently had large bruises on torso, no current or past thrombotic history. Factor VIII Activity <1% Confidential Do not copy or distribute 19
Testing Caveats: FVIII vs LA Factor VIII inhibitors: may yield false positive Hexagonal Phase Confirm (& no effect on DRVVT) Lupus Anticoagulants: may falsely decrease clotting based Factor Assays Besides clinical history, how to differentiate? Confidential Do not copy or distribute 20
FVIII Inhibitors vs Lupus Anticoagulant Chromogenic Factor VIII assay not affected by Lupus anticoagulants Sample Type Clotting FVIII Chromogenic FVIII Normal 95% 100% LA sample 30%/55%/86% 90% FVIII Inhibitor <1% <1% Confidential Do not copy or distribute 21
Case #2 Continued FVIII Activity Clotting <1% FVIII Activity Chromogenic <1% FVIII Inhibitor 461 Bethesda Units (Normal: less than 0.4 BU) Conclusion FVIII Inhibitor Detected. Hexagonal Phase Confirm false positive due to FVIII inhibitor (DRVVT negative) Confidential Do not copy or distribute 22
Acquired Factor VIII Inhibitors Autoantibodies in non-hemophiliac patients rare: 1-4/million/year high rate of morbidity/mortality Incidence increases with age, age distribution biphasic: 20-30 years (post-partum population) & major peak 68-80 years ~50% of cases: occur in patients lacking any disease Blood. 2008;112:250-255 Confidential Do not copy or distribute 23
Diagnosis of Acquired Hemophilia A Laboratory Findings: Isolated prolonged PTT which can t be corrected by incubated mixing study Reduced FVIII level FVIII inhibitor activity detected by Bethesda or Nijmegen modification Clinically: Hemorrhage into skin, muscles, soft tissues, mucous membranes; disease may manifest following trauma, surgery or by cerebral hemorrhage Confidential Do not copy or distribute 24
Case #2 Continued: 7 months later. Test VWF Antigen 324% (50-217%) FIX Activity FXI Activity Lupus anticoagulant & Antiphospholipid Antibodies FVIII Activity Chromogenic VIII 7% FVIII Inhibitor Result 62% - 95% (inhibitor pattern) 57% - 86% (inhibitor pattern) DRVVT negative, Hexagonal positive, Cardiolipin neg, Beta-2- glycoprotein I neg 11% (possible inhibitor pattern, values offcurve) 50.6 BU Confidential Do not copy or distribute 27
Case #3 59 year old female: sample submitted for PT/PTT mixing study and Lupus anticoagulant testing Test PT & PT Mix aptt & aptt Mix Thrombin time Staclot & DRVVT Result 80 sec / 48 sec, no correction Vmax / 184 sec, no correction 18 s (16-23 sec) Vmax Confidential Do not copy or distribute 28
Case #3: Factor Testing Sample Dilution FII FV FX FIX FVIII 1:10 16% <1% 12% Vmax 90% 1:20 62% 40% 173% 1:40 94% 99% 248% Differential diagnosis: Factor V Inhibitor or a monoclonal gammopathy. Confidential Do not copy or distribute 29
Case #3: Possibilities FV Inhibitors: extremely rare, <150 cases reported: ~50% reported (1955-97) developed in response to bovine FV (found in thrombin products, such as fibrin sealants applied to wound sites) Major surgery independent risk factor apart from use of bovine thrombin β-lactam antibiotics Blood Coagulation & Fibrinolysis 2011, 22:160-166. J Thromb Haemost 2005;2:1385-91. Confidential Do not copy or distribute 30
Case #3: Possibilities Monoclonal gammopathies: Multiple Myeloma, Waldenstron s macroglobulinemia, primary amyloidosis or other lymphoproliferative disoroders M protein interference with coag testing Rarely, anti-fv antibodies have been identified with these disorders In ~20% of cases of anti-fv formation, no underlying disease identified J Thromb Haemost 2005;2:1385-91. Confidential Do not copy or distribute 31
Case #3: Inhibitor Testing Inhibitor Factor V FII / X Result 23 BU / 25 BU With dilution, the factor activity is near normal, but can t precisely quantify. Since factor activity near normal, the inhibitor assay not performed as a specific inhibitor would almost certainly not be detected. Confidential Do not copy or distribute 32
Case #3 Conclusion Acquired Factor V Inhibitor interfering with PT and PTT based assays Most patients will have a bleeding diathesis Relationship to inhibitor titer and bleeding has not been studied Some patients do not bleed, possible differential recognition of plasma & platelet Factor V; or antibody specificity (i.e. interference of APC inactivation of FVa) Confidential Do not copy or distribute 33
Case #4 68 year old female, prolonged PT & APTT Possibilities: Common pathway factor deficiency or multiple factor deficiencies Thrombin Inhibitor (i.e. Pradaxa) Non-specific circulating inhibitor (i.e. monoclonal protein) Pre-analytic factor (i.e. underfilled tube) Lupus Anticoagulant (unlikely, unless high titer LA) Confidential Do not copy or distribute 34
Case #4 Continued Diln FII/FV/FVII FVIII FXI FXII 1:5 <5% 1:10 <10% 4% 6% 8% 1:20 <20% 45% 17% 21% 1:40 82% 38% 39% 1:80 70% 56% PT = 73 sec, aptt = 84 sec, Positive DRVVT Confidential Do not copy or distribute 35
Case #4 Continued Test Result Von Willebrand Factor Antigen 363% (50-217%) Ristocetin Cofactor Activity 473% ( 42-200%) Cardiolipin IgM Antibody Cardiolipin IgA Antibody Cardiolipin IgG Antibody >100 U/mL (<10 U/mL) 31 U/mL (<10 U/mL) <10 U/mL (<10 U/mL) Confidential Do not copy or distribute 36
Case #4 Conclusion PT based factors undetectable, inhibitor patterns on multiple PTT based factors DRVVT (a common pathway based test), strongly positive, Cardiolipin IgM strongly positive Elevated VWF antigen & activity Case conclusion: IgM monoclonal gammopathy (was this multiple myeloma?) Monoclonal paraprotein interfering with PT/PTT & factor assays, true LA positive?? Confidential Do not copy or distribute 37
Hemostatic Abnormalities & Multiple Myeloma Increased levels of VWF/FVIII with active disease, irrespective of treatment Increased levels of inflammatory cytokines (IL-6, TNF, CRP) Acquired protein C resistance Production of procoagulant auto-antibodies, i.e. Lupus anticoagulant Increased risk of venous thrombosis J Thromb Haemost 2011;9:653-63 Confidential Do not copy or distribute 38
Case #5 70 yr old woman with recurring massive nose bleeds. Her family Doctor became concerned when the CBC showed a hemoglobin of 5.5 g/dl Multiple tubes received for factor assays & fibrinogen Handwritten on all tubes tube was lipemic Confidential Do not copy or distribute 39
Case #5: Testing Test Factors VIII, IX, XI & XII Fibrinogen Result / Comment Sample appears to clot on the instrument. All factors >600% 600 mg/dl Confidential Do not copy or distribute 40
Case #5 Observation Sample clotted on the analyzer, but would go back into solution with reheating. Not a lipemic sample after all, but a cryoprecipitate. Confidential Do not copy or distribute 41
Case #5 Conclusion Suggested screen for multiple myeloma, which was positive Multiple myeloma patients may have: Cryofibrinogenemia: antibodies to fibrinogen complex that precipitate in the cold (also interferes w/ fibrin polymerization) Inability to polymerize fibrin: bleeding Confidential Do not copy or distribute 42
Case #6: A Rose by any Other Name Test Result Comment PT, PTT, TT Vmax Is this a serum DRVVT Screen Vmax sample or Heparin contamination? Hexagonal Phase Confirm Negative Negative for LA Heparin Level Less than 0.1 Negative for UFH Calcium = 18.2 Confidential Do not copy or distribute 43
Case #6: Riddle Solved Sample labeled as plasma was really URINE! BUN 1040 Total protein 11.0 Ca++18.2 Smell = urine Manneken Pis (located in Brussels), Flemish for Little Man Pee Confidential Do not copy or distribute 44
Case #6 Summary Why clot endpoint with the Hexagonal Phase Confirm? Sample is mixed 1:1 w/ normal pool plasma in test system How did we prove it was urine? BUN/Creatinine & calcium levels Whiff test Confidential Do not copy or distribute 45
Prolonged aptt: Thrombotic History Normal PTT-LA Stop - no further action Lupus Sensitive aptt (PTT - LA) Dilute Russell s Viper Venom Test (drvvt) Normal drvvt Stop - no further action Prolonged PTT-LA Prolonged drvvt Hexagonal Phase Neutralization (Staclot - LA) Confirmed Negative and Confirmed Negative Phospholipid Neutralization (drvvt Confirmation) Confirmed Positive Prothrombin Time (PT) Thrombin Clotting Time (TCT) Fibrinogen Confirmed Positive Consistent with Lupus Anticoagulant 2 nd Tier Testing at Medical Director Discretion: Specific Factor Assays Heparin Anti-Xa [30292X / 118623] D-Dimer, Quantitative [8659X / 38963] Confidential Do not copy or distribute 46
Prolonged aptt: Bleeding History Normal aptt Stop - no further action Routine aptt Minimally Prolonged aptt Prolonged aptt Factor VIII Factor IX Factor XI von Willebrand Factor Antigen Ristocetin Cofactor Activity aptt 1:1 Mixing Study Corrects Inhibitor Pattern aptt 1 hr Incubated Study Inhibitor Pattern Thrombin Clotting Time (TCT) Hexagonal Phase Neutralization (Staclot - LA) 2 nd Tier Testing at Medical Director Discretion: drvtt Reflex to Confirmation (Phospholipid Neturalization) [15780X / 33693] PT - Reflexive Pathway [19643X / 18180] Thrombin Clotting Time [883X / 2893] Factor XII Activity [362X / 29153] Fibrinogen Quantitative (Clauss Method)[461X/43053] Lupus Sensitive aptt (aptt-la)[17408x / 143573] Heparin anti-xa [30292X / 118623] Corrects Factor VIII Factor IX Factor XI Thrombin Clotting Time normal and Hexagonal Phase Neutralization Negative Confidential Do not copy or distribute 47
Prolonged aptt: Asymptomatic Normal aptt stop - no further action Routine aptt and Thrombin Clotting Time (TCT) Minimally Prolonged aptt Normal Thrombin Clotting Time Prolonged aptt Prolonged Thrombin Clotting Time Prolonged aptt Normal Thrombin Clotting Time Hexagonal Phase Neutralization (Staclot - LA) Factor VIII Factor IX Factor XI Heparin anti-xa Fibrinogen Corrects (18193 - non-orderable) aptt 1:1 Mixing Study Inhibitor Pattern Decreased Factor VIII Von Willebrand Factor Antigen Ristocetin Cofactor Activity aptt 1 hour Incubated Study Inhibitor Pattern Hexagonal Phase Neutralization (Staclot - LA) Corrects 2 nd Tier Testing at Medical Director Discretion: drvtt Reflex to Confirmation (Phospholipid Neuturalization) [15780X / 33693] PT - Reflexive Pathway [19643X / 18180] APTT / Hexagonal Phase Neutralization (Staclot -LA) [36573X / 143583] Reptilase Clotting Time [37700X / 9413] Fibrinogen Antigen [37801X / 127953] Specific Factor Assays Factor VIII Factor IX Factor XI Factor XII Confidential Do not copy or distribute 48
Conclusions Knowledge of test limitations & clinical history yields most cost-effective & clinically useful laboratory investigations Avoid test interpretation in isolation Questions? Confidential Do not copy or distribute 49