ISTH

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17 Poster presented at

ISTH2017 on:

Forty years of riddle solving to decipher puzzling test results in the coagulation laboratoryAriella Zivelin

Coagulation Laboratory, The Israeli National Hemophilia Center and Thrombosis Unit. Sheba Medical Center, Tel Hashomer, Israel

The coagulation laboratory is responsible for performing all the routine and specialized tests in the coagulation field. Occasionally the laboratory activity is “spiced up“ by puzzling results requiring out of the box thinking. Solving those issues contributes to enhancement of the professional level of the laboratory, expands clinicians' expertise and provides great benefit to the patients. This abstract illustrates my personal experience accumulated over 4 decades of work, starting as a young technician and stepping up to become the laboratory manager

• Each case will be presented including a short description, path of laboratory investigation and conclusions. References to published data are provided

• It should be noted that my experience represents long-term collaboration and team work with many laboratory personnel and clinicians, to whom I thank from the bottom of my heart

The detailed poster is available at the ISTH site. Any comments or questions can be addressed to Dr. Ariella Zivelin at azivelin@gmail.com

INR 5 to >10 how common and why?

The prevalence at Sheba Medical Center- 0.9% of PT measurements, (56% from ER) and the causes are:

1. Coumadin overtreatment (most common)

2. Poisoning with “super warfarin” – rat poison (self or not self)

3. Severe deficiency of a coagulation factor in the PT (extrinsic) pathway including afibrinogemia

4. Rarely strong Circulating Anticoagulant (Lupus Anticoagulant) is significantly prolonging the PT. It is reagent specific (i.e. Innovin) and is partially corrected by different reagents and Point of Care measurements

5. Hereditary combined deficiency of all Vitamin K dependent coagulation factors (Brenner et al. BJH 1990, 75:537) caused by a mutation in the Carboxylase gene (Brenner et al. Blood 1998, 92:4554)

6. Dysfibrinogenemia that affects the structure of the clot and its detection by optical detection method (i.e. Fibrinogen Longmont caused by the mutation FGB Arg166Cys)

7. Inhibition of fibrinogen polymerization. Thrombin time and Reptilase time are longer than detection range (not clotted)

8. A HIT positive patient receiving Bivalirudin- a direct thrombin inhibitor

9. Failure of the instrument to detect clotting that occurred in a time period shorter than the detection gate. (i.e. Hemophilia A patients with inhibitor receiving rFVIIa). This post analytical wrong interpretation could be avoided by looking at clotting graph of the sample!

10.Failure to detect a clot in the blood sample during the preanalytical state and eventually measuring PT of serum (preanalytical mistake)

“Pitfalls” in measurement of fibrinogen levels

• Presence of thrombin inhibitors, e.g. heparin and NOACs, may lower the level of fibrinogen measured

• We have added a permanent warning note regarding this possibility in our report of fibrinogen levels

• Does this effect on the results provide any benefit (i.e. awareness of over treatment, heparin in the sample)?

• The new direct anti-thrombin anticoagulant dabigatran (Pradaxa) may affect fibrinogen measurements even at therapeutic levels

• Different reagents show different sensitivity, which depends on the thrombin concentration of the reagent

• The effect is decreasing with increasing dilution of the plasma sample, in contrast to real fibrinogen deficiency

Lysis of unnoticeable small clot in a D-Dimer blood sample can produce very high D-Dimer levels

• Pre-analytical visual examination of blood samples for coagulation tests by inverting the tube fails to detect small clots

• Presence of a small clot in a tube can be suspected if the PTT is extremely short (shorter than the lower limit of the normal range). Its presence can be confirmed by mechanical testing, i.e. insertion of a wooden stick

• The level of D-Dimer in a sample is very stable for at least 24 hours

• However, if clot lysis occurs, a false positive result is obtained!

• Samples with small clots gave normal or slightly elevated D-Dimer levels when measured shortly after drawing, and levels of thousands ng/ml six to eight hours after drawing as the result of lysis of the clot

• As a warning, we have decided to routinely perform a PTT assay when D-dimer is requested. If the PTT value is very short and a clot is found, the sample is rejected. If the PTT value is very short and a clot is not found, we add the following warning note to the result: “possible presence of a clot”

Accidental diagnosis of reduced FVIII activity in females: What does it mean?(Dardik et al. ISTH 2011 Poster)

• A female with no family history of hemophilia, low FVIII and normal VWF may have a variant of vWD or be a carrier of hemophilia A

• Our strategy for evaluation of such cases, to prevent births of severe hemophiliacs, included measurements of FVIII and vWF levels in all available family members, testing for inversion mutations in the FVIII gene, testing for mutations in the FVIII binding region of the vWFgene, and establishing the segregation of haplotypes of FVIII gene markers in all available family members by analysis of STRs

• In four such females with reduced FVIII activity, we were able to establish the status of hemophilia A carriership:

1. MA (FVIII:25%, vWF Ag 50%) was compound heterozygote for vWD type Normandy (R91Q) and vWD type I (del C in exon 18)

2. For IH (FVIII:40-57%, vWF Ag 87%), hemophilia A carrier status was excluded at high probability by haplotype analysis in family members in correlation with FVIII activity

3. For DK (FVIII:36%, vWF Ag 100%), hemophilia A carrier status was excluded at high probability by haplotype analysis in family members in correlation with FVIII activity, followed by finding a mutation in the vWF gene

4. RZ (FVIII:20-25%, vWF Ag 70%) was a carrier-sister of previously undiagnosed brother with mild hemophilia A

• Poster is available upon request

Requirements for development of antibodies in factor XI (FXI) deficiency (Salomon, Zivelin et al. Blood 2003, 101:4783)

• Seven patients out of 118 unrelated severe FXI deficient patients developed FXI inhibitors

• All of them were homozygous for a null mutation Glu117stop with FXI activity of <1% in plasma

• All of them were exposed to exogenous FXI by transfusion of plasma for their clinical needs

• The prevalence of inhibitor development in those patients is very high -33% (7 out of 21)

• Antibodies (IgG) purified from FXI deficient patients with inhibitors showed inhibitory effects on the activation of FXI by thrombin or FXIIa and on the activation of the substrate FIX by FXIa

Patients-Severe FXI deficiencyGenotype II/II homozygous for nonsense Glu117Stop mutationGenotype III/III homozygous for missense Phe283Leu mutationGenotype II/III combined heterozygous for the 2 mutations

Induction of a FXI inhibitor in a patient with inherited severe FXI deficiency by Rh immune globulin (Zucker et al. Blood 2008, 111:1306)

• The prerequisite for inhibitor development is undetectable FXI and exposure to exogenous FXI

• The patient is homozygous for the null allele mutation Glu117stop and has undetectable FXI activity

• Unlike all previous cases of inhibitor development, the patient was not exposed to blood products, and her exposure to exogenous FXI occurred due to 3 injections of Rh immune globulin during pregnancy

• FXI analysis by Western blot (A) and ELISA (Table) revealed the presence of varying levels of FXI in 3 different brands of Rh immune globulins. Detection of bands on the gel was carried out by peroxidase labelled anti-FXI polyclonal antibody and ECL

• It was previously shown that IVIG preparations also contain FXI

• The inhibitory antibodies (IgG) from the patient inhibited activation of the substrate factor IX by factor XIa

The patient was injected with WinRho 3 times

FXI (ng) per injectionRh immune globulin

5000WinRho (W)

500KamrhoD (K)

20Rhophylac (R)

A

Severe bleeding due to a rare acquired Factor X inhibitor (Rao et al. Thromb Haemost 1994, 72:363)

Specific antibodies to factor X were demonstrated by:

1. Sole low FX activity that is only partially corrected by normal plasma

2. Presence of FX-Anti FX immune complexes evidenced by crossed immunoelectrophoresis

3. Binding of patient IgG to the FX light chain demonstrated by Western blot

4. Patient IgG inhibited activation of FX by factors VIIa/TF and by factors IXa/VIIIa/phospholipids (Fig A and C)

5. Patient IgG inhibited activation of prothrombin to thrombin by FXa

The patient was treated with steroids and the antibodies disappeared shortly after treatment

The effect of patient IgG on activation of factor X to generate factor XaA: Activation by factor VIIa/TFC: Activation by factors IXa/VIIIa/phospholipids• Patient IgG o Normal IgG

Acquired non-neutralizing antibodies causing bleeding

1. Prothrombin deficiency causing bleeding in a patient with Circulating Anticoagulant. (Lupus Anticoagulant)The Fig of crossed immunoelectrophoresis (CIE) gives evidence for the formation of immune-complexes of anti-prothrombin antibodies, present in the patient plasma, with prothrombin and elimination of the prothrombin from the patient plasma2. Acquired von Willebrand disease in a patient with angiodisplasia and severe bleeding displayed by the presence of VWF-anti VWF complexes in CIE and removal of the complexes by adsorption to immobilized Protein A Correction of VWF parameters and restoration of full range VWF multimers were observed after treatment with IVIG(Inbal et al. BJH 1997, 96:179)

CIE- First dimension: NP-Normal plasma, PP-Patient plasmaSecond dimension: Anti-prothrombin antibodiesNote the diminished prothrombin level in patient plasma and appearance of immune-complexes shoulder when patient plasma is mixed with normal plasma

Clinical significance of antibodies to bovine and human thrombin and factor V after topical use of commercial bovine thrombin (Rapaport et al. Am J Clin Path 1992, 97:84)

• A patient exposed to bovine thrombin during cardiac surgery developed antibodies reacting with bovine and, to a lesser degree, with human thrombin and factor V

• The antibodies emerged about 1 week following exposure to thrombin, but no bleeding was observed

Crossed immunoelectrophoresis of human plasma (1), bovine plasma (2) and commercial bovine thrombin (3) against heated patient plasma, as the source of antibodies, revealed many precipitin arcs indicative for vast immunization with bovine proteins

• Test results- TT with bovine thrombin >240/20 and TT with human thrombin 28/20. Factor V activity 3%, with neutralizing ability of factor V activity in human and bovine plasma

SDS polyacrylamide gel electrophoresis: 2 batches of the commercial bovine thrombin contained many bovine proteins in addition to thrombin (6,7) in comparison to purified bovine thrombin (8). Lanes 4,5 are size markers

Homozygous protein C deficiency manifested by massive venous thrombosis in the newborn(Seligsohn et al. NEJM 1984 310:359)

• We diagnosed homozygosity of protein C deficiency based on a home-made electroimmunoassay (see Figure)

• Protein C antigen was undetectable in plasma of the patient and the parents, who were first cousins; both had partial protein C deficiency

• The data supported the concept of hereditary protein C deficiency being an autosomal disorder in which the homozygous state may be manifested by virtual absence of protein C and fatal thrombosis in the neonatal period

• In 1996 the mutation T298M causing protein C deficiency was identified in this large family. Heterozygotes for the protein C mutation did not develop thrombosis! Heterozygotes that developed thrombosis were found to be double heterozygotes for the mutation in protein C and for factor V Leiden (Brenner et al. Blood 1996, 88:877)

Electroimmunoassay of protein C measured by home-made goat anti PC antibody. F-father P- Patient M-Mother

Standard curve %

Extensive venous and arterial thrombosis associated with a specific inhibitory antibody against Activated Protein C (APC) but not native protein C (Zivelin et al. Blood 1999, 94:895)

• Patient with APCR ratio of 1.2, equivalent to values in homozygosity for FV Leiden, with no mutation in the FV gene

• Patient IgG inhibited APC activity of normal plasma, as shown by a significant decrease in APCR ratio from 2.9 to 1.6

• Crossed immunoelectrophoresis: Panel A shows direct binding of patient IgG (2) to APC yielding formation of immune-complexes, indicated by mobility shift, in comparison to normal IgG (1). Panel B shows that Patient IgG had no effect on native protein C present in plasma (lanes 1-3). Commercial polyclonal anti Protein C antibodies were used for detection in the second dimension

• Patient IgG inhibited factor Va inactivation by APC in the presence or absence of either phospholipids or protein S

N-Normal, P-patient, NP-Normal Plasma

N IgG+APC P IgG+APC

P Plasma N IgG+NP P IgG+NP

Non-neutralizing antibodies causing coagulation factor deficiency by formation and clearance ofimmune complexes from the circulation (two examples)

Introduction

2116--PBAriella Zivelin Wednesday, July 12DOI: 10.3252/pso.eu.ISTH2017.2017

Laboratory Diagnostic