Laboratory Diagnosis of Inhibitors

ABSTRACT

The diagnosis of inhibitors of blood coagulation is often the most challenging problem in the clinical laboratory. Immediate attention must be given to the following patient groups whose principal laboratory abnormality is the prolonged activated partial thromboplastin time (aPTT): the patient with (1) hemophilia who previously responded to an adequate dose of clotting factor product and now fails to show effective clinical response to the same replacement concentrate; (2) previously benign clinical history who now presents with soft tissue bleeding or emergent internal hemorrhaging; (3) sudden onset of generalized ecchymoses who was previously well; (4) postpartum state; (5) malignancy, lymphoma, rheumatoid arthritis, or other autoimmune disorders; and (6) drug reactions.

Immediate attention must be given to the prolonged prothrombin time (PT), aPTT, and thrombin time (TT) in order to respond to urgent queries from a perplexed internist, hematologist, intensivist, or surgeon caring for a patient with unexpected bleeding. Sometimes the problem of a prolonged ``clotting time'' arises preoperatively, causing unanticipated delay in operative procedures. For this reason, the laboratory support, usually in the coagulation section of a clinical laboratory or reference laboratory, must be quick, unequivocal and precise.

The most common finding is an isolated mild, moderate, or severe prolongation of the aPTT with a normal PT, TT, and platelet count. The aPTT mixing study (The Mix), usually modified for time and temperature, along with appropriate controls, is the seminal test. This is the basis for all further testing. It may be supported by direct factor assays, and, therefore, the laboratory must know the reagent responsiveness and sensitivity for each clotting factor. By definition, complete correction of the aPTT in a 1:1 mix of patient and reference plasma is a factor deficiency. In this article, incomplete or minimal correction of The Mix will be characterized with particular attention to the various inhibitor assays, in other words, Oxford, Bethesda, and Nijmegen assays and the enzyme-linked immunosorbent assay (ELISA). An investigative approach to final characterization of the intensity (quantification) of the inhibitor and the exclusion of a lupus anticoagulant (LA) will be discussed.

KEYWORD

Inhibitors - hemophilia - lupus anticoagulant - antibodies factor VIII - Bethesda assay - Oxford assay - Nijmegen assay

REFERENCES

• 1 Shapiro S S, Hultin M. Acquired inhibitors to the blood coagulation factors.  Semin Thromb Hemost . 1975;  1 336-385
• 2 Anderson B R, Terry W D. Gamma G4-globulin antibody causing inhibition of clotting factor VIII.  Nature . 1968;  217 174-175
• 3 Brinkhous K M, Roberts H R, Weiss A E. Prevalence of inhibitors in haemophilia A and B.  Thrombosis et Diatheses Haemorrhagica . 1972;  51(suppl) 315-320
• 4 Lottenberg R, Kentro T B, Kitchens C B. Acquired hemophilia: A natural history study of 16 patients with factor VIII inhibitors receiving little or no therapy.  Arch Intern Med . 1987;  147 1077-1081
• 5 Green D, Lechner K. A survey of 215 non-hemophilic patients with inhibitors to factor VIII.  Thromb Haemost . 1981;  45 200-203
• 6 Mannucci P M, Lombardi R, Bader R. Studies of the pathophysiology of acquired von Willebrand's disease in seven patients with lymphoproliferative disorders or benign monoclonal gammopathies.  Blood . 1984;  64 614-621
• 7 Lackner H. Hemostatic abnormalities associated with dysproteinemias.  Semin Hematol . 1973;  10 125-133
• 8 Perkins H A, MacKenzie M R, Fudenberg H H. Hemostatic defects in dysproteinemias.  Blood . 1970;  35 695
• 9 Thompson A R, Counts R B. Removal of heparin and protamine from plasma.  J Lab Clin Med . 1976;  88 922-928
• 10 Ewing M P, Kasper C K. In vitro detection of mild inhibitors to factor VIII in hemophilia.  Am J Clin Pathol . 1982;  77 749-752
• 11 Scandella D, Mattingly M, de Graaf S, Fulcher C A. Localization of epitopes for human FVIII inhibitor antibodies by immunoblotting and antibody neutralization.  Blood . 1989;  74 1618-1626
• 12 Littlewood J D, Bevan S A, Kemball-Cook G, Evans R J, Barrowcliffe T W. Variable inactivation of human FVIII from different sources by human FVIII inhibitors.  Br J Haematol . 1991;  77 535-538
• 13 Arai M, Scandella D, Hoyer L W. Molecular basis of FVIII inhibition by human antibodies-antibodies that bind to the FVIII light chain prevent the interaction of FVIII with phospholipid.  J Clin Invest . 1989;  83 1978-1984
• 14 Orstavik K H, Miller C H. IgG subclass identification of inhibitors to FIX in haemophilia B patients.  Br J Haematol . 1988;  68 451-454
• 15 Briet E. Factor IX concentrates and inhibitor formation: Factor IX inhibitors in haemophilia B patients: Their incidence and prospects for development with high purity factor IX products.  Blood Coagul Fibrinolysis . 1991;  2 47-50
• 16 Hoyer L W. Inhibitor immunochemistry and detection: Immunochemical properties of factor VIII and factor IX inhibitors.  Blood Coagul Fibrinolysis . 1991;  2 11-15
• 17 Biggs R, Austen D EB, Denson D WG. The mode of action of antibodies which destroy factor VIII: I. Antibodies which have second-order concentration graphs.  Br J Haematol . 1972;  23 125-135
• 18 Martin P G, Sukhu K, Chambers E. Evaluation of a novel ELISA screening test for detection of factor VIII inhibitory antibodies in haemophiliacs.  Clin Lab Haematol . 1999;  21 125-128
• 19 Regnault V, Stoltz J F. Quantitation of factor VIII antibodies by an enzyme-linked immunoassay method.  Blood . 1994;  83 1155-1156
• 20 Exner T, Rikard K A, Kronenberg H. A sensitive test demonstrating lupus anticoagulant and its behavioural patterns.  Br J Haematol . 1978;  40 143-151
• 21 Triplett D A, Brandt J T, Kaczor D. Laboratory diagnosis of lupus inhibitors: A comparison of the tissue thromboplastin inhibition procedure with a new platelet neutralization procedure.  Am J Clin Pathol . 1983;  79 678-682
• 22 Kelsey P R, Stevenson K J, Poller L. The diagnosis of lupus anticoagulants by the activated partial thromboplastin time: The central role of phosphatidylserine.  Thromb Haemost . 1984;  52 172-175
• 23 Shapiro S S. The lupus anticoagulant/antiphospholipid syndrome.  Annu Rev Med . 1995;  47 533-553
• 24 Thiagarajan P, Pengo V, Shapiro S S. The use of the dilute Russell's viper venom time for the diagnosis of lupus anticoagulants.  Blood . 1986;  68 869-874
• 25 Rauch J, Tannenbaum M, Janoff A S. Distinguishing plasma lupus antifactor antibodies using hexagonal phase II phospholipids.  Thromb Haemost . 1989;  62 892-896
• 26 Biggs R, Austen D EB, Denson D WG. The mode of action of antibodies which destroy F VIII:II antibodies which give complex concentration graphs.  Br J Haematol . 1972;  23-137
• 27 Kelsey P R, Stevenson K J, Poller L. The diagnosis of lupus anticoagulant by the activated partial thromboplastin time: The central role of phosphatidyl serine.  Thromb Haemost . 1984;  52 172-175
• 28 Green D, Hougie C, Kazmier F J. Report of the working party on acquired inhibitors of coagulation: Studies of the ``lupus'' anticoagulant.  Thromb Haemost . 1983;  49 144-146
• 29 Kasper C K, Aledort L M, Counts R B. A more uniform measurement of factor VIII inhibitors.  Thrombosis et Diathesis Haemorrhagica . 1975;  34 869-872
• 30 Austen D EG, Lechner K, Rizza C R, Rhymes I L. A comparison of the Bethesda and New Oxford methods of factor VIII antibody assay.  Thromb Haemost . 1982;  47 72-75
• 31 Verbruggen B, Novakova I, Wessels H. The Nijmegan modification of the Bethesda assay for factor VIII:C inhibitors: Improved specificity and reliability.  Thromb Haemost . 1995;  73 247-251
• 32 Biggs R, Bedwell E. A method for the study of antihemophiliac globulin inhibitors with reference to six cases.  Br J Haematol. 1959;  5 379