Laboratory Diagnosis of Lupus Inhibitors: A Comparison of the Tissue Thromboplastin Inhibition Procedure with a New Platelet Neutralization Procedure

Laboratory Diagnosis of Lupus Inhibitors: A Comparison of the Tissue Thromboplastin Inhibition Procedure with a New Platelet Neutralization Procedure DOUGLAS A. TRIPLETT, M.D., JOHN T. BRANDT, M.D. JANIS SCHAEFFER, M.S., MT(ASCP) DANIEL KACZOR, MT(ASCP), AND The introduction of the activated partial thromboplastin time (APTT) as a screening test has resulted in increased recognition of circulating anticoagulants. The most frequently encountered inhibitor is the lupus-type anticoagulant. However, criteria for differentiation of this inhibitor are not well-established. We evaluated the ability of two procedures, tissue thromboplastin inhibition (TTI) and a new platelet neutralization procedure (PNP), to differentiate between various types of coagulation inhibitors. The TTI, widely used for the diagnosis of lupus anticoagulants, proved to be nonspecific. The PNP specifically separated lupus-type inhibitors from Factor, X, and V inhibitors. The PNP may be a useful test for the diagnosis of lupus anticoagulants. (Key words: Coagulation inhibitors; Lupus inhibitors; Coagulation tests) Am J Clin Pathol ; : - THE INTRODUCTION of the activated partial thromboplastin time (APTT) as a routine screening test has resulted in the increased recognition of circulating anticoagulants.,,,, These inhibitors occasionally are directed at specific factors, (usually Factor ), but most often are nonspecific in their inhibitory effect. This nonspecific type of inhibitor was first recognized in patients with systemic lupus erythematosus (SLE) and has thus been termed the "lupus anticoagulant." Subsequent studies have shown that this type of inhibitor can occur in a variety of clinical conditions other than SLE, including in otherwise normal individuals of all ages., Proper identification of inhibitors is essential for appropriate clinical management. Although initial studies suggested that patients with lupus inhibitors bled excessively, reevaluation demonstrated that the bleeding was associated with other concurrent hemostatic defects, such as thrombocytopenia or specific prothrombin inhibitors. Subsequent studies have documented that the lupus anticoagulant is not associated with a bleeding tendency.,, In contrast, patients with specific coagulation inhibitors such as Factor or IX inhibitors Received July, ; received revised manuscript and accepted for publication December,. Address reprint requests to Dr. Triplett: Department of Pathology, Ball Memorial Hospital, University Avenue, Muncie, Indiana. From Ball Memorial Hospital, Muncie, Indiana and Ohio State University, Columbus, Ohio have a significant risk of hemorrhage and often require specialized therapy for effective management. ' Criteria for the diagnosis of the lupus anticoagulant have not been well-established. The presence of a coagulation inhibitor is suggested by the failure of a prolonged prothrombin time (PT) or activated partial APTT to return to normal when the patient plasma is mixed : with normal plasma., In early studies of the lupus anticoagulant, it was recognized that dilution of the thromboplastin increased the sensitivity of the test system. In, Schleider and co-workers and Boxer and associates independently introduced the concept of the dilute thromboplastin test and suggested that it was a useful procedure for diagnosis of lupus-type anticoagulants., The tissue thromboplastin inhibition procedure (TTI) has been used extensively in clinical laboratories since its introduction., Although Boxer and colleagues claimed that it was not abnormal in patients with factor deficiencies or Factor inhibitors, no data has been published on the ability of the TTI to differentiate lupus inhibitors from specific factor inhibitors. Therefore, we evaluated the specificity of the TTI in the presence of lupus inhibitors, specific coagulation factor inhibitors, and heparin. In addition, we compared these results with a simple screening procedure based on the work of Thiagarajan and co-workers which suggested that the lupus inhibitor was directed at phospholipid. The results suggest that the TTI is not specific for lupus anticoagulants and that the new platelet neutralization procedure may be a more useful test for the identification of lupus inhibitors. Materials and Methods Samples for testing were collected into. M sodium citrate in a ratio of nine parts blood to one part anticoagulant, using standard venipuncture techniques. The samples were transported to the laboratory on ice -/// $. American Society of Clinical Pathologists Downloaded from https://academic.oup.com/ajcp/article-abstract//// on April

vol.. No. and centrifuged at, rpm for minutes at C. The platelet poor plasma was harvested using a plastic transfer pipette and stored in capped plastic tubes. If testing was to be delayed, the plasma samples were frozen at or below C until testing. Some samples were received from other laboratories transported frozen on dry ice. Pooled normal plasma was prepared by pooling platelet poor plasma from five female and five male normal donors who were free of medications. The plasmas were processed as described above and aliquots were frozen at - C. APTTs were performed using General Diagnostics auto APTT reagent on either a Bio/Data Coagulation Profiler CP- or MLA according to manufacturer's instructions. Prothrombin times were performed using General Diagnostics Simplastin on a Fibrometer according to manufacturer's instructions. Factor-deficient plasmas for specific factor assays were obtained from George King Biomedical, Inc., (Overland Park, KS) and General Diagnostics (Morris Plains, NJ). Either the Bio/ Data CP- or a Fibrometer was used to perform the factor assays. Detection of Circulating Anticoagulants All APTTs that were significantly prolonged (i.e., > seconds above the upper limit of the normal range) were screened for the presence of an anticoagulant. This screening procedure consisted of performing an APTT on an equal mixture of patient and normal pooled plasma. The failure of normal plasma to correct the prolonged APTT to within normal limits indicated the presence of a circulating anticoagulant. A TTI then was performed according to the method of Schleider and colleagues and the ratio of clotting times at : and :, dilutions of thromboplastin were determined. A ratio of. or greater was considered positive and indicative of a lupus anticoagulant. A two-hour incubation of patient-normal plasma mixtures and specific factor assays were performed to rule out concurrent specific factor inhibitors. Platelet Neutralization Procedure A newly expired unit of platelet concentrate (collected in CPDA,) was obtained from the blood bank. Twentymilliliter aliquots of platelet concentrate then were placed into large plastic screw cap test tubes, diluted with an equal volume of cold Tris buffered saline (. M NaCl,. M tris, pw A, TBS), and mixed. The mixture was centrifuged at g for - minutes to remove red blood cell contamination, and the supernatant (platelet rich) was transferred to a plastic centrifuge tube and centrifuged at, g for minutes. The LABORATORY DIAGNOSIS OF LUPUS INHIBITORS Table. Results of Tissue Thromboplastin Inhibition in the Presence of Specific Coagulation Inhibitors Inhibitor Type Factor Factor Factor Factor Factor Factor Factor V Factor IX Clinical Setting Hemophilia A Hemophilia A Hemophilia B Ratio of Clotting Times* : Dilution...... >.f. A ratio of. or greater is considered positive. t No clot observed in lest specimen: ratio essentially infinity. :...... >.f. supernatant was removed and discarded and the platelets were resuspended in - ml TBS and centrifuged at, g for minutes. This washing procedure was repeated two additional times. Following the third wash, the platelets were resuspended in TBS and the platelet count adjusted to,-,/mm. The platelet suspensions then were divided into -ml aliquots and frozen at C until further use. The platelet neutralization procedure (PNP) was performed as follows. A frozen platelet suspension was allowed to completely thaw at room temperature. Citrated plasma was obtained by standard technics. A reaction mixture of. ml APTT reagent (General Diagnostics),. ml patient plasma, and. ml frozen platelet suspension was incubated at C for minutes. Following incubation,. ml of prewarmed calcium chloride (. M) was added and the time to clot formation was determined using the BioData CP or MLA. The saline control mixture consisted of. ml APTT reagent,. ml NaCl (. M) and. ml patient plasma. Results were obtained in duplicate on all specimens. Patient Population Samples from patients previously identified as having a nonspecific or lupus inhibitor on the basis of a prolonged activated thromboplastin time and an abnormal TTI and no evidence of specific factor inhibitors Table. Results of Tissue Thromboplastin Inhibition in the Presence of Heparin Concentration of Heparin. U/mL. U/mL. U/mL. U/mL Ratio of Clotti ing Times* : Dilution.... * A ratio of. or greater is considered positive. : Dilution.. >. >. Downloaded from https://academic.oup.com/ajcp/article-abstract//// on April

TRIPLETT ET AL. A.J.C.P. June Table. Results of the Platelet Neutralization Procedure in the Presence of Lupus Inhibitors Patient QNS > > Saline-PNP _ > were available for study. A repeat APTT and PNP were performed on of these plasmas. When adequate sample was available, a repeat TTI also was performed. Nine patients with Factor inhibitors (four with hemophilia A, five spontaneous), one with a Factor IX inhibitor (occurring in a patient with severe hemophilia B), and one with an acquired Factor V inhibitor also were evaluated utilizing an APTT, the TTI procedure, and the PNP. In addition, varying concentrations of heparin were added to reconstituted lyophilized normal plasma and the following tests performed: APTT, APTT after : mix with normal plasma, the PNP, and the TTI. Results Results of Tissue Thromboplastin Inhibition Adequate samples for performance of the TTI were available in six of the nine patients with factor inhibitors (four spontaneous inhibitors and two occurring with hemophilia A) and in the patients with the Factor V and IX inhibitors. The test was considered positive (ratio greater than.) in the presence of three of the six Factor inhibitors, the Factor V inhibitor, and the factor IX inhibitor (Table ). A positive TTI was observed with Factor inhibitors in both hemophiliac and nonhemophiliac patients. In addition, the TTI was positive at heparin concentrations ranging from. to. U/mL (Table ). By prior definition, the TTI was positive in all patients with lupus inhibitors (data not shown). Results of Platelet Substitution Of the patients initially identified as having a lupus inhibitor, two were subsequently found to have a normal APTT following thawing at C. This normalization has been seen previously and is thought to result from the presence of platelets in the plasma prior to freezing. Further evaluation of these two plasmas could not be performed. The PNP resulted in shortening in each of the remaining specimens with lupus inhibitors. The PNP time ranged from to seconds shorter than the baseline APTT, with a median shortening of seconds. The PNP time ranged from to seconds shorter than the saline control time, with a median difference of seconds. In general, the longer the initial APTT, the greater the shortening with the PNP (Table ). In contrast, there was no shortening of the PNP APTT in of the patients with specific factor inhibitors Inhibitor V IX Table. Effect of Platelet Neutralization Procedure in the Presence of Specific Factor Inhibitors - - - - - < Sali rie-pnp < - - - - Downloaded from https://academic.oup.com/ajcp/article-abstract//// on April

vol.. No. Heparin Concentration. U/mL. U/mL. U/mL. U/mL Patient Patient Patient Patient Patient LABORATORY DIAGNOSIS OF LUPUS INHIBITORS Table. Effect of Platelet Neutralization Procedure in the Presence of Heparin Saline-PNP (Table ). One patient with a Factor inhibitor did show a -second shortening with the PNP, but the saline control also showed a shortening of seconds. The lack of difference between the PNP and saline control in this patient suggests the possibility of simple dilution of the inhibitor. In the patients with the Factor V and Factor IX inhibitors, the initial APTT was greatly prolonged ( seconds) and showed no evidence of shortening with the PNP. This is in marked contrast to the two patients with lupus inhibitors and greatly prolonged APTTs. The PNP resulted in significant shortening in both of these patients (Table, patients and ). The PNP resulted in significant shortening of the APTT at each of the heparin concentrations tested and in several patients receiving intravenous heparin therapy (Table ). No consistent effect of the PNP was observed when specimens with normal APTTs were tested (Table ), suggesting that the results obtained with heparin and lupus inhibitors were not a result of a nonspecific effect on the APTT. Discussion The results clearly demonstrate that the TTI is not an adequate procedure to differentiate between lupus and specific factor inhibitors. Use of this test as a diagnostic criterion could lead to a failure to recognize a hemostatically significant specific factor inhibitor with catastrophic consequences for the patient. These results are consistent with recent observations regarding the interaction of the intrinsic and extrinsic systems of coagulation." - - Extrinsic activation can lead to Factor X activation by at least two pathways, direct activation of Factor X by Factor Vila, and indirect activation resulting from activation of Factor IX by Factor Vila. Our results suggest that when the activator (tissue thromboplastin) is diluted, the PT system becomes sensitive to defects in either pathway. In accord with this hypothesis, Coots.and co-workers recently have presented evidence that the TTI is also abnormal in patients with single extrinsic system factor deficiencies. In contrast to the TTI, the PNP appears to adequately separate specific factor inhibitors from lupus type inhibitors. All cases of lupus inhibitors showed significant shortening of the APTT with the PNP, and this shortening was always greater than that obtained with the saline control. One Factor inhibitor plasma did show a slight shortening with the PNP, but this sample could be distinguished by the identical shortening obtained with the saline control. The data suggest that with most lupus inhibitors there will be greater than -second shortening with the PNP. Occasionally, less of an effect will be noted with low titer inhibitors. Neither test could distinguish the presence of heparin from a coagulation inhibitor. Clinically, this distinction is usually not difficult, as heparin also prolongs the thrombin time and can be neutralized by protamine sulfate or absorption with ion exchange resins. I, How- Patient Table. Results of the Platelet Neutralization Procedure in the Patients with Normal APTT Baseline : APTT (sec) - - - Sali ine-pnp - - Downloaded from https://academic.oup.com/ajcp/article-abstract//// on April

TRIPLETT ET AL. A.J.C.P. June ever, the laboratory occasionally encounters unsuspected heparinized specimens and should be aware of the possible interference of this substance with studies designed to differentiate coagulation inhibitors. The precise mechanism of the PNP is unknown. Early investigators noted that the lupus inhibitor was difficult to demonstrate in the presence of platelets. Yin and Gaston provided the first evidence that the lupus anticoagulant was directed at the prothrombin converting complex. Subsequent studies, summarized in several reviews, suggested that the inhibitor was directed at phospholipid. - Recently, Thiagarajan and associates studied a monoclonal IgM with characteristics of a lupus inhibitor. They demonstrated that this antibody was directed at specific phospholipids and could be bypassed by platelet membranes. Exner and colleagues developed a screening test for lupus anticoagulants based on the Kaolin clotting time of plasma. They found that platelets decrease the activity of the lupus inhibitor in their test system and that maximal sensitivity was obtained when platelet lipid substitutes (cephalin) also were omitted from the reaction mixture. Goldsmith and co-workers studied a labile circulating anticoagulant with many characteristics of lupus inhibitors. The inhibitor effect was neutralized by products of platelet lysis, but not by exogenous phospholipids or intact platelets. Our results support the concept that platelets or platelet products may provide a mechanism to bypass lupus inhibitors. The correct characterization of the nature of a circulating anticoagulant is exceedingly important in the clinical management of these patients. In contrast to the bleeding diathesis associated with specific factor inhibitors, a thrombotic tendency has been associated with the lupus inhibitor. ' - - - In addition, the lupus inhibitor can interfere with heparin monitoring during open heart surgery. The Til can no longer be accepted as a criterion for distinguishing these two groups of inhibitors. It appears that the PNP may be a useful technic for identifying the presence of a lupus inhibitor. Further prospective tests of its sensitivity and specificity will be necessary before it can be assumed to be the procedure of choice for evaluation of lupus-type inhibitors. Acknowledgments. The authors are indebted to Dr. Helen Glueck, Ms. Mary Ann Miller, and Mr. George King for acquisition of several of the patient specimens, to Ms. Judy Vanyo and Carol Orr for excellent technical assistance, and to Ms. Linda Glaze. Cindy Berner, and Christy Anderson for secretarial assistance. References. Angles-Cano E, Clauvel JP, Sultan Y: Thrombophlebitis in systemic lupus erythematosus. JAMA ; :. Bowie EJW, Thompson JH, Pascuzzi CA, Owen CA: Thrombosis in systemic lupus erythematosus despite circulating anticoagulants. J Lab Clin Med ; :. Boxer M, Ellman L, Carvalho A: The lupus anticoagulant. Arthritis Rheum ; :. Canciani MT, Maspero ML, Cattaneo M, Mannucci PM: Clinical and laboratory observations in eight patients with lupus-like ciculating anticoagulant. Haematologica ; :-. Carreras LO, Machin SJ, Deman R, et al: Arterial thrombosis, intrauterine death and "lupus" anticoagulant: Detection of immunoglobulin interfering with prostacyclin formation. Lancet : :. Conley CL, Hartmann RC: A hemorrhagic disorder caused by circulating anticoagulant in patients with disseminated lupus erythematosus. J Clin Invest ; :. Coots MC, Miller MA, Glueck HI: The lupus inhibitor: A study of its heterogeneity. Thrombosis and Haemostasis ; :-. Corrigan JJ: Coagulation inhibitors. Am J Ped Hem/Oncol ; :-. Exner T, Richard KA, Kronenberg H: A sensitive test demonstrating lupus anticoagulant and its behavioural pattern. Br J Haematol ;:-. Feinstein DI, Rapaport SI: inhibitors of blood coagulation. Progress in hemostasis and thrombosis, Vol.. Edited by Spaet T. New York, Grune and Stratton,, pp -. Firkin BG, Booth P, Hendrix L, Howard MA: Demonstration of a platelet bypass mechanism in the clotting system using an acquired anticoagulant. Am J Hematol ; :-. Goldsmith GH, Saito H, Muir WA: Labile anticoagulant in a patient with lymphoma. Am J Hematol ; :-. Green D, Lechner K: A survey of non-hemophilic patients with inhibitors to factor. Thromb Haemost ::-. King JF: Heparin neutralizationa new approach to the prolonged partial thromboplastin time. Lab Med ; :-. Lechner K: A new type of coagulation inhibitor. Thromb Diathes Haemorrh ; :. Lee SL, Miotti AB: Disorders of hemostatic function in patients with systemic lupus erythematosus. Semin Arthritis Rheum ;:. Mannucci PM, Canciani MT, Mari D, Meucci P: The varied sensitivity of partial thromboplastin and prothrombin time reagents in the demonstration of the lupus-like anticoagulant. Scand J Haematol ; :. Margolius A, Jackson DP, Ratnoff OD: Circulating anticoagulants: A study of cases and a review of the literature. Medicine ;:. Marlar RA, Griffin JH: Alternative pathways of thromboplastindependent activation of human X in plasma. Ann NY Acad Sci ;:-. Much JR, Herbst KD, Rapaport SI: Thrombosis in patients with the lupus anticoagulant. Ann Intern Med : :-. Okpara RA, Carabello J A, Day HJ: Spurious prolongation of the activated partial thromboplastin time. Thromb Haemost ; :-. Osterud B, Rapaport S: Activation of factor IX by the reaction product of tissue factor and factor VII: Additional pathway for initiating blood coagulation. Proc Natl Acad Sci USA ; :. Peck B, Hoffman GS, Franck WA: Thrombophlebitis in systemic lupus erythematosus. JAMA ; :-. Schleider MA, Nachman RL, JafFe EA, Coleman M: A clinical study of the lupus anticoagulant. Blood ; :-. Thiagarajan P, Shapiro S, DeMarco L: Monoclonal immunoglobulin M coagulation inhibitor with phospholipid specificity. J Clin Invest ; :. Triplett DA, Harms CS: Procedures in the coagulation laboratory. Chicago, Am Soc Clin Path Press,, p. Triplett DA: Heparin: Biochemistry, therapy and laboratory monitoring. Therapeutic Drug Monitoring ; :-. Wanger GP, Roberts W, Brandt JT: Misleading heparin monitoring leading to protamine sulphate overdose in a patient with a lupus anticoagulant. Lancet ; :. Williams H, Laurent R, Gibson T: The lupus coagulation inhibitor and venous thrombosis: A report of four cases. Clin Lab Haematol ; :. Yin ET, Gaston LW: Purification and kinetic studies on a circulating anticoagulant in a suspected case of lupus erythematosus. Thromb Diath Haemorrh ; :-. Zur M, Shastri K, Nemerson Y: Kinetics of the tissue factor pathway of coagulation: Activation of factor IX. Blood ; : (Suppl) Downloaded from https://academic.oup.com/ajcp/article-abstract//// on April