ARACHnase: An Evaluation of a Positive Control for ...

COAGULATION

AND

TRANSFUSION

Original

MEDICINE

Article

A R A C H n a s e An

Evaluation

Neutralization Activated

of Procedure

Partial

a Positive

Control

Testing Thromboplastin

With

for Seven Time

Platelet Commercial Reagents

DAVID L. McGLASSON, MS, CLS/NCA,1 MAJOR JAMES L. BABCOCK, MD, PHD, 1 LOIS BERG, MA, CLS/NCA,2 AND DOUGLAS A. TRIPLETT, MD3

The venom extract from the brown recluse spider contains an active protein component with an approximate molecular weight of 32,000 and sphingomyelinase-D activity. This venom extract, when added to normal PPP in a concentration of 3.5 fig/mL PPP, is the key component of ARACHnase.3 Seven APTT reagents were tested by an independent laboratory to determine whether the ARACHnase reagent would yield positive PNP results with each reagent, regardless of the activator or phospholipid source.

Lupus anticoagulants (LAs) can be denned as immunoglobulins (immunoglobulin G, immunoglobulin M, immunoglobulin A, or a mixture of all three) that can interfere with in vitro phospholipid-dependent tests of coagulation such as prothrombin time, activated partial thromboplastin time (APTT), and dilute Russell viper venom time.1 The platelet neutralization procedure (PNP) test is a confirmatory procedure used to differentiate the presence of LA from that of a specific factor inhibitor. The PNP test is based on the ability of platelets to "bypass" or "neutralize" the LA by significantly correcting a prolonged APTT.2 Outdated blood bank platelets washed in TRIS-buffered saline are frozen at —70 °C and stored until ready to use. The platelets are thawed and added to the platelet-poor plasma (PPP) for LA testing. If an LA is present, the APTT is shortened significantly. To date, there has not been a standardized positive commercial control for PNP testing. Individual laboratories often use a specimen from a known LA-positive patient for a control if it is available. Our study evaluated the use of ARACHnase (a commercially available, artificial positive control for LA testing) as a positive control for PNP testing with a variety of APTT reagents.

MATERIALS AND METHODS Collection and Processing of Donor Plasma

Whole blood was collected from 30 volunteers (15 males, 15 females; aged 20-45 years) into 3.8% citrated, siliconized Vacutainer tubes (Becton-Dickinson, Sunnyvale, CA) by nontraumatic venipunctures. The samples were centrifuged for 30 minutes at 2500g (3000 rpm). The PPP was separated carefully without disturbing the cell layer. A prothrombin time, APTT, and platelet count were performed on each sample. The platelet count of each sample was less than 15 X 109/L. The plasma was pooled and filtered through a 0.45-^m filter (Sartorius, Minisart, Gottinger, Federal Republic of Germany) to assure From ' Wilford Hall Medical Center, Lackland A FB, Texas:2 Hemex Laboratories, Department of Coagulation, Phoenix, Arizona;that andthe PPP was as platelet free as possible. An Unopette (Becton-Dickinson, Rutherford, NJ) platelet count was less than 5 ^Clinical Investigations. Ball Memorial Hospital, Department of PatholX 10'/L. ogy, Muncie. Indiana.

Received June 8, 1992; revised manuscript accepted for publication Preparation of ARACHnase February 11, 1993. The views expressed in this article are those of the authors and do not The pooled plasma was treated with 3.5 tig brown recluse reflect the official policy of the Department of Defense or other departspider venom extract per milliliter of PPP as previously dements of the United States Government. scribed.3 Address reprint requests to Mr. McGlasson: Clinical Investigation Directorate, 1255 Wilford Hall Loop, Lackland AFB, TX 78236-5319. The treated plasma was divided into 2-mL aliquots and fro576

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ARACHnase (Hemostasis Diagnostics International Co., Denver, CO) Thromboscreen-APTT LS (Pacific-Hemostasis, Ventura, CA). ARACHnase consistently displayed a positive PNP result of greater than 5 is a normal plasma that contains a venom extract from the brown recluse spider, Loxosceles reclusa, which mimics the presence of a lupus seconds correction of the initial baseline APTT. Thus, ARACHnase may provide a positive control for LA testing, regardless of the choice anticoagulant (LA). Seven activated partial thromboplastin time (APTT) reagents were used for platelet neutralization procedure (PNP) of APTT reagent and activator/phospholipid combination. (Key words: ARACHnase; Brown recluse spider; Activated partial thromboplastin testing with ARACHnase: Automated APTT (Organon-Teknika, Durtime; Lupus anticoagulants; Platelet neutralization procedure) Am J ham, NC); Thrombofax and Thrombosil (Ortho, Raritan, NJ); Actin Clin Pathol 1993;100:576-578. and Actin FSL (Dade, Aguado, PR); and Thromboscreen-Kontact and

McGLASSON ET AL. ARACHnase: A Positive Control for PNP Testing

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TABLE 1. ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) REAGENTS

Reagent

Activator

Phospholipid Source

Organon-Teknika (Auto APTT) Ortho (Activated Thrombofax) Ortho (Activated Thrombosil) DADEActin DADE Actin FSL Pacific Hemostasis (Thromboscreen-Kontact) Pacific Hemostasis (Thromboscreen-APTT LS)

Micronized silica Ellagic acid Colloidal silica Ellagic acid Ellagic acid Magnesiumaluminum silicate Ellagic acid

Coagulation Testing Seven commercial single lots of APTT reagents were obtained for testing (Table 1): (1) Automated APTT (OrganonTeknika, Durham, NC); (2) Activated Thrombofax (Ortho, Raritan, NJ); (3) Activated Thrombosil (Ortho); (4) Actin (Dade, Aguado, PR); (5) Actin FSL (Dade); (6) Thromboscreen-Kontact (Pacific Hemostasis, Ventura, CA); and (7) Thromboscreen-APTT LS (Pacific Hemostasis). The normal reference range stated in Table 1 for each APTT reagent was established at Hemex Laboratories, Phoenix, Arizona. Whole blood was drawn from 30 people (14 men, 16 women; aged 20-50 years) into 3.8% sodium citrate, with a two-syringe technique. The first 7 mL of blood was discarded. The remaining citrated whole blood was centrifuged with a "double-spin" procedure to obtain fresh PPP. A CoA Screener (American Labor) was used to establish the normal reference range for each APTT reagent. The clot-detection parameters were adjusted according to each APTT reagent. The PNP was performed on the CoA Screener with a method described by Triplett and associates.2 All of the tests were performed by personnel at Hemex Laboratories. Two lots of ARACHnase prepared in an identical manner at Wilford Hall Medical Center, Clinical Investigations, were assayed by PNP testing with each of the APTT reagents, similar to a previously described method.2 Two different preparations of freeze-thawed platelets were tested with each lot of ARACHnase. The freeze-thawed washed platelets used for PNP testing were prepared at Hemex Laboratories by a previously described method.2 Two runs of PNP testing were performed in duplicate, and the results of each duplicate pair of tests were averaged. RESULTS The results of the PNP testing are summarized in Table 2. The correction of the baseline APTTs and saline baseline APTTs by the addition of platelets was evident with each APTT reagent. The reagent sensitivity and responsiveness were based on the degree of correction that occurred when platelets were substituted for saline in the PNP and compared with the initial baseline APTT with each APTT reagent. The responsiveness of each reagent to ARACHnase can be ranked by groups.

Rabbit brain Bovine brain Rabbit brain Rabbit brain Soy and rabbit brain Rabbit brain

29.0-38.4 23.7-29.9 25.8-33.1 26.4-34.2 29.3-41.4 24.1-36.3

Rabbit brain

28.0-36.3

Activated partial thromboplastin time reagents E and G produced correction averages of the baseline APTT of 18.5 and 23.6 seconds, respectively. Reagent C had an average shortening of the APTT of 12.7 seconds. Reagents A, B, D, and F had corrections averaging 4.4-12.8 seconds. The reagent sensitivity was based on the greatest APTT correction on the addition of platelets to the PNP test. The source of the activator and phospholipid of the APTT reagents did not seem to exert a consistent influence on the shortening of the APTTs. DISCUSSION The PNP test is considered to have a positive result for the presence of an LA if the baseline APTT is shortened by greater than 5 seconds when platelets are substituted for the saline. On 2 separate days, different batches of the ARACHnase had positive results for LA with the PNP test when seven commercial APTT reagents were used, with one exception. The second run using Ortho Thrombofax APTT with the PNP test provided only a 3-second correction from the baseline APTT. The saline baseline APTT, however, had a 6.9-second correction. This exception emphasizes the need to view both the saline baseline APTT and baseline APTT to prevent misinterpretation of a false-positive PNP correction. If the addition of platelets does not shorten the baseline APTT, the results should be considered negative for the presence of an LA, regardless of the results of the saline baseline APTT. The sensitivity and degree of correction of baseline APTTs and saline baseline APTTs with platelet substitution APTTs vary between different APTT reagents. It should be observed in Table 2 that certain manufacturers' reagents showed increased baseline APTTs when saline was added to the system. This may suggest a factor deficiency caused by addition of the saline; however, mild LA inhibitors also may show small increases in baseline APTTs when saline is added to the test system. The shortening of the saline APTT by the addition of platelets still provides a system that can simulate a positive PNP test result. This can assure the laboratory of the viability of the freeze-thawed platelets and may provide a reference system. In a prior study conducted in three laboratories, we found that the ARACHnase system displays a classic LA response to coagulation factors of the intrinsic system.3 Levels of coagulation Factors VIII, IX, XI, and XIII; Prekallikrein; and high molecular weight kininogen (HMWK.) all were decreased initially but corrected to normal levels with progressive dilution. Our results show the importance of selecting an APTT re-

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zen for 2 hours at -70 °C. The plasma then was lyophilized and shipped on dry ice to the reference laboratory for testing.

Normal Range (Reference Lab Range) (n = 30)

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COAGULATION AND TRANSFUSION MEDICINE Original Article TABLE 2. PLATELET NEUTRALIZATION PROCEDURE (PNP) TESTING RESULTS

Reagent/Normal Range (/second) Organon-Teknika* 29.0-38.4 Orthot 23.7-29.9 Orthot 25.8-33.1 DADEActin 26.4-34.2 DADE Actin FSL 29.3-41.4 Pacific hemostasis§ 24.1-36.3 Pacific hemostasis1' 28.0-36.3

Baseline APTT 49.1 49.1 41.2 36.4 52.8 48.6 49.6 47.0 57.2 56.6 48.2 45.1 59.0 53.6

Saline APTT 50.3 50.3 49.5 40.3 57.3 54.4 48.8 47.0 64.1 61.9 43.5 44.4 57.8 55.1

Platelet APTT 43.2 41.0 35.4 33.4 38.6 37.4 39.8 40.4 37.5 39.3 33.9 33.8 32.1 32.3

Correction Saline-PNP (seconds) 7.1 9.3 14.1 6.9 18.7 17.0 9.0 6.6 26.6 22.6 9.6 10.6 25.7 22.8

Correction Baseline-PNP (seconds) 5.9 8.1 5.8 3.0 14.2 11.2 9.8 6.6 19.7 17.3 14.3 11.3 26.9 21.3

agent that is sensitive and responsive to the presence of an LA. Several publications have addressed the ability of different coagulation reagents to be responsive to the effects of an LA.4-5 It should be observed that only one lot number of each APTT reagent was tested. The sensitivity to LAs may vary with different lots of APTT reagents, so this should be considered when coagulation reagents are purchased.6 Our study shows that ARACHnase consistently yields a positive PNP result regardless of the source of the APTT reagents, with one exception on one run of PNP testing. Other models also have been proposed as positive controls for the presence of an LA, including polymyxin B and Annexin (lipocortin). Polymyxin B is a cationic antimicrobial detergent that binds to negatively charged phospholipid surfaces and appears to show a dose-dependent LA effect in preliminary studies.7 Annexins are a family of calcium-dependent proteins that bind phospholipids and may inhibit phospholipid-dependent coagulation assays.8,9 Both polymyxin B and Annexin are receiving scrutiny as possible agents for treating plasma to simulate an LA effect. ARACHnase is currently the only commercially available artificial control for LA control testing and has shown consistently reproducible results in a variety of assay systems.3 Acknowledgments. The authors acknowledge Hemostasis Diagnostics International, Denver, Colorado, for furnishing the ARACHnase controls. They also thank Anna Vasquez, Lou Ann Caywood, and Rachel Montez for preparing the manuscript.

REFERENCES 1. Triplett DA. Laboratory diagnosis of lupus anticoagulants. Semin ThrombHemost 1990;16:182-192. 2. Triplett DA, Brandt JT, Kaczor D, SchaefFer J. 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. 3. McGlasson DL, Babock JL, Patterson WR, et al. Evaluation of the lupus anticoagulant effects of brown recluse spider venom on normal plasma. J Mil Med Lab Sci 1989; 18:95-98. 4. Brandt JT, Triplett DA, Rock WA, et al. Effect of lupus anticoagulants on the activated partial thromboplastin time: Results of the College of American Pathologists Survey Program. Arch Pathol Lab Med 1991;115:109-114. 5. Brandt JT, Triplett DA, Musgrave K, Orr C. The sensitivity of different coagulation reagents to the presence of lupus anticoagulants. Arch Pathol Lab Med 1987; 111:120-124. 6. Kaczor DA, Bickford NN, Triplett DA. Evaluation of different mixing study reagents and dilution effect in lupus anticoagulant testing. Am J Clin Pathol 1991;95:408-411. 7. Uchman B, Triplett DA. Inhibitory effect of polymyxin B on the X-ASE and prothrombinase complex formation. Thromb Haemost 1989;62:375. 8. Thiagarajan P, Tait JF. Binding of Annexin V/placental anticoagulant protein I to platelets: Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J Biol Chem 1990;265:17420-17423. 9. Creutz CE. The Annexins and exocytosis. Science 1992;258:924931.

A.J.C.P. • November 1993


Saline = saline baseline APTT; Platelets = platelet addition in place of saline; Correction = platelet correction of saline baseline APTT/ baseline APTT. A >5.0 seconds correction of the baseline APTT is considered a positive PNP lest for the presence of an LA. * Auto APTT. t Activated Thrombofax. t Activated Thrombosil. §11 Thromboscrccn/Kontact. Thromboscrccn/APTT LS.