Multilaboratory Testing of Thrombophilia: Current and Past Practice in Australasia as Assessed through the Royal College of Pathologists of Australasia Quality Assurance Program for Hematology

ABSTRACT

We have conducted a series of multilaboratory surveys during the last 6 years to evaluate testing proficiency in the detection of congenital and acquired thrombophilia. For lupus anticoagulant (LA) testing, participant laboratories used a panel of tests, including activated partial thromboplastin time (aPTT; 100% of laboratories), kaolin clotting time (26 to 70%), and Russell's viper venom time (RVVT; 75 to 100%). Coefficients of variation (CVs) for assays ranged from 5 to 40%. RVVT assays appeared to be most sensitive and specific for detection of LA (fewer false-negatives or -positives), although laboratories performed best when they used a panel of tests. For congenital thrombophilia, tests evaluated comprised protein C (PC), protein S (PS), antithrombin (AT), and activated protein C resistance (APCR). Most participant laboratories performed PC using chromogenic (approximately 75%), or clot based (approximately 15%) assays, with few (< 10%) performing antigenic assessments. PS was most often assessed (approximately 60%) by immunological or antigenic assays, usually of free PS, or by functional or clot-based assays (approximately 40%). AT is usually assessed by functional chromogenic assays (approximately 95%). APCR was assessed using aPTT (approximately 50%) or RVVT (approximately 50%) clot-based assays, with the aPTT APCR typically performed using factor V-deficient plasma predilution, but the RVVT APCR typically performed without. Laboratories using the RVVT APCR generally performed better in detection of factor V Leiden-associated APCR, with the aPTT method group yielding higher false-negative and/or false-positive findings (approximately 5% of occasions). Some clot-based PC and PS assays appeared to be influenced by APCR status, and yielded lower apparent PC and PS levels with positive APC resistance. The overall error rate for PC, PS, and AT was approximately 2 to 8% (i.e., false-normal interpretations for deficient plasma or false-abnormal interpretations for normal plasma). The CVs for these assays ranged from 5 to 40%, with highest CVs typically obtained with PS assays.

KEYWORDS

Protein C - protein S - antithrombin - activated protein C resistance - lupus anticoagulant - laboratory assessment - quality control

REFERENCES

• 1 Bertina R M. Genetic approach to thrombophilia.  Thromb Haemost. 2001;  86 92-103
• 2 Simioni P. The molecular genetics of familial venous thrombosis.  Bailliere's Best Pract Res Clin Haematol. 1999;  12 479-503
• 3 Thiagarajan P, Shapiro S S. Lupus anticoagulants and antiphospholipid antibodies.  Hematol Oncol Clin North Am. 1998;  12 1167-1192
• 4 Van Cott E M, Laposata M. Laboratory evaluation of hypercoagulable states.  Hematol Oncol Clin North Am. 1998;  12 1141-1166
• 5 Rao A K, Kaplan R, Sheth S. Inherited thrombophilic states.  Semin Thromb Hemost. 1998;  24(suppl 1) 3-12
• 6 Field S L, Brighton T A, McNeil H P, Chesterman C N. Recent insights into antiphospholipid antibody-mediated thrombosis.  Bailliere's Best Pract Res Clin Haematol. 1999;  12 407-422
• 7 Haematology R CPA. QAP contact and program details. Available at: http://www.rcpaqap.com.au/. Accessed April 2, 2004
• 8 Favaloro E J, Smith J, Petinos P, Collecutt  M, Street  A, Hertzberg M. on behalf of the RCPA Quality Assurance Program (QAP) in Haematology Scientific Haemostasis Advisory Panel. Laboratory testing, diagnosis and management of von Willebrand's disease: current practice in Australasia.  Am J Clin Pathol. 1999;  112 712-719
• 9 Favaloro E J, Smith J, Petinos P, Hertzberg M, Koutts J. on behalf of the RCPA Quality Assurance Program (QAP) in Haematology Haemostasis Scientific Advisory Panel. Laboratory testing for von Willebrand's disease: an assessment of current diagnostic practice and efficacy by means of a multi-laboratory survey.  Thromb Haemost. 1999;  82 1276-1282
• 10 Favaloro E J, Thom J, Baker R. On behalf of the Australasian Society for Thrombosis and Haemostasis Emerging Technologies Group. Assessment of current diagnostic practice and efficacy in testing for von Willebrand's disease: results from the second Australasian laboratory survey.  Blood Coagul Fibrinolysis. 2000;  11 729-738
• 11 Favaloro E J, Bonar R, Sioufi J et al,. On behalf of the RCPA QAP in Haematology Haemostasis Committee. Laboratory diagnosis of von Willebrand disorder: current practice in the Southern Hemisphere.  Am J Clin Pathol. 2003;  119 882-893
• 12 Jennings I, Kitchen S, Cooper P C, Rimmer J E, Woods T A, Preston F E. Further evidence that activated protein C resistance affects protein C coagulant activity assays.  Thromb Haemost. 2000;  83 171-172
• 13 Jennings I, Kitchen S, Cooper P, Makris M, Preston F E. Sensitivity of functional protein S assays to protein S deficiency: a comparative study of three commercial kits.  J Thromb Haemost. 2003;  1 1112-1114
• 14 Meijer P, Kluft C, Haverkate F, De Maat M P. The long-term within- and between-laboratory variability for assay of antithrombin, and proteins C and S: results derived from the external quality assessment program for thrombophilia screening of the ECAT Foundation.  J Thromb Haemost. 2003;  1 748-753
• 15 Favaloro E J, Orsag I, Bukuya M, McDonald D. A nine-year retrospective assessment of laboratory testing for activated protein C resistance: evolution of a novel approach to thrombophilia investigations.  Pathology. 2002;  34 348-355
• 16 Favaloro E J, Mirochnik O, McDonald D. Functional activated protein C resistance assays: correlation with factor V DNA analysis is better with RVVT- than APTT- based assays.  Br J Biomed Sci. 1999;  56 23-33
• 17 Rylatt D B, Hohnen-Behrens C, Pilgrim R L, Dickenson L E, Neal M, Exner T. Detecting APC-resistant factor V: a functional method without plasma dilution.  Blood Coagul Fibrinolysis. 1999;  10 359-366
• 18 Quehenberger P, Handler S, Mannhalter C, Kyrle P A, Speiser W. The factor V (Leiden) test: evaluation of an assay based on dilute Russell viper venom time for the detection of the factor V Leiden mutation.  Thromb Res. 1999;  96 125-133
• 19 de Visser M C, Rosendaal F R, Bertina R M. A reduced sensitivity for activated protein C in the absence of factor V Leiden increases the risk of venous thrombosis.  Blood. 1999;  93 1271-1276
• 20 Graf L L, Welsh C H, Qamar Z, Marlar R A. Activated protein C resistance assay detects thrombotic risk factors other than factor V Leiden.  Am J Clin Pathol. 2003;  119 52-60
• 21 Jennings I, Kitchen S, Woods TA L, Preston F E, Greaves M. Clinically important inaccuracies in testing for the lupus anticoagulant: An analysis of results from three surveys of the UK NEQAS for Blood Coagulation.  Thromb Haemost. 1997;  77 934-937
• 22 Favaloro E J, Silvestrini R. Assessing the utility of anticardiolipin antibody assays: a cautious approach is suggested by high variation and limited consensus in multi-laboratory testing.  Am J Clin Pathol. 2002;  118 548-557
• 23 Favaloro E J, Silvestrini R. Assessing the utility of anticardiolipin antibody assays: a cautious approach is suggested by high variation and limited consensus in multi-laboratory testing.  Am J Clin Pathol. 2002;  118 548-557
• 24 Favaloro E J, Wong R, Silvestrini R, McEvoy R, Jovanovich S, Roberts-Thompson P. A multilaboratory peer assessment quality assurance program-based evaluation of anticardiolipin antibody, and beta₂-glycoprotein I antibody testing. Semin Thromb Hemost 2005 31: 73-84
• 25 Galli M, Luciani D, Bertolini G, Barbui T. Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome: a systematic review of the literature.  Blood. 2003;  101 1827-1832