Activated protein C resistance: the influence of ABO-blood group, gender and age.

We have evaluated the potential influence of ABO-blood group, gender and age, on laboratory procedures used for detection of Activated Protein C Resistance (APCR), using over 300 normal donor samples and two distinct laboratory test procedures, one based on an Activated Partial Thromboplastin Time (APTT) and the other on a Russell Viper Venom Time (RVVT). We observed a statistically significant influence of ABO-blood group on APTT test times, both in the presence and absence of Activated Protein C (APC), which was no longer evident when using assay ratios. This ABO effect was not observed using the RVVT-based assay procedure. We also observed a gender effect on the APTT-based procedure, such that females (compared to males) provided shorter APTT test times (both with and without APC). This effect was still evident when using APTT assay ratios, but was again not observed using the RVVT-based procedure. We also observed an age related increase in APTT ratios. Interestingly, some previous studies have reported some specific gender and age related effects on APTT-based testing, but reports using RVVT-based testing are lacking, as are ABO related studies. Such findings should be considered as potential variables when associating specific laboratory based findings of APCR to clinical thrombophilia conditions.

Cross-laboratory audit of normal reference ranges and assessment of ABO blood group, gender and age on detected levels of plasma coagulation factors.

We have assessed the influence of the ABO blood group, gender and age on plasma levels of the coagulation factors II, V, VII, VIII, IX, X, XI and XII as part of a quality audit of laboratory activities. There was no statistically significant difference in gender donor age (total normal donors: n = 406, mean/median age = 46.0/47.0 years, range = 16-77 years; females: n = 177, mean/median age = 44.7/46.0 years, range = 16-75 years; males: n = 229, mean/median age = 47.0/48.0 years, range = 17-77 years). With increasing age, we observed small but statistically significant rises (linear correlation; P < 0.01 for all parameters) in factors V, VII, VIII, IX and XI. With gender, we observed higher levels (P < 0.05) in females for factors II, VII, X, IX, XI and XII. With the ABO group, we observed lower levels in the O group (versus non-O group; P < 0.05) for factors VIII, IX and XII. We could therefore define differing normal reference ranges based on the differing study data. Study findings are compared with previously published literature, and this has identified a wide diversity in normal reference ranges both between different factors and between different studies. Finally, we also performed a cross-laboratory audit of peer laboratory practice and similarly show a wide diversity in normal reference ranges used between different laboratories.

Laboratory identification of familial thrombophilia: do the pitfalls exceed the benefits? A reassessment of ABO-blood group, gender, age, and other laboratory parameters on the potential influence on a diagnosis of protein C, protein S, and antithrombin deficiency and the potential high risk of a false positive diagnosis.

Laboratory testing for familial thrombophilia defines a large proportion of the modern hemostasis laboratory workload. As part of an ongoing assessment of our activities, we have re-evaluated our laboratory procedures for antithrombin (AT), Protein C (PC), and Protein S (PS), inclusive of normal reference ranges (NRR), the potential influence of ABO-blood group, gender and age, as well as other laboratory parameters, in order to help assess the effectiveness of testing as an aid to clinical diagnosis. We did not observe a significant influence of ABO-blood group on AT, PC, or PS. However, there were gender-related effects for PS (lower in females) and AT (higher in females), but not for PC. There were also age-related effects for AT, PC, and PS. Data is compared with literature findings. We also audited the positive detection rate for PC and/or PS deficiencies. In a 6-month period of testing, we identified that 18.9% of tested samples yielded low or near-low PC and/or PS levels. However, 33.3% of such samples were potentially derived from patients on oral anticoagulant therapy (ie, potential false positives). Additional pre-analytical variables, intra-assay, inter-assay, and inter-laboratory variability also contribute to the possibility of false positive detection. Thus, whilst NRR can be developed for test parameters, the likelihood of a false-positive test result can still be shown to exceed the likelihood of a true positive result, and this casts a shadow over the clinical value of such testing in some cases. In conclusion, laboratory testing for these markers of familial thrombophilia may or may not assist in the clinical diagnosis of this condition and clinical specialists should be made aware of laboratory test limitations, and consult with laboratories prior to making a definitive diagnosis of AT, PC, or PS deficiency.

Reassessment of ABO blood group, sex, and age on laboratory parameters used to diagnose von Willebrand disorder: potential influence on the diagnosis vs the potential association with risk of thrombosis.

We reassessed the influence of ABO blood group, sex, and age on plasma levels of von Willebrand factor (vWF) antigen, vWF:ristocetin cofactor, vWF:collagen binding assay, and factor VIII coagulant (FVIII:C). Data show that levels of vWF and FVIII:C increase with increasing age (P < .001 for all parameters) and that the ABO blood group influences plasma levels such that O group levels are significantly less than non-O group levels. There was no significant association with sex and Rh status. The selection of normal ranges based on ABO blood groups may influence the clinical diagnosis of von Willebrand disease (vWD) but might not be clinically relevant or help identify people at increased risk of bleeding. Differences in ABO-related ranges were more extensive at the high end of the ranges. This is of particular interest because high levels of vWF and FVIII are associated with thrombosis risk, and an ABO relationship also has been described. O group individuals may or may not be at greater risk for bleeding (they have lower levels of vWF and FVIII:C) and are more likely to be diagnosed with vWD. It also is possible that O group status may be protective for thrombosis.

Factor V inhibitors: rare or not so uncommon? A multi-laboratory investigation.

Acquired deficiencies of, or inhibitors to, factor V are considered rare events. We report a series of 14 acquired factor V deficiencies, 10 of which were confirmed to have inhibitors to factor V, as identified within Australia in the past 5 years following a multi-laboratory investigation. The initial index case seen by one laboratory was followed within 4 months by a separate similar case. This prompted local contact with colleagues (n = 20) working in other haemostasis referral laboratories to identify the current case series. In total, nearly one-half of all haemostasis referral laboratories contacted had seen a case within the past 5 years. Clinical features and the apparent associated risk of bleeding complications generally varied, as did laboratory findings and the likely causal event. There were three females and 11 males. Age ranged from 44 to 95 years (median, 81 years). The level of inhibitor ranged from undetectable to over 250 Bethesda units. The probable cause leading to development of the inhibitors ranged from exposure to bovine thrombin, exposure to antibiotics, surgery and malignancy. Of additional interest was the apparent association of anti-phospholipid antibodies in many of the cases. For example, in the two similar index cases, with factor V inhibitor titres > 200 Bethesda units, high levels of anti-cardiolipin antibodies (> 70 GPL units) were also detected. Although less clear because of inhibitor interference, many of the cases also showed evident co-associated lupus anticoagulant activity. In conclusion, we report a series of factor V inhibitors recently identified within our geographic region that would represent an annual incidence of around 0.29 cases per million Australians. Although considered a rare finding, there is a high likelihood that most haemostasis referral laboratories will see a case every five or so years.

Potential laboratory misdiagnosis of hemophilia and von Willebrand disorder owing to cold activation of blood samples for testing.

To assess the potential for misdiagnosis of von Willebrand disorder (vWD) and hemophilia A while following current National Committee for Clinical Laboratory Standards (NCCLS) guidelines and consequent to a poorly recognized cold-activation phenomenon, we processed 39 normal citrate-anticoagulated samples by standard procedures (reference) or stored at low (approximately 4 degrees C) or ambient (approximately 22 degrees C) temperature for 3.5 hours before centrifugation and processing. Samples were tested in parallel for several hemostasis factors, including von Willebrand factor (vWF). Similar results were obtained for all samples for factors II, V, VII, IX, X, XI, and XII. For factor VIII (FVIII) and vWF, only samples stored at ambient temperature had results comparable to reference sample results. In most cases, low temperature storage led to much lower results. Taking the lower reference limit as 50%, most would have been defined as "abnormal," and a misdiagnosis of vWD or hemophilia A could easily arise. ABO classification and age were associated with FVIII and vWF levels, but neither was associated conclusively with relative loss of plasma FVIII coagulant and vWF caused by the cold-activation phenomenon. We advise laboratories following current NCCLS guidelines not to store or transport whole blood samples for FVIII and vWF testing at 2 degrees C to 4 degrees C because of the risk of misdiagnosing vWD or hemophilia A. Storage and transport at ambient temperature seem acceptable and provide results comparable to freshly centrifuged samples.