Laboratory Assessment of von Willebrand Factor: Altered Interpretation of Laboratory Data, and Altered Diagnosis of von Willebrand's Disease

Abstract

Three laboratory assays for von Willebrand Factor (vWF), namely, (i) a standard "antigen" [antiseraenzyme-linked immunosorbent assay (ELISA)-based] (vWF :Ag), (ii) a standard ristocetin-dependent platelet agglutination procedure (vWF:RCof), and (iii) a newly developed ELISA-based functional vWF collagen binding assay (vWF:CBA), were coevaluated. We (a) assessed their ability to detect vWF under different assay conditions and (b) reevaluated normal vWF reference ranges using citrated plasma from >200 normal individuals. The following effective normal reference ranges were derived: vWF-Ag (40-200%), vWF:CBA (50-400%), and vWF:RCof (45-200%). Based on other laboratory data, caution is indicated in the interpretation of "borderline" or "equivocal" test results (e.g., 35-55%), since these may derive from normal individuals or from vWD patients. In addition, our study shows that selection of different assay conditions, different test plasma dilutions, or different test sample processing methods can also lead to differences in the "apparent" levels of detected vWF. For example, using plasma from 100 normal individuals processed as "test samples," we determined that higher sample dilutions tended to provide lower (dilution factor adjusted) assay results in the case of the vWF:Ag and vWF:CBA, and higher assay results in the case of the vWF.RCof, with greater differences noted in test samples containing higher levels of vWF. The explanation for these findings relate to the assay design. Thus, in the diagnostic laboratory, the standard vWF:Ag, vWF:CBA, and vWF:RCof assays procedures are designed to provide calibration curves that are "upward linear" in the (vWD) clinically important region of 0-50% of normal, with a plateau tending at higher vWF levels. Using these standard methodologies, and standard plasma dilutions, high-vWF level test samples will tend to fall on or near the plateau, and results will be unreliable, with a tendency to be exaggerated the higher the vWF. These findings have particular relevance to research studies measuring vWF in patients suffering various clinical conditions (e.g., thrombotic thrombocytopenic purpura, diabetes, renal failure) where higher than normal levels of vWF may be anticipated. In these cases, laboratoryderived vWF levels could be "misrepresented" if the study design did not take into account the need to revise the vWF testing procedure.

Potential Laboratory Misdiagnosis of Hemophilia and von Willebrand Disorder Owing to Cold Activation of Blood Samples for Testing

“…In most test cases, whole blood samples held at low temperature before centrifugation led to a detectable loss of FVIII:C and vWF and to the potential for misdiagnosis of these samples as vWD or hemophilia A, depending on the testing undertaken (ie, vWF studies ordered or only FVIII ordered, respectively). This finding, therefore, identifies a cause of possible misdiagnosis in addition to those previously reported 8,9 and extends the findings of previous reports regarding the cold-activation phenomenon. 10,11 A potential was shown for misdiagnosis or misclassification of vWD using a small number of samples processed according to the thencurrent NCCLS guidelines, 12 which recommended that coagulation specimens be "kept at 2 to 4°C or 18 to 24°C" until centrifuged and tested (within 4 hours of collection).…”

“…Potential issues relating to test results at borderline or equivocal cutoff points are particularly relevant because they frequently arise. For example, our laboratory has highlighted the potential for misdiagnosis of vWD or hemophilia A following inappropriate testing of filtered plasma, 8,9 in which levels of vWF and FVIII:C are reduced compared with nonfiltered plasma. This situation might occur when the clinician or laboratory is following up cases of prolonged routine coagulation test times, perhaps with vague clinical histories, to exclude vWD, hemophilia A, and the presence of lupus anticoagulant.…”

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

“…Reference ranges for all assays were calculated using either non-transformed (linear model) or log-transformed data (to achieve normality; logarithmic model) and AE 2SD to generate 95% confidence intervals, as previously described for VWF [5]. Blood samples for testing were obtained with permission from healthy individuals donating to a local Red Cross blood bank and collected into standard (3.2% sodium citrate) anticoagulant tubes (Greiner Vacuette brand, catalogue number 455322; Interpath, Sydney, Australia).…”

Modifications of protein Z and interleukin-6 during the acute phase of coronary artery disease

“…There is no doubt that other significant preanalytical events may occur of which we are aware, or unaware [12,13]. I have presented a summary of the information in the current report in Table 1.…”