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Potential misdiagnosis of von Willebrand disease and haemophilia caused by ineffective mixing of thawed plasma.

Author(s): Favaloro EJ, Oliver S, Mohammed S, Ahuja M, Grzechnik E, Azimulla S, McDonald J, Lima-Oliveira G, Lippi G

Publication: Haemophilia, 2017, Vol. 23, Page e436-e443

PubMed ID: 28750474 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper explored potential differences in the levels of von Willebrand factor (VWF) and coagulation factors in case-matched citrate plasma specimens that were stored at different temperatures (-20 or -80°C) for 2-7 d, and mixed post-thaw by either inversion, placement on a blood roller, or vortexed or analyzed without being mixed. In total, 48 residual citrate plasma samples were used for analysis; samples collected from a single individual during a single collection event were pooled, mixed by inversion (6 times), and aliquoted into four 1 mL samples that were stored at -20°C or -80°C for 2-7 d. Frozen aliquots were thawed for 5 min in a 37°C water bath, and either left unmixed, inverted six times, placed on a blood roller for 5 min at room temperature, or vortexed for 10 seconds. Each aliquot was then analyzed for VWF antigen and VWF collagen binding by ELISA, VFW ristocetin cofactor by automated platelet agglutination assay, and coagulation factors (FVIII, FIX, FXI, FXII) by one-stage clotting assays. The authors also performed retrospective analysis of VWF results from 49 plasma specimens, representing multiple tubes collected from individuals during the same collection event (the number of individuals was not specified).

   Summary of Findings:

   Retrospective analysis of VWF results from multiple case-matched plasma specimens collected during the same collection event displayed a reasonable level of variability (≤10%) for most specimens, although four samples had a substantial level of variability in VFW: Ag, VWF: CB, VWF:RCo, and FVIII.

   For prospectively collected citrate plasma samples, there were no significant differences between samples stored at -20°C and those stored at -80°C (for 2-7 d) on any of the VFW or cofactor levels measured, regardless of mixing method.  When citrate plasma samples stored at -20°C were compared, plasma samples that were not mixed after being thawed had significantly lower values for all VWF analytes (VWF: Ag, CB, RCo) and factors (FVII, FIX, FXI, FXII) than case-matched samples that were mixed by inversion, placement on a blood roller, or vortexing (P≤0.001 for all). However, only the VWF: RCo/Ag ratio (not the VWF: CB/Ag ratio) differed significantly when non-mixed samples were compared to those that were mixed on a roller (P<0.05). The authors note that some samples that initially yielded a normal VWF result displayed VWF and/or FVIII levels that were below the reference threshold in the unmixed sample, indicating a potential for misdiagnosis if citrate plasma samples are not properly mixed after being thawed; in these samples, VWF: CB/Ag ratio significantly differed between unmixed and mixed plasma samples after being thawed (P≤0.005), although VFW: RCo/Ag ratio was unaffected by sample mixing. 
    

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Hemophilia

   Platform:

   Analyte	Technology Platform
   Glycoprotein	ELISA
   Morphology	Hematology/ auto analyzer
   Protein	Immunoassay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Biospecimen mixing	Unmixed Inversion Blood roller Vortex
   Storage	Storage temperature	-20°C -80°C

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