The Importance of Pre-Analytic Conditions on the Determination of VWF Parameters

Determination of VWF Parameters

S. Täschner, M. Böhm, I. Stier-Brück, G. Asmelash, B. Putz, T. Vigh, C. Rabenstein, V. Meyer, G. Eckert and I. Scharrer

Introduction

It is well established that cold promotes a shortening of PT by FXII mediated acti- vation of FVII in whole blood and in plasma [5]. The effect of low temperature on Von Willebrand Factor (VWF) parameters has there against not been studied in detail. We therefore conducted a study to investigate the pre-analytic processing of blood samples on VWF:Ristocetin-Cofactor activity (VWF:RCo), VWF:Antigen (VWF:Ag) and FVIII activity (FVIII:C).

Patients and Methods

We collected 50 ml citrated blood from 10 apparently healthy individuals and from 8 patients with Von Willebrand Disease (VWD), type I and from 2 patients with VWD, type II. Individuals with conditions associated with increased VWF:Ag-levels such as surgery in the last 6 months, diabetes mellitus, renal insufficiency, chronic coronary heart disease, malignancy [4] and other severe disorders were excluded from the study. All participants gave their informed consent for blood sampling and research use. 30 ml of the blood was directly centrifuged (40 min, 4°C and 4000 g) and the separated plasma was either immediately frozen at –80°C (normally pro- cessed sample) or stored at Room Temperature (RT) or on crashed ice for 3 and 6 hours, respectively. The remaining citrated whole blood (CWB) was either stored at RT or on crashed ice for 3 and 6 hours prior centrifugation and storage at –80°C. All samples were tested for VWF:RCo, VWF:Ag and FVIII:C. VWF:RCo was measured with the BC von Willebrand Reagent from Dade Behring (Marburg, Germany) on the Behring Coagulation Timer (Dade Behring, Marburg, Germany) according to the manufacturers instructions. VWF:Ag was determined by enzyme immunoassay using rabbit anti-human VWF A0082 (DAKO A/S, Glostrup, Denmark) for capture and peroxidase-conjugated rabbit anti-human VWF P0226 (DAKO A/S, Glostrup, Denmark) for detection. FVIII:C was measured with FVIII deficient plasma from Instrumentation Laboratory (Lexington, USA). Results are expressed as percentage of the normally processed sample. Wilcoxon-matched pairs-test (Bias 7.01 software, Frankfurt, Germany) was used to analyse the difference between the test results of the various storage conditions. Values of p<0.05 were considered to be statistically significant.

I. Scharrer/W. Schramm (Ed.)

34

Hemophilia Symposium Hamburg 2003

” Springer Medizin Verlag Heidelberg 2005

Results

Storage on crashed ice as citrated whole blood induced in the samples from healthy individuals and patients with VWD, type I a drastic time dependent decrease of VWF:RCo, VWF:Ag and FVIII:C (Figs. 1–3, Table 1). The cold-induced loss of

0 20 40 60 80 100 120

3h 6h 3h

CWB Ice 6h

Plasma Ice 6h Controls vWD, Type 1

Plasma Ice 3h Plasma

RT 6h Plasma

RT 3h CWB Ice

CWB RT CWB RT

Table 1. Storage of citrated whole blood on crashed ice. Results (mean +/-SD) are shown in % of the normally processed sample

VWF:RCo VWF:Ag FVIII:C

3h 6h 3h 6h 3h 6h

Controls 51% 36% 53% 50% 73% 57%

(n=10) +/–30 +/–22 +/–21 +/–17 +/–22 +/–18

VWD, type I 54% 34% 69% 54% 60% 48%

(n=8) +/–23 +/–15 +/–21 +/–20 +/–16 +/–12 VWD, type II n.m. n.m. 93% 115% 86% 79%

(n=2) +/–20 +/–14 +/–4 +/–13

n.m. = not measurable

Fig. 1. VWF:RCo after storage of whole blood and plasma at RT and on ice

Results are expressed as percentage of the normally processed sample, the columns depict the

mean of the 8 investigated individuals

VWF:RCo was significantly correlated to the loss of VWF:Ag and FVIII:C (Spearman- Rang-Correlation: r=0.72; p=0.002 and r=0.70; p=0.002 respectively). The two pati- ents with VWD, type II showed in the normally processed sample VWF:RCo of <5%, VWF:Ag of 20 and 40% and FVIII:C of 16 and 53%, respectively. Storage of citrated whole blood on crashed ice from these two patients did not induce a significant loss of VWF:Ag and only a minor reduction of FVIII:C (Figs. 2–3, Table 1).

Statistical analyses confirmed the significant loss of VWF:RCo and VWF:Ag after storage as whole blood on ice (Table 1). Storage of whole blood at RT or stor- age of plasma at RT and on ice did there against not significantly change VWF:RCo and VWF:Ag-levels except for storage of plasma from healthy individuals for 6h on ice (Table 2). However, the decrease of VWF:Ag in the latter group was much lower than after storage of whole blood on ice (average reduction from baseline level was 20% and 49% respectively, Fig. 2).

FVIII:C was significantly reduced under all investigated storage conditions except for storage of whole blood from controls at RT for 3 hours (Table 1). The average reduction from baseline levels after storage of whole blood at RT and stor- age of plasma was always < 27%, whereas storage of whole blood on ice induced an average decrease of 40 and 52% (Fig. 3).

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0

C WB R T 3h

C WB R T 6h

C WB Ice 3h

C WB Ice 6h

P la s m a R T 3h

P la s m a R T 6h

P la s m a Ice 3h

P la s m a Ice 6h C o ntro ls vWD, Type 1 vWD, Type 2

Fig. 2. VWF:Ag after storage of whole blood and plasma at RT and on ice

Results are expressed as percentage of the normally processed sample, the columns depict the

mean of the respective individuals

0 20 40 60 80 100 120

C WB R T 3h

C WB R T 6h

C WB Ice 3h

C WB I c e 6h

P la s m a R T 3h

P la s m a R T 6h

P la s m a I c e 3h

P la s m a I ce 6 h C o ntro ls vWD, Type 1 vWD, Type 2

Fig. 3. FVIII:C after storage of whole blood and plasma at RT and on ice

Results are expressed as percentage of the normally processed sample, the columns depict the mean of the 8 investigated individuals

Table 2. Statistical comparison of the different storage conditions

Results from the stored samples were compared to the results of the normally processed samples with Wilcoxon-matched-pairs-test

Parameter Cohort CR3 CR6 CE3 CE6 PR3 PR6 PE3 PE6

VWF:RCo Controls n.s. n.s. p=0.02 p=0.02 n.s. n.s. n.s. n.s.

(n=10)

VWD, type I n.s. n.s. p=0.008 p=0.008 n.s. n.s. n.s. n.s.

(n=8)

VWF:Ag Controls n.s. n.s. p=0.002 p=0.002 n.s. n.s. n.s. p=0.04 (n=10)

VWD, type I n.s. n.s. p=0.008 p=0.008 n.s. n.s. n.s. n.s.

(n=8)

FVIII:C Normals n.s. p=0.04 p=0.006 p=0.02 p=0.04 p=0.02 p=0.01 p=0.04 (n=10)

VWD, type I p=0.03 p=0.02 p=0.008 p=0.008 p=0.02 p=0.008 p=0.008 p=0.008 (n=8)

n.s. = not significant (p>0.05)

Discussion

We found a drastic time-dependent loss of about VWF:RCo, VWF:Ag and FVIII:C after storage of citrated whole blood on crashed ice for healthy individuals as well as for patients with VWD, type I. Since we did not find a cold induced loss of the VWF in plasma and in samples from patients with VWD, type II (VWF:RCo < 5%) we assume, that the drastic cold-induced loss of VWF:RCo, VWF:Ag and FVIII:C is dependent on the presence of platelets and of high–molecular-weight-VWF. It is well established, that cold induces an extensive platelet shape change by intracellu- lar cytoskeletal rearrangement [1]. Hoffmeister et al. [3] recently found, that chilling of platelets induces a clustering of the Glycoprotein Ib (GPIb) receptor on the cell surface. We hypothesise, that the cold-induced loss of VWF in citrated whole blood is due to cold-promoted binding of VWF to platelets probably due to increased sus- ceptibility of GPIba subunit for VWF.

Our results demonstrate that blood destined for analysing VWF parameters should be stored at RT rather than at 4°C.

References

1. Berger B, Hartwell DW, WagnerDD: P-Selectin and platelet clearance. Blood 1998; 92:

4446–4452

2. Favaloro EJ, Nair SC, Forsyth CJ:Collection and transport of samples for laboratory testing in von Willebrands disease (VWD): time for a reappraisal? Thromb Haemost 2001; 86:

1589–1590

3. Hoffmeister KM, Felbinger TW, Falet H, Denis CV, Bergmeier W, Mayadas TN, von Adrian KH, Wagner DD, Stossel TP, Hartwig JH: The Clearance Mechanism of Chilled Blood.

Platelets, Cell 2003; 112: 87–97

4. Lutze G, Naumann C, Zawta B: Wissenswertes zur Gerinnung, Roche Diagnosties GmbH, Mnnheim, 1999

5. Palmer RN, Gralnick HR: Inhibition of the cold activation of Factor VII and the prothrom-

bin time American Journal of Clinical Pathology 1984; 81:618–622