Levels of factor VIII and factor IX in fresh-frozen plasma produced from whole blood stored at 4 °C overnight in Turkey

Abstract

Background

The aim of this study was to assess whether the quantities of factor VIII and factor IX in fresh-frozen plasma produced from whole blood stored at 4 °C for 24 hours are adequate for their intended purpose.

Materials and methods

Fresh-frozen plasma separated from whole blood after storage at 4 °C overnight (24 hours from donation) was compared with plasma prepared 8 hours after donation using a standard method. The amounts of factor VIII and factor IX obtained with the two methods were compared.

Results

Compared to the levels of factor VIII and factor IX in plasma prepared within 8 hours of blood collection, the levels in plasma prepared after 24 hours of storage at 4 °C were 25% and 9% lower, respectively. Ninety percent of the factor VIII and 100% of the factor IX levels were above 0.5 IU/mL (standard haematology reference range) after 24 hours of storage.

Discussion

These data suggest that there is good retention of coagulation factor activity in plasma produced from whole blood stored at 4 ºC for 24 hours and that such plasma would be an acceptable product for most patients requiring fresh-frozen plasma.

Introduction

Transfusion of fresh-frozen plasma (FFP) is indicated to replace deficiencies of multiple coagulation factors and control proteins in massive blood loss, liver disease, and disseminated intravascular coagulation¹. Production of frozen plasma after overnight storage of whole blood greatly increases operational flexibility, which can indirectly enhance component safety. There is increasing interest in producing components from blood that has been stored for 24 hours. Plasma from whole blood stored overnight at 4 °C can be made into plasma frozen within 24 hours after phlebotomy (FP24) but because of the 20 to 30% loss of factor VIII (FVIII) activity, this plasma does not consistently meet European or United Kingdom (UK) guidelines for transfusable plasma².

A growing concern regarding plasma transfusion is the risk of transfusion-related acute lung injury (TRALI)³. Typically the reactions causing these injury are mediated by donor anti-HLA or neutrophil-specific antibodies, which bind to recipient neutrophils leading to non-cardiogenic pulmonary oedema, hypoxia, and sometimes death⁴. The prevalence of anti-HLA antibodies is highest in donors who have previously been pregnant. UK haemovigilance data showed that HLA antibody-positive female donors are the principal cause of TRALI associated with FFP. Holding blood overnight could contribute to a reduction in the risk of TRALI by increasing the number of “male-only” donations available for FFP production^5,6.

Several studies have demonstrated that the activity of most clotting factors are well maintained in FP24⁵ and some transfusion services use FFP and FP24 interchangeably for the first 24 hours after thawing³.

The aim of this study was to compare the amounts of labile FVIII and stable factor IX (FIX) in plasma prepared from whole blood of Turkish donors which was processed either on the day of collection or after storage at 4 ºC for 24 hours.

Materials and methods

Thirty units of whole blood (450±50 mL) were collected into JMS bags (JMS Singapore Pte Ltd.) containing 63 mL of CPDA-1. Units were stored for approximately 24 hours at 4 ºC and then units were centrifuged at 2,800 g for 10 min (Hettich Holding GmbH & Co., Germany) and the red cells separated from the plasma with a manual blood component extractor (JMS Singapore Pte Ltd.). Another 30 units of plasma were prepared within 8 hours of whole blood collection and then processed in the same way as the 24 hour plasma samples. The units of plasma were frozen and stored at −30 ºC for FVIII and FIX analysis and comparisons of coagulation activity.

The coagulation factors FVIII and FIX were assayed using a coagulation analyser with a Factor VIII chromogenic test (Siemens AG, Marburg, Germany) and Factor IX Deficient test (Siemens AG, Marburg, Germany).

Considering the distribution of the data, results are presented as medians with ranges. The medians were compared using the Mann-Whitney U test. P values of less than 0.05 were considered statistically significant. Reference ranges were calculated from the geometric means with 95% confidence intervals.

Results

The levels of FVIII and FIX in FFP prepared from whole blood within 8 hours and those from blood stored for 24 h at 4 °C are shown in Table I. Plasma processed after 24 h showed losses of FVIII (25%, p< 0.05) and FIX (9%, not significant). Ninety percent of the 30 FFP units processed after 24 hours had FVIII levels above 0.50 IU/mL (the lower limit of the reference range calculated and the haematology standard range). However, 96.66% of the 30 FFP units processed after 24 hours had FIX levels above 0.70 IU/mL (the lower limit of the reference range) and 100% of them had levels above 0.5 IU/mL (the lower limit of the haematology standard range) (Table I).

Table I.

Comparison of coagulation factors in plasma prepared 8 and 24 hours after whole blood donation, with reference range.

			Reference range based on FFP separated <8 h (RR)		Standard haematology reference ranges (SHR)
	Whole blood storage time		RR	Units stored	SHR	Units stored
	<8h (n:30)	24h (n:30)		24 h in range		24 h in range
Factor	(IU/mL)	(IU/mL)	(IU/mL)	(%)	(IU/mL)	(%)
FVIII	1.0 (0.66–1.47)	0.75 (0.39–1.17)	0.50–1.75	50	0.50–2.00	90
FIX	0.89 (0.66–1.16)	0.81 (0.50–1.17)	0.70–1.20	96.66	0.50–2.00	100

Discussion

This study compared the effect of storing whole blood at 4 °C for 24 hours and less than 8 hours on the quality of plasma for transfusion. FVIII is known to be one of the labile coagulation factors in plasma and its level is a quality control requirement for FFP in Europe².

In the United States, plasma from blood stored in this way can be made into plasma frozen within 24 hours after phlebotomy² but because of the 20 to 30% loss of FVIII activity, this plasma does not consistently meet European or UK guidelines for transfusable plasma⁷.

In our study, holding blood at 4 °C overnight resulted in a 25% loss of FVIII (p<0.05) and in a 9% loss of FIX (not significant) compared to plasma frozen within 8 hours of donation.

Levels of FVIII studied in plasma separated within 8 hours were above the lower limit of the reference range and the haematology range (0.5–2 IU/mL) based on FP24 (Table I). This plasma meets current European and UK guidelines for FFP^8,9 since the levels of FVIII were more than 0.70 IU/mL in 75% of units.

Only 50% of the units prepared from whole blood stored overnight at 4 °C contained more than 0.70 IU/mL FVIII in our study. The levels reported by Cardigen⁵ and Naghadeg⁶ were 55% and 34.8%, respectively. The FVIII levels fell within the standard haematology range (0.5–2 IU/mL) in 90% of cases in our study, in 81.8% of those in the study by Naghadeg⁶ and in 92% of those in the study by Cardigan⁵.

In the present study the level of FVIII was lower in plasma prepared 24 hours after blood collection, as also reported by Cardigen⁵, Kakaiya¹⁰ and O’Neill¹¹ and Naghadeh⁶.

In the USA, FFP produced less than 8 hours or more than 24 hours after donation are issued as different products, although they may be used for the same indications (with the exception of FVIII replacement)¹².

Plasma from 24 hour donations cannot be guaranteed to contain more than 0.50 U/mL of FVIII, although some studies and our results show that good percentages of units do contain satisfactory levels. Although FFP is now never given for replacement of FVIII alone, it is important that FFP contains a reasonable level of FVIII (0.50 IU/ mL) for treatment of disseminated intravascular coagulation and massive transfusion. We, therefore, conclude that the loss of coagulation factors observed is unlikely to be clinically significant and that plasma produced from whole blood stored at 4 °C for up to 24 hours may be used for the same clinical indications as FFP or PF242.

Our study was performed to investigate extended storage of blood for FFP production, but we focused only two coagulation factors, FVIII and FIX, and tested a small number of units. Although more studies of coagulation factor levels in plasma are needed, our results demonstrate that there is good retention of FIX and FVIII in plasma produced from whole blood stored at 4 ºC for 24 hours and that such plasma would be an optional useful product for most patients except haemophiliacs.