A study of the quantity of some stable and labile coagulation factors in fresh-frozen plasma produced from whole blood stored for 24 hours in Iran

Abstract

Background

The aim of this study was to assess whether the quantities of some coagulation factors in fresh-frozen plasma (FFP) produced from whole blood stored at 4°C for 24 h are adequate for their intended purpose.

Materials and methods

The amounts of some coagulation factors (fibrinogen, FV, FVII, FVIII, FX and FXI) in FFP separated from whole blood after storage at 4°C for 24 h were compared with the amounts of the corresponding coagulation factors in FFP separated from whole blood within 8 h of donation.

Results

In 98% of the FFP units prepared after 24 h of storage, the levels of fibrinogen, FV, FVII, FX and FXI were greater than 0.5 IU/mL. The concentration of FVIII in the 24 h plasma units was 82% of that found in the FFP units prepared within 8 h of blood collection. In FFP, FVIII, FVII and FX were reduced by 38%, 8% and 3%, respectively, but FV, FXI and fibrinogen were not reduced.

Conclusion

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

Introduction

Extended storage of blood for the production of fresh-frozen plasma (FFP) greatly increases operational flexibility, which can indirectly enhance blood component safety¹. For example, because UK haemovigilance data have demonstrated that HLA antibody-positive female donors² are the principle cause of transfusion related acute lung injury (TRALI) associated with FFP, holding donations for 24 h could contribute to reducing the risk of TRALI by increasing the number of "male only" donations available for the production of FFP. It appears that FFP prepared after 24 h contains levels of coagulation factors that are equivalent to or higher than those seen in most studies evaluating plasma treated with solvent/ detergent (S/D), methylene blue (MB), or amotosalen pathogen reduction systems^3–5; both MB- and S/D-treated plasma are used in several European countries for all of the same clinical indications as FFP¹.

A number of studies have been performed to investigate extended storage of blood prior to FFP production^6–12. Given the clinical indications for FFP, it is important that a wide range of coagulation factors is preserved. The aim of this study was to measure the amounts of some stable (fibrinogen, FVII, FX, FXI) and labile coagulation factors (FV, FVIII) in FFP prepared from whole blood of Iranian donors stored at 4ºC for 24 h. Our hypothesis, based on published data, was that this plasma would be unlikely to meet current UK¹³ or European standards¹⁴ for FVIII content, yet retain substantial activity of this and other coagulation factors.

Materials and methods

Blood collection and processing

Sixty-six units of whole blood (450±50 mL) were collected into JMS bags (Singapore PTE Ltd.) containing 63 mL of CPDA-1. Because the levels of FVIII are lower in group O donors, an equal number of group A and group O donations were selected to avoid biases in the results. Units were stored for approximately 24 h at 4ºC. The units were then centrifuged at 2800g for 10 min (Jouan, KR 4-22 Ltd., France) and the red cells separated from the plasma with a manual extractor. The units of plasma were frozen and stored at −30ºC for later analysis and comparisons of coagulation activity. Another 66 units of plasma were prepared within 8 h of whole blood collection and then processed in the same way as the 24 h plasma samples. These units were used to establish the reference range and were also collected from a 1:1 mix of group O and group A donors.

Laboratory analysis

The coagulation factors FV, VII, VIII, FX and FXI were assayed using a coagulation analyser with a Stago kit (STA Liatest, Diagnostica Stago Ltd., Asnieres, France). Fibrinogen was measured by the Clauss technique with a Stago kit according to the manufacturer's instructions (STA Liatest, Diagnostica Stago Ltd., Asnieres, France).

Statistical analysis

Considering the non-Gaussian 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 fibrinogen, FV, FVII, FVIII, FX and FXI in FFP prepared from whole blood stored for 24 h at 4°C and in FFP processed from whole blood after 8 h are shown in Table 1. Plasma processed after 24 h showed losses of FVIII (38%), FVII (8%) and FX (3%). More than 95.5% of the 66 units produced from whole blood stored for 24 h had levels of fibrinogen, FV, FVII, FVIII, FX and FXI above the lower limit of the reference range calculated from units of FFP produced after 8 h. Furthermore, the levels of these clotting factors were above 0.5 IU/mL (the lower limit of the haematology standard range) in more than 98% of the 66 FFP units processed after 24 h, except for FVIII for which 81.8% of the units contained greater than 0.5 IU/mL (Table I).

Table I.

Comparison of coagulation factors of 8 h and 24 h plasma with reference ranges

	Whole blood storage time			Reference range based on 8 h plasma		Standard haematology reference range
Factor	<8 h (n=66)	24 h (n=66)	P valuea	8 h plasma range, IU/mL	24-h plasma units with factor levels within the range of 8 h plasmab (%)	Haematology standard range, IU/mL	24-h plasma units with factor levels within the standard haematology range (%)
Concentration, median (range), IU/mL
FV	0.93 (0.64–1.86)	0.98 (0.5–1.45)	NS	0.57–1.43	98.5	0.5–2	100
FVII	0.9 (0.49– 1.46)	0.83 (0.34–1.52)	NS	0.46–1.33	98	0.5–2	98
FVIII	1.03 (0.5– 2.06)	0.63 (0.38–1.55)	P< 0.05	0.40–1.74	95.5	0.5–2	81.8
FX	0.99 (0.52–1.31)	0.96 (0.59–1.37)	NS	0.66– 1.28	98	0.5–2	100
FXI	0.83 (0.48–1.54)	0.84 (0.48–1.26)	NS	0.44 –1.29	100	0.5–2	98.5
Fibrinogen (g/L)	2.37 (1.44–5.19)	2.6 (1.68–3.76)	NS	1.01– 4.08	100	0.5–2	100

^aComparison of the median values using the Mann-Whitney U test.

^bWithin the range is defined as above the lower limit of the range in 8 h plasma

NS, Not significant

According to UK specifications the level of FVIII should be greater than 0.7 IU/ml in at least 75% of FFP units. In our study only 23 units (34.8%) contained greater than 0.7 IU/mL FVIII activity. The mean levels of FV (0.98 IU/mL), FXI (0.84 IU/mL) and fibrinogen (2.6 g/L) were surprisingly high in the plasma processed from whole blood after storage at 4°C for 24 h.

The median levels of fibrinogen, FV, FVII, FX and FXI in the 8 h and 24 h FFP units are shown in table I. There were no significant differences in the levels of these factors between 8h and 24 h plasma, except for FVIII.

Discussion

Efforts are currently being made to lengthen the storage period of plasma in order to increase supplies; plasma stored for less than 8 h can be used for cryo-preparation and plasma stored for 24 h for the treatment of patients (except those with haemophilia) in need of coagulation factors.

Longer storage of plasma enables flexibility in practice, ensuring safer plasma for patients. For example, TRALI syndrome, one of the prevalent complications (1/5000) of blood transfusion, is due to the presence of HLA antibodies, which are frequent in plasma of multiparous women and multi-transfused donors¹⁵.

Given that a substantial proportion of blood donors in developed countries are women, 15% of whom have HLA antibodies, one of the most important causes of TRALI syndrome is the use of plasma from female donors^1,2. Long storage of blood gives the flexibility to exclude such female donors, as is done in England where FFP is mostly prepared from blood from male donors¹.

In the present study, there were no significant differences between the levels of coagulation factors in plasma prepared within 8 h of blood collection and that prepared at 24 h, except for FVIII: the level of this coagulation factor was lower in the plasma prepared 24 h after blood collection, as also reported by Cardigen, Kakaiya and O'Neill^1,10,12.

Despite the fact that the levels of FVIII in 24 h plasma did not meet the European Standard (> 0.7 IU/mL in at least 75% of units), they did fall within the haematology range (0.5–2 IU/mL) in 98% of cases in our study and in other studies. Thus, 24 h plasma can ensure the availability of coagulation factors, including FVIII, in almost all clinical situations, including massive transfusion, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, liver diseases, etc. It is also evident that most patients (except haemophiliacs) have normal levels of FVIII and that 24 h plasma can meet their needs for this factor and other coagulation factors. The situation in haemophiliacs is different, in that the quantity of FFP necessary to provide sufficient FVIII may be so great as to cause pulmonary oedema as a result of volume overload. For this reason, FVIII concentrates and cryoprecipitates are now used, instead of plasma, for the treatment of haemophilia. Thus, in the USA, 8 h and 24 h plasma are used for similar indications. Accordingly, solvent/detergent-treated plasma, with coagulation factor contents similar to those in 24 h plasma, is widely used in patients (except haemophiliacs)¹.

Conclusion

Our results demonstrate that there is good retention of coagulation factor activity in plasma produced from whole blood stored at 4ºC for 24 h and that such plasma would be an acceptable product for most patients requiring FFP.