Stability of coagulation factors in feline fresh frozen plasma intended for transfusion after 1 year of storage

Abstract

Objectives

The most common use of plasma transfusion is for haemostatic purposes, but coagulation factor activities in stored feline plasma are unknown. The concentration and stability of coagulation factors I (fibrinogen), II, V, VII, VIII, IX, X, XI and XII in feline fresh frozen plasma (fFFP) stored for 1 year were studied.

Methods

Fifty-five units of fFFP were produced from 55 fresh whole-blood donations obtained from indoor healthy blood donor cats. Twenty-one units were stored for <2 weeks (T0) and 34 were stored for 1 year (T1). After the completion of storage, specific coagulation factor activities for factors II, V, VII, VIII, IX, X, XI and XII were tested using modified one-stage activated partial thromboplastin or prothrombin time assays. Fibrinogen was determined using the Clauss method.

Results

Significantly decreased activities were observed for factors II (T0: 101.94% ± 19.06%; T1: 73.23% ± 39.06% [P = 0.001]), VII (T0: 102.78% ± 24.69%; T1: 60.08% ± 38.17% [P <0.001]), VIII (T0: 77.52% ± 30.39%; T1: 50.32% ± 23.8% [P = 0.001]), XI (T0: 88.76% ± 22.73%; T1: 66.28% ± 22.2% [P = 0.001]) and XII (T0: 89.50% ± 21.85%; T1: 55.46% ± 23.18% [P <0.001]) when comparing units at time 0 and after 1 year of storage. No significant difference was observed for factors IX (T0: 84.86% ± 29.35%; T1: 71.37% ± 22.23% [P = 0.064]) and X (T0: 96.24% ± 25.1%; T1: 83.91% ± 49.54% [P = 0.236]). Unexpectedly, a significant increase was observed for factor V (T0: 71.94% ± 24.14%; T1: 97.89% ± 62.33%; P = 0.046). Fibrinogen was 2.76 ± 1.09 g/l at T1. Factors VIII, XII and VII had the lowest mean activities after 1 year.

Conclusions and relevance

Although a decrease in most coagulation factors activities was noted with storage, 1-year-old fFFP was haemostatically active in vitro. The most suitable factors for quality control assessment of fFFP are factors VII and VIII. Approximately 13–20 ml/kg of fFFP is required to administer a minimum of 10 IU/kg coagulation factor activity.

Keywords

Transfusion medicine coagulation haemostasis plasma blood bank plasma storage

Introduction

The objectives of blood banks are to produce safe and efficacious blood products, and to establish quality standards that provide even safer and more efficient derivatives for transfusion.¹ Fresh frozen plasma (FFP) is a blood product obtained either from whole blood or from plasma collected by apheresis.^1,2 Feline plasma storage is common practice in blood banks and veterinary practices. Using plasma products rather than whole blood allows for longer storage times, increased availability and lower costs, and minimises the risk of transfusion reactions.^2,3 The most common reason for the administration of feline FFP (fFFP) is for haemostatic purposes, and FFP transfusion is the gold standard of care in patients with coagulopathies.^3,4 In a 2020 study by Lane and Sinnott-Stutzman,⁴ the most common indications for fFFP were suspected coagulopathy, haemorrhage and hypotension.

Coagulation factor activities in fFFP are unknown and thus, in many cases, plasma is given to effect. In the study by Lane and Sinnott-Stutzman,⁴ cats were significantly less likely to be coagulopathic post-transfusion after a median fFFP dose of 6 ml/kg. However, the optimal dose of fFFP to replace coagulation factors in cats has not been investigated. Coagulation factor activities are used as a standard to define blood plasma products. A general guideline is that 1 ml fresh plasma contains approximately 1 IU of each coagulation factor activity.⁵

Current veterinary convention is that FFP is termed frozen plasma (FP) when it exceeds the recommended 1-year storage time, and it is theoretically characterised by a reduced content of labile coagulation factors.^2,3,6 Quality control of human FFP is based on the measurement of coagulation factors V and VIII, which are considered the most labile.^2,7 A canine plasma study conducted in 2013 demonstrated that 5-year-old FP is haemostatically active, with a significant decrease in factor VIII activity but with a sustained stability of factor V, indicating that differences within species might exist.⁸ Recommended storage times for feline plasma have been adapted from human medicine, where many authors consider that plasma can be only classified as FFP within 1 year of storage based on the loss of factors V and VIII.³ However, to our knowledge, there have been no studies evaluating the stability of feline haemostatic proteins in plasma during storage.

The main aim of this study was to evaluate factor activities and stability over time of coagulation factors during storage of fFFP. The secondary aims were to identify the coagulation factors that are most affected by storage in feline plasma, and to facilitate understanding of the optimal volume of fFFP needed to administer 10 IU/kg of functional coagulation factors when administering units stored up to 1 year.

Materials and methods

In this prospective study, whole blood was collected from 55 indoor healthy cats weighing 4–9 kg that had been regularly vaccinated and dewormed. Complete blood counts and chemistry profiles were performed, and cats were included only if the results were within the normal reference intervals. All cats tested negative for feline immunodeficiency virus and feline leukaemia virus (Uranovet), and Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’ (PCR analysis by Genevet, Algés, Portugal).

No animals were utilised solely for the purpose of this study. All samples were obtained via the routine procedures of the Animal Blood Bank in the production of packed red blood cells (pRBCs) and fFFP; therefore, no unnecessary procedures were performed on the donors. All blood samples were collected after signed informed owner consent was obtained. This study was conducted according to European legislation (86/609/EU).

Whole-blood units were collected using a specific feline semi-closed system, and fresh plasma was separated from pRBCs as described elsewhere.⁶

Fresh plasma was frozen at −80°C and maintained at this temperature for 24 h. The fFFP units were then stored at a temperature between −18°C and −25°C. The pRBC units were used for medical purposes.

The fFFP units were then separated into two groups: T0 and T1. The units in T0 were stored for <2 weeks before testing, and those in T1 were stored for 1 year before testing. All units were stored at a temperature between −18°C and −25°C.

After the completion of their designated storage times, all units were thawed at room temperature and 1.5 ml samples were separated and refrozen at −80°C within 30 mins. Analysis of the coagulation factors in the latter samples was performed the next day.

Specific coagulation times for factors VIII, IX, XI and XII were tested using a modified one-stage activated partial thromboplastin time assay, and coagulation times for factors II, V, VII and X were tested with the modified one-stage activated prothrombin time assay (diagnostic stago ST4), with human-specific factor-deficient plasma. Fibrinogen (factor I) was determined using the von Clauss method. Each factor activity was measured twice per unit and repeated if more than a 10% discrepancy was observed. A four-dilutions curve of pooled fresh plasma from 10 different healthy cats was used to derive standard curves for all factors by diluting pooled plasma with plasma-poor in each specific factor (specific factor depleted plasma) respectively for every coagulation factor tested.

The variables under study were shown to follow the normal distribution using the Kolmogorov–Smirnov test. Thus, the parametric Student’s t-test for two independent samples was used to compare specific coagulation times for all analysed factors between groups T0 and T1. An alpha of 0.05 was used. The results were analysed with SPSS version 21.0 (IBM).

Specific factor recovery was calculated for each factor by dividing the observed plasma-specific factor activity after 1 year of storage (group T1) with the observed plasma-specific factor activity at the beginning of storage (group T0).

Results

A total of 55 units from different donors were included in the study, with 21 units being stored for <2 weeks (T0) and the remaining 34 stored for 1 year (T1).

Within the T0 group, the mean activity for each specific coagulation factor was higher than 70%. When comparing the values obtained from groups T0 and T1, significant specific activity decreases were observed for factors II, VII, VIII, XI and XII after 1 year of storage, with mean decreases of 28.7% (from 101.9% to 73.2%), 42.7% (from 102.8% to 60.1%), 27.2% (from 77.5% to 50.3%), 22.5% (from 88.8% to 66.3%) and 34.0% (from 89.5% to 55.5%), respectively, from the specific factor activity at T1 compared with T0. An increase in specific factor activity was only observed for factor V (26.0% increase). Although a reduction in most of the coagulation factor activities was identified, the majority of units after 1 year of storage (T1) were still within the minimally acceptable ranges for a healthy cat (>50%).⁹

The factors with lowest mean activities at T1 were VIII (50.3%), XII (55.5%) and VII (60.1%). All specific factor activities in the T0 and T1 groups, the variation between both groups and the percentage of recovery are detailed in Table 1.

Table 1

Comparison of coagulation factor activities from T0 to T1

Percentage coagulation factor activity	T0 (<2 weeks storage)	T1 (1 year storage)	P value	Reference interval	Percentage decreaseT0 to T1	Percentage recovery from T0 at T1
Percentage coagulation factor activity	Mean ± SD	Mean ± SD	P value	Reference interval	Percentage decreaseT0 to T1	Percentage recovery from T0 at T1
I (g/l)	–	2.76 ± 1.09	–	1.5–3	–	–
II (%)	101.94 ± 19.06	73.23 ± 39.06	0.001*	>50	−28.71	71.84
V (%)	71.94 ± 24.14	97.89 ± 62.33	0.046*	>50	25.95	136.07
VII (%)	102.78 ± 24.69	60.08 ± 38.17	<0.001*	>50	−42.7	58.45
VIII (%)	77.52 ± 30.39	50.32 ± 23.80	0.001*	>50	−27.2	64.91
IX (%)	84.86 ± 29.35	71.37 ± 22.23	0.064	>50	−13.49	84.10
X (%)	96.24 ± 25.10	83.91 ± 49.54	0.236	>50	−12.33	87.19
XI (%)	88.76 ± 22.73	66.28 ± 22.20	0.001*	>50	−22.48	74.67
XII (%)	89.50 ± 21.85	55.46 ± 23.18	<0.001*	>50	−34.04	61.97

Statistically significant differences for T0 vs T1

Discussion

This study was designed to evaluate the concentration of coagulation factor activities in fFFP and their stability over 1 year of storage. At the start of storage, mean coagulation activities for all factors exceeded 70%. After 1 year of storage, while decreased activities were observed for most factors, the majority of units remained within the minimally acceptable ranges for a healthy cat.⁹

Our study showed significantly decreased factor activities for factors II, VII, VIII, XI and XII in plasma units stored for 1 year compared with those stored for <2 weeks, most likely due to deterioration during storage. The factors with the lowest mean activities after 1 year of storage were factors VIII (50.3%), XII (55.5%) and VII (60.1%). These three factors were the only factors with a recovery of <70% of factor activity when comparing the units tested after 1 year of storage (T1) with the units tested at the beginning of storage (T0).

In one study of canine stored FFP, the factors with the lowest activities were VIII, X and IX, the last being <50% after 6 months of storage.⁵ In our study, changes in factor IX activity were considered not to be statistically significant (84.9% at T0 vs 71.4% at T1). This difference is probably due to species differences, test variability (10–15%), differences in sample size (with only seven units tested in the canine study) or the fact that factor IX mean activity was initially 61% before storage in the canine study. In another study performed in dogs, a good conservation of factor IX was observed over 5 years of storage at −30°C.⁸ Although a small decrease of factor X activity was observed in our study, the difference was not statistically significant. In humans, factors V and VIII are considered the most labile; however, a decrease in factor V was not detected in our study, and was also not detected in another study performed in canine plasma.^7,8 Conversely, a decrease in factor VIII has been commonly observed in canine and human FP, similarly to the feline FP in our study.

In the present study, factor V was the only specific factor to show increased activity after 1 year of storage. FFP units might contain certain amounts of non-functional platelets, and factor V is stored in platelets complexed with a polymeric alpha-granule protein multimerin.¹⁰ The physiology of feline platelets is different to those of humans or dogs. Therefore, a possible explanation for the increase in factor V during storage could be that feline platelets might contain more factor V, liberating it to plasma during storage. In human medicine, a thrombophilia named factor V resistance has been described, in which mutations in factor V make it resistant to inactivation by activated protein C, leading to the preservation of a higher specific factor activity.¹¹ In our study, the T1 group contained several units with factor V activity over 150%. A similar alteration to the one described in human factor V resistance could explain the high factor activity at T1, although this has not been yet described in cats. Another possibility could be that a technical error occurred during the analysis; however, every measurement was repeated twice and repeated if >10% discrepancy was observed. Furthermore, this phenomenon was not observed with specific factor activity measurements other than factor V.

The factors most affected by storage in our study were VIII, XII and VII. To decide on the critical factors for fFFP processing and storage, the clinical relevance of each factor has to be considered. This is especially important with regard to factor XII. Activated factor XII is the starting point of the contact or intrinsic pathway that leads to subsequent activation of factor XI.¹² While the rate of in vitro clot formation in the activated partial thromboplastin time (APTT) depends on this contact pathway activation, the contact system has no defined role in current models of in vivo haemostasis.¹² It has been observed in human medicine that deficiencies in the contact system proteins, including factor XII, prekallikrein and high-molecular-weight kininogen, are not associated with clinical signs of disease.¹³ In fact, the major physiological role of factor XII remains to be established.^12,13 In one study, 25 cats with factor XII deficiency were identified and none had experienced spontaneous haemorrhage or abnormal bleeding. Twenty of these cats had undergone ovariohysterectomy or castration, and none experienced haemorrhagic complications.¹⁴ Thus, factor XII deficiency is not considered an essential reason for transfusion in bleeding patients. In view of this information, factor VIII and factor VII, rather than factor XII, were considered the best candidates for being considered critical factors in the production and quality control of fFFP intended for transfusion in coagulopathic patients.

At the beginning of storage, the mean activities for all specific coagulation factors were >70%. The requirements and quality control of human products state that FFP must contain at least 70 IU of factor VIII per 100 ml (70% of specific factor activity) and similar quantities of the other coagulation factors and their natural inhibitors.¹ The Council of Europe states that 90% of FFP units must contain at least 70% of specific factor VIII activity in the first month of storage.¹⁵ Other guidelines, such as the ones of the British Society for Haematology, state that 75% of the units must contain at least 70% factor VIII activity.¹⁶ In our study, only 60% of units had >70% specific factor VIII activity in the first 2 weeks of storage (group T0). This could be due to variability between species (between human and feline plasma) or to differences in storage temperatures.

Two studies from human transfusion centres have addressed this fact: the first, published in 1992 and performed in six different transfusion centres, reported that only 40.9% of FFP units had <70% factor VIII, while the second study, published in 2018, reported that >70% of factor VIII activity was observed in 95% of the FFP units.^17,18 Both studies were performed in different countries, but the authors hypothesised that this could also reflect storage and management improvements over time, as 16 years separate both studies. In the future, the same improvements may be observed in feline plasma blood banking as more knowledge is gathered, allowing the improvement of storage protocols.

Multiple factors might influence specific coagulation factor activities in fFFP, but those that are considered most relevant are the temperature of storage, the delay until separation from cells, the anticoagulant solution and the speed and temperature of plasma freezing.⁷

Freezing is a critical step in the preservation of some plasma proteins. When the freezing rate is slow, solute diffusion adapts to the rate of ice crystal formation. Crystals are formed starting at the periphery of the unit and progressing to the interior, increasing the solute concentration in the centre of the unit. This phenomenon affects not only the coagulation factors, but also other solutes such as salts, reaching high central concentrations that, after prolonged contact, can inactivate factor VIII. If the freezing rate is high, ice formation overtakes solute displacement, forming small clusters of solutes trapped homogeneously in the ice, avoiding prolonged contact between highly concentrated salts and factor VIII molecules.¹⁹

FFP must be frozen within 24 h of extraction, and to achieve the highest concentration of coagulation factor activities, rapid freezing must be utilised, allowing for complete freezing within 1 h at a temperature lower than −25°C/30°C.^1,19 In our study all units were processed and frozen at −80°C within the first 24 h of extraction.

Plasma coagulation factor stability is also highly dependent on storage temperature.^{1,5,7,15,20,21} In a study of human FFP, the stability of factor VIII was tested by comparing four different temperatures over 6 months of storage.²⁰ Temperatures of −10°C, −17°C, −20°C and −30°C were applied to different samples, and a significant decrease in factor VIII activity was observed in units stored at −10°C and −17°C when compared with the units stored at lower temperatures.²⁰ In another study performed in human FFP stored at −40° C, the loss in factor V and VIII activities were 0.6% and 9%, respectively, over 3 years, showing that the recovery of coagulation parameters in FFP was not decreased below 70% of the starting activity and the factor levels remained in acceptable ranges.²¹

The Council of Europe recommendations (2017 version) are that human FFP units maintained under −25°C can be stored up to 36 months, but only 3 months is recommended if the temperature is maintained between −18°C and −25°C.¹⁵ In the 2020 edition of the American Association of Blood Banks guidelines, the recommended storage temperature was lower than −30°C, and the World Health Organization states that the optimal storage temperature is below −25°C.^15,22

In our study, following standard veterinary recommendations for 1 year of storage, while also trying to demonstrate what practitioners are able to do when storing plasma in their own practice, storage temperature was maintained between −18° C and −25°C.^5,23 Decreasing the storage temperature of feline plasma units could improve the conservation of coagulation factors, as previously observed in human plasma storage, and thus improve the therapeutic effect.²⁰ However, further studies are needed to confirm this hypothesis.

Recognition of coagulation factors that are most labile in feline plasma stored up to 1 year could help us to understand the optimal volume of fFFP needed to administer 10 IU/kg of functional coagulation factors. The effective control of active bleeding in dogs with factor IX deficiency using a dose of 10 U/kg of factor IX has been described in one study. In another study, where an increase of factor VIII activity was tested in non-bleeding dogs, a transfusion of approximately 15 U/kg of factor VIII in German Shepherd dogs with haemophilia A resulted in a factor increase of >30%.^5,24 To our knowledge, there is currently no corresponding published information in feline medicine.

One millilitre of fresh plasma is considered to contain approximately 1 IU of each coagulation factor activity.⁵ The loss of activity during storage could mean that the volume of stored FFP needed for transfusion may need to be larger than that of fresh plasma to attain the same therapeutic effect.

Considering that 10 IU/kg could be a target therapeutic dose for most cats with factor deficiency (information extrapolated from dogs), and on the basis of specific factor activities in the present study, it was calculated that approximately 10–15 ml/kg fFFP stored for up to 1 year could be necessary to provide a minimum of 10 IU/kg of factors II, V, IX, X and XI; 10–17 ml/kg for factors VII and XII; and approximately 13–20 ml/kg of fFFP for factor VIII, depending on the storage times (<2 weeks or 1 year, respectively). In each individual case, there are differences in target concentration, factor yield and potential pharmacokinetic properties, and thus these dose recommendations must be considered as guidelines only for the purpose of initial dosing.

These recommended doses are similar to those that have been reported in humans, in which 10–20 ml/kg is often recommended.^25–28 In a retrospective study in dogs, the reported mean doses used were 13.9 ml/kg in small dogs and 5.1 ml/kg in large dogs. Another study reported that a median of 16 ml/kg was administered to every dog, regardless of its size.^29,30 Veterinary textbooks recommend initial doses ranging from 6 to 20 ml/kg, without specifying the species.^3,31

In a recent retrospective feline study, the FFP doses commonly used (mean 6 ml/kg) were lower than those reported for coagulopathy in dogs and humans.⁴ Only 33% of the recipients were bleeding at the time of transfusion in this feline study, so most of the transfusions were performed with a prophylactic goal.⁴ In another recent study, doses of 2.15–10.85 ml/kg were effective for the treatment of prolonged prothrombin time and/or APTT in cats. Further in vivo prospective studies evaluating different fFFP doses are warranted.

This study had some limitations inherent to an in vitro study and secondary to its design. The measurement of specific coagulation times has been reported to be variable, with interassay coefficients of variation for factors VII and IX surpassing 10% reported in a previous study with canine plasma.⁵ Thus, the exact magnitude of their loss was difficult to define in our study. In an attempt to account for this variability, each factor activity was measured twice for every unit and repeated if >10% discrepancy was observed, and a single lot of reagents was used, as different reagents and substrate plasmas could contain different residual levels of coagulation factor activity, affecting the linearity of standard curves.³²

Another limitation was that when calculating the recommended dose, information on the number of IUs required for clinical effectiveness was extrapolated from canine publications and calculated along with in vitro results of the present study. Further feline in vivo studies should be performed to confirm our dose recommendation.

Additionally, for testing purposes, all units were thawed at room temperature and 1.5 ml samples were taken and refrozen at −80°C within 30 mins. We have to consider that this additional freeze–thaw cycle procedure may have affected haemostatic stability. In a previous article performed with feline and canine plasmas, a cycle of thawing and refreezing within 1 h was demonstrated to have no deleterious effect on haemostatic protein activity, including fibrinogen and factors II, VII, VIII, IX, X, XI and XII.³³ Furthermore, we performed a similar pilot study using pooled plasma from 10 healthy cats and compared the results with direct testing after thawing and after a cycle of thawing and refreezing. These results showed no differences and therefore it is considered unlikely that the freeze–thaw cycle had any major influence on the results of the present study.

Conclusions

Lower values were identified for specific coagulation factor activities for factors II, VII, VIII, XI and XII after 1 year of storage at a temperature between −18°C and −25°C when compared with T0, although most were still over the minimally acceptable ranges for a healthy cat. Considering the results of this study, the most suitable factors for fFFP quality control are factors VII and VIII, the latter having the lowest values after 1 year of storage at a temperature between −18°C and −25°C, with its recovery reduced to 65% of the starting activity. Considering this, a dose of approximately 13–20 ml/kg, depending on the storage time, is needed to ensure the administration of at least 10 U/kg of each of the specific coagulation factors tested in this study.

Footnotes

Acknowledgements

The authors thank all cats and owners attending the Animal Blood Bank.

Accepted: 26 June 2022

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

The work described in this manuscript involved the use of non-experimental (owned or unowned) animals. Established internationally recognised high standards (‘best practice’) of veterinary clinical care for the individual patient were always followed and/or this work involved the use of cadavers. Ethical approval from a committee was therefore not specifically required for publication in JFMS. Although not required, where ethical approval was still obtained, it is stated in the manuscript.

Informed consent

Informed consent (either verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work (either experimental or non-experimental animals, including cadavers) for all procedure(s) undertaken (either prospective or retrospective studies). No animals or people are identifiable within this publication, and therefore additional informed consent for publication was not required.

ORCID iD

Carles Blasi-Brugué

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