Abstract
Introduction
Originally developed as a form of factor VIII concentrate, cryoprecipitate’s primary clinical use has changed to treat fibrinogen deficiency as highlighted by recent approval of pathogen-reduced cryoprecipitated fibrinogen concentrates. The methodology by which frozen plasma is thawed during cryoprecipitate manufacturing is not standardized. This study compared plasma thawing techniques on cryoprecipitate fibrinogen and factor VIII levels.
Methods
A matched pairwise experimental design was employed across three experiments to compare plasma thawing approaches (water bath or 24–48 h refrigerator). Each experiment involved the creation of 10 sets of ten homogenous frozen plasma pools which were then used to manufacture 10 pairs of cryoprecipitate pools differing only by assigned plasma thawing method. Total cryoprecipitate fibrinogen and factor VIII content between plasma thawing methods were compared using matched t-testing within each experiment.
Results
Compared to water bath thawing, 24-h refrigerator thawing led to significantly higher cryoprecipitate fibrinogen content (2,554 mg vs. 1,824 mg; p < 0.001) and significantly lower cryoprecipitate factor VIII content (601 IU vs. 709 IU; p < 0.001). Longer refrigerator thaw times (36 and 48 h) led to significantly higher cryoprecipitate fibrinogen content than 24-h refrigerator thaw (3,180 mg vs. 2,956 mg and 2,893 mg vs. 2,483 mg, respectively; p = 0.01–0.03).
Conclusion
Using homogenous frozen plasma units in a matched pairwise experimental design, refrigerator plasma thawing led to superior cryoprecipitate fibrinogen yields and inferior cryoprecipitate factor VIII yields. When maximizing cryoprecipitate fibrinogen yields, refrigerator plasma thawing, and in particular longer thawing times (36–48 h), should be considered.
Keywords: Cryoprecipitate, Fibrinogen, Factor VIII
Introduction
In the mid-1960s, Pool and colleagues published their seminal work on a reproducible process to manufacture cryoprecipitate from frozen plasma as a concentrated form of antihemophilic factor (factor VIII) to treat patients with hemophilia A [1, 2]. In that work, fibrinogen was noted to be the only other clotting factor with any significant concentration present. Since then, it has been demonstrated that cryoprecipitate also contains concentrated amounts of von Willebrand factor, factor XIII, and fibronectin [3–6]. While national standards continue to require minimum content amounts of factor VIII and fibrinogen for cryoprecipitate, the primary clinical indication for cryoprecipitate has shifted over the subsequent 60 years away from factor VIII replacement to fibrinogen replacement [7]. This shift in clinical usage away from factor VIII replacement is highlighted by the fact that pathogen-reduced cryoprecipitated fibrinogen complex has been recently approved by the United States Food and Drug Administration for treatment and control of bleeding due to fibrinogen, von Willebrand factor, or factor XIII deficiency but not for factor VIII deficiency [8, 9].
Previous research on cryoprecipitate manufacturing optimization (e.g., use of thaw-siphon systems, use of additional plasma additives) has focused on bolstering cryoprecipitate factor VIII yields [10–18]. With this shift in traditional cryoprecipitate’s use and the new availability of cryoprecipitated fibrinogen complex, optimization of the cryoprecipitate manufacturing process to boost fibrinogen recovery would help provide more clinically efficacious products for patients. Currently, one aspect of the cryoprecipitate manufacturing process that remains variable across practices is the initial method by which frozen plasma is thawed to 1–6°C (e.g., refrigerator vs. water bath) [19]. In this study, we aimed to measure the impact of the thawing technique (water bath, 24–48 h refrigerator thaw) on final cryoprecipitate pool fibrinogen and factor VIII content. Direct comparisons of plasma thawing technique can be challenging given that plasma fibrinogen and factor VIII content can vary widely between donors and hence detected differences found in cryoprecipitate content may be due to donor factors instead of thawing technique. To eliminate any inter-donor variability in fibrinogen and factor VIII content, a pre-pooled plasma approach was utilized in this study to permit direct matched comparisons between plasma thawing techniques.
Methods
A matched pairwise experimental design was selected to directly compare total fibrinogen and factor VIII cryoprecipitate pool content between pairs of plasma thawing techniques (experiment #1: 1–6°C water bath vs. 24 h refrigerator; experiment #2: 24 h refrigerator vs. 36 h refrigerator; experiment #3 24 h refrigerator vs. 48 h refrigerator). In each of these three pairwise experiments, 100 unique plasma units derived from whole blood collections at an AABB-accredited hospital-based blood bank were used. These plasma units were predominantly from HLA antibody-positive donors, and all ABO and RhD blood types were included.
Preparation of Homogenous Ten Pools of Fresh Frozen Plasma
Ten units of whole blood-derived plasma (stored refrigerated as liquid plasma after the packed RBC product manufacturing process) were combined by sterilely attaching and draining each plasma unit together into a 2.5 L accessory bag (shown in Fig. 1). The 10-pool of plasma was then thoroughly mixed and weighed, and a sample was drawn off and stored separately at ≤–20°C for fibrinogen and factor VIII concentration testing. This homogenous 10-pool of plasma was then sterilely attached back to the original ten plasma unit bags and separated back into a set of 10 homogenous plasma units of equal weight. This set of 10 homogenous plasma units was then completely frozen simultaneously using a blast freezer at ≤–20°C. Ten homogenous 10-pools of plasma were created, and each 10-pool was separated into a set of 10 identical frozen plasma units for each pairwise experiment.
Fig. 1.
Overview of experimental design. Ten whole blood-derived liquid plasma units are combined, mixed, sampled, and then redivided back into a set of ten homogenous plasma units and frozen for up to 2 days. From each set of 10 homogenous plasma units, five frozen plasma units each underwent one of the two assigned plasma thawing techniques and then manufactured into a cryoprecipitate pool of 5. Each of the two resulting cryoprecipitate pools was mixed and sampled. This process was repeated a total of ten times for each of the three pairwise experiments in this study. *Fibrinogen testing samples were diluted ×10 prior to testing.
Plasma Thawing Approaches
Sets of 10 homogenous frozen plasma units were thawed either the same day or within 1–2 days after freezing for the creation of cryoprecipitate. Five homogenous plasma units from each set were assigned to one of the two plasma thawing approaches being directly compared within each pairwise experiment (shown in Fig. 1).
Water bath thawing was performed by placing each frozen plasma unit into a plastic bag and then placing the bagged plasma unit into a 1–6°C circulating water bath with minimal manipulation until thawed [10, 11, 15]. Refrigerator thawing was performed by placing each frozen plasma unit flat on a shelf within a 1–6°C walk-in refrigerator with minimal manipulation for the assigned thawing time. Once all 5 units of plasma were thawed using the assigned thawing technique, existing institutional standard operating procedures were followed for manufacturing of whole blood-derived cryoprecipitate pools and completed within 1 h (11 min centrifugation at 5,000 g and 4°C to separate the cryoprecipitate and cryoprecipitate-poor plasma, siphon all but 25–30 g of plasma into satellite bag for use as cryoprecipitate-poor plasma, resuspend cryoprecipitate into remaining 25–30 g of plasma, and then sterilely pool 5 cryoprecipitate units into a single pooling bag) [10, 11, 15, 19]. After cryoprecipitate pooling, each cryoprecipitate pool was weighed, and a sample was collected and stored at ≤–20°C for fibrinogen and factor VIII concentration testing (shown in Fig. 1).
Fibrinogen and Factor VIII Testing
Frozen homogenous 10-pool plasma and cryoprecipitate pool samples were thawed and tested on clinical platforms for fibrinogen concentration (HemosIL fibrinogen-C, Werfen, Bedford, MA) and factor VIII activity (activated partial thromboplastin time with factor VIII deficient plasma, Werfen, Bedford, MA) by a CLIA-accredited coagulation reference laboratory. Cryoprecipitate pool samples required 10-fold sample dilution prior to fibrinogen measurement, and the diluted fibrinogen concentration obtained was then multiplied by a factor of 10.
Statistical Analysis
Homogenous 10-pool plasma and cryoprecipitate pool net weights were converted to volumes using a 1.03 g/mL conversion factor. Total product fibrinogen concentration and factor VIII content (100% factor activity = 1 IU/mL) were then calculated from measured fibrinogen and factor VIII activity using these calculated product volumes. A total factor VIII content of 400 IU and fibrinogen content of 750 mg was deemed to meet national minimum content requirements for cryoprecipitate pools of 5 [19].
Percent cryoprecipitate recovery of total plasma fibrinogen and factor VIII was calculated as (total cryoprecipitate pool content)/(0.5 * total plasma 10-pool content) * 100%. Matched t tests were performed using R version 4.3.1 to compare cryoprecipitate pool total fibrinogen and factor VIII content between plasma thawing techniques within each pairwise experiment. A p value <0.05 was considered statistically significant.
Results
Thirty pairs of cryoprecipitate pools of 5 were manufactured across the three different matched pairwise experiments and involved the use of plasma of all ABO blood types (experiment #1: 52 O, 45 A, 13 B; experiment #2: 61 O, 27 A, 9 B, 3 AB; experiment #3: 50 O, 30 A, 14 B, 6 AB). Across all three pairwise experiments, the starting homogenous plasma 10-pools had overall similar total fibrinogen and factor VIII content (shown in Tables 1–2) and all cryoprecipitate pools met the national minimum requirements for total factor VIII content and twice the national minimum requirement for total fibrinogen content (shown in Fig. 2).
Table 1.
Total product fibrinogen content (mg) for initial homogenous plasma 10-pools and subsequent cryoprecipitate pools of 5 by assigned plasma thawing technique across 3 matched pairwise experiments
| Experiment | n | Total fibrinogen content, mg | p value | ||||
|---|---|---|---|---|---|---|---|
| plasma 10-pool | water bath | 24-h refrigerator | 36-h refrigerator | 48-h refrigerator | |||
| 1 | 10 | 9,120±786 | 1,824±225 | 2,554±375 | <0.001 | ||
| 2 | 10 | 9,342±557a | 2,956±443 | 3,180±469 | 0.03 | ||
| 3 | 10 | 9,238±684 | 2,483±236 | 2,893±575 | 0.01 | ||
Results are given as mean ± SD unless indicated otherwise.
p values calculated using matched t test.
aFibrinogen concentration not measured in one plasma 10-pool (n = 9).
Table 2.
Total product factor VIII content (IU) for initial homogenous plasma 10-pools and subsequent cryoprecipitate pools of 5 by assigned plasma thawing technique across 3 matched pairwise experiments
| Experiment | n | Total factor VIII content, IU | p value | ||||
|---|---|---|---|---|---|---|---|
| plasma 10-pool | water bath | 24-h refrigerator | 36-h refrigerator | 48-h refrigerator | |||
| 1 | 10 | 2,685±651 | 709±101 | 601±79 | <0.001 | ||
| 2 | 10 | 2,785±358a | 573±88 | 529±98 | 0.005 | ||
| 3 | 10 | 2,769±478 | 612±67 | 590±41 | 0.1 | ||
Results are given as mean ± SD unless indicated otherwise.
p values calculated using matched t test.
aFactor VIII concentration not measured in one plasma 10-pool (n = 9).
Fig. 2.
Total cryoprecipitate pool fibrinogen (top) and factor VIII (bottom) content across three matched pairwise plasma thawing experiments.
In experiment #1, the 24-h refrigerator plasma thawing technique led to significantly higher cryoprecipitate total fibrinogen content and significantly lower cryoprecipitate total factor VIII content compared to the water bath plasma thawing technique (shown in Tables 1–2). These differences in cryoprecipitate total fibrinogen and factor VIII content were observed across all 10 pairs of cryoprecipitate pools manufactured in this experiment (shown in Fig. 2). On average, the 24-h refrigerator thaw technique yielded 56% and 45% cryoprecipitate recovery of total plasma fibrinogen and factor VIII, respectively, while the water bath thaw technique yielded 40% and 53% cryoprecipitate recovery of total plasma fibrinogen and factor VIII, respectively.
In experiment #2, the 36-h refrigerator plasma thawing technique led to significantly higher cryoprecipitate total fibrinogen content and significantly lower cryoprecipitate total factor VIII content compared to a 24-h refrigerator plasma thawing technique (shown in Tables 1–2). These differences were more modest than the differences seen in experiment #1 and not seen consistently across all pairs of cryoprecipitate pools (shown in Fig. 2). On average, the 36-h refrigerator thaw technique yielded 68% and 38% cryoprecipitate recovery of total plasma fibrinogen and factor VIII, respectively, while 24-h refrigerator thaw technique yielded 63% and 41% cryoprecipitate recovery of total plasma fibrinogen and factor VIII, respectively.
In experiment #3, the 48-h refrigerator plasma thawing technique led to significantly higher cryoprecipitate total fibrinogen content compared to a 24-h refrigerator plasma thawing technique (shown in Tables 1–2), and this increase in total fibrinogen content was seen in 9 of the 10 pairs of cryoprecipitate pools manufactured in this experiment (shown in Fig. 2). On average, the 48-h refrigerator thaw technique yielded 63% and 43% cryoprecipitate recovery of total plasma fibrinogen and factor VIII, respectively, while 24 h refrigerator thaw technique yielded 54% and 44% cryoprecipitate recovery of total plasma fibrinogen and factor VIII, respectively.
Discussion
With the primary use of cryoprecipitate shifted away from factor VIII replacement to fibrinogen replacement and the recent development of pathogen-reduced cryoprecipitated fibrinogen concentrates, optimizing the cryoprecipitate manufacturing process to maximize fibrinogen recovery can help provide more effective blood product support for patients [8, 9]. Twenty-four-hour refrigerator plasma thawing consistently provided higher cryoprecipitate fibrinogen content than water bath plasma thawing and increasing the refrigerator plasma thawing process to 36 h and 48 h led to, on average, additional cryoprecipitate fibrinogen content gains. In an opposing fashion though, cryoprecipitate factor VIII content was significantly lower with refrigerator thawing than water bath thawing.
All plasma thawing techniques explored in this study led to cryoprecipitate pools meeting the minimal national requirement for factor VIII (400 IU) and twice the minimal national requirement for fibrinogen (1,500 mg) for traditional cryoprecipitate. While traditional cryoprecipitate manufacturing requires balancing both minimum fibrinogen and factor VIII content requirements, pathogen-reduced cryoprecipitated fibrinogen concentrate manufacturing only needs to meet minimum fibrinogen content requirements [8, 9]. Since the package insert for pathogen-reduced cryoprecipitated fibrinogen concentrate does not specify a plasma thawing approach in the manufacturing process, this work demonstrates that refrigerator plasma thawing will likely bolster fibrinogen content recovery compared to water bath thawing and create a more potent product for patients [9].
One strength of this work is the use of pre-pooled plasma to create identical homogenous plasma units for freezing and cryoprecipitate manufacturing between experimental arms. Such an approach eliminates any donor variability in plasma fibrinogen and factor VIII content that can easily confound cryoprecipitate content comparisons and permits more powerful matched statistical comparisons that can better detect any true differences between experimental groups. Furthermore, this study was conducted within an AABB-accredited blood component manufacturing laboratory following long-established and quality-controlled clinical standard operating procedures for cryoprecipitate manufacturing from whole blood-derived plasma. The main weakness of this study is that these matched experiments comparing plasma thawing approaches were performed in a pairwise fashion instead of simultaneously for all 4 plasma thawing approaches. Such an approach was utilized because of volume limitations on the size of pre-pooled plasma bags available for these experiments (2.5 L) which would not permit the pre-pooling and mixing of 20 whole blood-derived plasma units.
In summary, 24-h refrigerator plasma thawing leads to significantly higher cryoprecipitate fibrinogen content and significantly lower cryoprecipitate factor VIII content than water bath thawing. Moreover, longer refrigerator plasma thawing to 36 or 48 h further boosts on average cryoprecipitate fibrinogen content.
Statement of Ethics
All blood donors signed a consent form with each blood donation permitting deidentified unused components of their donation to be used by Transfusion Medicine divisional staff for quality and transfusion-related research. This work was determined to not require ethics approval by the Mayo Clinic Institutional Review Board (documentation provided to the journal editorial board). Ethical approval was not required for this study in accordance with local/national guidelines.
Conflict of Interest Statement
The authors have no conflicts of interest to disclose.
Funding Sources
There were no funding sources for this work.
Author Contributions
J.E.J., J.A.S., and M.A.S. were involved in the conception and experimental design of this work. J.A.S. and M.A.S. were involved in the acquisition of the case report data. All authors were involved in the analysis and interpretation of the case report data. J.E.J. prepared the original draft of this research article, and all authors then participated in manuscript revisions before submission. All authors approved the final version of this manuscript for publication and agree to be accountable for all aspects of this work.
Funding Statement
There were no funding sources for this work.
Data Availability Statement
All datasets generated during this work contain only deidentified data and therefore will be made available to anyone upon request to the study authors.
References
- 1. Pool JG, Gershgold EJ, Pappenhagen AR. High-Potency antihaemophilic factor concentrate prepared from cryoglobulin precipitate. Nature. 1964;203:312. [DOI] [PubMed] [Google Scholar]
- 2. Pool JG, Shannon AE. Production of high-potency concentrates of antihemophilic globulin in a closed-bag system. N Engl J Med. 1965;273(27):1443–7. [DOI] [PubMed] [Google Scholar]
- 3. Bennett E, Dormandy K. Pool’s cryoprecipitate and exhausted plasma in the treatment of von Willebrand’s disease and factor-XI deficiency. Lancet. 1966;2(7466):731–2. [DOI] [PubMed] [Google Scholar]
- 4. Barrett KE, Israels MC, Burn AM. The effect of cryoprecipitate concentrate in patients with classical haemophilia. Lancet. 1967;1(7483):191–2. [DOI] [PubMed] [Google Scholar]
- 5. Allain JP. Non Factor VIII related constituents in concentrates. Scand J Haematol Suppl. 1984;41:173–80. [DOI] [PubMed] [Google Scholar]
- 6. Caudill JS, Nichols WL, Plumhoff EA, Schulte SL, Winters JL, Gastineau DA, et al. Comparison of coagulation factor XIII content and concentration in cryoprecipitate and fresh-frozen plasma. Transfusion. 2009;49(4):765–70. [DOI] [PubMed] [Google Scholar]
- 7. AABB . Circular of information for the use of human blood and blood components; 2021. Available from: https://www.aabb.org/docs/default-source/default-document-library/resources/circular-of-information-watermark.pdf [Google Scholar]
- 8. FDA . Approval order for INTERCEPT blood system for plasma. In: Administration FD; 2020. [Google Scholar]
- 9. FDA . INTERCEPT blood system for cryoprecipitation package inset. In: Administration FD, editor. 2021. p. 25. [Google Scholar]
- 10. Mason EC. Thaw-siphon technique for production of cryoprecipitate concentrate of factor VIII. Lancet. 1978;2(8079):15–7. [DOI] [PubMed] [Google Scholar]
- 11. Mason EC. Thaw-siphon technique for factor VIII cryoprecipitate. Lancet. 1978;2(8090):636–7. [DOI] [PubMed] [Google Scholar]
- 12. Prowse CV, McGill A. Evaluation of the “Mason” (continuous-thaw-siphon) method for cryoprecipitate production. Vox Sang. 1979;37(4):235–43. [DOI] [PubMed] [Google Scholar]
- 13. Rock GA, Cruickshank WH, Tackaberry ES, Palmer DS. Improved yields of factor VIII from heparinized plasma. Vox Sang. 1979;36(5):294–300. [DOI] [PubMed] [Google Scholar]
- 14. Kang EP. An improved thaw-siphon method for the cryoprecipitate preparation. Vox Sang. 1980;38(3):172–7. [PubMed] [Google Scholar]
- 15. Mason EC, Pepper DS, Griffin B. Production of cryoprecipitate of intermediate purity in a closed system thaw-siphon process. Thromb Haemost. 1981;46(02):543–6. [PubMed] [Google Scholar]
- 16. Rock G, Smiley RK, Tittley P, Palmer DS. In vivo effectiveness of a high-yield factor VIII concentrate prepared in a blood bank. N Engl J Med. 1984;311(5):310–3. [DOI] [PubMed] [Google Scholar]
- 17. Cumming AM, Wensley RT, Winkelman L, Lane RS. A simple plasma anticoagulant-exchange method to increase the recovery of factor VIII in therapeutic concentrates. Vox Sang. 1990;58(4):264–9. [DOI] [PubMed] [Google Scholar]
- 18. Yousef H, Neurath D, Freedman M, Rock G. Cryoprecipitate production: the use of additives to enhance the yield. Clin Lab Haematol. 2006;28(4):237–40. [DOI] [PubMed] [Google Scholar]
- 19. AABB . AABB standards: blood banks and transfusion services. 33rd ed. AABB; 2022. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All datasets generated during this work contain only deidentified data and therefore will be made available to anyone upon request to the study authors.