In vitro evaluation of novel FFP
Suggested study design
Because of the wide normal range of some clotting factors and potential inter-batch variation of assays, it is suggested that initially 20 novel units and 20 controls be produced and assayed in parallel, with the novel technology being the only variable. A less costly alternative, if logistics permit, is to do a pooled paired comparison, where two units are pooled, and one half processed by the novel technique. This provides greater statistical power for fewer units assayed, and is particularly important for storage studies. For LD or pathogen reduction systems it is recommended that assays are performed on samples collected before and after the process under investigation. Ideally provision should be made for storing and testing aliquots from each pack at every time point, as thawing out three or four different packs at each time point introduces excessive variation. However, a prevalidation should be done to ensure that the behaviour of the aliquotted component during storage is the same as that in the main pack.
Assays required
The extent of any evaluation depends in part on the degree of novelty of the component. The list of assays below need not be applied in every setting. The attached table gives a summary of which assays are recommended in different situations. All evaluations must include the routine quality control parameters such as FVIII:C.
Before freezing:
volume, platelet count, WBC*
prothrombin time (PT), activated partial thromboplastin time (APTT)
factors I (fibrinogen), II, V, VII, VIII, IX, X, XI, XIII von Willebrand factor (vWf):Ag, vWf:RiCof, which measures the functional activity or an assay validated as yielding equivalent results, vWf multimeric analysis, vWF cleaving protease
inhibitors of coagulation – antithrombin, protein C, protein S, α2-antiplasmin
markers of unwanted activation of coagulation* – prothrombin fragment 1.2, fibrinopeptide A, factor XIIa, thrombin-antithrombin (TAT) complexes
markers of unwanted activation of kinins/complement* – C3a, C5a, bradykinin.
*Particularly relevant to plasma which has been collected by any filtration technique, in which case the assays should be performed before and after filtration or to packs made of novel materials.
During storage:
Consideration should be given to performing storage studies at >–20°C in addition to those at <–30°C to reflect hospital storage conditions. Samples should be taken at 6, 12 and 24 months. Ideally, all clotting factors should be assayed at each time point, if only in a few packs. FVIII should be assayed at each time point as a minimum in addition to the proteins most severely affected by the initial process
Storage parameters may be assayed after the date of implementation of routine production, provided data 'keep ahead' of the age of any clinical product which might be issued.
In vitro evaluation of novel cryoprecipitate
It is assumed that this will be produced from a 'novel' start plasma so that investigators will be aware of any specific losses of clotting factors which should be particularly considered.
Assays to be performed before and after production, and during storage: fibrinogen, FVIII:C.
Cryosupernatant
This component is increasingly used for plasma exchange procedures for patients with thrombotic thrombocytopenic purpura. Analysis of von Willebrand factor multimers and cleaving protease is therefore appropriate. vWf multimeric and cleaving protease analysis should be performed in a laboratory recognised to be proficient in this technique and which is performing the assay regularly.
In vivo studies
Whether or not in vivo studies are needed depends on the degree of novelty of the component, e.g. this may not be necessary for plasma which has been leucocyte depleted in the course of producing leucocyte depleted red cells, but would certainly apply in the case of a novel pathogen reduced plasma which had been exposed to chemicals. Unlike red cells and platelets, administration to normal volunteers has not been traditional. Suitable patient groups to consider would be:
For FFP:
correction of prolonged INR prior to liver biopsy
liver transplant recipients
plasma exchange for TTP
DIC.
It is difficult to get permission to study neonates and usually considerable experience has to have been gained with the product in adults.
A randomised design is preferred, with standard FFP as control.
For cryoprecipitate:
DIC
liver disease/transplant
congenital hypofibrinogenaemia, if maintained on cryoprecipitate.
Table 9.5 Evaluation of novel plasma components| | Fresh Frozen Plasma | Cryo-precipitate | Cryo-supernatant |
|---|
| Novel filter | New centrifuge/component extractor | Novel anticoagulant | Novel apheresis system | Novel apheresis + anticoagulant | Pathogen reduction |
|---|
| Volume |  | | | | | | | |
| Leucocyte content | | | | | | | | |
| FVIII:C | | | | | | | | |
| Platelets | | | | | | | – | – |
| PT ratio | | – | | – | | | – | – |
| APTT ratio | | – | | – | | | – | – |
| Fibrinogen | | – | | – | | | | |
| II, V, VII, IX, X, XI, XIII | | – | | – | | | – | – |
| vWf: Ag | | – | | – | | | | |
| vWf: RiCof | | – | | – | | | | |
| AT III, Prot C, Prot S | | – | | – | | | – | – |
| TAT/Frag1.2/FPA + FXIIa | | – | | | | | Omit if not elevated in source plasma |
| C3a + C5a | | – | | | | | | |
| C1 inhibitor | | – | | – | | | | |
| vWf Multimers | | – | | – | | | | |
| vWF cleaving protease | | – | | – | | | | |
| alpha-2 anti-plasmin | | – | | – | | | | |
| Pathogen reduction* | | | | | | | | |
| PrP c/microvesicles | ? | – | | | | | | |
| Clinical trial | – | – | # | # | # | | # | # |
Key: recommended. – = not needed. # = consider individually. * = normally undertaken by the manufacturer.