JPAC Joint United Kingdom (UK) Blood Transfusion and Tissue Transplantation Services Professional Advisory Committee

8.2: Evaluation of new red cell components for transfusion

8.2.1: Introduction

In establishing any novel component, the development process is expected to involve three stages:

  • Investigation: Initial intensive investigation of a range of parameters on a relatively small number of units to establish concepts. This should involve in vitro studies with serial sampling, and may also involve in vivo studies. Components produced during this phase should not be used for transfusion.
     
  • Validation: Operational validation on a larger number of units (e.g. 50 to 100) to establish routine operation of the technique, normally testing for those parameters listed in the current edition of the Red Book. These tests may be supplemented by a limited set of assays selected from the investigational phase to allow setting of routine quality parameters. This may involve in vivo studies and normally would involve sampling at the times shown below for routine testing.
     
  • Routine: Ongoing routine validation using a small set of parameters selected on the basis of the above studies. This will not normally involve in vivo studies. For clarity the guidance on which tests need to be performed is as shown in Table 8.2. Advice may be sought from SACBC on the validation requirements for red cells produced from automated processing of whole blood or other technologies that are not specified in Table 8.2.

Red cell components may be derived either from whole blood or collected by apheresis and, in either case, the standard requirements for donor selection and for mandatory donation microbiological testing should be fulfilled. When well prepared, there is no evidence that the clinical performance of any of these products is different, and the guidance provided below applies equally to the various approaches.

In vitro assays should be performed on samples representative of the pack contents taken by an aseptic technique that does not appreciably alter the gross volume of the pack contents (must be kept to a minimum but in any case no greater than 10%). Parallel testing of units prepared by a well-established method is recommended, and the use of a split-pool or crossover design will increase the power of such comparisons. If required, in vivo studies, preferably with parallel testing of ‘standard’ components, should be performed on the last day of the proposed storage period. The number of units studied should be determined by statistical analysis based on the difference between test and control units to be detected. A sample size of at least 12 tests or controls would be required to detect a 30% difference in ATP and potassium at Day 35 of storage using an unpaired study. Fewer units will be required if a pooled and split study design is used, but should not be less than four.

Red cell components will be stored for the recommended storage period or longer in the case of experimental additive solutions (AS) that are designed to extend the shelf life of red blood cells (RBC). Samples will be taken weekly (or minimally at Days 1, 21, 35 and at the end of storage if this is >35 days) for in vitro studies. If required, autologous in vivo recovery studies should be undertaken at the end of the storage period.

8.2.2: In vitro studies

The measurements described below and in Table 8.2 will be made at the time of component production (Day 0/Day 1) or other relevant stages of component preparation. An equal number of appropriate control components (e.g. standard AS RBC) should be tested in parallel. Greater consistency of information may be obtained if two or more group-compatible components are pooled and divided prior to processing for in vitro studies only. The number of units to be studied should be based on the study objectives and design.

8.2.3: On the day of component production/collection

Weight, volume, haematocrit (L/L), haemoglobin (Hb, g/unit), platelets (x 109/unit), red cell loss* (%), platelet loss* (%), leucocyte depletion (given as residual WBC × 106/unit) and log depletion*. These results should be obtained by validated test procedures and be within the limits defined by the preliminary component specification.

* Relevant to procedures involving integral filtration or other methods that are likely to result in loss of cellular components during production. Validated techniques using flow cytometry or cell counting chambers should be used to count leucodepleted components and would currently be expected to exhibit a sensitivity of less than or equal to 1 leucocyte per microlitre.

Table 8.2 Evaluation of new red cell components for transfusion: recommended tests

New characteristic parameter New pack Leuco-depletion New centrifugation/component extractor (e.g. Optipress, Compomat etc.) Novel anti-coagulant Novel apheresis system Novel additive solution Irradiation Pathogen reduction

Unit volume (mL)

Haematocrit (L/L)

Haemoglobin
(g/unit)

MCV

WBC (x 106/unit) (post-leucodepletion)

 

 

 

 

 

 

 

Leucocyte subsets (%) (post-leucodepletion)

 

?

 

 

 

 

 

 

Residual platelets
(x 109/unit)

 

 

 

 

 

 

Hb loss (g)
(post-filter)

 

 

 

 

 

 

K+ (mmol/L)

Haemolysis (%)

pH

 

 

 

 

Lactate (mmol/L)

 

 

 

 

Glucose (mmol/L)

 

 

 

 

ATP (µmol/g Hb)

 

 

2,3-DPG
(µmol/g Hb)

 

 

 

 

Na+ (mmol/L)

 

 

 

 

pCO2 (kPa)

 

 

 

 

pO2 (kPa)

 

 

 

 

Pathogen reduction*

 

 

 

 

 

 

 

 

Prion protein (PrPc) and microvesicles

 

?

 

 

 

 

 

?

24-hour recovery (%)

 

 

 

?

 

?

?

Some components may need to be tested for a combination of parameters, e.g. apheresis red cells in a novel/experimental additive solution (AS) that are also leucodepleted. In this case the sampling requirement includes that of a leucodepleted red cell component and that of an experimental AS component.

Key: = recommended; ? = optional; other tests are not excluded. * = normally undertaken by the manufacturer.

At the end of the storage period components should be checked for sterility and a representative sample labelled with 51Cr (single label method) or 51Cr plus 131I-albumin (dual label method), washed, reinjected and blood samples taken at 5, 7, 10, 12.5, 15 minutes and 24 hours to calculate the 24-hour recovery. Results will be considered acceptable if the mean 24-hour recovery is >75% with a standard deviation of <9%. Alternative methods to 51Cr labelling may be used if shown to yield equivalent results.

8.2.4: During storage

Parameters to be studied during storage of red cells include: haemoglobin, haematocrit, MCV, ATP, 2,3-DPG, glucose, lactate, potassium, haemolysis (soluble haemoglobin as a percentage of total haemoglobin per mL of whole product), pH, pO2, pCO2, cytokines. These may include interleukin-1α, IL-1β, IL-6, IL-8, TNF-α and TGF-β. Measurements should, wherever possible, be by bioassay (seek advice from SACBC). Cytokine measurements are complex and may be considered optional. As red cell components are leucocyte depleted, measurement of leucocyte-derived cytokines is probably not informative. Advice should be taken from SACBC on the selection of cytokine tests.

These results should be obtained by validated test procedures. Where manipulation of components during processing might increase the risk of bacterial contamination, microbiological sterility testing should be performed at the end of storage.

8.2.5: Autologous in vivo studies

See Table 8.2 for details of testing. An equal number of appropriate control components obtained from healthy volunteer donors with ethical approval (e.g. standard AS RBC) should be tested in parallel. The number of components transfused should be justified based on the study objectives and design.