Official Journal of the British Blood Transfusion Society
Quality control of buffy coat removed red cell concentrates – a Croatian experience T. Vuk,T. Oˇci´c, M. Strauss Patko & I. Juki´c Croatian Institute of Transfusion Medicine, Zagreb, Croatia Received 3 October 2013; accepted for publication 16 November 2014
SUMMARY Objectives: Results are presented of the statistical quality control of red cell concentrate buffy coat removed in additive solution (RCC/BC/AS) and red cell concentrate buffy coat removed and leucoreduced in additive solution (RCC/BC/LR/AS) produced at the Croatian Institute of Transfusion Medicine during an 8-year period (2005–2012). The aim was to assess quality conformity of these products with specified requirements, as well as the suitability and justification of current regulations on the minimal quality requirements.
Key words: buffy coat, quality control, red cell concentrates.
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The procedures for blood product preparation and quality control differ substantially from those in pharmaceutical industry. The initial materials from which blood products are prepared are characterised by their hereditary variability, manifest as different cell and protein concentration and function. The quality and safety of final products are influenced not only by this variability, but also by an array of factors related to blood collection, manufacture, storage, transportation and blood product manipulation. Therefore, quality assurance and hemovigilance in all segments of transfusion service are preconditions for safe and efficient transfusion treatment, with blood product quality control as an important tool to reach these goals. Continuous and comprehensive quality control enables timely recognition of deviations in product quality, which is necessary for implementation of appropriate corrective/preventive measures. At the Croatian Institute of Transfusion Medicine (CITM), quality control is performed according to legal regulations, which have been harmonised with the Directive 2004/33/EC (European Commission, 2004) and Council of Europe Recommendations (EDQM, 2008). Red cell concentrates (RCCs) are the most frequently used blood products. They are essential drugs because of their unique role in the correction of acute and chronic anaemia. According to the method of preparation, there are several RCC types, which generally differ by the content of cell elements and type of anticoagulant and additive solutions. One of the most common methods of RCC preparation is separation of plasma and buffy coat from whole blood with the addition of additive solution to red cells, thus reducing leucocyte and platelet content while losing a portion of red cells. At CITM, the preparation of this RCC type was introduced in 2001, accounting for 94% of the primary RCC production in the 2005–2012 period (the remaining 6% was filtered CPDA-1 RCC). In the same period, the rate of filtration of these products was continuously increased. Thousands of products tested during the study period enabled relevant assessment of their quality, as well as of the standard requirements used in this assessment. The results reported here may prove to be useful to those working in transfusion medicine for comparison or in deciding on the introduction of
© 2014 British Blood Transfusion Society
First published online 3 December 2014 doi: 10.1111/tme.12167
Methods: The measurements of all the study parameters of the products analysed are expressed using descriptive statistics and graphs showing the distributions of observed parameters. Results: In RCC/BC/AS, the mean (±SD) volume was 279 ± 17 mL; haematocrit, 0·60 ± 0·03 L L−1 ; haemoglobin content, 55 ± 5 g; leucocyte count, 0·65 ± 0·41 × 109 ; and haemolysis at expiry date, 0·16 ± 0·13%. In RCC/BC/LR/AS (post-production filtration), the mean (±SD) volume was 255 ± 14 mL; haematocrit, 0·60 ± 0·02 L L-1 ; haemoglobin content, 51 ± 4 g; leucocyte count, 0·11 ± 0·16 × 106 ; and haemolysis at expiry date, 0·11 ± 0·07%. In RCC/BC/LR/AS (inline filtration), the mean (±SD) volume was 254 ± 15 mL; haematocrit, 0·61 ± 0·02 L L−1 ; haemoglobin content, 51 ± 5 g; leucocyte count, 0·04 ± 0·06 × 106 ; and haemolysis at expiry date, 0·16 ± 0·10%. The standards were just met for leucocyte count in RCC/BC/AS (90%), whereas for all other parameters satisfactory results were obtained in at least 99% of products analysed. Total incidence of bacterial contamination was 0·23% for all products. Conclusion: Results of the RCC/BC/AS and RCC/BC/LR/AS quality control showed very high conformity with the specified requirements in the majority of study parameters, suggesting that the current requirements could be redefined and improved at the institutional level.
Correspondence: Tomislav Vuk, Quality Assurance Department, Croatian Institute of Transfusion Medicine, Petrova 3, HR-10000 Zagreb, Croatia.
386 T. Vuk et al. this method in routine use, whereas clinicians can use them to evaluate the therapeutic effect of these products.
MATERIALS AND METHODS Results of the statistical quality control of RCC produced at CITM by buffy coat method during an 8-year period (2005–2012) are presented, including products submitted to inline or post-production leucofiltration.
Preparation of RCC/BC/AS Donor blood (450 ± 45 mL) was collected into multiple (PVC3 and PVC4 with inline RBC filter) TAB bags (top and bottom) with 63 mL of CPD (citrate, phosphate and dextrose) anticoagulant solution in the collection bag and 100 mL of SAGM (saline, adenine, glucose and mannitol) additive solution for RBC resuspension. Whole blood was kept at room temperature without cooling plates until production. Within 6 h of collection, the blood in PVC3 CPD-SAGM TAB bags was centrifuged for 15 min at 2998 × g and 22 ∘ C, with acceleration grade 5 and stopping grade 3, and the blood in PVC4 CPD-SAGM TAB bags (with filter) for 15 min at 4138 × g and 22 ∘ C, with acceleration grade 7 and stopping grade 3. The Cryofuge 6000i (Heraeus Instruments, Osterode, Germany) blood centrifuges were used. The Optipress II (Baxter, Fenwal Division, Deerfield, IL, USA) device was used to separate blood into components. RBC filtration was performed inline (PVC4 bags) or within 24–48 h of collection (PVC3 bags). Post-production filtration was performed by use of BPF4 filter (Pall Corporation, Westbury, NY, USA) and inline filtration by using Leucolab LCG2 filters (Macopharma, Tourcoing, France). Shelf life of all RCC types was limited to 35 days from blood collection, with storage at 4 ± 2 ∘ C.
Quality control planning and sampling Blood products were sampled according to the quality control plan, designed to enable continuous and comprehensive surveillance of the manufacturing process. Bacteriological testing of blood products was carried out using the German specification described in Minimal Requirements for Sterility Testing of Blood Components (Arbeitskreis Blut, 1997), where the frequency of bacteriological testing of blood products is calculated using the √ formula n × 0·4, n standing for the number of RCC produced per month. The frequency was calculated for each type of RCC separately. Products within 3 days of the expiry date were used for bacteriological testing. On the day of testing, a retention sample was taken in a 150-mL sterile welded bag and kept until the end of bacteriological testing or an initial signal of a positive result. Sampling for testing of other parameters was performed in line with the EU Directive 2004/33/EC (European Commission, 2004) and Guide to the Preparation, Use and Quality Assurance of Blood Components (EDQM, 2008), which defines the time and frequency of testing. For determination of haematological
Transfusion Medicine, 2014, 24, 385–391
parameters (haemoglobin, haematocrit and leucocytes), samples were taken into 150-mL sterile welded bag upon completion of the manufacturing process. Testing for stability (determination of haemolysis at expiry) was performed on whole units of blood products at expiry date (storage day 35). Table 1 shows the quality requirements for measurement of particular parameters and frequency of their testing in all types of RBC products analysed. The specified requirement of leucocyte count should be met in 90%, and the requirement of microbiological purity in 100% of products tested. According to regulations, quality requirements for other parameters should be met in at least 75% of products tested.
Methods of testing During the study period, all tests were performed at CITM Department of Quality Control, with the exception of microbiological testing performed at CITM Department of Microbiology since 2008. Volume determination. For RCC volume determination, the mass of empty blood bag and plastic tube segments for cross-matching was subtracted from the product total mass measured on a precise SB 8000 Mettler Toledo scale, and the result was divided by the blood component specific weight. A specific weight of 1·06 g mL−1 was used for all products. Determination of haematological parameters. Determination of haematological parameters in the RBC product samples was done using an Abbott Cell-Dyn 3200 haematology analyser (Abbott Diagnostics, Santa Clara, CA, USA). The products were analysed after 10-min incubation and mixing in 3-mL tube with 5·4 mg K2 EDTA. Haemoglobin content in the unit was calculated as haemoglobin concentration determined in the sample (g L−1 ) multiplied by unit volume (L). The original haematocrit value determined by the analyser was used as the product haematocrit value and expressed as L L−1 (litre of packed cells per litre of whole blood). The procedure of validation confirmed counting of leucocytes in RCC/BC/AS samples on the Cell-Dyn 3200 analyser as reliable, precise and linear. When the measurement result was accompanied by the suspect ‘WBC’ flag, the sample was retested using the respective programme on the analyser (programme for resistant RBCs or fragile leucocytes), and if the suspect flag persisted, the leucocyte count was verified in the Nageotte chamber. Determination of residual leucocytes in RCC/BC/LR/AS. Residual leucocytes in filtered RCC were determined by using the Nageotte chamber and light microscopy. Samples were diluted at 1 : 10 ratio with Na-oxalate solution to which crystal violet was added, and were incubated on a sample mixer for 10 min (the time needed for RBC lysis and staining of leucocyte nuclei). Leucocytes were counted after filling the 2 × 50-𝜇L chamber and allowing 10 min for the cells to settle down. Leucocytes were
© 2014 British Blood Transfusion Society
Quality control of buffy coat removed red cell concentrates
Table 1. Quality assurance of RCC prepared using buffy coat method Quality requirement Parameter Volume Haematocrit (L L−1 ) Haemoglobin Leucocyte count Haemolysis Sterility 1 Minimal
240–340 mL 0·50–0·70 Min. 43 g unit−1