Toshio Mazda, Japanese Red Cross Central Blood Institute, Tokyo, Japan E-mail: firstname.lastname@example.org
The Japanese Red Cross is now emphasizing an intense quality control procedure for testing blood bags and for leukocyte counts in blood components. The inspections and tests are described and the effects and significance these have on blood product management of the supply of safe blood are presented.
The Japanese Pharmaceutical Affairs Law and governmental ordinances, especially the Regulation for Manufacturing Control and Quality Control of Drugs, require many control procedures for blood components. Quality control tests for blood components are usually conducted by sampling inspections of the final blood components. Each blood centre at the Japanese Red Cross (JRC) conducts these quality control measures, but to provide better quality control for blood components, the laboratory of the JRC Central Blood Institute has centralized its quality control measures so as to include acceptance inspections of raw materials and other materials, such as blood bags and solutions, and sampling inspections of leukocyte counting of blood components.
Centralized quality control of acceptance inspections of blood bags and solutions started in 2002. For protection against adverse effect on blood transfusions, the JRC introduced supply pre-storage leukocyte reduced blood components. The process to reduce the leukocyte number in platelet concentrates derived from aphaeresis donations (PC) started in October 2004, then in fresh frozen plasmas derived from aphaeresis donations (FFP) in March 2006, and lastly in blood components derived from whole blood donations (WB) in January 2007.
The methods, procedures and data for these centralized quality control inspections at the JRC Central blood Institute will be discussed in this paper.
Acceptance inspections of blood bags and solutions
Acceptance inspections and the standard requirements of blood collection sets containing transfer tubes and needles were determined according to the ministerial ordinance on the Standard Test Methods for Vinyl Chloride Resin Blood Sets. These tests were performed by physical, chemical and biological tests as follows:
• Physical requirements: visual inspection, size, air content, blood collection capabilities, transparency, thermal stability, water vapour transmission, resistance to leakage, marking and labelling, shape of needles, silicon content of needle surfaces, etc.
• Chemical requirements: residue on ignition test, heavy metal test, lead test, vinyl chloride monomer and extractable substances (pH, potassium-permanganate reducing substances, residue on evaporation, ultraviolet absorption spectrum and heavy metals).
• Biological requirements: sterility test and endotoxin test.
Polyolefin blood bags were tested by the Standard Test Methods for Vinyl Chloride Resin Blood Sets with the exception of the vinyl chloride monomer test.
Qualitative and quantitative analysis of anticoagulant-preservative solutions (CPD and ACD) in blood bags was tested according to the Minimum Requirements for Biological Products. Additive solutions (MAP) in blood bags and solutions for use in the preparation of blood components (saline, solutions using blood cryopreservation, etc.) were tested using our own test methods based on the Japanese Pharmacopoeia.
Lead test (combustion test)
Two grams of blood bags or tubing were cut into small pieces and then crushed. After the sample was moistened with 2 ml of sulphuric acid in a porcelain crucible, they were heated gradually from 100 to 300°C and then allowed to dry (1–2 days). After drying, samples were heated to 500°C for 5–6 h until combustion. To the combusted samples, 2 ml of hydrochloric acid and 2 ml of distilled water were added and the residue dissolved at 70–80°C for 2–3 min. After adding water to a volume of 20 ml, the sample was measured using inductively coupled plasma optical emission spectrometry, Vista MPX (Seiko Instruments Inc., Chiba, Japan).
Vinyl chloride monomer test
One gram of blood bag or tubing was cut into small pieces and then crushed. After adding 20 ml of tetrahydrofuran, samples were dissolved with cooling using a dry ice-methanol bath. Then samples were analyzed using gas chromatography GC-14B (Shimadzu, Kyoto, Japan) with flame ionization detector. The column was Ucon LB-550X 20% chromosorb W 80/100 AW (Shinwa Chemical Industries Ltd, Kyoto, Japan) at 60°C.
Determination of di-(2-ethylhexyl)phthalate (DEHP) content
The ethanol extracted DEHP from blood bags and tubing were assayed using a high performance liquid chromatography (HPLC) system. Blood bags or tubing were cut into small pieces by scissors and then DEHP extracted with 10 ml of 47·5% ethanol for 60 min at 37°C.
DEHP content in blood (plasma or packed red cells) was measured after deproteinization. One ml of blood sample and 2 ml of acetonitrile were mixed well. The mixture was centrifuged at 1000 g for 5 min. After filtering using a 0·45 μm membrane filter, ADVANTEC (Toyo Roshi Kaisha, Tokyo, Japan), the supernatant solution was injected into the HPLC system.
For DEHP determination, the HPLC Alliance 2695 system (Waters Corporation, Milford, MA, USA) with UV detection (272 nm) was used. The HPLC column was packed with Atlantis 4·6 × 150 (Waters Corporation). The elution fluid was methanol/H2O (95/5) at a flow rate of 1 ml/min at 30°C.
Determination of leukocyte count
Twenty-six JRC blood centres currently prepare blood components. Sampling inspection for leukocyte counts were conducted on 1 sample per 100 samples of components. The samples for leukocyte counting were fixed at these blood centres using Pallfix (Nihon Pall Ltd, Tokyo, Japan). After fixing, the samples were sent to the JRC Central Blood Institute, where they were stored at 2–8°C until tested.
Leukocyte counts were determined at the JRC Central Blood Institute by flow cytometry – FACS Calibur (BD Biosciences, San Jose, CA, USA) or Cytomics FC500/BC (Beckman Coulter Inc., Miami, FL, USA). LeucoCOUNT Kits from BD Biosciences were used to prepare the samples and to stain for flow cytometry. LeucoCOUNT Kits contain TruCOUNT Tubes and LeucoCOUNT Reagent. TruCOUNT Tubes contain an internal standard of TruCOUNT beads. One hundred μl samples were mixed with the TruCOUNT beads in the TruCOUNT Tubes before staining. Then, 400 μl LeucoCOUNT reagent containing a nucleic acid dye, i.e. propidium iodide is added to the TruCOUNT Tubes. After standing over 5 min at room temperature samples were applied to a flow cytometry. Leukocyte counts were determined using a simple calculation based on bead counts and sample volume.
The regulation for the residual leukocyte number is 95% of the blood components must be under 1 × 106/bag.
Acceptance inspections of blood bags and solutions
All test results for blood bags and solutions from 2002 to 2010 were within standard requirements. For example, the results of lead tests were as follows: 139 out of 149 blood bag samples and 125 out of 135 transfer tube samples contained less than 0·1 μg lead/g of sample. The remaining samples had from 0·1 to 0·2 μg lead/g of sample. Within the lead tests, results for polyolefin blood bags had 27 out of 28 blood bags with less than 0·1 μg lead and one bag with 0·1–0·2 μg lead/g sample. The standard level of lead in plastic bags and transfer tubes is less than 1 μg lead/g sample.
Vinyl chloride monomer was tested in 121 blood bag samples and 142 transfer tube samples. All samples had less than 0·025 ppm of vinyl chloride monomer. The standard level of vinyl chloride monomer in plastic bags and transfer tubes is less than 0·05 ppm.
Quantitative test results for CPD solution and for MAP are shown in Table 1.
Table 1. Results of quantitative tests of CPD and MAP solutions (2002–2010), n = 35. The data shown as w/v%, except pH (Y. Ishii, A. Takano, T. Mazda, unpublished data)
Citric acid (monohydrate)
0·32 ± 0·02
0·329 ± 0·005 (0·320–0·340)
Trisodium citrate (monohydrate)
2·63 ± 0·13
2·634 ± 0·021 (2·572–2·690)
Sodium dihydrogen- phosphate (dihydrate)
0·25 ± 0·01
0·250 ± 0·002 (0·246–0·255)
2·32 ± 0·12
2·319 ± 0·033 (2·249–2·377)
Citric acid (monohydrate)
0·020 ± 0·003
0·0216 ± 0·0007 (0·0200–0·0230)
0·15 ± 0·01
0·151 ± 0·004 (0·146–0·160)
Sodium dihydrogen- phosphate (dihydrate)
0·094 ± 0·005
0·0949 ± 0·0010 (0·0928–0·0976)
0·50 ± 0·03
0·502 ± 0·006 (0·492–0·520)
0·72 ± 0·07
0·707 ± 0·026 (0·687–0·790)
1·46 ± 0·07
1·449 ± 0·018 (1·390–1·483)
0·014 ± 0·001
0·0139 ± 0·0001 (0·0136–0·0142)
Blood samples in blood bags (packed red cells and plasma) were stored at 4°C and DEHP was determined after 3, 10, 17 and 24 days storage. DEHP content in blood samples are shown in Fig. 1. DEHP content increased during storage, but amounts differed by blood components.
DEHP content in blood in transfer tubes (segments) had the highest, 26·1 ± 5·3 mg/100 ml (range: 21·9–36·6, n = 6) for packed red cells and 44·7 ± 9·3 mg/100 ml (range: 33·1–51·3, n = 6) for plasma respectively when stored for 24 days at 4°C. This level is seven to eight times higher than for DEHP content in blood in blood bags, 3·23 ± 0·26 mg/100 ml (range: 2·94–3·59, n = 6) for packed red cells and 6·88 ± 0·49 mg/100 ml (range: 6·29–7·38, n = 6) for plasma.
However, DEHP in ethanol extracts from blood bags and transfer tubes were the same content. DEHP extracted by 47·5% ethanol from blood bags and transfer tubes was 7·61 ± 0·33 mg/100 ml/cm2 (range: 7·08–8·05, n = 12) and 7·18 ± 0·49 mg/100 ml/cm2 (range: 6·21–7·53, n = 6), respectively. These data were similar to the content in plasma stored for 24 days at 4°C, but not in packed red cells stored for 24 days at 4°C. In Japan, plasma in packed red cells is diluted with additive solution approximately 1/6. DEHP extraction levels depend on the plasma concentration and the surface area of contact with plastics.
Sampling inspections of leukocyte counts
Residual leukocyte counts in blood components were within acceptable limits and conformed to the requirement that the residual leukocyte number in 95% of the blood components must be under 1 × 106/bag. From 2008 to 2010, leukocyte counts in 124 samples out of 65 399 PCs (0·19%), 58 samples out of 121 544 FFPs (0·05%), and 138 samples out of 110 898 WBs (0·12%) were over 1 × 106/bag.
However for blood components, leukocyte counts varied depending on the blood collection method and the aphaeresis machine employed. Almost all blood components (over 99% of the PCs and FFPs and 96·6% of WBs) had a leukocyte count under 5 × 105/bag. Especially the leukocyte counts in over 70% of the PCs for all aphaeresis machines were low (under 5 × 104/bag) and it appears that the type machine employed makes little difference (Fig. 2A–C).
By contrast, leukocyte counts in FFPs varied with the aphaeresis machine used: 63·0% of the FFPs prepared using one brand aphaeresis machine had leukocyte counts of under 5 × 104/bag while another brand showed only 16·8% of the FFPs had counts under 5 × 104/bag (Fig. 2D and E).
Counts in the WBs were widely distributed; 39·9% had counts of (1–5) × 105/bag, 20·2% had count of (5–10) × 104/bag, and 36·5% had counts of under 5 × 104/bag. There were little differences in leukocyte counts among the various blood bag brands (Fig. 3).
The leukocyte counts in all blood components were within the standard limits. 99·8% of blood components had counts of under 1 × 106/bag. The limits of regulation are 95% of the blood components must be under 1 × 106/bag. Leukocyte counts did vary however depending on the blood collection method and aphaeresis machine employed. Plateletpheresis machines and plasmapheresis machine of manufacturer D removed leukocyte well but these machines had a higher percentage of over 1 × 106/bag leukocytes in blood components than plasmapheresis machine of manufacturer E (Fig. 3).
Manufacturers of medical equipments and solutions of prepared blood components need premarketing approval from the Ministry of Health, Labour and Welfare (MHLW) in Japan. Quality control tests are used to obtain approval by the MHLW. The JRC tests the same products again using the same procedures as their acceptance inspection.
Nowadays there are many types of blood bags: blood bags for whole blood donation, for aphaeresis donation, blood bags with attached leukocyte removal filters and also there are many kinds of materials used for blood bags. Government regulations and/or ministerial ordinances do not always keep up with these new products. For example, we have Standard Test Methods for Vinyl Chloride Resin Blood Sets but not for polyolefin bags, and also no standards for leukocyte removal filters.
The JRC performs quality control inspections according to regulations and ministerial ordinances. However, there are many kinds of raw materials and products. Thus if the materials have no regulations, we have decided on our own standards according to the Japanese pharmacopoeia, the Japanese Industrial Standards (JIS) and ministerial ordinances. These testing methods can be complicated. However, our system is basically well established. We have never obtained values violating the standard values for acceptance inspections of blood bags and solutions and sampling inspections of leukocyte counting.
We have to move next step. We still have a space. Many regulations and ministerial ordinances were drawn up for manufacturers of general materials, but not for blood programmes. Even if these regulations are for blood programmes, they are described for manufacturers that cannot use blood for testing. Recently, the MHLW called for the determination of DEHP in separating transfer bags. However, the test uses extraction by ethanol according to the JIS. In our data DEHP content differed according to the extraction method and blood components. These data show that the tests at the blood centre should use blood samples even though blood bag manufacturers cannot use blood. We have no tested lead and vinyl chloride monomer levels in blood components using extraction by blood samples, only by the contents in blood bags and tubes directly and also heavy metals test using water extraction. These tests also need to assay not only contents in blood bags and tubes directly and in distilled water extraction, but also extraction with blood components.
Further additional examinations using extraction by blood samples need to be done as a quality control tests. These centralized tests are effective and significant in blood components management and in safe blood supplies.
The author expresses his gratitude to Dr. Kenji Tadokoro and Dr. William L. Gyure for reviewing the manuscript.