Sixty-six patients who succeeded in storing a sufficient volume of autologous blood (mean±SD; 1097±452ml) using KL-3R3 solution contained in blood bags before elective surgery were included in this study, a five week phase 3 clinical trial of the whole blood preservative solution KL-3R3, commonly known as CPDA-1 (citrate phosphate dextrose adenine) solution. The mean time required for sufficient storage of autologous blood was 23.4±6.9 days, with 40 of the 66 (60.6%) requiring more than 21 days, a period of time that would have resulted in time expiration had we used the citrate-phosphate-dextrose (CPD) or acid-citrate-dextrose (ACD) solution bags which are presently commercially available. Sixty of the 66 patients (90.9%) completed their operations using only autologous blood units. After eliminating three patients who were administered recombinant erythropoietin, the efficacy of KL-3R3 was evaluated as effective in 61 (96.8%) of the 63 patients evaluated (markedly effective 57 (86.4%) and effective 4 (6.1%). Furthermore, in 48 of 56 patients (85.7%), in whom we were able to measure the autologous blood recovery rate after 24 hours of transfusion were estimated effective (markedly effective 29 (51.8%) and effective 19 (33.9%)). In sixty-three patients who had hematological and biochemical laboratory data, vital signs, and urinary data recorded both before and after receiving autologous blood units. RBC counts and hemoglobin levels and platelets counts decreased, and WBC counts increased after returning autologous blood at operation, changes observed commonly under surgical stress. Hemolysis markers such as GPT, indirect bilirubin, and LDH were slightly increased the day after operation, but returned to normal the second day after operation. Other data moved within normal limits expect urine occult blood. Hematuria was observed immediately after transfusion of autologous blood units, but this finding was observed transiently (the first day after transfusion 45.7% and the second day 13.3%). The safety was evaluated in sixty-five patients' blood bags by sight observations and bacterial culture tests, and no abnormal findings or bacterial propagation were detected. One patient was excluded from the safety evaluation because he was operated on within a week of initial predeposition. Two-hundred-thirty-eight KL-3R3 preserved blood bags were transfused in the 66 patients, in one (1/238, 0.4%) which had be discarded because of a large clot formation. In sixty-one patients, both the efficacy and the safety tests of KL-3R3 were rated for usefulness, with all 61 confirmed useful. The laboratory data of the 26 patients whose autologous blood were used within 21 days and the 40 patients whose autologous blood contained at least one unit that exceeded 21 days preservation was compared, with no significant differences observed.
Our results confirmed that, in clinical trial of KL-3R3 preserved autologous blood units, all the criteria of effectiveness, safety and usefulness were satisfied.

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The origin and formation mechanism of macroaggregates (MA) observed in RC-MAP, red cell preparation with additive solution MAP, were investigated here.
MA were formed in all of RC-MAPS which were stored for 5 weeks at 4°C. In experiments of aggregates formation using the mixtures of each blood cell component and MAP solution, only the mixture of granulocytes and MAP solution formed experimental macroaggregates (exp-A) which was very similar to spontaneous MA in terms of appearance, elasticity and adhesiveness. Although both MA and exp-A were rapidly disintegrated by deoxyribonuclease I digestion, they were not digested at all by plasmin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions showed that most of proteins of MA were below 60kD and the band pattern was similar to that of granulocytes. In SDS-PAGE under reducing conditions of MA, fibrin bands were not detected.
Leukocytes in RC-MAPs gradually decreased after 2 days of storage and the size distribution of leukocytes showed granulocytes to be decreased in number. On the other hand, platelets did not decreased until 14 days of storage. DNA contents of dried MA were constant, giving about 5.0%. The ratio of DNA of MA to total DNA in RC-MAP at 0 day of storage was inversely correlated with the ratio of leukocyte count in RC-MAP after separation of MA to 0 day of storage. But there was no relation between the DNA ratio and platelet count.
Examinations by light microscopy and scanning electron microscopy showed that MA were composed of degraded leukocytes at large and variable numbers of leukocytes and erythrocytes, but no fibrin fibers.
These results suggest that MA is formed by DNA fibers which are released from nuclei of granulocytes, taking up leukocyte debris and other blood cells. It does not seem that any coagulation factor or coagulation system is involved.

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Macroaggregates are found frequently in stored red cell concentrates with MAP solution (RC-M·A·P).
Incidence and causative factors of macroaggregate formation were investigated compared to concentrated red cells containg CPD (CRC). The number of microaggregates was also compared.
The incidence of macroaggregate formation in RC-M·A·P increased during storage. They were found in 36 or 67% of RC-M·A·P at 3 weeks of storage and in more than 70% at 5 to 6 weeks, while in 8.6% of CRC at 3 weeks.
The macroggregate formation was not sufficiently affected either by mannitol or citrate concentrations in MAP solution.
Such macroaggregates were composed of leukocytes, platelets and fibrin like substances. The leukocytes in RCM·A·P drastically decreased during storage. Fibrinopeptide-A (FAP) in RCM·A·P gradually increased during storage, which significantly correlated to the decrease of leukocytes, but not in CRC. These results suggested that macroaggregate formation was not due to mannitol, but may be related to the degeneration of leukocytes and the activation of coagulation system.
The number of microaggregates which pass through transfusion filter was statistically less in RC-M·A·P than in CRC. Therefore, the incidence of side effects caused by them might be decreased by using RC-M·A·P.

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The growth of Yersinia enterocolitica in the stored red cell MAP (RC-MAP) units and the effect of leukocyte-depletion with filters on Y. enterocolitica growth were examined. Y. enterocolitica was inoculated into whole blood units just before RC-MAP units were prepared. These units were divided into two portions, the one stored at various times after removal of leukocyte through the filters (LPRC group), and the other stored without any process (control group). Inoculated amount of Y. enterocolitica were 0.1-4.4CFU/ml (S group, n=4), 78-82CFU/ml (M group n=3) and 200CFU/ml or more (L group, n=4).
In S group, Y. enterocolitica did not grow, probably due to phagocytosis and bactericidal activity of leukocytes. In M group, the growth of Y. enterocolitica was observed after storage of 14 days and total amount of Y. enterocolitica reached 5.3×10⁸-6.8×10⁸CFU/ml on 42nd day. In Lgroup, the growth of Y. enterocolitica was found only in 2 out of 4 inoculated samples at 7th day and later observed in the rest of the samples, up to the amount of 2.6×10⁸-1.4×10⁹CFU/ml on 42nd day. On the other hand, each control group of S, M and L group in which leukocytes were removed through the filters 24 hours after blood collection, showed no bacterial growth during 42 days, regardless of inoculated amount of Y. enterocollotica. This means that Y. enterocolitica together with leukocytes are eliminated through the filter.
This study suggest that the filtration of blood through leukocyte-depleted filters at least within 24 hours after blood collection is an effective measure to prevent Y. enterocolitica infection.

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Recent studies have shown that residual leukocytes, especially lymphocytes in the huffy coat-poor red cell concentrate (BPRCC) prepared by the “top and bottom system” were significantly lower in number than those prepared by the conventional system. In this study we investigated the effects of different anti-coagulants on the composition of blood components prepared by the “top and bottom system”.
We compared the characterization of whole blood collected in a citrate-phosphate-dextrose solution (CPD-WB) to that collected in an acid-citrate-dextrose solution (ACD-WB). The mean corpuscular volume of red cells in CPD-WB was smaller than that in ACD-WB. Although the same number of red cells existed in whole blood, the hematocrit (%) in CPD-WB was significantly lower than that in ACD-WB. After centrifugation, the red cells in CPD-WB were more concentrated than those in ACD-WB. The condition of concentrated red cells in the different anti-coagulants was also observed by freeze-replica electron microscopy. The recovery rate of plasma were 88.1±1.7% (n=25) and 83.8±2.1% (n=36) in platelet poor plasma prepared from the CPD-WB and the ACD-WB, respectively.
The removal rates of leukocytes in BPRCC prepared from CPD-WB and ACD-WB were 78.4±10.4% (n=25), 70.4±8.9% (n=36), respectively. We conclude that the CPD-WB is more suitable than ACD-WB to prepare BPRCC with a high removal rate of leukocytes and high recovery of plasma when the BPRCC is prepared by the “top and bottom system”.

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We evaluated 3 types of newly developed leukocyte-removal filters made for platelet concentrates (PCs), the Sepacell PLS10A, PLS5A, PLS5N, to determine their effectiveness in removing leukocytes. The former two filters are made for filtration at the bedside and the latter is developed for use at blood centers. The leukocyte removal rate, platelet recovery and platelet functions were studied. The leukocyte removal rates of the Sepacell PLS10A, PLS5A, PLS5N with 1-day-stored PCs were 3.52±0.18log(n=5), 3.95±0.52log(n=4), 3.83±0.26log(n=4), respectively. The platelet recovery was higher than 80% with three filters, and filtered platelets maintained their normal functions. Thus, these high performance filters for PC should help to prevent the alliommunization on platelet transfusion.

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We have developed an automatic flow cytometer, the Sysmex RU-2 (the tentative name), able to measure the small number of residual leukocytes in both filtered platelet concentrate (PC) and red cell concentrate (RCC). The flow cytometer is also able to count pre- and post-filtered red cells and platelets. Leukocytes were stained with a fluorescent dye, auramine O, and red cells were lysed in the flow cytometer simultaneously. Mixing of the sample and fluorescent dye was performed automatically in the machine. The number of leukocytes in the pre-filtered sample was first measured in a hemocytometer and serially diluted with leukocyte-free PC or RCC prepared by repeated filtration. The number of leukocytes measured on the Sysmex RU-2 as a function of cell number expected from the hemocytometer count can be expressed by regression lines: y=0.961x+0.373 (r=0.998) for PC and y=0.913x-1.903 (r=0.994) for RCC. When PC and RCC had been filtered with several kinds of leukocyte removal filters, the automated electronic cell counter failed to detect leukocytes in the filtrate, whereas the Sysmex RU-2 could detect leukocytes at concentrations as low as 1 cells/μL. The Sysmex RU-2 should be useful to count the residual leukocytes in leukocyte-depleted blood products to count red cells and platelets, which are required for the quality control of leukocyte-depleted blood products.

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The utility of gel centrifugation method for blood grouping, irregular antibody screening, antibody titrating, and crossmatching of patients' specimens was compared with conventional tube methods. There were no discrepancies in blood grouping (ABO, Rho) between the gel method and the tube method. The detection rates of irregular antibodies were enhanced compared with the tube method except anti-M and anti-Fy^b, and the number of non-specific antibodies detected was reduced using the gel system. Antibody titers examined using the gel system were more sensitive (2-16 times) than with the tube method. We recognized that the gel test was sensitive, reliable, reproducible, and easy to perform. The gel method will be used as the standard method of blood transfusion test in the near future, because the strength of red cell agglutination can be objectively determined using the gel method.

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The CD34+ hematopoietic stem cells have been shown to be able to reconstitute hematopoiesis in vivo. We concentrated CD34+ cells from bone marrow (BM) of normal adults or mobilized peripheral blood (PB) of patients with breast cancer using the MicroCELLector (Applied Immune Sciences). The purified BM CD34+ fractions were enriched in CFU-GM (9.6-153-fold, average 64-fold) and BFU-E (25-61-fold, average 44-fold), with 7-95% recovery of these colony-forming cells in the CD34+ fraction. Similar results were observed for PB CD34+ fractions. CD34 phenotypic analysis using the FITC-conjugated CD34 monoclonal antibody 8G12 showed that the purifed CD34+ fractions were 18-93% (average, 71%) positive using lymphoblast gate. These preclinical data form part of the basis for future clinical studies of stem cell transplantation and gene transfer.

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Purpose:
Red blood cells added MAP as a additive solution (RC-MAP), developed by the Japanese Red Cross Society, is reported to be superior to the conventional CRC in that the good quality of red blood cells can be preserved for a long term. Another advantage is that the removal of huffy coat serves to alleviate transfusion-induced adverse reactions. We used RC-MAP (preserved 10-12 days after collection) and FFP (obtained from the same donors) in a total of 10 cases requiring cardiac surgery (3 for ASD, 1 for VSD+RSV, 1 fort AVR, 1 for MVR and 4 for AC bypass) from the start of extracorporeal circulation to postoperative management. This report describes the clinical courses in these cases.
Results:
The mean time of extracorporeal circulation was 112.5±71.3min. The volume of bleeding from the postoperative drain ranged from 4.6 to 20ml/kg except 35ml/kg in the AVR case, showing little difference from that in the cases of blood component and fresh whole blood transfusion. Fibrinogen and prothrombin time were within the normal ranges. Free Hb and BUN returned to the normal in a short time. Renal disturbance was not found in any case. The red blood cells of RC-MAP filled in the artificial heart-lung apparatus were lableled with Cr⁵¹. The half life proved to be about 26 days with little difference from that in the cases of fresh whole blood transfusion.
The time of extracorporeal circulation was longer in one of the four AC bypass cases and the AVR case than in others. The total volume of RC-MAP used throughout the course was accordingly larger in these cases. This suggested that some appropriate measures should be taken for such cases, because hypoproteinemia and relative shortage of coagulation factors might arise from MAP-induced hyperhemodilution. With this in mind, we decreased MAP content in RC-MAP immediately before use for the cases treated after the above two cases. This measure could bring a good result.

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It was well known that the first generation anti-HCV assay, based on the C100-3 antigen, a nonstractural gene product of HCV, was found to be an effective, but still an insufficient marker of infectivity in blood donors.
In order to clarify an effort to prevent posttransfusion hepatitis (PTH), anti-HCV associated screening markers (C100-3, GOR, N-14, EIA II, PHA, Imucheck) were investigated in 450 transfusional cases followed between August 1988 to March 1993. In addition to evaluate those markers, total 682 stored sera from PTH patients and HVC seropositive blood donors were tested for the presence of antibody against HCV by 2nd generation (2nd) markers, mainly PHA and Imucheck, compared with anti-C100-3.
One of 105 cases (0.95%) after screening for HCV by 2nd PHA was diagnosed as a typical PTH, even though it was not confirmed as PTH type C without the detection of HCV-RNA by PCR as described in the text. According to our follow-up study on PTH retrospectively using 147 stored sera of 13 defined cases, PTH type C was still remained at least 1-2% of all transfusinal cases after screening for 2nd PHA. Twelve of defined 13 cases were strongly related to HCV with seroconversion, whereas all of suspected 8 ones were not found to be correlation to HCV. Of them, two cases with seropositive 2nd HCV antibody (both PHA and Imucheck) before transfusion indicated the re-infection of HCV seropositive patient by rising in an antibody titer after transfusion. The existense of the neutralizing antibody was suggested in those cases, because of complete recovering from PTH with single ALT peak.
The specificity of HCV related markers, 2nd PHA, 2nd EIA II, and Imucheck, were 81.8%, 71.7%, and 73.0%, respectively. They were higher than that of C-100-3 (51.8%) extremely. Finally it was suggested that anti-core antibody in HCV seropositive donor remained continuously as past exposure to HCV and the core antibody titer reflected the existence of HCV.

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This is a first report in Japan, showing that anti-HPA-3b antibody was detected in a 7-year-old patient with myelodysplastic syndrome. The detection was made by a MPHA (mixed passive haemagglutination) test.
The patient had neither anti-HLA antibody nor anti-HPA-3b antibody at the onset. After 221 units of transfusion, the patient produced both anti-HLA antibody (Score: 8) and anti-HLA-3b antibody (titer: 1x). Refractriness against platelet transfusion developed after 495 units of transfusion, when both anti-HLA antibody and high titer anti-HPA-3b antibody (10-20x) had been produced. The production of anti-HPA-3b antibody lasted for about 1 month, while the production of anti-HLA antibody was kept longer.
Both HLA antibody and anti-HPA-3b antibody were considered to be the causes of the refractriness against platelet transfusion, although the main cause might be anti-HLA antibody.

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We report here a case of Hydrops foetails found in a pregnant woman with low titer anti-Jr^a antibody. The antibody titer was 1:8, and the immunoglobulin subclass was IgG1 and IgG4. The newborn had the same antibody as mother's in its serum, and its red cells were positive for direct anti-globulin test. The elution of antibody bound to baby's red cells was also confirmed by polyethylene glycol (PEG)-indirect anti-globulin test.
The direct evidence, in which Hydrops foetalis was originated from immunological reaction between low-titer anti-Jr^a antibody and Jr^a-positive red cells, was not obtained. However, it was presumed that an altered circulatory condition by congenital left heart hypoplasia of the foetus accerelated its hemolytic reaction.

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