We concentrated mononuclear cells (MNCs) containing hematopoietic stem cell from bone marrow (BM) or peripheral blood (PB) of patients and MNCs fraction were cryopreserved in cryoprotectant solution (final concentration: 6% hydroxyethyl starch, 5% dimethyl sulfoxide and 4% human serum albumin) without controlled-rate freezing. In preparation of bone marrow MNCs were concentrated by density gradient centrifugation using the COBE 2991, the recovery of MNCs and granulocytemacrophage colony forming unit (CFU-GM) were 56% and 83%, respectively, of the original BM. On the other hand, in preparation of peripheral MNCs were concentrated by low speed centrifugation of lymphocyte fraction collected using the COBE spectra, the recovery of MNCs and CFU-GM were 89% and 90%, respectively. After thawing of both cryopreserved preparations, the recovery of CFU-GM were 58% of original BM and 68% of original PB fraction. These results indicate that the techniques for the separation and storage of blood components in blood center are applicable for the preparation and storage of hematopoietic stem cells from BM and PB.

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Transfusion-associated graft-versus-host disease (TA-GVHD) was described as occurring after nonirradiated blood tranfusion not only in immunosuppressed but also in immunocompetent recipients who share one HLA haplotype with HLA-homozygous blood donors.
From the distribution of 1% or more frequencies of HLA-A, -B, -C, -DR, -DQ haplotypes obtained at the 11th International Histocompatibility Workshop (1991), using the fomulae of Kanter, we tried to calculate the potential risk of TA-GVHD as a result of directed donations from relatives in the Japanese and other ethnic groups.
The Japanese have the greatest risk of TA-GVHD than any other populations because are more homogeneous in HLA and have a common haplotype: A24-CBL-Bw52-DRw15-DQw1 (7.5%).
Of noteworthing, the risk of transfusion of HLA-homozygotes to heterozygotes among 1st-degree relatives in the Japanese was 4.7-folds compared with that of transfusion in the U. S. A. population. And, the risk of TA-GVHD among the Japanese unrelated was near to that of transufusion among the French first degree relatives.
Therefore, we recommend that donations of blood components from recipient's relatives should be irradiated essentially, and as possible even from unrelated donors in the Japanese population.

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A sensitive immunoradiometric assay (IRMA) was used to quantitate the amount of red blood cell-associated IgG (RBC-IgG). The mean RBC-IgG of 91 healthy volunteers was 33±13 (SD) molecules per one RBC. The correlation between the agglutination strength in antiglobulin test and the number of IgG were very satisfactry. We also tested the amount of RBC-IgG of 27 patients with immune hemolytic anemia of warm type (AIHA). (AIHA, n=17; RBCs with positive antiglobulin test, n=2; RBCs with negative antiglobulin test, n=8; eluates from patients' RBCs). The mean RBC-IgG of 17 patients with antiglobulin test positive AIHA was 998±930 (SD) molecules per one RBC. It was ranged from 257 to 3000 molecules per one RBC. Two patients with antiglobulin test negative AIHA (7.4%, 2/27) had 138 and 172 IgG molecules per one RBC, respectively. The indirect antiglobulin tests of RBCs sensitized with 8 eluates showed positive. The RBC-IgG of sensitized RBCs was higher than that of RBCs from healthy volunteers. Using this assay, quantitative studies of RBC-IgG was extremely useful for the detection of small amount of IgG on RBC in case of the antiglobulin test negative AIHA.

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Long term prognosis of posttransfusion hepatitis (PTH) in patients undergoing open heart surgery was studied by a questionaire and liver function tests. The diagnosis of PTH was made according to the criteria of the Yoshitoshi committee (PTH research comittee of the Japanese Ministry of Health and Welfare) for 224 of 1218 patients (18.3%) who underwent open heart surgery between 1965 and 1987 at the Nagasaki University Hospital. The questionaire was related to recent physical condition and quality of life and was sent to 147 PTH patients. Hundred and twenty-two patients (83.3%) made answer and 76 of whom underwent further examination and liver function tests.
The results obtained were as follows; (1) There were 7 late deaths, none related to liver disease. (2) The liver function tests showed elevated S-GPT in 17 patients (22.3%). (3) Seventy patients (61%) had jobs and 10 could not work due to illness being liver disease in one case. (4) Hundred and nine patients (94%) were quite or moderately active in their daily lives (0-1 grades of performance status).

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Anti-hepatitis C virus (HCV) core antibodies in blood donors were detected in enzyme-linked immunosorbent assay with oligopeptides (CP9 and CP10) corresponding to the putative core protein of HCV and their performances were compared with C100-3 antibodies. HCV-RNA was detected by nested polymerase chain reaction in fresh plasma samples from blood donors positive for anti-core antibodies in hihg-titers and/or anti-C100-3 antibodies. In plasma samples from 2, 034 apparently healthy blood donors, anti-CP9 was detected in 133 (6.5%), anti-CP10 was detected in 95 donors (4.7%) and anti-C100-3 was detected in 33 donors (1.6%). Among plasmas positive by at least one test, 31 were found to contain HCV-RNA; 19 of them were negative for anti-C100-3, but positive for anti-CP9 and/or anti-CP10 in high titers (OD≥1.000). These results indicated that anti-core antibodies would be useful in complementing anti-C100-3 for further decreasing the incidence of post-transfusion non-A, non-B hepatitis.

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The objective of this study is to evaluate the effects and problems of the maximum surgical blood order schedule (MSBOS) and type and screen (T & S) system on the usage of blood for surgical procedures.
Although 303 units of blood were prepared and cross-matched for 142 cases of the selected 8 types of surgical procedures assigned by T & S during a year before the initiation of the system, only 31 units of blood were prepared for 130 cases of those during a year after the initiation. In some of the surgical procedures assigned by various units of MSBOS, the average units of blood prepared were decreased. In the procedures of radical mastectomy and total gastrectomy, the system of MSBOS was not effective in reducing the units of blood prepared. The method of anesthesia such as deliberate hypotension that can reduce the volume of blood loss or precise classification of the surgical procedure should be considered to decide the units of MSBOS for radical mastectomy or total gastrectomy, respectively. In addition, the units of MSBOS should be re-evaluated for the procedures for which autologous blood are preoperatively prepared. The units of autologous blood prepared are thought to be reduced from the previous MSBOS units.
These results indicate that the system is effective in reducing the units prepared and that the MSBOS and T & S should be re-evaluated depending on the changes of anesthetic method or surgical procedures and the application of autologous blood donation programs.

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Fifty children (26 girls and 24 boys) scheduled for autologous blood donation before elective surgery were studied. As a rule, about 10% of intravascular blood volume estimated by the Morse formula was drawn once a week. Before surgery, an average of 3.5 units of autologous blood per patient were prepared during the 25 days of donation period. Two patients excited at the thought of phlebotomy failed to donate autologous blood, but none of the autologous blood donors was deferred due to phlebotomy-induced anemia. Since an appropriate endogenous erythropoietin response was observed, which might stimulate erythropoiesis.
The ages of the donor-patients ranged from 4 to 15 years with a mean of 9.8 years, and weighing from 15 to 70kg with a mean of 34kg. All patients received about 50mg of iron sulphate per day orally. Of the 48 patients who donated autologous blood, 48 underwent surgery without using homologous blood. Three units of homologous blood were transfused to each of 2 orthopedic patinets.
We conclude that a predeposit autologous transfusion program in children can be performed safely when patients are cooperative.

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To clarify the current status of hepatitis C virus infection in children in Japan, investigation was performed in aspects of blood transfusion and mother-to-child infection.
The subjects were the cases before a routine test for HCV antibody in the Red Cross Blood Center in Japan started. A total of 1, 468 samples were obtained from 959 cases of under 18 years old and 509 cases of cord blood. C100 antibody was measured as HCV antibody.
C100 antibody positive case was not observed in 669 children with no history of blood transfusion. Thirty-two cases (14.2%) were positive in 226 cases with the history of blood transfusion. C100 antibody positive state lasted for 3 to 41 months in 21 cases among these cases, and 10 cases of them progressed to chronic hepatitis.
Although C100 antibody was positive in 4 cases out of 509 cases of cord blood, it disappeared spontaneously in 4 to 8 months after birth. These findings suggested that the possibility of mother-to-child infection was actually very low.
In 8 cases, C100 antibody was negative in spite of a large quantity of blood from more than 300 donors was transfused and liver dysfunction had been found. Although these results were obtained from using C100 antibody test, it will be possible to explain the current status and the nature of HCV infection in children by using the 2nd generation HVC test and PCR method.

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Anti-HCV (c100-3, Abbott 2nd, N-14 and AR-142) were assayed in the sera of 531 medical employees (196 physicians, 300 nurses and 35 technologists). Positive rate of anti-HCV were 1.1% (c100-3), 0.9% (Abbott 2nd), 0.6% (N-14) and 0.8% (AR-142) respectively. Anti-HCV was positive by at least one of these 4 tests in 11 cases (2.1%). Only 3 cases out of these 11 were proved positive for HCV-RNA. These data indicate that medical employees do not belong to the high-risk group of HCV infection.

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Rodgers antigen (Rg^a) has been reported at first (1986) to be present in erythrocyte and plasma and subsequently also in lymphocyte.
Recentry, we examined a case of Rg^a antibody, which seemed to be the second case reported in Japan.
The present case is IgG type antibody, which was produced in washed erythrocyte transfusion.
Erythrocyte life-span test using Rg^a-positive erythrocytes revealed neither side effect nor increase in antibody titer, but erythrocyte short life-span (13 days).

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(Case report) A 29-year-old male with mild hemophilia A (FVIII activity: 10.5%) and chronic hepatitis.
In 1984, he suffered from epistaxis and persistent hemorrhoidal hemorrhage, with occasional massive bleeding. He began using highly purified but non-heated factor VIII concentrates imported from foreign countries to treat the bleeding. He did not visit the hospital thereafter, and there was no major bleeding episode until 1988.
When he visited our hospital in 1988, HIV antibody was negative (ELISA method) and he was clinically in good health with almost normal immunological function.
However, he was subsequently identified as positive for HIV antibody (ELISA and Western blot methods) when examined before surgical operation for severe hemorrhoidal hemorrhage in 1991. He is now clinically in good condition and shows normal immunological function.
The patient's course has remained uneventful for more than 8 years, and he is now working and living normally. It is thought that he was infected by HIV around 1984 during frequent and massive transfusion of non-heated blood preparations. CD-4/CD-8 ratio and CD-4 cell count were 0.73, 1.1 and 1.12 and 1127, 1317, and 1612/μl, respectively, in August, 1988, February, 1989 and May, 1991. These data are considered to be normal and furthermore to show gradual improvement of immunological function.

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With the increase in demand for platelet concentrate (PC), how to meet it and side effects due to WBC mixed in platelet preparations have become great problems. We studied a total of eighty four platelet donations done by the donors (male 80, female 4), who met all the criteria for platelet donation, during the period of April 1990 to August 1991.
1) According to the initial count of platelets, 28 donors were divided into Group A (less than 25×10⁴cells/μl), 22 into Group B (25×10⁴/μl to 30×10⁴/μl), 22 into Group C (30×10⁴/μl to 35×10⁴/μl) and 12 into Group D (more than 35×10⁴/μl). The collection rate of PC of more than 15 units (3.0×10¹¹) was 25.0%, 50.0%, 81.8% and 91.7% in respective group.
2) As for the time-course change in concentration of platelets collected, it was 122.3±19.0×10⁴cells/μl in Group A 30 min after the start of collection and 177.8±32.6×10⁴cells/μl in Group B and C 50 min after the start; these were the, highest values in each group.
3) The average count of WBC mixed in was as few as 3cells/μl and 6.9×10⁵cells/bag.
4) As for the time-course change in count of WBC mixed in, it was highest at the start of collection, 14.3±14.5cells/μl, and thereafter decreased in the course of time.
5) We tried to take photos of the interface forming process in centrifuge channel. Whole blood flow rate was maintained at 40ml/min up to 500ml blood volume processed to obtain proper interface, since the establishment of interface directly affects platelet collecting efficiency and white blood cell contamination.
6) The volume of citric acid injected was 21.9mg/TBV-1/min on an average and 29.4mg/TBV-1/min at the maximum, causing no side effects, and the Ca⁺⁺ reduction was only 20.8%.

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The Autopheresis-C apheresis set (Baxter) can collect platelet-rich plasma (PRP) and subsequently separate it into platelet concentrate (PC) and platelet-poor plasma (PPP). The set consists of two devices. One is the Platelet-cell set for separation of PRP from whole blood. The other is the Plasmacell-C for separation of PRP into PC and PPP. This set can automatically adjust the suspended plasma volume of PC during the concentration process. Thus, the PC obtained is not pelleted and need not be resuspended. To evaluate the efficacy of PRP collection with this device and the separation and function of PC, we drew and separated blood from eight donors. About 450ml of PRP was collected in 55.5±1.8 min with a high PC yield. For comparison, PRP collected by an apheresis machine (Ultralight-PCS, Haemonetics) was processed using the Plasmacell-C. PC recovery was 95% and preparation time about 14min. The PC maintained their normal function for three days of storage. No significant increase of anaphylatoxins was observed in the PC. We conclude that the Autopheresis-C system is simple to use and gives higher PC recovery than centrifugation.

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Platelets aggregates or floating matters are occasionally observed in the bags of platelet and granulocyte concentrates collected from donors using cell separator. With this in mind, we studied this modality of blood collection with special attentions on donors, platelet activating factor (PAF), fibronectin (FN), blood viscocity and serum protein (albumin/gammaglobulin ratio=A/G ratio) as platelets and granulocytes within the blood collection bags. FN before collection was tended to be higher in donors with higher leukocyte counts. During collection of blood components, both PAF and FN tended as well as blood viscocity and A/G ratio to decreased compared to the baseline level before collection. In the platelet concentrate bags, PAF tended to increase lightly, and FN tended to decrease slightly, compare to those in the circulating blood. Where platelets aggregates or floating matters are observed in the bag, high level of PAF before collection in the donor and also of whose PAF was higher and FN was lower than the baseline values.
The decrease in PAF and FN observed during collection of blood components seemed to be attributable to ACD-A injected during blood collection and to the dilution of blood in the body caused by injection of saline. It was also presumable that platelet activation during extracorporeal circuration could induce platelet aggregation, as evidenced by the increases in PAF and decreases in FN after blood collection and within the collection bags.

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We analyzed discard of blood and its components for transfusion in our hospital to reduce the ineffective expenditure. During five years since 1986, our hospital purchased 159, 198 units of blood from Japanese Red Cross Blood Center, of which 999 units (0.6%) were abandoned through 269 cases (3.7 units per a case). Discard rate and units per a case for each product were as follows: whole blood and packed red cells 0.8% and 2 units, platelet concentrate 0.5% and 9.1 units, and FFP 0.5% and 6.4 units. About one third of the discard was regarded avoidable, for at least 31% of the cases (35% of the units) were chiefly attributed to mishandling of blood or carelessly delayed communication to our section about a change of transfusion schedule.
Return of blood products to the Center with approval lessened our discard rate of whole blood and packed red cells appreciably. They were only occassionaly discarded on account of acute change in patient's status, while such change frequently led the “unreturnable” components such as FFP and platelet concentrate to discard. Doctor's ignorance or indifference on blood products made it difficult to reduce blood discard.

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