Banking of human placental/umbilical cord blood (PCB) requires volume reduction for prevention of hemolysis-related side effects and cost-effective storage. We collected, separated, and cryopreserved PCB following the New York Blood Center's protocol, and obtained PCB with an mean volume of 70.0±28.1ml (n=100). Red cells were sedimented by addition of 1% hydroxyethyl starch with gentle centrifugation. Recovery of nucleated cells (NC) and colonyforming units (CFU) was 82.9±13.7% and 90.4±18.9%, respectively (n=25). Volume of the NC suspension was reduced to 20ml by centrifugation and 5ml of 50% dimethylsulfoxide and 5% Dextran 40 solution were added slowly. The frozen cells were washed with a solution containing 10% Dextran 40 and 5% human serum albumin. NC and CFU recovery was 88.8±7.13% and 81.2±20.3%, respectively (n=18). No aggregation of cells occurred after the washing procedure.
We studied the effect of storage after collection, cell separation, and cryopreservation under various conditions. Results suggested that collected PCB should be prestored at 25°C for less than 24 hours, and that HES of moleculer weight 400, 000 should be used at the concentration of 1-4% for efficient cell separation. Further, the cryoprotective solution could maintain the number of NCs, CFUs, and CD34⁺ cells under slow cooling.

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Hepatitis B-type virus (HBV) screening at Japanese Red Cross Blood Centers is carried out by agglutination method. The risk of post-transfusion hepatitis B (PTHB) has markedly decreased, especially since the hemagglutination inhibition test (HI) for anti-HBc and the passive hemagglutination test (PHA) for anti-HBs were introduced to screening in addition to the reversed passive hemagglution test (RPHA) for HBsAg in 1989. However, PTHB had not been completely eliminated even using the current system (RPHA, HI & PHA) which is rather complicated and time-consuming. We therefore evaluated the PRISM-HBsAg assay (Abbott), a fully automated assay based on chemiluminescence technology, in comparison with EIA-HBsAg (Auszyme overnight protocol, Dainabot) and our present HBV system.
Sensitivity of PRISM-HBsAg (0.08ng/ml in ad panel and 0.06ng/ml in ay panel) was higher than that of EIA-HBsAg (0.23ng/ml in ad panel and 0.11ng/ml in ay panel). In 87 HBV-DNA-positive panels that were RPHA-negative (or weakly positive) and HI-positive, 59 and 50 were positive with PRISM-HBsAg and EIA-HBsAg, respectively.
A total of 20, 041 donor specimens were tested simultaneously by PRISM-HBsAg, EIA-HBsAg and RPHA. The number of disqualified samples was 98, 91, 71 and their determination ratio (disqualified samples/initially positive samples×100) was 96.1%, 20.8% and 19.7%, respectively. These results show that the determination ratio of PRISM-HBsAg assay is clearly higher than that of EIA-HBsAg and RPHA. There were 19 RPHA-negative and PRISM-HBsAg-positive samples, 17 of which were HI-positive and disqualified according to the criteria of our present HBV system. Of 313 samples disqualified because of their high HI titer despite their negative PRISM-HBsAg and EIA-HBsAg, 10 were PCR-positive with DNA preparation concentrated from 10ml samples. These results indicate that the sensitivity and specificity of RPISM-HBsAg assay are significantly higher than those of EIA-HBsAg and RPHA. However, anti-HBc screening is still necessary, because some HBV-DNA-positive samples with high anti-HBc activity are PRISM-HBsAg-negative.

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In patients with cold-reactive antibodies (CRAbs) undergoing transfusion no consensus has yet been reached as to whether or not the corresponding antigen-negative red blood cells (RBC) should be selected.
In our hospital, we routinely perform unexpected antibody screening tests by the saline method (S), bromelin technique (Br) and indirect anti-globulin testing (IAT). Of 401 unexpected antibodies detected between January 1995 and June 1996, 263 (66%) were CRAbs. Of these 213 CRAbs (81.0%) were not reactive in IAT with IgG-specific antisera (IAT-IgG) or at 37°C. Among patients with CRAbs, 172 were corns-matched with Br at 37°C regardless of CRAbs. Of these, 27 were transfused with 255 units of RBC compatible with the corresponding antigens; and 11 patients with anti-Le^a, 16 with anti-Le^b, 10 with anti-P₁ and 1 with anti-M were transfused 25, 181, 47 and 2 units of antigen-positive RBC, respectively. None of these patients exhibited increaed levels of antibodies or developed transfusion reactions.
Over the past six and a half years, further more, 17 other cold-reactive anti-Le^a-positive patients given Le(a+) RBC were followed to examine antibody levels, and 71 cold-reactive anti-Le^a-positive patients transfused with Le(a+) RBC were observed with respect to clinical course without monitoring of antibody titers. None of these patients showed any increase in antibody values or transfusion reactions.
We conclude that antibodies non-reactive at 37°C and/or IAT-IgG are of no clinical significance and can be ignored in transfusion testing.

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We evaluated the effect of repeated apheresis on storage iron status in 82 males and 138 females. Storage iron was calculated according to the formula proposed by the National Health and Nutrition Examination Survey (NHANES) as previously described. Calculated storage iron after repeated apheresis was 362±282mg in male donors and 128±176mg in females. These values were significantly lower than those in normal subjects and 400-ml repeated whole blood donors. A total of 12.2% of males and 45.7% of females were in negative iron balance before donation. The high prevalence of storage iron deficiencfy may be due to residual blood in the circuit or other factors.

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The ProCOUNT™ kit (Becton-Dickinson Immunocytometry Systems) has recently been developed for the measurement of CD34-positive cells. Here we describe a comparative study of CD34-positive cell counting by ProCOUNT™ and standard two-dimensional side scatter-fluorescence representation (SSC-FL) using nucleated cells from peripheral blood (PB: n=15) and cord blood (CB: n=20). A high correlation was found between the two methods: r=0.997, Y=0.991X+0.044 for the PB cells and r=0.944, Y=0.891X+0.02 for the CB cells. The percentages of CD 34-positive cells in the PB cells were 1.49% and 1.46% by the two methods, and both 0.25% in the CB cells. Further, a high correlation (r=0.945-0.960) between the percentage of CD34-positive cells and CFU-GM cell counts was also found in PB cells measured by these two methods. However, this correlation coefficient in CB cells by SSC-FL was 0.684, even though a high correlation (r=0.819) in the same CB cells was found by ProCOUNT™. This difference appears to be due to a higher percentage of non-hemolytic red blood cells in the SSC-FL (mean±1SD: 32.45±25.10%) than in the ProCOUNT™ (15.17±10.49%). As a consequence, we consider that ProCOUNT™ is preferable in the measurement of CD34-positive cell counts in PB and CB cells for standardization, especially in cord blood bank use.

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We performed large-volume leukapheresis (LVL) to collect sufficient numbers of hematopoietic stem cells for peripheral blood stem cell transplantation (PBSCT) and examined the efficiency and safety of this procedure in patients and stem cell donors. Forty-four rounds of LVL were carried out from patients with various malignancies (n=16) and healthy allogeneic PBSCT donors (n=6). Hematopoietic stem cells were mobilized with granulocyte colonystimulating factor (G-CSF) after myelosuppressive chemotherapy in 12 patients (group A) or G-CSF alone in 4 patients (group B) and 6 normal adults (group C). Blood was drawn at the rate of 68ml/min (range, 40-95ml/min), with an average of 12L (range, 5-15L) processed per procedure. The time for the procedure averaged 177min (range 69-238min). A total of 2×10⁶ CD34⁺/kg, the minimal number of stem cells required to ensure engraftment, could be harvested by single LVL in 60% (15 out of 25) of patients in group A, 25% (2 out of 8) in group B, and 82% (9 out of 11) in group C. Thrombocytopenia and prolonged APTT were observed in all individuals after single LVL. The platelet count decreased by 40% of the preleukapheresis value and APTT was prolonged to approximately twice the preleukapheresis value in some cases. One patient bled at the access point to the femoral vein one hour after the completion of LVL. Twenty-three percent of the blood donors complained of dysethesia of the lips during LVL. One patient complained of chest pain that necessitated discontinuation of leukapheresis. No subject showed abnormalities in serum levels of Na, K, Cl, CPK, AST, ALT, or LDH. We conclude that LVL can be performed to obtain a sufficient number of hematopoietic stem cells from peripheral blood in patients with malignancies and in healthy donors.

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