In order to confirm engraftment of sibling-donor marrow cells or lekuemia relapse from the patient leukemic cells, DNA from nucleated cells in peripheral blood or bone-marrow fluid from patients who had received bone marrow transplantation (BMT) was examined using the MCT118 gene (D1S80) (a variable number of tandem repeats: VNTR) and 6 other genes and 9 restriction enzymes before and after BMT. In 5 of 10 patients (Case 1, 3, 4, 7, and 9) a difference between the DNA from donor cells and recipient cells was confirmed using the MCT118 gene, and engraftment of the donor marrow cells could be demonstrated. In Case 7, not only the engraftment of donor cells but also the leukemia relapse from recipient cells were confirmed using MCT118. Among the remaining 5 cases, Case 5, 8, and 10, the engraftment of donor cells was confirmed using the COLA2A1 gne (12q14.3). In Case 5, and 10, the engraftment could be detected using a gene in the CA2 locus (8g22). In Case 2, the presence of cells from the sibling was identified with the THRB gene (3p24) alone. In Case 6, the presence of cells from the donor could be confirmed using the DMD gene (Xp21.3-21.1) alone. In Case 8, the AK1 gene (9q34.1-34.2) could identified the difference between DNA from donor cells and recipient cells only, but the difference could also be detected using the COLA2A1 gene. In none of the cases, the INS gene (11p15.5) could identify the difference between the DNA from donor and recipient cells. Although only 10 cases were investigated, the findings suggest the use of first the MCT118 gene, then the COLA2A1 gene and the other genes when trying to determine engraftment of donor marrow cells or leukemia relapse after BMT.
The normal viable platelets display a “streaming or swirling pattern” when the container is agitated under the appropriate lighting condition. The swirling patterns of platelet concentrates (PC) gradually disappeared with lowering pH of PC stored for 3 to 4 days. The change in platelet morphology from disc from to sphere forms was also closely related with decreasing pH of PC. We classified the swirling patterns into two categories, (-) or (+), or into three categories, (-), (+), or (++). PC with a pH level of 6.0-6.2 had a score of (-) in the 2 categories classification. The pH of PC with a score of (+) ranged from 6.2 to 7.1. In the 3 categories classification PC with a pH of 6.2-6.8 had a score of (+) and pH of PC with the score of (++) was 6.8-7.1. In PC with the score of (-) platelet swelling and decreased aggregation responses to collagen were apparent. Red cell contamination did not affect seriously the evaluation of swirling patterns. Now we have obtained a simple and easy method utilizing swirling patterns of platelets for the non-invasive evaluation of the quality of stored platelets.
Donors showing a weak positive reaction for antibody to hepatitis C virus (anti-HCV) by PHA (25-211) are not subject to notification as HCV carriers. In order to estimate the frequency of HCV-RNA positive donors in low titer PHA positives, serume samples of PHA positive donors were tested for anti-HCV using different EIA test kits (Abbot EIA-2, JCC-2, UBI), and for HCV-RNA by nested PCR. All HCV-RNA positive samples in this study were also positive by all three ETA tests. On the other hand, HCV-RNA were not detected in the samples showing negative in some second generation tests. All three second generation tests were able to identify all of the HCV-RNA positive samples and showed 100% sensitivity. We therefore propose that a new HCV screening and confirmation method using other second generation anti-HCV test (s) and PCR be accepted for notification purposes. First, blood donors should be screened for anti-HCV by PHA, with positive samples showing a PHA titer of 25-211 subject to a second generation confirmation test (s) with which we identify donors likely to be HCV carriers. Second, a PCR test would be done to samples positive by the second generation confirmation test (s). Using this screening method, the persentage of serum samples subject to PCR test were estimated to be 38% of PHA positive donors and 0.1% of the overall donors in Okinawa. Hence, it would seem to be feasible to use the PCR test to determine donors to be notified as HCV carriers.
The HLA class II (DQA1, DQB1, DRB1 and DPB1) genes of 28 unrelated Japanese insulindependent diabetes mellitus (IDDM) patients and 27 healthy control subjects were typed by PCR-RFLP (polymerase chain reaction-restriction length polymorphism). DQA1*0301, DQB1*0303, DQB1*0401 and DRB1*0405 were positively, and DQA1*0103 and DQB1*0601 were negatively associated with IDDM. There was no specific association between IDDM and the DPB1 allele. Haplotypes determined according to the published linkage disequilibrium in the Japanese population showed that DQA1*0301-DQB1*0303-DRB1*0901 and DQA1*0301-DQB1*0401-DRB1*0405 were increased in IDDM patients. Twenty-four of the IDDM patients had either of the two haplotypes, while only six of the control subjects did. The homozygosity of non-aspartic acid at position 57 of the DR beta chain increased susceptibility to IDDM, while aspartic acid at position 57 of the DQ beta chain did not protect Japanese against IDDM. Homozygosity of arginine at position 52 of the DQ alpha chain was increased in IDDM. These findings suggest that, *DQA1*0301 and arginine at position 52 of the DQ alpha chain increase the susceptibility to IDDM in Japanese as in caucasian and black populations, but that DRB1*0405 and non-aspartic acid at position 57 of the DR beta chain, rather than DQB1 allele and non-aspartic acid at position 57 of the DQ beta chain, also influence the susceptibility of Japanese to IDDM.
The activation of anaphylatoxins during hemapheresis procedures including 3 plateletapheresis and 2 plasmapheresis procedures was studied. The membrane filtration plasmapheresis procedures using APC-4000 (Asahi) and Autopheresis C (Baxter) generated very high levels of C3a of 1254±332ng/ml and of 1435±310ng/ml, respectively. While, the plateletapheresis procedures including the blood centrifugation steps with MCS (Haemonetics), CS-3000 (Baxter), and Spectra (Cobe) elicited much less C3a generation of mean values of 287ng/ml, 471ng/ml and 418ng/ml, respectively. The obtained plasma products from 5 hemapheresis procedures contained high levels of C3a of 303 to 1116ng/ml. All the hemapheresis procedures did not induce C5a but most platelet concentrates and plasma products contained increased C5a levels of 22 to 29 ng/ml. On the other hand, C4a also increased with two membrane-plasmapheresis procedures for 114 to 606ng/ml even if there is no evidence of classical pathway activation. Finally C3a is a most reliable indicator for complement activation during hemapheresis procedures.
Changes in ATP content and P50 of the blood during storage play an important role in oxygen transport and red blood cell viability. Three types of stored blood, namely CPD whole blood, CPD-CRC and MAP-CRC, with respect to blood gases, pH and l-lactate concentration ware examined. The number of days of storage correlated with blood acidity and l-lactate concentration, and P50 correlated with pH. On the basis of these observations, the efficacy of high CO2 permeability bags for blood preservation was tested. Blood PCO2 remained lower and pH and P50 higher on storage in these bags than on storage in conventional bags. I also examined the effects of pH change on ATP and 2, 3-DPG levels by addition of l-lactate to the stored blood. When pH of the blood was higher than 6.9, ATP level decreased significantly. When pH range was lower than 6.8, 2, 3-DPG level decreased. These results suggest that pH between 6.8 and 6.9 is optimal for the simultaneous maintenance of adequate ATP and 2, 3-DPG (and thus also P50) levels.