A leukocyte removal filter (Pall PL Filter) were widely believed to show no significant effect on platelet function and platelet count of platelet concentrates. In this paper, we evaluated platelet function of platelet concentrates in detail. In the first 6ml filtered samples, ADP, collagen and epinephrine-induced aggregations were depressed and platelet count showed significant decrease with platelet recovery rate of 72%. These abnormalities returned to normal in more than 18ml filtered samples. In pooled samples, no significant changes were observed in platelet function before and after filtration. In twelve patients with hematological disorders, post-transfusion side reactions were significantly low incidence in those with the filter (25%) compared with those without filter (75%). Our results demonstrated that leukocyte removal filter was effective for the prevention of post transfusion side reactions but could give some possibility that very small amount of platelet transfusion might not be useful for preventing the bleeding.

View full abstract

Recently, recombinant human erythropoietin (rHuEPO) has been administrered in order to increase preoperative blood store. Otherwise, the effect of rHuEPO for postopertive anemia is unknown. We used rHuEPO to improve postoperative anemia. The patients were twenty-one person who were not transfused homologous blood but autologous blood perioperatively, since April 1988 to March 1991. They were divided into two groups. There were no statistical significance in sex, age, body weight, and extra corporeal circuration time between non rHuEPO group and rHuEPO group. Autologous blood collection was started several months before operation and separated into concentrated red cell (CRC) and plasma to freeze. They were thawed on the day of operation to transfuse. 6.000 IU rHuEPO was administrered intervenously three times per week for two weeks pre-and postoperatively. Iron was administrated more than 100mg per day P. O. or more than 40mg per day I. V. The standard for homologous blood transfusion were, 1) less than 7.0mg/dl in Hb concentration, 2) unstable hemodynamics, and 3) deterioration of general condition. In our method of rHuEPO administration, rHuEPO group showed higher Hb concentration level than that of non rHuEPO group at 14 POD and 21 POD, so that rHuEPO administration is effective to improve postoperative anemia.

View full abstract

Incidence of hepatitis C after blood transfusion at an operation was compared between before and after Japanese Red Cross Blood Centers applied HCV antibody test to donor blood. As subjects, 48 cases (9.5 units were transfused on average) before the introduction of the test and 75 cases (7.7 units on average) after it were investigated.
The ratio of the subjects seroconverted for HCV antibody decreased to a quarter after the introduction. Five cases (11%) seroconverted for C100-3 antibody before the introduction of the test, while only 2 cases (3%) seroconverted after introduction. With the introduction of screening, incidence of hepatitis decreased from 23% (7 cases of definite diagnosis and 5 cases of suspected diagnosis) to 6% (Including 3 cases of definite diagnosis and 2 cases of suspected diagnosis), that is about a quarter of the pre-screening level. These showed that the introduction of HCV antibody test was effective to decrease the incidence transfusion-associated of hepatitis. However, in spite of the supply of HCV antibody negative blood, hepatitis C occurred in 6% of 75 transfusion cases.

View full abstract

We experienced 4 cases whose red cells showed negative reaction with polyclonal anti-D reagents and positive with monoclonal anti-D reagents after the immediate spin. The reactivities of 4 cells were between those of D positive and D^U cells. The reactivities of R₁R₂ cells (n=44), which were used as a positive control when D^U testing, with polyclonal anti-D reagents were distributed from 2⁸ to 2¹¹. Four cases were typed D^U or D positive depending on the reactivity of R₁R₂ cells used. The reactivities of 4 red cells determined by ELISA or FCM with polyclonal anti-D reagents were also between those of D^U and D positive cells. We considered that the type of our 4 cases was weak D.

View full abstract

Irradiation of blood products is an effective way to prevent post-transfusion graft-versus-host disease (GVHD). However, the characteristics of platelet concentrates (PC) irradiated with X-ray were still unknown. Therefore, in order to study the effects of X-ray irradiation on PC, we examined the mitotic activity of lymphocytes and in vitro characteristics of PC stored for 5 days after X-ray irradiation. The results showed that Mixed Lymphocyte Culture (MLC) response of lymphocytes contaminating PC was completely abolished by irradiation with only 5 Gy, and that there were no significant differences between the irradiated and unirradiated PC in pH, platelet count, aggregability, ability of shape change and hypotonic shock response. Furthermore, no differences were found in the amount of lipid peroxides and di-(2-ethylhexyl) phthalate in plasma of PC and morphology of platelets between the irradiated and unirradiated PC.
In conclusion, we showed that X-ray irradiation of PC with 15 Gy was sufficient for preventing post-transfusion GVHD, and that irradiation below 50 Gy had no deleterious effects on in vitro qualities and functions of PC stored for 5 days after the treatment.

View full abstract

The Nak^a antigen is an isoantigen on platelet GPIV (CD36) and Nak^a negative platelets are deficient of GPIV. The Nak^a epitope defined by anti-Nak^a serum is also recognized by OKM5, a monoclonal anti-GPIV (CD36). This was shown by blocking experiments, in which the reactivity of OKM5 was completely blocked by the pretreatment of the platelets with anti-Nak^a serum and vice versa. Quantitative studies of the Nak^a-GPIV epitope on platelets and monocytes were performed both with the OKM5 and anti-Nak^a serum. Amounts of the Nak^a-GPIV epitope on monocytes were 4 times as many as those on platelets. Monocytes from Nak^a negative platelets donors could express the epitope, which varied quantitatively from the one comparable to that of Nak^a negative platelets to the other to that of Nak^a positive platelets. Two distinct Nak^a-GPIV positive patterns of Nak^a positive platelets were also observed in monocytes and appeared to be genetically determined. Since mRNA for CD36 is present in Nak^a negative platelets as well as monocytes, the genetic control of Nak^a-GPIV expression must be at post-translation level.

View full abstract

We developed the syncytium induction assay in which peripheral blood lymphocytes from HTLV-I healthy carrier donors were co-cultivated with the human epidermoid cancer cells (ME180).
A significant increase of multinucleated syncytium formation was observed in ME180 cells along with HTLV-I expression after co-cultivation with 1×10⁶ HTLV-I carrier lymphocytes. Co-cultivation with HTLV-I negative lymphocytes did not induce a significant syncytium formation. The syncytium formation activities varied widely among carrier lymphocytes and did not correlate with the expression of HTLV-I antigen in the lymphocytes. By freeze-thawing treatment to lymphocytes this syncytium formation was inhibited.
This syncytium induction assay using ME180 cells can be utilized for detection of infectious HTLV-I in peripheral lymphocytes in healthy carriers and thereby for their follow up study whose natural history is largely unknown.

View full abstract

To evaluate the effect of recombinant human erythropoietin (r-HuEPO) on a preoperative blood donation and on hematological influence after surgery, we studied patients undergoing elective orthopedic surgery. r-HuEPO was administered intravenously twice a week from one week before the first donation except with the 12 controls. All patients received 200mg iron sulfate a day orally and 40mg twice a week intravenously. 400ml of autologous blood was donated at weekly intervals up to 1200ml. A dose-dependent hemoglobin increase for three weeks was observed in the 3000U (2.02g/dl), 6000U (2.22g/dl), and 9000U (2.89g/dl) groups respectively, as compared with 1.34g/dl in controls.
We also observed the delay in recovery from postoperative anemia in patients using r-HuEPO compared to the controls and a suppressive effect of r-HuEPO on endogenous erythropoietin secretion. In conclusion, although preoperative administration of r-HuEPO in collection of autologous blood is very useful, the preoperative anemia induced by repeated phlebotomy may accelerate the postoperative secretion of endogenous erythropoietin.

View full abstract

In recent years, it has been expected that UV light irradiation on PC may prevent HLA-alloimmunization following repeated platelet transfusion. For the purpose to evaluate this technique, we tested the UV light permeability of blood bags and inactivation effect of UV light on lymphocytes in PC.
UVB light (280-320nm) permeability of blood bags, available from Japanese mamufactures, was 60% on average. Among them, PVC bag plasticized with DnDP showed most excellent permeability of 73%.
In MLR testing, UBV light irradiation at proper doses of 1×10⁴J/m² was recommended for complete lymphocytes inactivation. Plasma thickness of 16mm and RBC contamination of 4×10⁴μl did not disturb the inactivation effect of UVB light.
These data support our conclusion that UVB light irradiation on PC can be performed without changing any present procedures of blood separation, blood bags, PC volume and red cell contamination. UVB light irradiation is simple and inexpensive technique, and so it has a practical usefulness.

View full abstract

Biochemical properties of Factor VIII (FVIII) in highly-purified FVIII concentrates (CROSS EIGHT M) produced by monoclonal antibody column were studied and compared with those of Hemofil M which was already used clinically. The FVIII clotting activity (FVIII:Cclot) levels obtained by one-stage method using severe hemophilia A plasma as a substrate were 614-729U/vial and identical with those indicated on the label (500U/vial). The FVIII light chain antigen (FVIIIL:Ag) levels measured by Enzyme Immunoassay were 540-760U/vial. The ratio of FVIIIL:Ag to FVIII:Cclot (FVIIIL:Ag/FVIII:Cclot) were 0.88-1.04. The ratio of FVIIIL:Ag to von Willebrand Factor antigen (vWF:Ag) (FVIIIL:Ag/vWF:Ag) were 49.0-60.8. No difference was observed between CROSS EIGHT M and Hemofil M on the FVIII:Cclot levels, FVIIIL:Ag levels, FVIIIL:Ag/FVIII:Cclot and FVIIIL:Ag/vWF:Ag.
SDS-PAGE patterns showed almost no contamination except albumin being added as a stabilizer. Western blot analysis revealed FVIII heavy chain antigen of M. W. 90kDa-220kDa and FVIII light chain antigen of M. W. 80kDa. No degradates of MW. 43kDa-50kDa derived from FVIII heavy chain antigen were observed. CROSS EIGHT M lacked the large multimers of vWF which were detected in normal human pooled plasma. No difference was observed between CROSS EIGHT M and Hemofil M by western blot analysis of FVIII and multimer analysis of vWF.
These results suggest that CROSS EIGHT M preserves the fundamental structure of FVIII molecule for coagulant activity. Therefore, CROSS EIGHT M as well as Hemofil M may be clinically used with safety as FVIII concentrates.

View full abstract

The prevalence of antibody to hepatitis C virus (HCV) and HCV RNA were tested in blood doners with ELISA and reverse transcription-polymerase chain reaction (RT-PCR). In this study, four ELISA Kits were used to screen for antibodies to HCV (Ortho-1st:c100-3 viral peptide, TND-2:c-7 and c-11 viral peptide, GOR: host derived peptide and Ortho-2nd: c200 and c22-3 viral peptide). Among 998 doners, the seropositivity rate was 1.2% (Male 1.6%, Female 0.8%) for Ortho-1st, 1.5% (M 1.6%, F 1.4%) for TND-2 and 1.2% (M 1.4%, F 0.8%) for GOR. Also, HCV RNA was detected in 6 samples from 12 positive to Ortho-1st (50.0%), 10 samples from 12 positive to TND-2 (83.3%) and 7 samples from 12 positive to GOR (58.3%).

View full abstract

In this experiment, the commercial T98G and U373MG cells were used as the target cells. The T98G cells were markedly destroyed by the lymphokine activated killer (LAK) cells induced from the peripheral blood lymphocytes of normal volunteers. The cytolysis of the U373MG cells by the LAK cells, however, was very low. The T98G cells exhibited high adherent tendency against the LAK cells and the adhesion was further enhanced by adding of the recombinant interferon-r (IFN-γ) to the medium longer than 3 hours. On the other hand, the U373MG cells showed low adherence and the tendency was not affected by adding of IFN-γ or OK432. It seemed that the low adherence of the cells may be a cause of the low cytolysis. These results are suggestive that the tumor cells with low adherent tendency against LAK cells are unsuitable for the adoptive immunotherapy by using LAK cells.

View full abstract

We report a case of low-titer cold agglutinin disease with ant-Pr₂ specificity and ataxic polyneuropathy associated with IgM_κ monoclonal gammopathy, that is the first report in Japan.
The cold agglutinin (CA) reacted with untreated OI and Oi cells, and untreated and proteasetreatd dog RBCs. Both protease and sialidase treatments of human RBCs abolished its reactivity, and these findings confirmed ant-Pr₂ specificity. Agglutination with the anti-Pr₂ CA was inhibitted by beaf brain gangliosides and GM₃, and weakly by G1a, GD1b, GT1b and glycophorin. The anti-Pr₂ could play a pathogenetic role not only in hemolytic anemia, but also in peripheral nerve demyelination in this case, because gangliosides GM₃, GD1a, GD1b and GT1b are constituents of human peripheral nerve myelin.

View full abstract

Case No. 1: A 24-year-old man, complaining of fever, skin eruption, jaundice, brown urine and lymphnodes swelling, had hemolytic anemia caused by cold agglutinin following rubella infection. Laboratory tests revealed low Hb (4.6g/dl), high GOT (60U/L), LDH (2050U/L), T-bilirubin (bil) (7.4mg/dl), D-bil (1.7mg/dl), and high cold agglutinin titer (over×2048), undetectable amount of haptoglobin, and elevated anti-rubella antibody (IgM-c×6.64). Because the cold agglutinin of the patient strongly reacted against a group O red blood cell (RBC), O umbilical RBC, O adult i RBC and neuraminidase treated RBC, and weakly against enzyme (papain, ficin, bromelin) treated RBC, it was identified as anti-Pr^a. Glucocorticoid administration of the patient improved his clinical and laboratory findings.
Case No. 2: A 44-year-old male patient of malignant lymphoma, blood group A₁, Rh-positive, suffered from S. pneumonia. He had never received blood transfusion. Laboratory tests revealed anti-H, composed of IgG (IgG1, IgG2) and IgM, the titer being 1:64 by the saline method at room temperature, 1:4 by the albumin method at 37°C, and 1:32 by the antiglobulin method. The antibody titer fell to 1:1 along with remission of the pneumonia. These cases suggest viral or bacterial infections may induce some anti-RBC antibodies.

View full abstract

Here reported are two patients having anti-Wr^a antibody, which have been uncommon in Japan. Further, we have found 38 such cases so far reported in our country. The 40 cases in all have been examined on their backgrounds which might be related to the anti-Wr^a production.
1) Case report: Case No. 1: A 78-year-old female, complaining of dizziness and palpitation, was found to have autoimmune hemolytic anemia.
She had never received blood transfusion. Her blood group was B, CcDEe, Wr (a-). Direct antiglobulin test (DAT) using anti-IgG was positive, and the eluate from RBC reacted against all of the panel red cells, showing presence of panreactive warm-type anti-RBC autoantibody. The patient's serum, even after its autoantibody was absorbed by her own RBC, still showed presence of anti-Wr^a.
Case No. 2: A 68-year-old male had been suffering from the myelodysplastic syndrome, which thereafter transformed to myelocytic leukemia.
His blood group was A, Rh (D)-positive. The screening test for unexpected anti-RBC antibodies was initially negative, which turned to positive along with DAT after frequent blood transfusions. Further laboratory tests detected panreactive warm-type anti-RBC autoantibody in the eluate from the patient's RBC as well as alloimmunized anti-E and anti-Wr^a in his serum.
2) Results obtained by the survey on the anti-Wr^a in Japan: In the majority of the 40 cases with anti-Wr^a in Japan, including our above-mentioned two, the anti-Wr^a seemed to be a natural antibody, while, in 3 of them, anti-Wr^a was coexistent with the warm-type anti-RBC autoantibody. Besides, other 2 cases seemed to be drug-induced. These findings raise interests in the etiology of anti-Wr^a. However, the anti-Wr^a itself may be clinically innocent here in Japan, because any individuals having the blood group antigen Wr^a have not yet been found among Japanese. In addition, if Wr^a-positive RBC will be hereafter more commonly included in panel red cells for screening unexpected anti-RBC antibody, anti-Wr^a may, probably, be more frequently detected in Japan.

View full abstract