The changes of hemostatic molecular markers such as beta-thromboglobulin, platelet factor 4, fibrinopeptide A, soluble fibrin monomer complex, fibrinopeptide B beta 15-42, fibrin degradation product and fibrinogen are measured during donor plateltphereses using cell separators. They are most sensitive markers of platelet, coagulation system, and fibrinolysis. Plateletphereses were performed using Phenwall CS3000 in 13 cases and IBM2997 in 8 cases. Platelet and coagulation system were activated during plateletpheresis, although any side effect was not recognized. From the results we perform that processed volume of whole blood should be limited 2, 000ml, approximately half of ordinary apheresis, in cases of donors from the age of 45 to 54. In addition donors who are considered to have any risk factor for thrombosis also should be omitted for plateletpheresis.

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Three methods, indirect immunofluorescence test (IF), gelatin particle agglutination (PA) and enzyme immunoassay (EIA) were examined for the detection of anti-HTLV-I. Of 57, 816 donor sera tested, 1, 320 PA⁺ samples were also tested by IF and EIA. Only 306 of those samples were positive both by IF and EIA. Additional 3 samples were IF^-EIA⁺ and 2 were IF⁺EIA^-.
The serological specificities of PA⁺IF^-EIA^- samples were then tested by the PA inhibition test. Specific PA inhibition by HTLV-I positive antigens were observed in 89 of 114 such samples.
Since most of positive samples by IF with MT-2 which is a strongly positive cell line for HTLV-I antigens, were also positive with MT-1, a standard test cell of IF, the low positive rate by IF did not appear due to the antigen deficiency of the MT-1.
Thus, a large number of PA⁺IF^-EIA^- samples appeared to contain anti-HTLV-I.

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The use of the citrate-phosphate-dextrose solution (the CPD solution) in place of the acidified citrate-dextrose solution (the ACD solution), as an anticoagulant solution has led to the slow resuspension of packed platelet sediments. Then, we studied for the state of resuspension of packed platelet sediments using the platelet concentrate (PC) prepared with supplement of the CPD solution, and obtained good results. That is, the rapid resuspension of packed platelet sediments was observed by adopting this new method. The platelet yield on the preparation of PC according to the present method was at least better than that of PC prepared by the standard procedure. In the PC obtained by the proposed method, the platelet functions were kept satisfactory; for example, both the aggregation, using collagen as a aggregating agent, and the percent recovery from hypotonic shock (%HSR) of 48-h stored platelet were remained about 80% of the initial activities. And a special triple bag system containing 26ml of the CPD solution in the primary bag and 10.6ml of the CPD solution in one of the two satellite bags, allows preparation of the PC under the closed condition.

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In the platelet apheresis and granurocyte apheresis used by the cell-separater: IBM 2997, the changes of absolute number of lymphocytes and lymphocyte subsets were investigated in comparison with pre- and post-apheresis in the peripheral blood of donors. Also, lymphocyte subsets into the bags collected from donors were examined.
In the platelet apheresis, absolute number of lymphocytes and precentages of lymphocyte subsets were not changed in the peripheral blood of donors.
However, in the granulocyte apheresis, after injection of hydrocortizone (200mg), percentages of lymphocyte, OKT3 and OKT4 positive cells in the peripheral blood of donors were decreased significantly compared to pre-apheresis.
On the other hand, OKT4/8 ratio into bags collected by granulocyte was more decreased significantly compared to platelet apheresis bags.
From these results, we strongly feel that the prevention of lymphocyte contamination and calculation of OKT4/8 ratio into the collected bags will be important to avoid the allergic reaction and GVHD.

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In response to the concept of component blood transfusion, collection of individual components of blood directly from healthy donors needs to be popularized. Especially, a safe and effective method of plasma collection, donation plasmapheresis, is requested to be urgently developed. Reported herein is a donation phasmapheresis technique utilizing a membrane filter for separation of plasma in connection with a single needle for venipunture. The result obtained from ten actual donation plasmapheresis procedures showed that the average amount of 453ml of plasma was collected in the average period of 56 minutes without any unfavorable untoward effects in ten volunteer donors, and the solutes including important proteinous fractions such as immunoglobulins and coagulation factors were sufficiently contained in their concentration to be used as plamsa components for transfusion.

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When granulocytes were exposed in cryoprotectants such as glycerol, dimethyl sulfoxide and ethylene glycol, and removed from them, cell plasma membranes were osmotically stressed and injured. Membrane integrity was determined by number of cells, the pattern of cell volume distribution, mean cell volume and the continuous measurement of cell volume in hypotonic solution. At the same molecular concentrations of cryoprotectants, the degree of membrane integrity was in the order as follows: glycerol>>etylene glycol>dimethyl sulfoxide. Dimethyl sulfoxide was suitable for cryoprotectant even at the concentration of 7v/v% (about 1M). By adding and removing of cryoprotectants at various temperature, membrane integrity was less with the rise of temperature from 4°C to 37°C. The presence of sucorose and mannitol in ethylene glycol or dimethyl sulfoxide showed to have a protective effect against osmotic stress. 200mM sucrose or mannitol diminished the membrane integrity of cells which were added to and removed from 10v/v% (about 1.5M) of dimethyl sulfoxide.

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The series was collected in 1978-1984 from 20 hospitals in Gunma prefecture where all pregnant women were Rh-typed and blood samples were checked for antibodies by this laboratory.
The total number of Rh₀(D) negative pregant women was 499 and of these, 28 had developed antibodies. Three of these 28 patients had recieved blood transfusion. Six of the antibody containing patients were first immunized during the period of observation. Four of these 6 produced the antibody in the course of a first pregnancy and 2 in second preganacy. The antibody titers increased after 20th gestational week, especially after 30th through 40th week in most of patients.
Three out of 7 infants, whose mothers' antibody titers increased no more than 32 folds by papain treated red cells recieved phototherapy. In the cases of antibody titers more than 32 folds, 20 out of 26 babies received exchange blood transfusion. The mature infants were all alive healthily.
There were four Rh₀(D)-negative women who repeated intrauterine fetal death or newborn death by hemolytic disease of the fetuses.
IgG anti-D was used in only 3 cases in order to prevent production of anti-Rh₀(D) antibody. Amniocentesis was executed in 9 cases.

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We report a case of naturally occurring anti-Sc2 found in a Japanese pregnant woman with Rh (D) negative phenotype. She has no history of blood transfusion, and experienced two normal deliveries.
Anti-Sc2 was found in the serum at the 13th week of gestation in her third pregnancy. Antibody titer by indirect antiglobulin test was 1: 2. The antibody was also detected by low isonic strength polybrene (LISP) method in the AutoAnalyzer (Technicon-AAII).
In addition to anti-Sc2, anti-D appeared at the 17th week (1: 8 by indirect antiglobulin test) and reached the maximum (1: 32) at the 36th week. In contrast, anti-Sc2 decreased to the level at which only LISP method can detect it after the 20th week.
A boy was born at the 38th week and received phototherapy for neonatal jaundice. Cord blood was positive for direct antiglobulin test. Only anti-D was detected on the red cell surface.
Red cells from this woman, her husband and their children including a new born were all negative tested for Sc2 antigen.
It is suggested that this woman has developed anti-Sc2 in the course of pregnancy without exposure to Sc2 antigen. The mechanism of this antibody production remains to be investigated.

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Autoimmune hemolytic anemia (Hb 4.1g/dl, reticurocyte count 18.1%) developed in a 42-year-old woman who took any drug before admission. The supernatant of her red cell's eluate prepared from the procedure of adsorption technique with Jk (a- b-) normal red cells, contained antibody with anti Jk^a specificity. Her antibody was confirmed to have the characterization of IgG₁.

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In 1985, at the Hokkaido Red Cross Blood Center, a carrier clinic was set up for the HB virus carriers who were found through blood donations. The main purposes of the clinic are to contribute to the health control for the carriers and to get high titered HBs positive blood for HB vaccines. A total of 903 carriers attended the orientation in fiscal 1985, at which they were explained about HB and advised to consult the doctors at the carrier clinic regularly; the average number of carriers present per orientation was 21. The total number of carriers who consulted the clinic was 682; the average number of the attendance was 18per clinic. The carriers who were suspected of having liver disease were 67; 52 were advised to go to the medical specialists. The percentage of the HBe-Ag positive carriers whose liver function was abnormal was 43%, which was much higher than the percentage among the whole carriers in the clinic (16%). It can be said that HBe-Ag positive carriers form a high-risk group for liver disease. The HBs positive blood for vaccine preparation showed an increase of 20% by the donations from the carriers. All the carriers' records are stored in a personal computer, enabling us to manage them effectively for a long time.

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The constitutional nature of hemostatic plug is important for hemostasis, because hemostatic plug might be functioning a direct role to cease bleeding at the site of hemorrhage. Therefore, the structual abnormalities of the plug in patients with hemophilia have been reported recently by us and others. However, there are many unknown mechanisms to produce the qualitative abnormalities of plug structure, which might be constructed mainly fibrin-closslinked mass and appregated platelets.
In this paper, the authors observed the time course on the release of factor XIII a subunit from the platelet aggregates to the factor XIII deficient plasma by the use of our ELISA technique with polyclonal antibody against factor XIII a subunit, and it was shown a positive relationship between the release phenomenon of platelet factor XIII and the autolytic destruction process of platelet aggregates, and the 50% release of factor XIII from platelet aggregates was measured about 4hrs after platelet aggregation induced by ADP or thrombin.
In the experiments of hemophilia A cases the amount of crosslinked von Willebrand factor and fibronectin to fibrin clot tended to increase but the crosslinkage of α₂ plasmin inhibitor to fibrin showed a decreasing tendency in the fibrin clot obtained from the recalicified patient plasma mixed with ¹²⁵I-labeled α₂ plasmin inhibitor. Those data might indicate the reason for rapid fibrinolysis by urokinase added on the recalicified clot from the plasma collected before and after the administration of factor VIII reparation to hemophiliacs and also the reason for low values of the maxmum amplitude in thrombelastgraphy performed on nine patients with hemophilia A.
According to the results above mentioned it might be suggested that the qualitative abnormalities of crosslinked fibrin structure of the hemostatic plug in the patients with hemophilia might effect on the disfunction of the plug, but the reaction mechanisms inducing such abnormal changes of the clot structure are still not elucidated in detail. However, the prolongation of blood clotting time due to low activation of thrombin with untimely supply of platelet factor XIII might be possible as a reacting condition for this abnormal distribution of crosslinkage.

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The interaction of Factor VIII and von Willebrand Factor in plasma has a major impact on hemostasis. These two proteins contribute to hemostasis by supporting platelet adhesion to the damaged vessel wall and by accelerating fibrin formation. While the absence of Factor VIII in hemophilia A has no detectable effect on vWF levels, normal plasma vWF levels are necessary for normal Factor VIII content. In addition, complex formation with vWF stabilizes plasma Factor VIII and concentrates Factor VIII, a trace protein, at the site of vessel injury.
There has been remarkable progress during the past decade in our understanding of Factor VIII and von Willebrand Factor structure and function. The genes responsible for the synthesis of these two proteins have now been characterized and we are beginning to learn about the molecular defects responsible for hemophilia A and von Willebrand's Disease. While the binding sites that lead to complex formation have not yet been characterized, future studies will undoubtedly clarify how vWF protects Factor VIII from proteolysis, how vWF modifies Factor VIII synthesis, and how the two proteins interact in a way that concentrates Factor VIII at the site of vessel injury. Answers to these important questions will further establish the critical role of the Factor VIII/von Willebrand Factor complex in hemostasis.

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