HLA antibodies in multiple transfused patients.

Totally 86 patients with multiple transfusions were followed up regularly for more than six months. Their sera were screened for HLA antibodies every two to four weeks. Subsequently 28 (33%) of them developed HLA antibodies. The specificities of identified antibodies were anti-A2, All, B16 and B60. However, most of the antibodies were either multiple or undetermined. The panel reactive activity (PRA) was correlated with the amount of blood components transfused. We concluded that about one third of the patients will develop HLA antibodies after multiple transfusions and the reactivity of the antibodies is correlated to the amount of blood components transfused.

[A rapid and simple method for direct genotype determination of ABO blood group].

We report the use of an allele-specific polymerase chain reaction (ASPCR) format together with low melting temperature agarose gel electrophoresis which allows rapid identification of the six major genotypes of the ABO blood group. Four sequence specific primer sets, each specific for a different set of ABO alleles, were used. Twenty individuals, whose ABO genotypes were previously determined by serological and family analysis, were typed with this new approach. A 100% correlation between serology and the ASPCR was found. This method is rapid, simple, and reproducible. Potential applications include identification of ABO subgroups and variants, paternity testing, as well as forensic science.

HLA, GM, KM, and Diego blood group typing of Chippewa Indians.

This is the first report characterizing HLA antigen distribution in North American Indians of the Chippewa tribe. One hundred seventy-four Chippewa from Minnesota underwent HLA-A,B,C,DR, and DQ typing in a search for a single unrelated bone marrow donor. The high matching rate of this successful search is attributed to homozygosity and the extreme frequency of certain antigens in this small ethnic community. It is emphasized that smaller donor pools are required in searches within a minority population. GM and KM allotype typing as well as blood group Diego typing show patterns similar to those reported in other North American Indian groups.

Clinical application of therapeutic hemapheresis.

Hemapheresis has been used in a variety of clinical states, primarily for its ability to remove an offending component, likely be either plasma or cellular elements. We have reviewed 43 cases of therapeutic hemapheresis over the past two years at Veterans General Hospital. There were 20 cases of plasmapheresis, 7 cases of leukapheresis and 16 cases of thrombocytapheresis. Most of them had satisfactory and desirable effects except a few cases in therapeutic plasmapheresis. Among 20 cases of therapeutic plasmapheresis, all but 2 cases of systemic lupus erythrematosus had transient clinical improvement. Average decrease of leukocyte count in 11 procedures of leukemic patients was 37%. As for the myeloproliferative disorders with thrombocytosis, the average drop of platelet count was 41%. The side effects of therapeutic hemapheresis were not infrequent. There were 56 recorded side effects with one sudden death among 94 procedures. Hemapheresis is useful in certain clinical conditions but more judicious application should be considered.

Detection of antibodies to human immunodeficiency virus (HIV-1) with the particle agglutination test.

This study was designed to evaluate the sensitivity and specificity of the particle agglutination test (PA) for antibody detection and to determine whether it is possible to use this test as a screening routine for anti-human immunodeficiency virus type I antibodies instead of the enzyme linked immunosorbent assay (ELISA). Serum samples were collected from 5,142 subjects and tested with the above two tests. A total of 83 samples were observed to be ELISA-positive. However, only 36 were found to be seropositive by the PA test. These samples were then confirmed by the Western blot analysis and 28 were positive for HIV-1, 33 were negative and 22 gave indeterminate results. The most common indeterminate band was p15/17 and the p24 band came next. The sensitivity and specificity of the PA test were 100% and 99.9%. The results indicate that the PA test is not only a convenient technique but also has a high sensitivity and specificity. It can be used as a primary screening test for antibody against HIV-1 instead of the ELISA.

HLA haplotype frequencies of Taiwan-Chinese families.

HLA haplotype frequency was studied by typing 201 members of 32 unrelated families. Linkage disequilibrium was determined by observed and expected haplotype frequencies. The two-locus haplotype frequencies with most significant linkage disequilibrium were A30-B13, Aw33-B17, Bw46-Cw11, B12-Cw8, A1-Cw6 and A33-Cw3. No locus recombination was noted among 137 children.

Hemolysis after ABO-incompatible platelet transfusions.

An 18 year old girl, with acute myeloid leukemia, developed progressive hemolysis after receiving multiple transfusions with ABO-incompatible platelets. It was caused by passive transfusion of anti-A and -B isoagglutinin from the donor plasma. Her hemoglobin level returned to normal after giving group compatible or pooled and reduced volume platelet concentrates. Transfusing group-incompatible platelets is not contraindicated, but donor plasma reduction should be considered for those patients who need prolonged platelet support. Testing for isoagglutinin titer in group O donors is an alternate method to reduce the incidence of plasma-induced hemolysis in group-incompatible platelet transfusions.

Family study of Rh haplotype frequencies in Taiwan.

Two hundred and two families (404 parents and 448 children) were typed for Rh antigens. The most common Rh haplotype is R1R1 followed by R1R2 and R1r. R1r', R2r', R0r', R0r and r'r are all very rare. The results of observed and expected haplotype frequencies do not differ significantly except few rare phenotypes. However, there are some major differences between Caucasian and Chinese populations in certain Rh genotypes. Although r is the second common genotype in Rh system among Caucasian but is rather rare in Chinese.

[Setting up a CMV negative donor file].

Cytomegalovirus (CMV) infection is serious to immunocompromized individuals such as recipients of bone marrow transplantation, preterm infants and other immunosuppressed patients. Since there is no effective treatment as yet, prevention is the most effective way so far to avoid CMV infection. Blood transfusion is one of the dangerous sources and screening for CMV negative donors is mandatory, therefore, to provide blood components for immunocompromized patients. We have screened 2,015 donors at various ages for CMV antibody by using passive latex agglutination test (PLA). The results showed that the CMV antibody positive rate was 91.7% in Taiwan. It differs from those of the western countries (40-79%) and the countries of Africa and Asia (96-100%). It has also shown that the positivity rate is closely related to the age of donors. The seronegative rates of ages among 18-20, 21-30, 31-40, 41-50 and 51-60 years old were 10.7%, 9.8%, 3.9%, 1.2% and 0% respectively. It has suggested thus that the older the donor is, the higher the seropositive rate will be. Therefore, we should choose the donors of younger age as candidates for making CMV negative donor file.

Alloantibodies other than anti-D in pregnant women.

Alloantibodies other than ant-D in two primigravidas have been reported. There was no history of exposure to risk factors. One of these primigravida developed anti-E and anti-c of IgM specificity and another developed anti-Jkb of IgG specificity. No evidence of hemolytic disease of the newborn was noted in both cases. From the family studies of both cases, the maternal alloantibodies seem to have been induced by the fetal red cell antigens. Although alloantibodies can occur in primigravidas, the incidence is very low. It seems unnecessary to do routine prenatal antibody screening in our population.

Blood group phenotypes in Taiwan.

This report describes a study of 31 red cell antigens in 13 blood group systems tested over a period of 3 years in the Chinese population of Taiwan. The study provides evidence that major differences exist between Taiwanese and whites or blacks in five blood group systems: Rh, MNSs, Duffy, P, and Xg.

Platelet crossmatching with lymphocytotoxicity test: an effective method in alloimmunized Chinese patients.

Fifty-three patients receiving long-term platelet transfusions were regularly screened for platelet-associated antibodies by a platelet suspension immunofluorescence test (PSIFT) and a lymphocytotoxicity test (LCT). Subsequently, 24 patients became alloimmunized; all of their antibodies were of HLA specificity. Eighty-two single-donor platelet transfusions were given, and the clinical responses were considered satisfactory if the 18-hour corrected count increment was 7.5 x 10(3) per microL or higher. In the meantime, 82 pairs of patient sera and donor lymphocytes were crossmatched. Among 63 crossmatched transfusions, 53 (84%) resulted in a satisfactory increment, with a mean (+/- SEM) of 17.71 +/- 1.96 (x 10(3)/microL), and 10 did not result in a satisfactory increment. The increments after 19 unmatched transfusions and 25 random-donor (uncrossmatched) transfusions were 0.7 +/- 0.3 and 2.39 +/- 0.66, respectively. The difference was not significant (p greater than 0.05). The agreement between the LCT results and clinical response was 88 percent. Retrospectively, the corrected count increments showed no significant differences (p greater than 0.05) among three groups of HLA grading: the increments for A/BU/BX, C/D, and random HLA matches were 22.97 +/- 4.07, 15.1 +/- 1.97, and 14.85 +/- 2.04, respectively. These results suggest that platelet crossmatching by LCT is an effective method for use in alloimmunized patients, especially Chinese patients.

Successful management of alloimmunized patients by transfusing HLA compatible and crossmatched platelets.

An average of three (range 0-22) perfect matches for 65 patients could be found in 1490 filed HLA-typed donors. It could increase to 8.7 (0-45) if strong cross-reactive antigens were added. Three methods of platelet crossmatching, i.e, lymphocytotoxicity test (LCT), platelet suspension immunofluorescence test (PSIFT) and solid phase red cell adherence test (SPRCA), were used to search for compatible donors. Forty episodes of apheresis platelet transfusion were evaluated in seven patients. Using one-hour posttransfusional corrected count of increment (CCI), greater than 7500 microliters as a guideline to assess the reliability of these three methods, we found that the efficiency of PSIFT, LCT and SPRCA were 67.5%, 97.5% and 97.5% respectively. Retrospectively, we did the HLA-A, B typing of those matched donors. There was no significant difference in CCI among different HLA match grades. The concordance of LCT and SPRCA was 100%, indicating that all the antibodies were of HLA specificity and there was no platelet specific antibody. We concluded that HLA donor-file and platelet crossmatching technique could be used together in selecting suitable donors for alloimmunized patients.

Platelet antibody screening in patients with leukemia and aplastic anemia.

Twenty-three patients with leukemia and ten patients with aplastic anemia who needed long-term platelet transfusion were regularly screened for platelet associated antibodies by a combination of platelet suspension immunofluorescence test (PSIFT) and lymphocytotoxicity test (LCT). Subsequently 13 of the patients (56.5%) with leukemia and 7 of the patients with aplastic anemia (70%) became alloimmunized. The overall incidence was 60.6% (20/33). The concordance of PSIFT and LCT was 100%, suggesting that all the platelet associated antibodies were of HLA specificity. The identified antibodies were anti-A2, A11, A24, B5, B40, B46, B57, B60 and B62. Most of them were antibodies against the high frequency HLA antigens in the Chinese population. There was no dose-response relationship in the development of alloimmunization. The interval between the initiation of platelet transfusion and the development of antibody varied from 10 to 192 days. The immunization is of all or none response. In our study group, about 40% of the patients who did not develop alloantibody within six months will never do so. We concluded that platelet transfusion should not be withheld for fear of alloimmunization and that HLA matched or lymphocytotoxic compatible platelet-donors may be helpful to alloimmunized patients.

Do the reticulocyte maturation fractions and bone marrow reticulocyte count further help the classification of anemias?

BACKGROUND: Reticulocyte count plays a major role in anemic evaluation. The conventional method done by the manual supravital staining cannot subclassify the group of less than 2% of corrected reticulocyte count. The newly-developed flow cytometer provides different maturation fractions by measuring its fluorescent intensity. The reticulocytes are believed to shift to the circulation from the bone marrow earlier in more severe anemia. Therefore, the purpose of this study is to evaluate the role of reticulocyte maturation fractions and bone marrow reticulocyte in anemia classification. METHODS: By using a fully automated counter, the roles of the reticulocyte with maturation and their shifting from bone marrow were evaluated in anemias. Different groups of subjects (243 in total) including aplastic, nutritional, and infiltrative anemias and anemia due to excess destruction and blood loss were studied. Each subject had bone marrow examination for morphologic diagnosis and reticulocyte evaluation. RESULTS: Both the absolute count and the maturation fractions of reticulocytes showed significant difference among marrow infiltration, aplastic anemia, and hemolytic anemia. Both the absolute reticulocyte count and less mature fractions were lowest in aplastic group. The marrow reticulocyte counts and shift ratio to circulating blood added little benefit in the classification of anemias. CONCLUSIONS: The automated reticulocyte count with maturation fractions helps classify anemias, particularly for those with low reticulocyte count by the manual method.

[Blood group phenotyping and their application in Taiwan].

During the 1950's and 60's as new blood group systems were identified, antigen distribution studies were performed in Europe and North America among Caucasians and American Blacks. However, to date only limited studies have been performed in Africa and Asia. Because of lack of knowledge of the antigen distribution of most other than ABO and Rho (D) blood group systems within these areas and among the people of non-Caucasian races, questions of testing needs and problems have occurred. In recent years, three big matters have been encountered off and on in blood banking in Taiwan. First, multiple-transfusion recipients develop so many alloantibodies that finding compatible donors becomes a difficult task. Second, since bone marrow transplantation technology is being instituted in many teaching hospitals, it is a task of blood banks to monitor the antigen changes of other blood group systems (including of Rh system other than D) before and after transplantation. Third, more than enough disputed paternity cases that can not be resolved by simple ABO testing. Therefore, blood banks should be staffed with suitable backgrounds to cope with the procedures needed for analysing all blood group antigens. In order to resolve all the problems effectively, we ran the tests for blood group antigens other than ABO and D in our blood bank from 1984 to 1986. A total of 31 sets of antisera were used to identify the specificity of 13 blood group system antigens of the Chinese population ranging from 99 to 2257. Based on the datum obtained, we found a significant difference between Chinese and Caucasians in the distribution of eight blood group systems (Rhesus, MNSs, Kell, Duffy, Kidd, P, Lutheran and Colton). The antigen frequency of Fya and s are 99.74, 99.91 respectively. The results are higher than those of the Caucasian population. On the other hand, Fyb and S are 9.22 and 6.56, much lower than those in Caucasians. We found no K, Lua and Cob antigens among the Chinese. We conclude that this study is a significant contribution to the knowledge of blood group antigen systems and antigen distribution, and also will benefit this population in many areas of medical care.

Antigen expression of frozen platelets.

Platelet antigens of platelet samples from 36 donors, frozen for different intervals, were evaluated by the platelet suspension immunofluorescence test (PSIFT). A, B, PLA1(HPA-1a) and various HLA antigens were tested by their corresponding antisera. The antigen could be detected in almost all the samples after one month of freezing. After 3 and 6 months, the platelet antigens could only be detected in 29.2% and 3.7% of the samples, respectively. There was no difference in decay of antigen expression among A, B, PLA1 and HLA antigens. When compared with the freshly prepared platelets, frozen platelets presented stronger antigen expression after 2 to 4 weeks of storage. This may suggest that the frozen platelets could be used for platelet crossmatching procedures without loss of their antigenicity within one month.

Neonatal alloimmune thrombocytopenia due to HLA-A2 antibody.

A male, full-term baby with thrombocytopenia was born by a G3P2A1 mother who was not associated with autoimmune disease. Platelet antibody screening was positive by using lymphocytotoxicity test, platelet suspension immunofluorescence test and solid-phase red cell adherence test. The identified HLA antibody was of A2 specificity. It was confirmed by testing the mother's and the baby's sera against the lymphocytes and platelets of 10 HLA-A2-positive donors. The possibility of platelet-specific antibody as the cause of neonatal alloimmune thrombocytopenia was ruled out by testing against platelets of 10 HLA-A2-negative donors and the known platelet-specific antigens utilizing immobilized, purified platelet glycoprotein as targets. The mother's serum reacted strongly with both the father's and the baby's platelets and lymphocytes. This neonatal thrombocytopenia was most likely due to the maternal HLA antibody, which was induced by her antecedent gestations.

Anti-Nform antibody in hemodialysis patients.

The anti-Nform antibody is produced by dialysis patients following reuse of dialyzers sterilized with formaldehyde and it has been implicated as a cause of hemolytic anemia. Formaldehyde is one of the common disinfectants used for reprocessing capillary hemodialyzers. The safety of formaldehyde and the clinical significance of anti-Nform antibody need further evaluation. Amongst 45 patients practising dialyzer reuse, anti-Nform antibody was detected in 5 (11.1%), but not amongst 111 patients not reusing their dialyzer (p < 0.005). The presence of anti-Nform was not related to the sex, or duration of dialysis with positive anti-Nform antibody. Direct Coombs' test was positive amongst 80% of all tested patients with anti-Nform antibody, and in 38% of patients reusing dialyzers but without anti-Nform antibody. No tests of hemolysis (including direct Coombs' test) discriminated between anti-Nform antibody-positive and -negative patients, nor between anti-Nform antibody patients with and without overt hemolysis. The best diagnostic test for hemolysis in anti-Nform antibody-positive patients was hematocrit rise after cessation of dialyzer reuse. It appears that despite the induction of anti-Nform antibody, hemolysis is rarely a serious consequence of dialyzer reuse.