Mutations at the activated protein C cleavage sites Arg336 and Arg562 of factor VIII in Thai patients with venous thrombosis.

Venous thrombosis is a multicausal disease, more than one genetic risk factor may cooperate to effect thrombotic risk. Factor V Leiden is found to be an important hereditary risk factor for venous thromboembolism. Analogous to factor V Leiden, a point mutation at amino acid positions Arg336 and Arg562 in factor VIII may predispose patients to thrombosis. Eighty-one Thai patients with venous thrombosis and 100 Thai healthy volunteers have been studied. Neither heterozygous nor homozygous mutations were detected both thrombosis patients or normal volunteers. However, further studies with larger samples of venous thrombosis patients are recommended.

HLA alloimmunization in patients receiving multitransfusions of red blood cells.

HLA antibodies were studied in 88 patients with chronic hemolytic anemia who received multitransfusions of red blood cells prepared by conventional (PRC-C), inverted centrifugation (LR-I) and leukocyte filter (LR-F) techniques. Their mean age was 8 years and 4 months with a duration of transfusion of 8 years. The patients were divided into five groups: group 1, receiving PRC-C (n=20); group 2, receiving LR-I (n=33); group 3, receiving LR-F (n=11); group 4, subsequently receiving LR-I and LR-F (n=10); and group 5, receiving PRC-C followed by LR-I and LR-F (n=14). The HLA class I antibodies were found in 30 out of 88 patients (34%). All were against HLA antigens commonly found in the Thai population. The patients receiving PRC-C exhibited HLA antibodies of 65%, which was significantly higher than those of patients receiving LR-I (24%) and LR-F (0%). Consequently, the transfusion reactions of fever, chill, rash and urticaria were also commonly found in patients receiving PRC-C (13.4%), which was significantly higher than patients receiving LR-I (0.4%) and LR-F (0%). The leukocyte filter technique has been shown to be effective in preventing HLA alloimmunization and transfusion reactions but the price is rather high. For the inverted centrifugation technique, only transfusion reactions were effectively prevented and the HLA alloimmunization continued to develop. A more effective and low-cost method for the removal of leukocytes should be investigated for these multitransfusion patients.

A preliminary study of the distribution of blood group systems in Thai blood donors determined by the gel test.

Two hundred blood samples obtained from volunteer blood donors at the Blood Bank, Army Institute of Pathology were studied for red cell groupings in the ABO, Rh, MNSs, Duffy, Lewis. P. Kell, Lutheran and Kidd Systems. Each sample was tested by the gel test using five cards; the ABO-Rh card, Diaclon Rh sub groups + K card, Antigen profile I card (P, Le(a), Le(b), Lu(a), Lu(b)), Antigen profile II card (k, Kp(a), Kp(b), Jk(a), Jk(b)) and Antigen profile III card (M, N, S, s, Fy(a), Fy(b)). For the ABO System, group O is the most common (40.5%) followed by group B (30.5%), group A (20.5%) and group AB (8.5%). The most common Rh gene complex was CCDee (51.5%), which was similar to other studies. The incidence of MMss and MNss gene complexes were the most common in the MNSs System. Fy(a) is very common as in other Asians. In the Lewis System, the incidence of Le (a-b-) was 23.5%, which is consistent with other findings in the Thai population. Sixty (30%) were positive with anti-P1. For the Kell System, only kk and Kp(b) positive types were observed in this study, as well as Lu (a-b+) in the Lutheran System. Jk (a-b-) was not found, which is considered a rare phenotype among Thai people. This study reveals the blood group distribution in 200 Thai volunteers using the gel test. Because of its simplicity and efficacy, this test is practical in population studies. Moreover, it is useful for mass screening and application in emergency situations.

HLA class II alleles in Japanese patients with non-Hodgkin's lymphoma.

The distribution of HLA-DRB1 alleles and DQB1 alleles in 30 Japanese patients with non-Hodgkin's lymphoma (NHL) was analyzed using polymerase chain reaction with the sequence-specific primer (PCR-SSP) method, and the association between the disease and the presence of certain HLA class II alleles was investigated. The frequencies of HLA-DRB1*0803, DRB1*0802 and DRB1*1502 were increased while those of DRB1*1501 and DRB1*0405 were decreased. On the other hand, the incidence of HLA-DQB1 alleles was similar to that in the normal population. However, none of these HLA class II alleles showed significant positive or negative associations with NHL. In addition, when allele frequencies of NHL Japanese patients were compared to Thai patients, only DRB1*0803 was significantly increased in Japanese patients. These results indicate that DRB1*0803 may not contribute to NHL susceptibility in the Japanese population. However, further studies with larger numbers of NHL Japanese patients are needed to confirm our preliminary findings.

Detection of factor V Leiden in Thai patients with venous thrombosis.

The molecular defect underlying activated protein C resistance (APC-R) is caused by a G to A point mutation in the codon for arginine 506 in the factor V gene (factor V Leiden) which is a major risk factor for venous thrombosis, especially in Caucasian populations. This study is an analysis of the Thai population to determine the prevalence of the factor V Leiden mutation. Twenty-seven patients with apparent venous thrombosis were divided into two groups according to APC-R test. Thirteen patients were diagnosed as positive for n-APC-SR, ratio < 0.8 and fourteen patients were diagnosed as negative for n-APC-SR, ratio > 0.8. Two of thirteen APC-R positive patients and one of fourteen APC-R negative patients were found to have the heterozygous allele for the factor V Leiden mutation but the homozygous allele was not detected in these groups of patients. Neither the heterozygous nor homozygous Leiden mutation was detected in 200 healthy volunteer blood donors. In conclusion, our findings indicate that factor V Leiden mutation is related to venous thrombosis in Thai people. Moreover, a further study of other mutations at the activated protein C cleavage sites of factor V and factor VIII is recommended.

Comparison of the RPHA and EIA techniques for the detection of HBs antigen among pregnant Thai women.

Five hundred serum samples obtained from pregnant women attending an antenatal clinic in Bangkok were tested for HBsAg by reverse passive hemagglutination assay (RPHA) and enzyme immunoassay (EIA). It was found that 21 (4.2%) and 28 (5.6%) of the sera were positive by RPHA and EIA, respectively. The sensitivity and specificity of the RPHA were 75% and 100%, respectively, when using EIA as the standard method. The RPHA positive predictive value was 100% and the negative predictive value was 98.5%. Accuracy was 98.6%. This study showed that the RPHA was simple and required inexpensive equipment, making it suitable for mass screening. However, the possibility of false negative readings due to low levels of HBsAg should be kept in mind, especially in the blood transfusion practice.

Comparative analysis of serological and molecular results for HLA-DR typing in 120 Thai subjects.

In this study, serological HLA-DR typing results were compared to typing results obtained with sequence-specific primers in the polymerase chain reaction (PCR-SSP). HLA-DR typing was performed on 120 random Thai individuals. Differences in HLA-DR typing results were found in 18 out of 120, which were due to cross reactive antibodies and the lack of potent antisera to define proper HLA-DR splits by serology. Furthermore, PCR-SSP is fast and easy to perform as HLA-DR typing results can be obtained within 2 hours. From the results of this study it can be concluded that PCR-SSP is a reliable and promising technique for HLA-DR typing which can replace the serological technique in routine clinical practice.

HLA class I antibodies in thalassemic patients.

This study was undertaken to demonstrate the prevalence of HLA class I antibodies among 62 polytransfused patients. The diagnosis included beta-thalassemia major, beta-thalassemia/Hb E disease and severe Hb H disease. Their ages ranged from 1 year to 23 years with the mean age of 10.7 years. The number of packed red cell transfusions ranged from 3 to 235 with the mean of 60 episodes per patient. The standard microlymphocytotoxicity test was performed using 50 panels of lymphocytes which specifically identified the majority of HLA class I antibodies. 31/62 cases (50%) were positive for HLA class I antibodies. The detection of single or multiple antibodies depended upon the number of blood transfusions and the patients' ages. These antibodies were induced by the leukocytes present in the transfused packed red cells. Therefore, leukocyte-reduced packed red cells prepared by either additional inverted centrifrugation or leukocyte filter is suggested for the routine blood bank service.

A comparative study of three techniques for eluting red cell antibodies.

Sixty-seven eluates obtained from the heat, ether and acid elution techniques were tested with the specific red blood cells (RBCs) and were compared according to their reactivities using the indirect antiglobulin test (IAT). It was found that the ether elution technique was superior in eluting Rh antibodies except for anti-e while the acid elution technique was superior in eluting Miltenberger (Mi(a)) antibodies (P < 0.05). The heat elution technique gave the lowest reactivity among the three techniques. In conclusion, the reactivities of the eluates obtained from the acid elution technique were overall comparable to those from the ether elution technique. The acid elution technique is practical for routine use in most blood banks because it is less time consuming and reduces the risk of exposing hazardous chemicals.

The benefit of the direct antiglobulin test using gel technique in ABO hemolytic disease of the newborn.

The direct antiglobulin test (DAT) using the gel technique was included in the investigation of infants with hyperbilirubinemia in the first week of life. Twelve cases were preterm and 48 cases were full term infants. The patients were divided into 2 groups: the study group comprised 22 cases of blood group A or B infants born to blood group O mothers; the control group comprised 38 cases of ABO blood groups compatible with those of their mothers. The mean +/- SD (39 +/- 26 hours) of the age at the onset of hyperbilirubinemia induced by ABO hemolytic disease of the newborn (HDN) was significantly earlier than that due to other causes. The positive rate of DAT in the ABO incompatible group was similar by both the conventional technique and the gel technique, 54.5% and 50% respectively. However, the scores by the gel technique were higher than those of the conventional technique. The gel technique is simple, reliable, involves less technical error and requires a small amount of blood sample. The grading system is clear-cut, especially grade 1 + or weak positive as compared to the conventional technique which requires examination under a microscope. Therefore, the DAT using the gel technique is beneficial to the diagnosis of ABO HDN. It should be included in the investigation of infants with hyperbilirubinemia especially in case of suspected ABO HDN.

Detection of thalassemia genes using smeared blood film or leukocytes adhering to polysthylene fibers.

Presently genetic analyses for thalassemia types require relatively large amounts of heparinized blood (5 to 10 ml), and transport as well as degeneration of these sample is a problem in the developing world. We have developed a new method to simplify this procedure and obtain DNAs from small specimens. As experimental materials, thinly smeared blood on a glass slide or blood filtered with and adhered on polysthylene telephtalate (PST) fibers were used. These materials could be safely stored without interfering with DNA extraction for up to 3 months. The slide materials were digested with proteinase K, and DNA was extracted with Tris-EDTA-phenol:chloroform and precipitated with absolute ethanol. The PST specimens were washed with physiologic saline and treated in the same manner as described above. Products were easily amplified by PCR and digested with restriction endonucleases for beta thalassemia typing as well as for HLA-DQA1 gene typing. Results obtained by this method correlated well with previously reported incidences for thalassemia and HLA-DQA1 types in Thailand. This method can be used in the routine laboratory because it allows for stable and biosafe genetic analyses.

Cost effectiveness in screening of class I HLA antibodies in Thai pregnant women.

Fifty-seven sera obtained from pregnant women who attended antenatal clinic were screened for class I HLA antibodies by standard microlymphocytotoxicity test. The age ranged from 20 to 40 years (mean +/- SD = 30.4 +/- 5). The lymphocytes obtained from 98 HLA-typed persons were used as the panel which would include all common HLA-A and HLA-B antigens and as many rare antigens as possible. Eight out of 57 sera (14%) had HLA antibodies which reacted as follows: 3 sera had nonspecific antibodies, 3 sera had multispecific antibodies (B60 + B48 + B7, B60 + B61 + B13 and B51 + B35 + B53), one serum had a monospecific antibody (A2) and one serum was unidentified. The identified antibodies showed strong reactivity (r > 0.8). These sera will be used as HLA typing sera and exchanged with other HLA laboratories. The screening test for class I HLA antibodies in pregnant women attending the antenatal clinic is useful and practical. Several HLA antisera can be obtained at low cost. The cost of self-screening antisera was US$90/ml while the cost of commercial HLA typing sera is approximately US$250/ml, a factor to be considered by a new HLA laboratory with limited budget.