Multiple miscarriages in two sisters of Thai origin with the rare Pk phenotype caused by a novel nonsense mutation at the B3GALNT1 locus.

OBJECTIVES: To determine the genetic background underlying the Pk phenotype in two Thai sisters suffering from multiple spontaneous abortions. BACKGROUND: The P antigen is carried by globoside, an abundant glycosphingolipid in the red blood cell (RBC) membrane. Inactivating mutations in the 3-ß-N-acetylgalactosaminyltransferase gene (B3GALNT1) give rise to the rare Pk phenotype, which lack the P and PX2 antigens. Consequently, naturally occurring anti-P may cause recurrent miscarriages following the cytotoxic attack of the globoside-rich fetal portion of the placenta. METHODS/MATERIALS: P/P1/PX2/Pk antigens on RBCs and their corresponding antibodies were detected by haemagglutination and flow cytometry. The B3GALNT1 coding region was sequenced, and an allele-specific polymerase chain reaction (PCR) was developed. RESULTS: The two sisters had suffered 8 and 11 miscarriages, most of which occurred in the first trimester. Anti-P and anti-PX2 were identified in their plasmas, and the RBCs typed as P-PX2-Pk +, i.e. had the Pk phenotype. Sequencing revealed homozygosity for a nonsense mutation, c.420T>G, in B3GALNT1. This substitution introduces a premature stop codon, p.Tyr140Ter, which is predicted to abolish enzymatic activity. Screening of 384 Thai donors indicated an allele frequency of 0·13%. CONCLUSION: We describe a novel nonsense mutation (c.420T>G) in B3GALNT1 (GLOB*01N·13), which was added to the 12 alleles already known in the GLOB system.

Detection of MNS hybrid molecules in the Thai population using PCR-SSP technique.

We developed a polymerase chain reaction-sequence-specific primer (PCR-SSP) technique to screen for hybrid molecules in the MNS blood group in the Thai population using two sets of newly designed primers specific for four GYP(B-A-B) hybrids, GP.Mur, GP.Hop, GP.Bun and GP.HF, and two GYP(A-B-A) hybrids, GP.Vw and GP.Hut. One thousand and forty-one blood samples were tested with human anti-Mi(a) by conventional tube technique, and 598 samples of these were tested by the PCR-SSP technique. Ninety-four samples (9.03%) were strongly positive with human antisera by conventional tube technique. For PCR-SSP test results, the GP.Hut, GP.Mur, GP.Hop, GP.Bun and GP.HF genotypes were amplified with the first set of primers, whereas GP.Vw genotype was amplified with a second set of primers. The GYP(A-B) hybrids (GP. Hil and GP.JL), GYP(A-B-A) hybrids (GP.Nob, GP.Joh and GP.Dane), GYPA, GYPB and GYPE were not amplified by either set of primers. Results of testing 94 Mi(a+) and 504 Mi(a-) by conventional tube technique and PCR-SSP were concordant. This study shows that analysis by PCR-SSP is simple and convenient; therefore, it can be used as an alternative to conventional tube technique for mass screening for MNS hybrids, especially when specific antisera are not available.

External quality assessment scheme in red blood cell serology: a 5-year experience in Thailand.

From 2000 to 2004, 36, 58, 72, 78, and 86 laboratories participated in an external quality assessment scheme (EQAS) organized by the Department of Transfusion Medicine, Faculty of Medicine Siriraj Hospital. Each year the staff was requested to perform ABO grouping, D typing, antibody screening, antibody identification, and DATs on eight blood samples. Each participant received information on the correct test results and a coded summary. Regarding ABO grouping, the error rate ranged from 0.3 to 1.3 percent, mostly due to human errors. Error rates in D typing ranged from 0.7 to 5.7 percent, the most problematic being weak D phenotype interpretation. Although every sample was negative by the DAT, error rates due to false positive test results were determined to be 0.4 to 2.1 percent. Antibody screening errors were also found; however, errors steadily decreased from 4.2 percent in 2000 to 0.3 percent in 2004. Only 69.4 to 87.2 percent of laboratories performed antibody identification; however, correct results increased from 78.4 to 91.0 percent. In conclusion, an EQAS in RBC serology should be used to compare results from different laboratories and to identify those laboratories that need improvement in testing procedures.

Analysis of SERF in Thai blood donors.

The Cromer blood group system consists of nine high-prevalence and three low-prevalence antigens carried on decay-accelerating factor (DAF). We recently described one of these Cromer highprevalence antigens,SERF, the absence of which was found in a Thai woman. The lack of SERF antigen in this proband was associated with a substitution of nucleotide 647C>T in exon 5 of DAF, which is predicted to be a change of proline to leucine at amino acid position 182 in short consensus repeat (SCR) 3 of DAF. This study reports on PCR-RFLP analysis of the SERF allele with BstNI restriction endonuclease on more than one thousand Thai blood donor samples. One new donor homozygous (647T) and 21 donors heterozygous (647C/T) for the SERF allele were found. Among this cohort of random Thai blood donors, the SERF allele frequency was 1.1 percent. Thus, like other alleles in the Cromer blood group system, SERF is found in a certain ethnic group.

HLA class I serotypes and cytotoxic T-lymphocyte responses among human immunodeficiency virus-1-uninfected Thai volunteers immunized with ALVAC-HIV in combination with monomeric gp120 or oligomeric gp160 protein boosting.

Antigen-induced cellular immunogenicity may vary between populations due to differences in human leukocyte antigen (HLA) diversity and, hence, may play a critical role in the protection afforded by vaccines. In the setting of two, phase I/II human immunodeficiency virus-1 vaccine trials of a recombinant canarypox prime, and boosting with either recombinant monomeric gp120 or oligomeric gp160, we assessed the association between specific human leukocyte antigen (HLA) class I serotypes and the presence of cytotoxic T-lymphocyte response measured by 51Cr-release assay. HLA class I serotypes A11, A24, A33, B46, and B75 were the most common, present in 10% or more of 245 individuals studied. Forty of 187 (21.4%) Thai adults who received either ALVAC-HIV with gp120 or oligomeric gp160 or ALVAC alone had a precursor cytolytic CD8 T-cell response (pCTL). HLA-B44 was positively and significantly associated with a pCTL response (odds ratio 7.6, 95% CI: 2.7-21.2), whereas B46 was negatively associated but not robust when adjusted for multiple comparisons. Responses to Env proteins accounted for the majority (nine of 11) of pCTL activity among those persons with B44. This HLA class I serotype occurred in 9.4% of participants overall (including the placebo group), less commonly than what is reported from populations of European ancestry. These results strengthen the importance of assessing HLA class I distributions in conjunction with studies of vaccines designed to elicit cellular immunity in different populations.

Association of Fcgamma receptor IIb and IIIb polymorphisms with susceptibility to systemic lupus erythematosus in Thais.

We recently reported association of a newly identified polymorphism of Fcgamma receptor (FcgammaR) IIb, I232T, with systemic lupus erythematosus (SLE) in Japanese. To date, information on FcgammaR genotypes and their association with SLE is limited in South-east Asian populations. To gain further insight into the role of FcgammaR polymorphisms in the genetic predisposition of SLE, association of FcgammaRIIa-H131R, IIb-I232T, IIIa-F176V and IIIb-NA1/NA2 (HNA-1a/1b) polymorphisms with SLE was analyzed in the Thai population, using case-control association analysis. FcgammaRIIb-232T/T and IIIb-NA2/NA2 genotypes were associated with SLE with the odds ratio of 2.55. Genotype relative risk analysis revealed significant association of IIb-232T/T and IIIb-NA2/NA2, and a tendency of association of the IIIa-176F/F genotype. Moreover, carriers of FcgammaRIIa-131R were significantly increased in patients with lupus nephritis. Significant linkage disequilibrium was present among FcgammaRIIb, IIIa and IIIb, and two-locus analyses suggested that the tendency of association of FcgammaRIIIa could derive from linkage disequilibrium with IIb and IIIb. These results provided evidence that FcgammaR polymorphisms may be an important predisposing factor also in Thais in a complex manner.

Association of HLA-DRB1*1502-DQB1*0501 haplotype with susceptibility to systemic lupus erythematosus in Thais.

In this study, we investigated the association of HLA-DRB1 and DQB1 with Thai patients with SLE. A highly significant increase in the frequency of DRB1*1502 and DQB1*0501 was found in SLE patients compared with normal controls. DRB1*1501 and DRB1*1602 were also slightly increased. In contrast, DRB1*1202 and DQB1*0301 were decreased, and DRB1*0406 and DRB1*1401 were not found in the patients' group. The haplotype analysis revealed that DRB1*1502 - DQB1*0501 was most strongly associated with SLE, and also suggested a primary role for DRB1 rather than DQB1. Taken together with the previous report which demonstrated the association of the same haplotype in Taiwan, our present observations strongly suggested that DRB1*1502 - DQB1*0501 is the major HLA haplotype that confers susceptibility to SLE in the South-east Asian populations.

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.

HLA class II allele frequencies in northern Thais (Kamphaeng Phet).

The polymorphism of HLA class II genes (HLA-DRB1, -DQA1, -DQB1 and -DPB1) was investigated in 97 normal Northern Thais (NT) from Kamphaeng Phet province using PCR-SSO typing. Allele frequencies (AF) have been determined. DRB1*1202 (17.5%), DRB1*1502 (16.5%), DQA1*0101 (25.8%), DQA1*0102 (21.7%), DQB1*0502 (22.7%), DPB1*0501 (23.2%) and DPB1*1301 (22.7%) showed the highest frequencies in each locus. These results were more similar to those observed in Present-day Thais (PDT) and Central Thais (CT) than Northern Thais from Chiang Mai (CM) and Dai Lue (DL). However, the data presented in this population study should be useful in many fields, such as anthropology, organ transplantation, disease susceptibility and evolutionary genetics.

HLA-A, -B, -DRB1, -DQA1, and -DQB1 polymorphism in Thais.

In this study we examined HLA-A, -B, -DRB1, -DQA1, and -DQB1, gene allele, and haplotype frequencies in two ethnic Thai populations. We compared these frequencies to the known HLA class I and II allele profiles of non-Thai mainland and insular Southeast (SE) Asians. HLA-A locus gene and allele frequencies, are comparatively homogeneous in both Thai and non-Thai SE Asians. In contrast, HLA-B; -DRB1, -DQA1, and -DQB1 gene and allele frequencies, show more ethnic and geographic variation in SE Asians. Conserved haplotypes, or combinations of linked HLA class I and II alleles were detected in Thais, but at relatively low frequencies. It would appear that ethnic Thais, reflect an admixture of peoples from both the northern mainland and southern island groups of SE Asia.

Angiotensin converting enzyme gene polymorphism in healthy Thais and patients with non-insulin dependent diabetes mellitus.

Insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene has been shown to be associated with various cardiovascular disorders in diabetic and non-diabetic patients. Its association with the development of non-insulin dependent diabetes mellitus (NIDDM) has been raised. This study was aimed to examine I/D polymorphism of ACE gene in healthy Thai subjects and patients with NIDDM. The I/D ACE genotypes were determined by polymerase chain reaction technique. Healthy unrelated subjects were 151 males and 147 females, 17-70 year old (mean +/- SD = 37.5 +/- 10.4). The unrelated diabetic patients were 42 males and 66 females, 20-79 years of age (mean +/- SD = 54.7 +/- 12.0). In healthy subjects, the ACE genotypes were DD 10.1 per cent, ID 39.2 per cent and II 50.7 per cent. Diabetic patients had similar distribution of ACE genotypes. The frequency of I and D alleles in diabetic patients was 0.69 and 0.31, similar to 0.70 and 0.30, respectively, in healthy subjects (p = 0.69). The frequency of I and D alleles in healthy Thai subjects was similar to the Japanese (I = 0.66 & D = 0.34) but different from Caucasians (I = 0.44-0.46 & D = 0.54-0.56). We conclude that I/D ACE gene polymorphisms may possess a racial difference. The similar frequency of both alleles in diabetic patients and healthy subjects suggests that there is no association between I/D polymorphism of ACE gene and diabetes mellitus in Thai individuals.

Estimated rate of HIV-1-infectious but seronegative blood donations in Bangkok, Thailand.

OBJECTIVES: To determine HIV seroprevalence and incidence among various blood donor types, and to estimate the rate of window-period blood donations. DESIGN: Retrospective cohort from computerized donor records. METHODS: Records were analysed from all 60,483 donors (contributing 97,464 donor units) at a public university teaching hospital blood bank in Bangkok, Thailand, from 1 January 1990 to 30 June 1993. Annual HIV incidence among 14,482 repeat donors who were HIV-seronegative on their first donation was calculated assuming equal probability of seroconversion between last seronegative and first seropositive donations. To estimate the probability of window-period donations, we assumed that the time from HIV infectivity to onset of detectable antibody was 45 days. RESULTS: In 1990, HIV incidence calculated for all repeat donors was 307 per 100,000 person-years; the probability of a window-period donation was 38 in 100,000 donations or one in 2644 donations. During 1991-1993, this probability decreased by one-half. However, one-time donors were more than twice as likely as repeat donors to be HIV-1-seropositive. CONCLUSIONS: The rate of HIV window-period blood donations among Thai repeat donors was relatively high compared with that in developed countries and was probably even higher among one-time donors. Improved donor deferral criteria are needed in Thailand.

PIP: In countries with a high incidence of human immunodeficiency virus (HIV), blood donations made during the "window period" (time between infection and the development of detectable antibody) pose a serious risk. To assess the extent of this risk in Thailand, records of all 60,483 blood donors at Bangkok's Mahidol University from January 1, 1990, to June 30, 1993, were reviewed. A retrospective cohort of 14,482 repeat donors was created by identifying those who were HIV-negative at their first screening and made one or more subsequent donations (mean, 3.6). Among these repeat donors, 40 (0.3%) seroconverted during the study period. The time from HIV infectivity to onset of detectable antibody was assumed to be 45 days. The HIV transmission rate among repeat donors resulting from donations during the window period was estimated to be 1 in 2644 units transfused in 1990 and 1 in 5000 units transfused during 1991-93. Extrapolation of these estimates suggests that, during the 1991-93 period, 57 units in Bangkok (194 units nationwide) were from donors in the HIV window period. Voluntary 1-time donors were 1.9 times more likely to be HIV-infected than voluntary repeat donors, while paid one-time donors were 13.7 times more likely to be seropositive than paid repeat donors. In 1994, the hospital initiated HIV p24 antigen screening of all donated blood; although this technique shortens the window period and should improve blood safety, improved donor referral criteria are needed.

HLA-DPB1 polymorphism in the Thais of Southeast Asia.

In this study we examined DPB1 allele frequencies in five ethnic Thai populations resident in different regions of Thailand and neighboring countries. In contrast to other Asian and Pacific populations such as the Japanese, Chinese, Korean, and Papua New Guineans, where DPB1*0501 has consistently been shown to be the most frequent allele, NE Thais and Thai-Khmers demonstrate a prevalence of DPB1*1301. Comparison of DPB1 allele frequencies in the Thais of SE Asia, with known frequencies in the Chinese and Japanese populations of E Asia, would appear to confirm previous calculations of genetic divergence between these Oriental populations.

Potential deferral criteria predictive of human immunodeficiency virus positivity among blood donors in Thailand.

BACKGROUND: To develop deferral criteria to prevent human immunodeficiency virus (HIV) transmission by recently infected blood donors in the seronegative "window" phase, routine data on donors at a university hospital were examined for factors predicting seropositivity. STUDY DESIGN AND METHODS: Records of all 281 HIV-positive blood donors from August 1987 through September 1991 were retrospectively compared with those of 1076 randomly selected control donors matched only by year of donation. Four controls were selected for each HIV-positive donor. RESULTS: The prevalence of HIV in 102,684 donor units during the period rose from 0.02 percent in 1987 to 0.52 percent in 1991. Multivariable analysis revealed that male sex (odds ratio [OR] = 26.4), VDRL test positivity (OR = 3.0), age 21 to 30 years (OR = 2.2; referent: 16-20-year-old group), and replacement donorship (OR = 1.4; referent: voluntary donors) were independent factors significantly associated with HIV positivity among these donors (p < 0.05). Since replacement donorship cannot be avoided, only male sex, age 21 to 30 years, and VDRL test positivity were considered as potential criteria. When these findings were extrapolated to all donors in 1990 and 1991, those with all three or only two (excluding VDRL test, because the results are known only after donation) of these high-risk factors had HIV positivity probabilities of 2.2 and 1.0 percent, respectively. These probabilities were, respectively, 4.9 times (95% CI: 2.9 8.3) and 4.1 times (3.1, 5.4) the risk among other donors. However, applying such criteria would have eliminated 1.5 and 31.2 percent, respectively, of all HIV-negative donors in 1990 and 1991. The latter deferral proportion is too high to be acceptable. CONCLUSION: In Thailand, improved donor deferral criteria addressing sexual risk factors could lead to decreased probability of window-period donation, with an acceptable rate of deferral. Additional p24 antigen testing may be indicated for donors at increased risk for HIV infection, specifically, men aged 21 to 30.

PIP: Researchers compared August 1987 and September 1991 data on 281 HIV-positive blood donors, 16-59 years old, with those of 1076 randomly selected controls to determine factors associated with HIV infections they could develop simple and inexpensive deferral criteria to prevent donations from high-risk persons who may transmit HIV during the seronegative window period. The blood bank records were at Siriraj Hospital in Thailand. HIV-positive donors were significantly more likely to be men than were controls (98.9% vs. 76.3%; odds ratio = 2.64). They were also more likely to test positive for syphilis (VDRL test) (8.2% vs. 2.3%; OR = 3). A higher proportion of HIV-positive blood donors were 21-30 years old than were controls (66.2% vs. 40.5%; OR = 2.2). A greater percentage of blood donors who were asked by friends or relatives of transfused patients to donate blood (i.e., replacement donors) were HIV positive than controls (55.5% vs. 40.4%; (OR = 1.5). All these independent risk factors were significant at the 5% level. Paid donors were less likely to be HIV positive than controls (2.8% vs. 9.9%; odds ratio [OR] = 0.6). Since 40.4% of all HIV-negative donors were replacement donors, the researchers decided not to include replacement donorship in the analysis for potential deferral criteria. They used the records of 214 HIV-positive donors with all 3 other risk factors in 1990 and 1991 to determine the potential deferral criteria. The probabilities of HIV positivity of blood donors with 2 and 3 risk factors were 1 and 2.2% (respectively), which meant that they were 4.9 and 4.1 times (respectively) more likely to be HIV positive than were other donors. Using these criteria would have eliminated 1.5% and 31.2% (respectively) of all HIV-negative donors. Elimination of 31.2% is too high to be acceptable. The researchers suggest an additional p24 antigen testing for donors at high risk for HIV infection, particularly 21-30 year old men.

Preoperative autologous blood collection and haemodilution with gelatin solution (gelifundol).

Autologous blood collection and haemodilution with gelatin solution had an effect on the decrease in red blood cells, haemoglobin, haemotocrit, fibrinogen and platelets; however, this technique had no effect on coagulograms, platelet function and haemostasis. In conclusion, this technique is suitable and possibly practical in obtaining sufficient blood for elective surgical patients and is without any undesirable side effects.

Factor VIII, factor IX and fibrinogen content in cryoprecipitate, fresh plasma and cryoprecipitate-removed plasma.

In order to provide accurate information for physicians, factor VIII, factor IX and fibrinogen content were determined in 40 bags of cryoprecipitate, fresh plasma and cryoprecipitate-removed plasma. A cryoprecipitate bag with a volume of 21.8 +/- 5.3 ml contained 139.5 +/- 42.9 units of factor VIII and 200.0 +/- 80.0 mg of fibrinogen. Fresh plasma with a volume of 208.0 +/- 22.5 ml contained 180.9 +/- 45.3 of factor IX, significantly higher than in cryoprecipitate-removed plasma. It was also found in this study that group O blood showed a significantly lower level of factor VIII.

Unexpected red cell antibodies in donors and patients at Siriraj Hospital.

The incidence of unexpected red cell antibodies in patients and donors at Siriraj Hospital were found to be 4.91% and 4.33% respectively. Anti Lewis, anti P1 and anti Mia were the most common antibodies detected. Antibodies of Rh system were the next, in which anti E was more common than anti D. Anti Jka, Jkb, Fyb, Dia, S and rare antibodies such as anti K, Ge1, Mta and H were also detected in this study.