Development of Control Material for Exhaled Breath-Alcohol Testing and its Application.

Background: Breath analyser tests are used worldwide to obtain proof of alcohol intoxication and often used in the conviction of traffic violators. These tests are conducted to quickly and painlessly determine the existing concentration of alcohol in arterial blood by measuring the amount of ethanol in exhaled breath, which can be identified with an electrochemical sensor.At present, the calibration and maintenance of analysers used for these tests are typically performed regularly but lack quality control. Consequently, test results may not be accurate because of calibration deterioration.The aim of this study was to develop and evaluate the uncertainty of control materials used in breath-alcohol testing at the Bangkok Metropolitan Police Station. Material and methods: Ethyl alcohol (99.99%; Certified Reference Material grade) diluted at three different concentrations was kept under design conditions. The concentrations were 28, 67, and 134 mg/dL, determined by performing headspace gas chromatography, and the uncertainty was set as ±1.3925, ±2.8736, and ±1.8231 mg/dL (±4.97%, ±4.29%, and ±2.72% for the concentrations, respectively), as per ISO Guide 35:2017. Results: The total error percentages of the developed control materials were 4.97%, 4.29%, and 2.72% for concentrations of 28, 67, and 134 mg/dL, respectively. Each concentration of the materials was tested by using measurements from 70 breath-alcohol analysers belonging to the Bangkok Metropolitan Police Station. Conclusion: These control materials are applicable to quality assurance and standards tests and may help to ensure the accuracy of breath-alcohol testing in the future.

Glycoproteomics analysis of plasma proteins associated with Opisthorchis viverrini infection-induced cholangiocarcinoma in hamster model.

OBJECTIVE: To apply lectin affinity chromatography and glycoproteomics-based LC-MS/MS to preliminarily investigate the possible potential plasma biomarkers of Opisthorchis viverrini (OV)-associated CCA in OV/dimethylnitrosamine (DMN)-induced CCA hamster model. METHODS: Nine Syrian hamsters were divided into 3 groups as follows (n = 3 each): normal (healthy control group); OV group; and OV/DMN group (CCA group). Pooled plasma samples collected from animals in each group at the 6th month post-infection with OV metacercarae were subjected to glycoproteomics analysis. Glycoproteins in the pooled sample from each group were initially isolated by concanavalin A (ConA)-based affinity chromatography. The expression of glycoproteins isolated by both enrichment methods were determined using LC-MS/MS. RESULTS: Among the 24 ConA-binding glycoproteins isolated, two proteins, N-myc downstream regulated gene 1 (NDRG1) and fetuin-B (FETUB) were found up-regulated only in the samples from the OV and control groups, but not in the OV/DMN (CCA) groups. On the other hand, one protein, i.e., NSFL1 cofactor p47 isoform ×3 (NSFL1C) was found only in the samples from OV/DMN (CCA) and control groups, but not in the OV group. The remaining 21 proteins were upregulated in the samples from all groups. CONCLUSIONS: NDRG1, FETUB and NSFL1C glycoproteins isolated by ConA-based affinity chromatography could be potential biomarkers for CCA. Plasma samples with negative for NDRG1 and FETUB proteins but positive for NSFL1C are likely to be OV-associated CCA. Nevertheless, this conclusion remains to be confirmed whether this battery test can discriminate OV-associated CCA from other risk factors.

Improved allele-specific PCR technique for Kidd blood group genotyping.

BACKGROUND: We developed an allele-specific polymerase chain reaction (AS-PCR) technique for Kidd blood group genotyping. METHODS: Altogether, 340 blood samples from Thai blood donors at the National Blood Centre, Thai Red Cross Society, were tested with anti-Jk(a) and anti-Jk(b) using the gel technique and the direct urea lysis test was used for screening Jk(a-b-) phenotype. For AS-PCR technique, different types of primers were used for JK*01 and JK*02 allele detections in known DNA controls. RESULTS: Regarding JK*02 allele detection, the pseudopositve amplification products were found when using correctly matched forward primer and a single mismatch forward primer. Interestingly, one type of two mismatch pairing at the 3' end of the forward primer can be used together with the newly designed reverse primer for Kidd blood group genotyping. It was found that the typing results in all samples obtained by serological techniques and newly developed AS-PCR technique were in agreement and this PCR technique also gave 100% concordance of results in 30 samples randomly tested twice and demonstrated reproducible results. CONCLUSION: This study shows that the in-house AS-PCR is simple, cost-effective, and convenient for Kidd blood group genotyping in routine laboratories, especially, in resolving serologic investigations.

The characteristics of ABO antibodies in group O Thai blood donors.

This study aimed to characterize anti-A and anti-B hemolysins, IgM, and IgG titers in Thai blood donors. Altogether, 300 serum samples from group O donors at the National Blood Centre, Thai Red Cross Society, were screened for anti-A and anti-B hemolysins and treated with 0.01 M dithiothreitol to characterize IgM and IgG titers by standard tube technique. Antibody titers were compared with hemolysis grade. Male and female ratio = 1:1.3 and ages ranged from 17 to 60 years. The overall prevalence of anti-A and anti-B hemolysins was 69%. Anti-A and anti-B hemolysins comprised 18.3% and 16.7%, respectively and 34% had both antibodies. High titers of anti-A hemolysins were associated with females (P< 0.05), and only anti-B IgM titers were associated with age (P< 0.05). Interestingly, the association of anti-A IgM titers, anti-A IgG titers, and hemolysin grade was demonstrated (P< 0.05). A significant association between hemolysin grade and anti-B IgM titers was found (P< 0.05). The prevalence of anti-A and anti-B hemolysins and high titers of IgM and IgG in Thais are high. Hemolysin grade showed significant associations with IgM titers; therefore, when providing ABO-incompatible platelet transfusion, especially for female plateletpheresis donors, IgM high titers of anti-A and anti-B screening is suggested.

The Association Between HLA Class II Alleles and the Occurrence of Factor VIII Inhibitor in Thai Patients with Hemophilia A.

OBJECTIVE: This study aimed to investigate the association between HLA class II alleles and the occurrence of FVIIIinhibitor in Thai hemophilia A patients. MATERIAL AND METHODS: The distribution of HLA-DRB1 alleles and DQB1 alleles in 57 Thai hemophilia A patientsand 36 blood donors as controls was determined using the PCR sequence-specific primer (PCR-SSP) method, and theassociation between the occurrence of factor VIII (FVIII) inhibitor and the presence of certain HLA class II alleles wasinvestigated. RESULTS: The frequency of HLA-DRB1*15 was higher in the hemophilia A patients with and without FVIII inhibitor,whereas that of DRB1*14, DRB1*07, and DQB1*02 was lower in the hemophilia A patients with FVIII inhibitor, ascompared to controls. Interestingly, only the frequency of DRB1*15 was significantly higher in the patients with inhibitorthan in the controls (P = 0.021). Moreover, the frequency of DRB1*15 in the patients with inhibitor was higher than inthose without inhibitor (P = 0.198). CONCLUSION: The study's findings show that the DRB1*15 allele might have contributed to the occurrence of inhibitorin the Thai hemophilia A patients; however, additional research using larger samples and high-resolution DRB1 typingis warranted.

Genomic characterisation of the Jk(a-b-) phenotype in Thai blood donors.

BACKGROUND: The Kidd (JK) blood group antigens are encoded by the JK gene. The rare Jk(a-b-) phenotype can be caused by homozygosity for a silent JK allele. Currently, JK(null) alleles have been identified among different populations; however, information on its presence among Thais is not available. MATERIALS AND METHODS: Screening for the Jk(a-b-) phenotype by the urea lysis test was performed in 25,340 blood samples from Thai blood donors. The Jk(a-b-) phenotypes were confirmed by an indirect antiglobulin test (IAT). Additionally, polymerase chain reaction amplification and sequence analysis of the JK gene were performed using previously described methods. RESULTS: Five samples were confirmed as having a Jk(a-b-) phenotype by a urea lysis test and IAT; four of these samples were investigated. Two samples of JK*02 alleles were homozygous for a g>a mutation at the 3' acceptor splice site of intron 5 of the JK gene, as in previous studies in Asians and Polynesians. Moreover, one sample of JK*02 alleles was homozygous for an 896G>A mutation at exon 9 (Gly299Glu), as in a previous study in Polynesians. Interestingly, missense dual mutations of JK*01 alleles from a female blood donor were identified. The first mutation was 956C>T (Thr319Met) in exon 10, as in a recent study in African-Americans. The second mutation was 130G>A (Glu44Lys) at exon 4, as in previous studies among Caucasians. CONCLUSION: There are various different molecular bases of the Jk(a-b-) phenotype. This is the first report of JK(null) alleles among Thais. The information presented in this study could be beneficial in planning genotyping strategies for blood donors and patients.

Proteomics analysis and evaluation of biomarkers for detection of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a rare but devastating neoplasm that accounts for about 3% of all gastrointestinal cancers and about 15% of all primary liver cancers worldwide. The lack of early detection and limited therapeutic options are major problems in controlling CCA. The current study attempted to identify novel serum markers which can substitute the carbohydrate antigen CA19-9, or can improve, when measured together, the diagnostic accuracy of CA19-9. Differentially expressed proteins in pooled and individual plasma samples obtained from patients with CCA and control subjects (10 each) were identified by using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MALDI-TOF). Out of a total of 21 protein spots separated and identified, five spots were found to be up-regulated in plasma from CCA patients. The up-regulation of α1-antitrypsin (AP1) was observed in all of the ten samples from CCA patients with protein intensity significantly higher than control subjects. Based on results of binary logistic regression analysis of the three serum biomarkers (CA19-9, AP1 and α-fetoprotein: AFP), serum levels of at least CA19-9 together with AP1 were the minimum requirement to obtain prediction accuracy of greater than 80% in a battery test for diagnosis of CCA. However, in order to obtain high predictability of 100% or approaching, an addition of at least one of the three liver function enzymes (alkaline phosphatase: ALP; aspartase transaminase: AST; alanine trasaminase: ALT) is required. Serum biomarkers may be a useful diagnostic or prognostic monitoring tool for CCA. Further evaluation of larger number samples is needed to support their applicability in a clinical setting as diagnostic and prognostic tools. Determination of clinical utility of these marker models in early diagnosis of CCA requires study in animal models with disease progression.

Antibody elutions in Thai patients with a positive direct antiglobulin test.

BACKGROUND: The direct antiglobulin test is performed to determine whether an anaemic patient with evidence of haemolysis has autoimmune or alloimmune haemolytic anaemia. MATERIALS AND METHODS: We determined the antibody specificity of eluted IgG antibodies from patients' blood samples with a positive direct antiglobulin test. Overall, 134 Thai patients were included in this study. EDTA blood samples were obtained from recently transfused patients, patients with unexplained anaemia and patients who had serum antibodies detected during routine pre-transfusion tests from different hospital blood banks. These complicated samples were sent to the National Blood Centre of the Thai Red Cross Society for investigation and to find compatible blood components. Each blood sample underwent a direct antiglobulin test with the gel technique using polyspecific antihuman globulin and mononospecific anti-IgG and anti-C3d. Acid eluates were prepared from the samples for which the direct antiglobulin test was positive and the specificities of the eluted antibodies were determined by the gel technique. RESULTS: Of the samples tested, 101 showed a positive direct antiglobulin test result (75.4%) using polyspecific antihuman globulin sera whereas only 95 samples (70.9%) were positive with anti-IgG or anti-IgG and anti-C3d. Moreover, 54 of 95 eluates (56.8%) were positive for antibody screening and tested with the reagent panel cells. Twenty-one eluates had specific alloantibodies, which were concordant with the findings in the patients' sera and all patients had a history of blood transfusion. Additionally, 33 eluates contained pan-agglutinins. Interestingly, alloantibodies could be determined using titration studies in 5 of 26 eluates with pan-agglutinins. CONCLUSION: Although the direct antiglobulin test is not routinely performed in pre-transfusion screening, this test and elution studies would be useful in patients with a history of previous transfusions, and in those for whom compatible blood cannot be found.

Jk(a-b-) phenotype screening by the urea lysis test in Thai blood donors.

BACKGROUND: The Jk(a-b-) phenotype is rare in most populations and often detected after transfusion or pregnancy. After immunisation, anti-Jk3 forms and it can be difficult to find compatible Jk(a-b-) donors. Using anti-Jk(a) and anti-Jk(b) in a conventional tube method is unsuitable for identifying Jk(a-b-) in mass screening of blood donors. Jk(a-b-) phenotypes are associated with the absence of urea transporters on erythrocytes, making red blood cells (RBC) resistant to lysis by 2M urea, while Jk(a+b-), Jk(a-b+) and Jk(a+b+) phenotypes are susceptible to lysis. MATERIALS AND METHODS: We screened for Jk(a-b-) phenotypes in blood donors by the urea lysis test using a 96-well microplate. The Jk(a-b-) phenotypes were confirmed by the indirect antiglobulin test (IAT). RESULTS: Altogether, 20,163 blood samples from Thai blood donors were tested and only RBC from five samples were resistant to lysis by 2M urea, while 20,158 samples were completely lysed within 5 min. In an IAT, both anti-Jk(a) and anti-Jk(b) failed to agglutinate RBC from all five samples. Using a micro-titre plate, the direct urea lysis test, costs * 0.01, about 480 times less than IAT. Moreover, the test time for each plate (94 samples) is about 18 times less than that for IAT. CONCLUSION: Jk(a-b-) phenotype screening by the direct urea lysis test on samples in a micro-titre plate is simple, cost-effective and practical for mass screening of blood donors.