Medicine in Malaysia: haematology.
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Biomedical subjects
Publications and source records attributed to S K Cheong.
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HBV-DNA were analysed in 330 HBsAg-positive carriers in Malaysia by dot-blot hybridization and polymerase chain reaction. Seventy-three (22.12%) were positive for the virus. Of these, 65 (89%) were males and 8 (11%) were females. Statistically, there was no significant difference (P = 0.13). No significant decline in HBV-DNA with age in the Malay and Chinese males was observed (P = 0.2). Prevalence of HBV-DNA was higher in the Chinese carriers than in the Malay carriers for most age groups in both sexes. Sixty-one HBV-DNA-positive carriers were also positive for HBeAg. However, three individuals were positive only for anti-HBe, one was positive for both HBeAg and anti-HBe, and eight were negative for both HBeAg and anti-HBe. Fifty-seven were positive for HBeAg but negative for HBV-DNA. No relation was observed between raised alanine aminotransaminase and aspartate aminotransaminase levels and the presence of HBV-DNA (P = 0.4).
A 35-year-old man from Bangladesh, who had been in Malaysia for approximately a year, was extensively investigated for more than two months in a state hospital for pyrexia with hepatosplenomegaly. However, no obvious cause of his illness was found. He was treated with multiple antibiotics with no resolution of pyrexia and hepatosplenomegaly. He was later referred to the Haematology Unit, Universiti Kebangsaan Malaysia for further assessment as a case of lymphoma. On carefully reviewing his bone marrow aspirate smears, the diagnosis of leishmaniasis (kala-azar) was finally made. The patient responded to treatment with pentamidine.
G6PD deficiency is the most common human enzymopathy and affects 200 million people worldwide. To date more than 400 biochemical variants and at least 60 different point mutations in the G6PD locus have been discovered. In Malaysia the overall incidence of G6PD deficiency among males is 3.1%, being more prevalent among the Chinese and Malays and less common among the Indians. As part of our initial effort to characterise G6PD deficiency in the Malaysian population, we investigated 18 G6PD deficient Chinese male neonates for the G6PD mutation G-->T at nt 1376, a common mutation seen among the Chinese in Taiwan and mainland China. The mutation was detected by a PCR-based technique using primers that artificially create a site for restriction enzyme Xho I. We found 61% (11 out of 18) of the Chinese G6PD deficient male neonates positive for this mutation. Study of enzyme electrophoretic mobility in 7 of the cases positive for this mutation revealed three different patterns of mobility. 107% (5 out of 7), 103% (1 out of 7) and 100% (1 out of 7). This study shows that mutation G-->T at nt 1376 is a common allele causing G6PD deficiency in Malaysians of Chinese origin. The finding of different patterns of electrophoretic mobility among the 7 cases positive for 1376 G-->T mutation supports the notion that diverse biochemical variants may share the same mutation.
A system for computerising full blood picture reporting developed in-house using dBASE IV on IBM-compatible microcomputers in a local area network environment is described. The software package has a user-friendly interface which consists of a horizontal main menu bar with associated pull-down submenus. The package captures data directly from an automatic blood cell counter and provides options to modify or delete records, search for records, print interim, final or cumulative reports, record differential counts with an emulator, facilitate house-keeping activities which include backing-up databases and repairing corrupted indices. The implementation of this system has helped to improve the efficiency of reporting full blood picture in the haematology laboratory.
An in-house enzyme-linked immunoabsorbant assay (ELISA) for SP-A was successfully developed using in-house polyclonal anti SP-A and a commercial polyclonal anti-rabbit immunoglobulin horseradish peroxidase conjugate system. The standard curve, generated by using 50 ng of SP-A to coat the plate and 1:500 dilution of polyclonal anti SP-A as a primary antibody, was linear for concentrations of SP-A ranging from 4 micrograms/l to 4000 micrograms/l and reproducible. Results of recovery study of SP-A from a known sample of tracheal aspirate ranged from 94%-114%. Intra- and inter-assay coefficients of variations were 2.7% and 5.6% respectively for a known sample of tracheal aspirate. Interference study showed that tracheal aspirate did not interfere with the assay. The assay developed was intended to be used for SP-A measurement in tracheal aspirates obtained from neonates with and without respiratory distress syndrome.
Surfactant protein A (SP-A) is one of the four known surfactant-associated proteins found in human lungs. It plays a major role in determining regulation of surfactant uptake and resecretion. Qualitative and quantitative deficiencies of SP-A may contribute to neonatal respiratory distress syndrome. The measurement of its level in amniotic fluid or neonatal tracheal aspirate may be useful in the assessment of replacement therapy using natural or synthetic surfactants. In order to develop an in-house immunoassay to detect the level of SP-A, we used a discontinuous sucrose density gradient to isolate SP-A from amniotic fluid. Polyacrylamide gel electrophoresis was carried out on the isolates with low molecular weight markers. We successfully isolated SP-A from 12 out of 31 samples of amniotic fluid. The isolates were found to be relatively pure and have a molecular weight of about 35 kD. The isolated SP-A were used as immunogens to raise antibodies in rabbits for the immunoassay.
The cord blood glucose-6-phosphate dehydrogenase (G6PD) enzyme activity of 262 normal term Malaysian neonates (92 Malays, 96 Chinese and 74 Indians) was quantitatively determined by the World Health Organisation method. Analysis of variance for the levels of G6PD enzyme activity by ethnic origin and sex showed that there was a significant difference between mean levels of enzyme activity in the three ethnic groups (P = 0.03) but no difference between the sexes (P = 0.36). Multiple range analysis showed that Malays had significantly higher mean levels of G6PD enzyme activity than those of Chinese (P = 0.01). There was no significant difference between the mean levels of G6PD enzyme activity of Chinese and Indians (P = 0.52), nor was there any difference between those of Malays and Indians (P = 0.08). The difference in levels of G6PD enzyme activity among the different ethnic groups could be due to the existence of different G6PD variants.
In Malaysia, alpha-thalassaemia, beta-thalassaemia, haemoglobin (Hb) E, deltabeta-thalassaemia and Hb Constant Spring are prevalent. It has been estimated that 1 in 4 persons carries one of the above genetic abnormalities. In clinical practice, the major problems are: Hb Bart's hydrops fetalis (homozygous alpha(o)thalassaemia), homozygous 3(o)-thalassaemia, E-alpha thalassaemia and HbH disease. The laboratory procedures for diagnosis are standardised and the molecular basis of most of these genetic abnormalities are characterised. Thus it is possible to formulate a strategy for the detection and prevention of these disorders. The steps include the setting-up of population screening and genetic counselling service for the affected individuals, Society of Thalassaemias for public education and group support, and prenatal diagnosis with selective abortion of affected pregnancies. We embarked on such a programme between 1988 and 1992 in Kuala Lumpur General Hospital and hope to kindle similar effort in other state hospitals.
Several fixation and permeabilization techniques that enable the flow cytometric analysis of the cell contents have been introduced in recent years. These methods allow sensitive detection of intracellular antigens that facilitates the diagnosis of certain diseases. We have undertaken in this study to evaluate a simple method of fixation and permeabilization using 2% paraformaldehyde and Tween 20. Intracellular antigens in three different leukaemia cases were analysed. We found that the method was reliable and easy. Intracellular kappa light chains were found in abundance in a case of plasma cell leukaemia. CD3 and CD22 were found in greater amount intracellularly than on the surface in pre-T-ALL and pre-pre B-ALL respectively.
A 33-year-old Malay lady with chronic myeloid leukaemia (CML) became amenorrhoeic during therapy with busulphan. Pregnancy was diagnosed via a urine pregnancy test and an ultrasound confirmed a viable foetus at 16 weeks. The busulphan was stopped. Her pregnancy was unremarkable and continued till term. She delivered a healthy child.
All-trans retinoic acid therapy induces maturation in acute promyelocytic leukaemia. We document in vivo evidence of differentiation by all-trans retinoic acid in a case of acute promyelocytic leukaemia which was characterized by cytoplasmic vacuolations.
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The applications of antibodies, be it monoclonal or polyclonal, in the diagnostic and research fields are well established. The disadvantage is the high cost of commercially available antibodies. In a diagnostic establishment like ours which also functions as a training ground for laboratory related personnel, it is beneficial to be able to produce in-house reagents. Therefore, we have undertaken this project to produce a rabbit polyclonal antibody against B lymphocytes. We found that the rabbit was a good choice because the titre of antibody produced was high and positive reactions were still detected at a dilution of 1:38400. The antibody showed significant positive reaction only with the lymphocyte subpopulation. A positive reaction was observed between the immunized rabbit serum and B lymphocytes but not T lymphocytes. This shows that the antibody was B lymphocyte specific. There was a positive correlation between the percentage of B lymphocytes labelled using the commercial anti-CD19 monoclonal antibody and the in-house polyclonal antibody (n = 13, r = 0.7, p = 0.02). However, the percentage of cells labelled by the in-house polyclonal anti-B was lower than that by the commercial monoclonal anti-CD19. The fluorescence intensity of the polyclonal antibody was lower than that of the monoclonal. In general, the performance of the in-house polyclonal antibody can be considered as satisfactory. The rabbit serum was stored at -20 degrees C and no significant loss of activity was detected for over a period of 19 months.
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DNA analysis for the diagnosis of beta-thalassaemia is a relatively new technique in Malaysia. This, combined with chorionic villus sampling, has enabled us to offer prenatal diagnosis in the first trimester for this common condition. To the best of our knowledge, this has not hitherto been reported in Malaysia.
The distribution of immunoregulatory cells in the peripheral blood of an individual has now been established as an important tool in helping the management of several diseases. It is necessary to set the normal ranges of these cells for the laboratory. We have undertaken in this study to establish the reference ranges for normal Malaysian adults. We found that the mean percentages of T cells, B cells, T Helper cells (CD4), T suppressor cells (CD8), NK cells and the ratio of CD4/CD8 were 70.91%, 11.38%, 38.15%, 37.76%, 17.45%, and 1.00 respectively. There was no significant difference between the sexes. In certain parameters, there was significant differences between Malay, Chinese and Indians. The Chinese and Indians were significantly different in the distribution of B cells and in the CD4/CD8 ratio. In the case of CD4 and NK cells, the Indians were different from the other two groups.
With the advent of new monoclonal antibodies that are applicable to formalin-fixed, paraffin embedded sections, immunophenotyping is becoming increasingly important in the diagnosis and classification of lymphomas. However, multiple factors such as fixation, trypsinization and even type of antibodies used have certain effects on the final outcome of the staining procedure. In this paper we report our experience and the problems encountered in our laboratory when we first tried to establish a workable immunostaining protocol for formalin-fixed, paraffin embedded tissue sections using the immunoalkaline phosphatase technique.