Comparison between flowcytometry and immunoperoxidase staining for the enumeration of lymphocyte subsets.

Immunoperoxidase staining was compared with flowcytometry for the enumeration of lymphocyte subsets. The percentages obtained for peripheral blood lymphocytes using immunoperoxidase (CD3 = 76 CD4 = 27.9, B = 10.7 CD4/CD8 = 1.8) differed significantly from those obtained by flowcytometry (CD3 = 65.7 CD4 = 39.4, CD8 = 25.6, B = 16.7, HLA DR = 11.9 CD4/CD8 = 1.54) for certain subsets (CD3, CD4, B). There was no significant difference in lymphocyte subsets between children and adults using the same method. These differences are probably due to the different methods used to prepare lymphocytes for analysis. Other factors that should also be considered are the presence of CD4 antigen on monocytes and CD8 on natural killer cells.

A double antibody sandwich micro-ELISA kit for the rapid diagnosis of snake bite.

A micro ELISA assay was established to diagnose systemic poisoning for the rapid administration of specific antivenom. Rabbit anti venom IgG was bound to the solid phase to enable detection of venom from both the Malayan Pit Viper (Agkistrodon rhodostoma) and the Common Cobra (Naja naja). This assay is read visually and takes 35 to 45 minutes to perform. It can detect 15.6 ng/ml of viper venom in 75 minutes and 7.8 ng/ml of cobra venom in 55 minutes. Tests on sera from snake bite patients showed detectable levels of snake venom in the serum even though administration of antivenom was not necessary. Furthermore, results from these clinical cases were obtained in less than 45 minutes. It was found that the most suitable washing media was saline/Tween, the assay could be performed at room temperature and plates stored for 6 months showed no loss of activity.

HLA-A and HLA-B antibodies of pregnant mothers in Malaysia.

The aim of this study was to determine the frequency and specificity of HLA-A and B antibodies in multiparous mothers in the Malaysian population. 1,100 maternal serum samples obtained during normal childbirth were screened against a panel of 100 lymphocytes with known HLA antigen types for HLA antibodies by the complement dependent lymphocyte microcytotoxicity dye exclusion test. From the total number of 1,100 samples of maternal serum that were screened for HLA antibodies only 205 specimens (18.6%) tested positive for antibodies. The percentage of maternal sera which contained HLA-B specificities (10.6%) were significantly higher than those which contained HLA-A specificities (3.0%). Sixty maternal serum samples (5.5%) had high enough titres to be utilised as tissue typing reagents. Thirty nine maternal serum samples (3.5%) contained monospecific HLA antibodies. In this study the most common monospecific HLA antibodies characterised included the following specificities: A2, B5, B17 and B40. Malaysian multiparous mothers of gravida 3, 4 and 5 had a higher frequency for producing HLA-antibodies.

Inappropriate responses to Mycobacterium leprae infections--C reactive protein in man and serum amyloid P in mice.

In a study of C-reactive protein (CRP) levels in the sera of 77 patients with leprosy, it was found that in the majority of newly diagnosed patients, the level was within the normal range for a healthy Malaysian population. Elevated levels did occur, but were usually found in patients with complications, and were more likely to occur in patients who had been receiving drug treatment for some time. This suggested that Mycobacterium leprae infection by itself does not stimulate CRP synthesis and could reflect a failure of synthesis by macrophages of interleukin-1, or related molecules. This was supported by the study of an analogous acute phase protein, serum amyloid P (SAP) in mice bearing M. leprae from human sources in their hind footpads. Such mice showed no significant difference in SAP levels from control mice.

Chronic mucocutaneous candidiasis with deficient CD2 (E receptor) but normal CD3 mononuclear cells.

A case of chronic mucocutaneous candidiasis in a Malaysian child who subsequently developed disseminated tuberculosis and toxoplasmosis is described. The phenotype of her peripheral blood mononuclear cells showed discordance for her T cell markers. The presence of a subpopulation of CD2-/CD3+ mononuclear cells leading to an immunodeficiency state is consistent with failure of activation of CD2-mediated alternative pathway resulting in immunodeficiency. Such abnormal CD2-/CD3+ subpopulations have been described in lepromatous leprosy and foetal abortuses.

HLA antigens in Malay patients with systemic lupus erythematosus.

Many studies have shown an association between human leucocyte antigens (HLA) and systemic lupus erythematosus (SLE) in the various study populations. Although SLE is not an uncommon disease in the Malaysian Archipelago, and appears to affect all three major racial groups equally (i.e. Southern Chinese, Malays and Southern Indians), very little information is available on the HLA profiles in the two latter groups. In phase I of our study of the HLA profiles in Malaysian SLE patients, the HLA phenotypes (class I: A, B, C; Class II: DR, DQ) of Malay patients with confirmed SLE and 91 normal Malay controls were determined using the microcytotoxicity assay. The strong association between DR (RR 3.28, P = 0.008) concurs with that reported among Chinese and Japanese populations. Moderate to strong associations with HLA-B 7 (RR 4.99, P = 0.02) and Cw 7 (RR 2.94, P = 0.003) were also found. We believe this is the first report of the association of HLA and SLE in the Malay population.

Effect of irradiation on the precursor, activated and memory suppressor T cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

The relative radiosensitivities of precursor (Tsp), activated (Ts) and memory (Tsm) suppressor T cells for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) were investigated in mice. Spleen cells from CBA mice, primed i.v. with 10(9) SRBC 3-4 days previously, contain specific Ts cells which substantially impair the induction of DTH to SRBC in normal syngeneic recipients. Exposure of mice to 400 rad irradiation 1 day before the priming completely eliminated the subsequent development of Ts cells. In contrast, 3 days after the priming injection, Ts cell activity in mice is resistant to doses higher than 600 rads. Mice primed 40 days previously with 10(9) SRBC contain Ts-cell memory which can be readily recalled by i.p. injection of 10(8) SRBC. The secondary Ts cells which specifically inhibit DTH induction can be demonstrated adoptively in normal recipients. Mice were exposed to various doses of irradiation 40 days after the priming and 1 day before the i.p. injection. Ts memory was significantly reduced by 300 rads and was completely abrogated by 400 rads. The relative radiosensitivities of the three subsets of suppressor T cells are in the order of Tsm = Tsp greater than Ts.

A double antibody sandwich micro-ELISA kit for the rapid diagnosis of snake bite.

A micro ELISA assay was established to diagnose systemic poisoning for the rapid administration of specific antivenom. Rabbit anti venom IgG was bound to the solid phase to enable detection of venom from both the Malayan Pit Viper (Agkistrodon rhodostoma) and the Common Cobra (Naja naja). This assay is read visually and takes 35 to 45 minutes to perform. It can detect 15.6 ng/ml of viper venom in 75 minutes and 7.8 ng/ml of cobra venom in 55 minutes. Tests on sera from snake bite patients showed detectable levels of snake venom in the serum even though administration of antivenom was not necessary. Furthermore, results from these clinical cases were obtained in less than 45 minutes. It was found that the most suitable washing media was saline/Tween, the assay could be performed at room temperature and plates stored for 6 months showed no loss of activity.