Phagocytic Activities of Neutrophilic Leukocytes

This study evaluated the phagocytic activities of neutrophilic leukocytes in both sexes during reproductive age and in women in different phases of the menstrual cycle and in pregnancy. In this study, 9 male and 12 female medical students, 17 to 20 years of age, 23 pregnant women and 9 women after delivery were evaluated. Heparinized blood was incubated with heat killed bacteria Staphylococcus aureus at 37°C for 20 minutes. The blood films were stained with Leishman stain and the phagocytic index was found out by the number of neutrophilic leukocytes ingested with organisms in 100 cells and the total number of organisms counted therein. At the interval of 14 days, on 3 consecutive occasions, the phagocytic index was studied on the same male and female subject. The phagocytic activities of neutrophilic leukocytes were found slightly higher in females than in males; more so in pregnancy than in non-pregnant women, woman controls and after delivery. During the expected ovulation period, the phagocytic activities were observed more than during the menstrual and premenstrual phases. The phagocytic activities were found more in pregnancy and during the ovulatory period in women. is LH surge just before ovulation. The functions of LH are similar to that of hCG. At the termination of pregnancy hCG secretion ceases. If hCG stimulates the phagocytic activities in pregnancy, there may be variation in phagocytic activity during the different phases of the menstrual cycle and also at the end of pregnancy (Shibuya et al, 1991). MATERIALS AND METHODS The phagocytic activities were observed in an in vitro experiment using whole blood. The subjects were taken from 21 healthy medical students (9 male, 12 female) 17-20 years of age at Nepal Medical College, 23 healthy pregnant women of all trimesters, and 9 women who had a normal delivery at the college hospital. Preparation of suspension of organisms In this study, the Gram-positive bacterium, Staphylococcus aureus, was used for the phagocytic experiment. A suspension of this organism was prepared in nutrient broth and incubating it for 24 hours at 37°C. The suspension was centrifuged and washed with normal saline; then the SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 878 Vol 34 No. 4 December 2003 suspension of organism was matched to get the desired concentration. A heat killed suspension of organisms was prepared by keeping it in a water bath at 56°C for one hour. The same suspension was preserved in refrigerator and used for all the experiments. In order to detect proper heat killing of organisms, one drop of suspension was cultured on nutrient agar and growth was not detected after 48 hours of incubation at 37°C. Determination of the phagocytic index Two parameters were considered for the phagocytic index: a) the number of neutrophilic leukocytes with engulfed bacteria in 100 cells, and b) total number of organism present therein. The incubation for 20 minutes was preferred after evaluating results from 5, 10, 15, 20, 25 and 30 minutes incubation. Both parameters were found highest after 20 minutes incubation. On three occasions; at the interval of 14 days, studies were done on the same male and female medical student (with a regular menstrual cycle). Pregnant women with normal pregnancy, without any history of drug intake, were selected irrespective of para and trimester. At the termination of pregnancy, during the first week, but two days after normal delivery, blood was collected for study. One milliliter of blood was collected in a heparinized vial and 50 μl of saline suspension of heat killed Staphylococcus aureus was added having the concentration of 500 million per ml. (ratio of neutrophil: organism is 1:4). The sample was thoroughly mixed and incubated for 20 minutes at 37°C. Immediately after incubation, two smears of blood were prepared from each sample and stained with Leishman stain. Counting of ingested cells and organisms From each film, 200 neutrophils were observed by two independent observers. The number of neutrophils with ingested organisms among the two hundred neutrophils and the total numbers of organisms in them were counted. The average number of cells with ingested organisms and the number of organisms per cell, from 400 cells, were evaluated in two independent experiments. The organisms took the Leishman stain nicely and were easy to count. Occasional monocytes were found with ingested organisms but these were not included in the results. RESULTS The results of the phagocytic index: the number of neutrophilic leukocytes engulfed with bacteria among 100 cells and the total number of bacteria therein are summarized and presented in Figs 1, 2, 3 and 4. The mean value with standard deviation of the numbers of leukocytes marked with organisms among 100 cells and the total number of organisms therein, between the male and female reproductive age groups were observed and found to be 18.18 ± 4.58; 56 ± 14. 86 and 19.6 ± 6.28; 61.33 ± 28.21 respectively. The higher value in females than in males is not statistically sigMale Female 100 90 80 70 60 50 40 30 20 10 0 No. of cells with organisms/100 cells Total no. of organisms therein