Micromethod for PMN function studies in neonates.
Polymorphonuclear neutrophils play an important role in host defense mechanism. To assess PMN function in neonate a micromethod using only a few drops of blood was standardised. PMN adherent to coverslip were incubated with bacteria and using a differential staining with Acridine Orange (AO) phagocytic and bactericidal capacity were estimated.
Plymorphonuelear cells (PMN) form an important part of cellular response in host defence mechanism. Functional disorders of peripheral blood phagocytes are characterised by chronic and recurrent bacterial or fungal infections, as manifested in chronic granulomatous disease. Infections in neonatal period account for a sizeable portion of mortality and morbidity Assessment of PMN function in neonate is therefore important. Susceptibility of neonates to infections may be due to impaired immunological functions. For assessment of PMN function in neonate, which may at times be required serially during course of infection, drug or any other therapeutic interventions, it is necessary to carry out the test with minimum amount blood. We describe a method to assess phagocytic and bactericidal capacity of PMN using just a few drops of blood. This method is a modification of nuorochrome microassay as described by Horn et al.
PMN cells adherent to cover slip are incubated with bacteria. Using the differential staining of dead and live bacteria with acridine orange (A0) phagocytic and bactericidal capacity of PMN is estimated. The details of the method are as follows:
Preparation of bacterial suspension: An 18 hour old culture of S. aureus (ATCC 6538) in nutrient broth was taken and optical density adjusted to correspond to 2 x 10 bacteria/nil. This bacterial suspension was centrifuged at 2000 RPM for 10 min., washed with cold physiological saline and opsonised at 37ºC for 30 min, with 10% pooled NHS. This suspension was again centrifuged at 2000 RPM washed in DPBS and diluted with DPBS to give a final concentration of 2 x 10 bacteria/ml. To this bacterial suspension was added 0.5 ml. of 1.25 mg/1 of acridine orange (AO) and allowed to react for 1 min. Suspension was centrifuged and washed twice with DPBS. DPBS was used for washing excess of A.O. instead of gelatin as in the original method as DPBS was found to give satisfactory results.
Preparation of PMN monolayer :
Two-three drops of blood were taken and spread on a previously (Bovine scrum albumin) BSA coated cover slip. The cover slip was incubated at 37ºC for 30 min., in 100% humidity. After ½ hr. the blood clot was gently removed with the help of forceps. Cover slip was washed gently with DPBS. One hundred, ?l of pre-stained bacterial suspension as prepared earlier was layered on the PMN which were adherent to the cover slip. The PMN and bacteria were incubated at 37ºC for 45 min. The excess of bacteria was removed by tilting the cover slip. The cover slip was then fixed in 3% formaldehyde for 30 min at room temperature then washed with DPBS twice and stored in DPBS at 2ºC-4ºC. The cover slips were then mounted on slide in glycerine-DPBS (1:1) mixture, sealed with varnish and observed under fluorescent microscope using oil immersion lens.
Percentage phagocytosis was calculated by noting the number of PMN that had engulfed bacteria and a hundred PMN were counted. Percentage bactericidal activity was calculated by the formula given below after noting the number of living and dead bacteria. The living and dead intracellular bacteria were distinguished by their colour. The live bacteria fluoresced green dead bacteria fluoresced green and red. This was possible as Acridine Orange fluorosces green when binding with double stranded DNA and orange or red with single stranded DNA.
No. of PMN that have engulged bacteria
% phagocytosis = ---------------------------------------------- x 100
No. of PMN counted
No. of dead intracellular bacteria
% bactericidal = ------------------------------------------ x 100
activity No. of bacteria engulfed
The standard tube techniques was also carried out in 10 normal neonates to assess phagocytic index. Here, 4-5 nil of venous blood was withdrawn and added to dextran. Granulocyte rich plasma was allowed to separate from whole blood. This plasma was then layered on to Ficoll density gradient and centrifuged at 1500 RPM for 25 min. The supernatent was discarded and the pellet consisting of polymorphs was used for the estimation. The polymorphs were counted and bacteria were added to polymorphs (1 : 10) ratio. Bacteria were prepared as described earlier, only Acridine orange was not added.
The cell and bacteria mixture was incubated at 37ºC for 20 min in a shaking water bath, centrifuged and slides were prepared from pellet. Slides were fixed in methanol, stained with Glemsa stain and observed under 100 ml immersion lens. Phagocytic index was obtained as described earlier.
The phagocytic and bactericidal indices of normal neonates by fluorochrome assay were found to be 76.3 + 0.965 and 69.88 + 1.166 respectively. The phagocytic index of normal neonates by standard tube technique was 75.8 + 1.28.
The phagocytic index and bactericidal index of healthy normal adults was 60.2 + 0.70 and 60.0 + 0.84.
As can be seen from the results that phagocytic index by fluorochrome microassay method (76.3%) and by standard tube method (75.8%) were comparable. Thus the fluorochrome microassay gives comparable results and was also advantageous to use in neonates as it required a minute quantity of blood as compared to 4-5 ml in tube method. It also assessed two PMN functions i.e. phagocytic index and bactericidal index at one time. The phagocytic index and bactericidal index were observed to be higher in neonates than in adults when using 10% normal human scrum by Miller as quoted in Davies and Gothefors . Our results are in agreement with his findings.
The method can be used for investigating opsonization and effect of drugs. PMN function in premature neonates assessed by this method was found to be significantly reduced.