Modified Leishman Stain: The Mystery Unfolds

Abstract

Background: Current Leishman staining technique for staining thin blood films for differential leukocyte count is too time consuming to meet emergency needs in hospitalized patients with infectious and other deadly diseases. This study aimed at discovering optimal phenol: Leishman powder ratio appropriate for modified Leishman stain and finding an optimized staining reaction and facilitating rapid cellular analysis of blood without alteration in quantity and quality. Methodology: Leishman stain was modified using phenol crystals and liquefied phenol. Various ratios of phenol and Leishman powder were experimented in absolute methanol. Fixing and staining times of staining process were manipulated to develop new staining procedures that gave optimal staining reaction on thin blood films prepared within two hours of receipt. Results were presented as photomicrographs of stained slides. Results: 30mg and 50mg of phenol crystals or 30µL and 50µL of liquefied phenol were required to give 1:5 and 1:3 phenol: Leishman powder ratios respectively. Two modified Leishman staining techniques were developed. The first fixed thin blood films for 25 seconds and stained for 50 seconds while the second technique fixed slides for 1 minute 4592 and stained for 3 minutes. Photomicrographs of thin blood films showed excellent staining results that compared well with the conventional technique. Conclusion: Unlike the conventional method which requires a total of 10-12 minutes, to complete the staining process, modified Leishman staining techniques require only 75.0 seconds and 4.0 minutes! Batches of blood films can be stained within a short time thus facilitating rapid diagnosis and treatment of patients. ABBREVIATIONS WBC: White blood cell; RBC: Red blood cell; DMSO: Dimethyl sulphoxide; Polymorphonuclear cells: Cells having two or more nucleus separated by mitotic strands. While the term generally describes neutrophils, eosinophils and basophils it is sometimes used to refer to neutrophils; Mononuclear: Having one nucleus; pH: This is a measure of hydrogen ion concentration; a measure of the acidity or alkalinity of a solution. Aqueous solutions at 25ºC with a pH less than seven are acidic, while those with a pH greater than seven are basic or alkaline. A pH level of 7.0 at 25ºC is defined as 'neutral' because the concentration of H 3 O + equals the concentration of OH − in pure water; mg: milligram; µL: Microlitre