Prior Incubation as a Tool to Overcome Understaining of Leishman Stain: A Cross-sectional Study

Abstract

Introduction: A well stained peripheral smear is an integral part in the diagnosis and management of many clinicohematologic conditions. Leishman stain is recommended universally by the International Committee for Standardisation in Haematology (ICSH). However, a commonly encountered problem in tropical countries, especially during the rainy season, is under staining by Leishman stain as a result of interference by the atmospheric moisture. Hence, incubation of slides and/or buffer prior to standard Leishman staining protocol could bring out best staining qualities. Aim: To assess the effects of prior incubation of slides and/or buffer on standard Leishman staining technique. Materials and Methods: The cross-sectional study was conducted over a period of 8 weeks in the Central Laboratory, Kempegowda Institute of Medical Sciences, Bangalore in the months of October 2020 to November 2020. Blood samples were received in the central laboratory for routine haematological investigations. Total of 100 samples of left over non haemolysed Dipotassium Ethylenediaminetetraacetic Acid (K2-EDTA) anticoagulated blood irrespective of age, gender and cell counts were included in the study. Slides were stained following prior incubation of slides and/or buffer at 37° C and compared with the standard staining technique. The staining characteristics of nucleus, cytoplasm and granules of White Blood Cells (WBCs), platelets and erythrocytes were observed. One-way Analysis of Variance (ANOVA) test and p-value of <0.05 was considered statistically significant and higher f value (>104.35152) rejected the null hypothesis. Results: Prior incubation of slides and/or buffer resulted in better stained smears which were found to be statistically significant (p-value<0.05) as compared to standard Leishman staining technique. Conclusion: Minor modifications when applied to standard Leishman staining technique such as prior incubation of slides and/ or buffer at 37° Centigrade produced best staining characteristics to overcome the effects of humidity which normally interfere with staining of slides.