Alteration in Eosinophil’s Granular Content in Stained Film as an Index for Estimating the Shelf-lives of Modified Leishman Stains: Implications on Disease Diagnosis

Abstract

Modified Leishman stain is a newly discovered stain which is composed of Leishman powder, absolute methanol and phenol. It is not ususual sometimes in diagnostic haematology laboratories to have improperly stained thin blood films, without any clear-cut reason, leading to misdiagnosis of disease. Notably due to lack of published data on the shelf-lives of modified Leishman stain, we hypothesize that misidentification of cells may be due to expiration of the stain. Four modified Leishman staining solutions were prepared using phenol crystals and liquefied phenols as the sole modifying component ingredient. Thin blood smears were made in multiples of four from  each of the patients requiring peripheral blood film review especially those with high eosinophil counts. The smears were stained with the prepared dyes for a total of 75.0 seconds or 4.0 minutes  and examined for cellular elements of the blood by the researchers. At expiration, while other cellular elements of the blood stained normally, eosinophil granular content altered from orange-red to orange/reddish-brown and shades of brown and that was used to estimate  modified Leishman stains’ shelf-lives. Estimated shelf-lives of the modified Leishman stain were 25 days (21-29 days), ≤ 28 days, up to 52 days and ≤ 18 days  when the first, second, third and fourth methods of preparation respectively were used. Modified stain prepared by initial dissolution of phenol in absolute methanol before using resultant mixture to dissolve Leishman powder has the longest shelf-life- up to 52 days. It was ≤28 days when liquid phenol was used in similar pattern to conventional technique.When phenol crystals were used, the average shelf-life were 25 days (21-29 days) and ≤ 18 days respectively. Conclusively, modified Leishman stain should be prepared and used with the consciousness of the expiry dates. This mystery unfolded prevents misclassification of eosinophils during differential leukocyte counts, thus enhancing disease diagnosis.