Characterization of pseudobasophilia on Sysmex-XT 1800i automated hematology analyser

Background: Pseudobasophilia is a common automation related phenomenon which requires manual peripheral smear study in an era of complete automation. This study has attempted to evaluate the reasons for pseudobasophilia and in-turn suggest measures to eliminate the errors.Methods: A sample size of 207 cases showing pseudobasophilia on automation were studied by manual peripheral examination to categorize the possible cause for its occurrence. Descriptive and inferential statistical analysis was carried out. Results on continuous measurements are presented on Mean SD and results on categorical measurements are presented in Number (%). Significance is assessed at 5% level of significance. Student t test has been used to find the significance of study parameters on continuous scale within each group.Results: Atypical/ reactive lymphocytes were present in 86.5% cases contributing to pseudobasophilia phenomenon on automation, which also showed falsely increased absolute basophil count with more percentage of lymphocytes showing reactive changes. Temperature and storage effects did not contribute to their occurrence in this study. Another finding was an associated pseudomonocytosis with pseudobasophilia on automation which was statistically significant (p<0.001).Conclusions: Pseudobasophilia, and pseudomonocytosis are automation related phenomenon. Atypical/ reactive lymphocytes, which are cytoplasmic strip resistant, contribute to their occurrence. Hence, newer modalities like multicolour flow cytometry coupled with antibody tagging, multiangle polarised scatter separation and volume conductivity scatter may reduce the chances of pseudobasophilia, thereby reducing the overall turnaround time

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

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 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

Traversing Their Path to the Peripheral Smear: The Journey of Traumatized Red Blood Cells

Background Thrombotic microangiopathy encompasses a wide range of conditions, of which thrombotic thrombocytopenic purpura being a medical emergency requires prompt intervention, with schistocytes being a reliable morphological indicator of microvascular injury. However, there are conditions other than thrombotic microangiopathic anemia where schistocytes can be seen in large numbers. These nonthrombotic microangiopathic conditions are broadly grouped under cytoskeletal abnormalities, mechanical damage, and thermal injuries. Automated methods in schistocyte evaluation have shown varied reproducibility requiring manual identification. International Council for Standardization in Hematology (ICSH) recommends standardized morphological criteria and quantitative assessment as a percentage after counting at least 1,000 red blood cells in optimal areas of smear to reduce interobserver variability. Objectives The aim of this study was to evaluate and quantitate schistocytes in thrombotic microangiopathic and nonthrombotic microangiopathic groups using ICSH guidelines and to evaluate interobserver reproducibility of manual schistocyte count. Materials and Methods Overall, 157 peripheral blood smears showing schistocytes were studied by two independent observers using ICSH recommendations on light microscopy. The hematological findings were correlated with clinical diagnosis and other relevant investigations. Results Schistocytes were observed in five cases of thrombotic microangiopathic anemia and 152 cases of nonthrombotic microangiopathic anemia. Schistocyte count in thrombotic microangiopathic anemia and nonthrombotic microangiopathic anemia groups with mean (±standard deviation) value was 2.28 ± 2.65% and 0.76 ± 0.67%, respectively (p < 0.001). The correlation coefficient between the two observers was 0.59 (confidence interval = 0.966–1.346) showing an excellent agreement on the reproducibility of schistocytes by application of ICSH guidelines. Conclusion Percentage of schistocytes more than 1% is a robust morphological indicator for diagnosis of thrombotic microangiopathic anemia in adults. Strict application of ICSH guidelines reduces interobserver bias