Differential immunophenotyping is essential for diagnosis and follow-up. Routine analysis requires a minimum of 250 γl blood, and neither a detailed immunophenosubtyping nor absolute cell count are available. We present two concepts using slide-based cytometry in order to break these limitations. Materials and Methods: 10 γl EDTA blood were stained with DRAQ5 and either CD3-PE, CD4-Alexa Fluor 488 and CD8-PE-Cy5 or CD45-FITC and CD14-PE. A ‘no-lyse-no-wash’ versus a‘lyse-no-wash’ method (Quicklysis) was performed. 20 γl of the suspension were applied to a Neubauer chamber. Leukocytes were analyzed by laser scanning cytometry(LSC) twice with a time interval of 15 min. Another aliquot of blood was taken for routine analysis. Erythrocyte count was performed after another dilution. Results:Brévais’ regression coefficient showed a good internal correlation of LSC (r > 0.85) with no significant difference between routine analysis and LSC (‘lyse-no-wash’: r = 0.9,p = 0.009, α = 0.05; ‘no-lyse-no-wash’: r = 0.98, p < 0,0001,α = 0.05). Immunophenosubtyping by LSC (CD3/CD4/CD8, CD45/CD14) was unequivocal. Conclusion: A detailed immunophenosubtyping and absolute cell count is possible by using the Neubauer chamber for slide-based analysis. This method requires minimal amounts of blood, is very cost-efficient, and data show no significant difference to routine laboratory. This concept might prove versatile in patients with low blood volume and where absolute cell count is essential.

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2007
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