Immunophenotyping of acute leukemia by flow cytometric analysis: Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis

Michael J Borowitz, K. L. Guenther, K. E. Shults, G. T. Stelzer

Research output: Contribution to journalArticle


This article describes a procedure for performing routine three-color flow cytometric analysis for acute leukemia on lysed whole bone marrow preparations. This technique uses the combination of CD45 intensity and right-angle light scatter (RALS) to distinguish leukemic cells from normal lymphocytes, monocytes, neutrophils, eosinophils, and nucleated red blood cells. On this display, leukemic cells occupy a unique blast region characterized by intermediate CD45 density and low RALS, which, in normal marrows, contains less than 5% of the total cells. This approach was applied to 39 cases of acute leukemia and 8 cases of myelodysplasia or myeloproliferative disorders. The estimate of blasts by flow cytometric analysis was correlated highly with morphologic leukemic cell counts over a wide range. Moreover, the pattern seen on the CD45-RALS display was different for different French-American-British subtypes of leukemia, suggesting that this pattern might be useful for categorization. When CD45-peridin chlorophyll α protein was combined with other pairs of fluorescein isothiocyanate- and phycoerythrin-conjugated reagents, it was possible to set an analysis window on the leukemic blasts and display dual-parameter (ie, green vs. red fluorescence) data regarding expression of two additional markers on the leukemic population. This gating strategy was superior to traditional forward-angle versus RALS displays in that it did a better job of isolating the leukemic cells analytically.

Original languageEnglish (US)
Pages (from-to)534-540
Number of pages7
JournalAmerican Journal of Clinical Pathology
Issue number5
Publication statusPublished - 1993



  • Acute leukemia
  • CD45
  • Flow cytometry
  • Immunophenotyping

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

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