An example of Flow Cytometric DNA Analysis as

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An example of Flow Cytometric DNA Analysis as a diagnostic tool J. Chezar Western

An example of Flow Cytometric DNA Analysis as a diagnostic tool J. Chezar Western Galilee Hospital-Nahariay Israel

History: A 49 year old male referred to the hematology department because of pancytopenia.

History: A 49 year old male referred to the hematology department because of pancytopenia. After 2 dry taps, a bone marrow aspirate resulting in barely 200 u. L of material was obtained and sent for flow cytometry. The sample was stained with the following antibody combinations. CD 45 PC 7/CD 34 ECD/CD 33 PC 5/ CD 117 PE/ HLA-DR FITC, CD 45 PC 7/CD 3 ECD/CD 56 PC 5/CD 8 PE/CD 4 FITC+CD 19 FITC, CD 45 PC 5/CD 13 PE/CD 16 FITC, CD 45 PC 5/CD 64 PE/CD 14 FITC. DNA cell cycle analysis was performed alone and in combination with CD 45 FITC and glycophorin A FITC, CD 13 FITC. In addition a bone marrow biopsy was sent to pathology. Immunohistochemistry was done with peroxidase conjugated LCA, MPO, CD 34, Td. T, CD 15, CD 117, PAS, CD 43, CD 138, CD 31, CD 79 a, Factor VIII, Ker, CD 68, CD 3, B and T cell antibodies

BLASTS

BLASTS

Aberrant population Normal Bone Marrow Patient Bone Marrow

Aberrant population Normal Bone Marrow Patient Bone Marrow

Myeloid phenotype = Blasts?

Myeloid phenotype = Blasts?

MPO: GRAN+ / BLASTS -

MPO: GRAN+ / BLASTS -

PAS NEG IN BLASTS

PAS NEG IN BLASTS

Normal 2 n Aneuploid (A)

Normal 2 n Aneuploid (A)

To verify identity of aberrant population – double stain with PI for DNA and

To verify identity of aberrant population – double stain with PI for DNA and relevant FITC labeled antibody

A Aneuploid population NOT leukocytic

A Aneuploid population NOT leukocytic

A Aneuploid population NOT thrombocytic

A Aneuploid population NOT thrombocytic

Glycophorin A + A Aneuploid population IS erythroid !!

Glycophorin A + A Aneuploid population IS erythroid !!

Glycophorin

Glycophorin

Erythro Leukemia DNA analysis and subsequent double staining with appropriate antibodies lead to the

Erythro Leukemia DNA analysis and subsequent double staining with appropriate antibodies lead to the correct diagnosis.