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. After 2 dry taps, a bone marrow aspirate resulting in barely 200 uL of material was obtained and sent for flow cytometry. The sample was stained with the following antibody combinations.

CD45PC7/CD34ECD/CD33 PC5/ CD117 PE/ HLA-DR FITC, CD45PC7/CD3ECD/CD56PC5/CD8PE/CD4FITC+CD19FITC, CD45PC5/CD13PE/CD16FITC, CD45PC5/CD64PE/CD14FITC.

DNA cell cycle analysis was performed alone and in combination with CD45 FITC and glycophorin A FITC, CD13 FITC.

In addition a bone marrow biopsy was sent to pathology. Immuno-histochemistry was done with peroxidase conjugated LCA, MPO, CD34, TdT, CD15, CD117, PAS, CD43, CD138, CD31, CD79a, Factor VIII, Ker, CD68, CD3, B and T cell antibodies

BLASTS

Normal Bone Marrow Patient Bone Marrow

Aberrant population

Myeloid phenotype = Blasts?

MPO: GRAN+ / BLASTS -

PAS NEG IN BLASTS

Normal 2n

Aneuploid (A)

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 thrombocytic

A

Aneuploid population IS

erythroid !!

Glycophorin A +

Glycophorin

Erythro Leukemia

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