an example of flow cytometric dna analysis as a diagnostic tool j. chezar western galilee...
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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.