Estimation of fetal hemoglobin levels in individual red cells via fluorescence image cytometry.
A method for estimating fetal hemoglobin (Hb F) levels in individual red blood cells was developed. Cell smears were prepared using a slide maker to ensure uniform thickness and were then stained with immunofluorescence. An antifading gel was applied to preserve a stable fluorescence. The total fluorescence intensities from the same number of red cells in different slide specimens correlated with their hemolysate Hb F levels, which were determined via column chromatography (R = 0.95). Hb F level in individual cells was estimated from fluorescence intensity and cell area, which were determined via image analysis techniques and the hemolysate Hb F level. Blood from a normal subject, a subject with hereditary persistence of fetal hemoglobin, and from sickle cell patients with varying Hb F levels was analyzed. Our analyses showed a wide distribution of Hb F among cells for the normal subject and a gaussian distribution with a peak at the hemolysate Hb F level for the subject with hereditary persistence of fetal hemoglobin. The Hb F distributions were unique to the patients with sickle cell disease. Because Hb F level in individual sickle cells is crucial to the inhibition of cell sickling, the unique hb F distribution may be important in determining the clinical course of this disease.
Horiuchi
K
K
Department of Pediatrics, University of Pennsylvania School of Medicine, Children's Hospital of Philadelphia 19104, USA.
Osterhout
M L
ML
Kamma
H
H
Bekoe
N A
NA
Hirokawa
K J
KJ
D000755:Anemia, Sickle Cell / Q000097:blood*; D004912:Erythrocytes / Q000737:chemistry / Q000166:cytology* / Q000473:pathology*; D005319:Fetal Hemoglobin / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006453:Hemoglobinopathies / Q000097:blood*; D006461:Hemolysis; D006652:Histological Techniques; D006801:Humans; D012016:Reference Values
D005319:Fetal Hemoglobin