Estimation of fetal hemoglobin levels in individual red cells via fluorescence image cytometry

Precise determination of reticulocytes (young red blood cells) containing fetal hemoglobin (Hb F), F-reticulocytes, is important for assessment of the efficacy of drugs such as hydroxyurea and butyrate in elevating the levels of Hb F in patients with sickle cell disease (SCD) and beta-thalassemia. We developed a reliable and easily applicable method for determining F-reticulocytes using fluorescence image cytometry. Reticulocytes were first identified by preparing a monolayer smear of blood stained by acridine orange. Images of reticulocytes and of all cells were obtained for a selected area on the smear. After removing the acridine orange, cells containing Hb F (F-cells) in the same area were then identified by immunofluorescence. Using images of F-cells, reticulocytes, and all cells for the same fields it was possible to identify F-reticulocytes. To assess the validity of our two-stage staining method, we compared our results with those obtained by traditional methods. There was significant correlation of our method with the conventional immunofluorescence staining method for F-cells (r2 = 0.99; slope = 0.99) and with the accepted brilliant cresyl blue method for reticulocytes (r2 = 0.97; slope = 0.96). Heretofore, the ability to determine F-reticulocyte levels has been limited to a small number of laboratories possessing special equipment and techniques. The method presented here should be of great interest to many basic science and clinical investigators involved in studies evaluating synthesis of Hb F.

Download Full-text

Glycerol-3-phosphate dehydrogenase activity in the red cells of patients with thalassemia

Blood ◽

10.1182/blood.v55.4.564.bloodjournal554564 ◽

1980 ◽

Vol 55 (4) ◽

pp. 564-569

Author(s):

P Fessas ◽

NP Anagnou ◽

D Loukopoulos

Keyword(s):

Cord Blood ◽

Fetal Hemoglobin ◽

Thalassemia Major ◽

Red Cells ◽

Beta Thalassemia ◽

Gamma Chain ◽

Cord Blood Cells ◽

Chain Synthesis ◽

Glycerol 3 Phosphate Dehydrogenase ◽

Normal Adults

L-alpha-Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) has been reported to be absent in the erythrocytes of normal adults, but can be found in those of cord blood and of thalassemia major. The aid of this study was to investigate whether there is any relation between GDH and gamma-chain synthesis. Erythrocyte GDH activity was determined on 118 different blood samples. It was undetectable in normal adult erythrocytes and definitely high in cord blood cells (23.6 UI/10(11) RBC). Considerable GDH activity was also noted in patients with thalassemia major (11.0 IU10(11) RBC) as well as in cases with pronounced reticulocytosis (11.4 IU/10(11) RBC). Red cells from beta- thalassemia heterozygotes exhibited moderate but distinct GDH activity (5.2 IU/10(11) RBC). After fractionation into young and old erythrocyte populations, clearly higher GDH activity was found in the younger cells; however, there was no significant correlation with the reticulocyte count. Presence of reticulocytes alone appears insufficient to explain the values obtained in cord blood and the thalassemias, especially heterozygous. Furthermore, no direct correlation between GDH and fetal hemoglobin (HbF) was obtained in cord and thalassemic erythrocytes.

Download Full-text

The effect of alpha-thalassemia on the expression of the beta- thalassemia/HPFH heterozygote in a black family

Blood ◽

10.1182/blood.v57.6.1132.1132 ◽

1981 ◽

Vol 57 (6) ◽

pp. 1132-1134 ◽

Cited By ~ 6

Author(s):

E Beutler ◽

E Turner ◽

W Kuhl

Keyword(s):

Black Family ◽

Clinical Manifestations ◽

Fetal Hemoglobin ◽

Pedigree Analysis ◽

Red Cells ◽

Beta Thalassemia ◽

Hemoglobin Gene ◽

Alpha Thalassemia ◽

Alpha Globin ◽

Hereditary Persistence

Abstract A 2-yr-old black girl presented with a thalassemic clinical picture and was found to have nearly 100% fetal hemoglobin in her red cells. Pedigree analysis indicated that she was a heterozygote for the hereditary persistence of fetal hemoglobin gene and for a beta O- thalassemia gene. A brother, who also had nearly 100% fetal hemoglobin in his red cells, manifested, in contrast to his sister, no anemia and only minimal splenomegaly. Examination of the family's alpha-globin loci using the restriction endonuclease Eco Rl demonstrated that the brother had a single alpha-locus deletion that he had inherited from his mother. The mild clinical manifestations of this boy are consistent with the often expressed view that excess alpha chains may contribute significantly to the hematologic manifestation of beta-thalassemia.

Download Full-text

Fetal hemoglobin-containing cells have the same mean corpuscular hemoglobin as cells without fetal hemoglobin: a reciprocal relationship between gamma- and beta-globin gene expression in normal subjects and in those with high fetal hemoglobin production

Blood ◽

10.1182/blood.v69.4.1109.1109 ◽

1987 ◽

Vol 69 (4) ◽

pp. 1109-1113 ◽

Cited By ~ 29

Author(s):

GJ Dover ◽

SH Boyer

Keyword(s):

Globin Gene ◽

Fetal Hemoglobin ◽

Reciprocal Relationship ◽

Transmitted Light ◽

Red Cells ◽

Mean Corpuscular Hemoglobin ◽

Beta Globin ◽

Corpuscular Hemoglobin ◽

Normal Individuals ◽

F Cells

Abstract We have developed methodology that allows comparison of the mean corpuscular hemoglobin (MCH) of fetal hemoglobin (HbF)-containing red cells (F cells) with the MCH of non-F cells from the same individual. To do this, suspensions of peripheral blood erythrocytes and their internal contents are fixed with an imidodiester, dimethyl-3,3′- dithiobispropionimidate dihydrochloride (DTBP). Thereafter fixed cells are made permeable to antisera by treatment with Triton X-100 and isopropanol, reacted with a mouse monoclonal antibody (MoAb) against HbF, and then with fluorescein-conjugated antimouse IgG. No appreciable hemoglobin is lost during such manipulation. Red cells from a diversity of subjects were thus treated and examined microscopically, first by transmitted light and then by epifluorescence. A direct correlation between Coulter-derived MCH and mean absorbance of 415 nm transmitted light was found for 100 unfixed (r = 0.96) and for 100 antibody-treated fixed-permeabilized red cells (r = 0.99) among individuals selected so as to provide a range of Coulter MCH values between 20 and 35. Comparisons of microscopically derived MCH of F cells and non-F cells were statistically nondistinguishable (P greater than 0.05) in all subjects. Such comparisons included normal individuals (less than 1% F cells), SS patients (7% to 48% F cells), subjects with congenital anemia (22% to 65% F cells), individuals with heterocellular hereditary persistence of HbF (HPFH) (12% to 21% F cells), and heterozygotes for beta + thalassemia (11% to 31% F cells). We conclude that gamma- and beta-globin production within F cells is regulated in a reciprocal fashion both among normal individuals and among individuals with elevated HbF production.

Download Full-text

Reticulocyte Survival in Sickle Cell Anemia: Effect of Cyanate

Blood ◽

10.1182/blood.v40.5.733.733 ◽

1972 ◽

Vol 40 (5) ◽

pp. 733-739 ◽

Cited By ~ 5

Author(s):

Blanche P. Alter ◽

Yuet Wai Kan ◽

David G. Nathan

Keyword(s):

Cell Survival ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Red Cells ◽

Red Cell ◽

Hemoglobin Molecule ◽

Red Cell Survival ◽

And Control

Abstract Cyanate prevents sickling in vitro and apparently prolongs the survival of 51Cr-tagged sickle erythrocytes in vivo. Cautious interpretation is required because the effects of cyanate on 51Cr binding to sickle and fetal hemoglobin-containing red cells are unknown, and comparison of the effect of cyanate on sickle red cell survival to control red cell survival must be performed sequentially. We have studied the survival of sickle reticulocytes utilizing radioactive amino acids that are incorporated into hemoglobin. Two informed adult patients with sickle cell disease were studied. In each study, two 50-ml samples of blood were incubated separately with 14C- and 3H-leucine for 2 hr, after which 50 mM cyanate was added to one aliquot for 1 hr. The cells were then washed and reinfused. Frequent venous samples were obtained, and the specific activities of 14C and 3H in the hemoglobin were followed. The t ½ of the carbamylated cells was tripled, but remained below normal. This method provides a generally useful measurement of the influence of drugs bound to red cells on reticulocyte lifespan. The labels are incorporated into the hemoglobin molecule of the reticulocyte, and simultaneous comparison of the survivals of the same cohort of drug-treated and control cells is achieved.

Download Full-text

The transition from HK to LK phenotype in the red cells of newborn genetically LK lambs.

The Journal of General Physiology ◽

10.1085/jgp.79.5.893 ◽

1982 ◽

Vol 79 (5) ◽

pp. 893-915 ◽

Cited By ~ 5

Author(s):

E M Tucker ◽

C E Smalley ◽

J C Ellory ◽

P B Dunham

Keyword(s):

Transport Properties ◽

Developmental Process ◽

Fetal Hemoglobin ◽

Kinetic Studies ◽

Red Cells ◽

Small Cells ◽

Adult Hemoglobin ◽

K Concentration ◽

Cell Volumes ◽

K Transport

Red cells from newborn lambs were separated into different age populations by centrifugation, and cells with fetal hemoglobin (Hb) were distinguished from those with adult Hb by an acid elution technique. Changes were followed during development in rates of K+ transport (active and passive), numbers of Na+/K+ pump sites per cell, cell volumes, and numbers of Lp and L1 antigen sites per cell. These changes were correlated with the percentage of cells with adult hemoglobin. (The Lp and L1 antigens are associated with K+ transport in that specific alloantibody against Lp, anti-Lp, stimulates active transport, and anti-L1 inhibits passive transport.) Active K+ transport decreased during development because of a decline in number of Na+/K+ pumps (from measurements of ouabain binding) and because of an alteration in the affinity of the pumps for intracellular K+ (from kinetic studies in which the intracellular K+ concentration was varied). Cells with fetal Hb had fewer Lp sites and were larger than cells with adult Hb. As transport properties changed, the number of Lp sites increased and continued to increase after all the cells had adult Hb Cells with fetal Hb had as many L1 sites as lamb cells with adult Hb, but the number of L1 sites was less than those found previously for adult sheep. A population of small cells with intermediate K+ concentration and intermediate numbers of Lp sites appeared soon after birth. The various points of evidence suggested that the developmental process leading to cells with adult transport properties was a gradual one and did not coincide precisely with the switch from fetal to adult Hb.

Download Full-text

Paroxysmal Nocturnal Hemoglobinuria with Elevated Fetal Hemoglobin

Blood ◽

10.1182/blood.v43.2.233.233 ◽

1974 ◽

Vol 43 (2) ◽

pp. 233-238 ◽

Cited By ~ 4

Author(s):

Anne L. Rassiga-Pidot ◽

Gibbons G. Cornwell ◽

O. Ross McIntyre

Keyword(s):

Paroxysmal Nocturnal Hemoglobinuria ◽

White Male ◽

Acid Stress ◽

Fetal Hemoglobin ◽

Acetylcholinesterase Activity ◽

Red Cells ◽

Peripheral Smear ◽

Heterogeneous Distribution ◽

Mixed Blood ◽

Persistent Elevation

Abstract A persistent elevation of the fetal hemoglobin (Hgb F) level (5%-15%) was observed in a 22-yr-old white male with paroxysmal nocturnal hemoglobinuria (PNH). Acid treatment of the peripheral smear (Betke-Kleihauer technique) demonstrated a heterogeneous distribution of Hgb F in the red cells. As expected, the lowest acetylcholinesterase activity and the most hemolysis after acid stress were found in the low-density, reticulocyte-rich cell fraction. In contrast, the highest Hgb F levels were found in the high-density, reticulocyte-poor fraction. Further evidence for the segregation of these two abnormalties was obtained from the observation that less Hgb F was present in the hemolysate obtained after acid lysis than was present in the mixed blood sample or the remaining nonhemolyzed red cells.

Download Full-text