Characterization of in vitro BFU-E colony growth in response to hydroxyurea

A prediction of in vivo fetal hemoglobin induction

D. M. Kitchens, B. S. Baliga, Betty Sue Pace, Y. M. Yang

Research output: Contribution to journalArticle

Abstract

Cytotoxic agents such as hydroxyurea have been shown to induce fetal hemoglobin (Hb F) production and reduce the frequency and severity of vasoocclusive pain crisis in 50-75% of sickle cell patients. Therefore, a way of determining whether or not a patient will respond to hydroxyurea following the treatment is administered would be helpful. An inverse linear relationship between total number of BFU-E colonies and Hb F levels in sickle cell patients treated with hydroxyurea has been demonstrated in our laboratory (Am J Hematol 46:259-263). We conducted a study to assess the relationship between the BFU-E colony growth in response to hydroxyurea in vitro and the fetal hemoglobin induction by hydroxyurea treatment in vivo in sickle cell patients. We performed in vitro cell cultures by using erythroid precursor cells isolated from sickle patients and incubated these cells with hydroxyurea in cell culture systems. Mononuclear cells from peripheral blood of each patient were incubated in both the absence and presence of hydroxyurea (100 uM) in multiwell tissue culture plates. BFU-E colonies were counted on day 14. The Hb F levels were determined by alkaline denaturation procedure followed by spectrophotometric analysis in cells harvested from culture plates on day 14. After the patients were started with hydroxyurea, blood samples were drawn every 2-4 weeks for BFU-E assay and Hb F levels. Seven patients with sickle cell anemia not previously treated with hydroxyurea were included in this study. The in vitro experimental results show that the number of BFU-E colonies is decreased from 173.5 ± 46.0 to 9.5 ± 10.8 with concomitant increase in Hb F levels from 4.0 ± 1.2 to 22.6 ± 9.2 percent. One patient completed in vivo study. The results show that the number of BFU-E colonies is decreased gradually from 180 to 2 where as Hb F levels are increased from 0% to 18.5% over a 12-week period. These data suggest that the change in the number of BFU-E colonies and Hb F levels after in vitro hydroxyurea treatment in cell culture systems may predict the in vivo response to hydroxyurea in sickle cell patients. Further studies that include more patients are underway.

Original languageEnglish (US)
JournalJournal of Investigative Medicine
Volume44
Issue number1
StatePublished - Jan 1 1996
Externally publishedYes

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Fetal Hemoglobin
Erythroid Precursor Cells
Hydroxyurea
Growth
Cell Culture Techniques
Cell culture
In Vitro Techniques
Blood
Tissue culture
Denaturation
Cytotoxins
Sickle Cell Anemia
Assays
Blood Cells
Therapeutics

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Characterization of in vitro BFU-E colony growth in response to hydroxyurea : A prediction of in vivo fetal hemoglobin induction. / Kitchens, D. M.; Baliga, B. S.; Pace, Betty Sue; Yang, Y. M.

In: Journal of Investigative Medicine, Vol. 44, No. 1, 01.01.1996.

Research output: Contribution to journalArticle

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abstract = "Cytotoxic agents such as hydroxyurea have been shown to induce fetal hemoglobin (Hb F) production and reduce the frequency and severity of vasoocclusive pain crisis in 50-75{\%} of sickle cell patients. Therefore, a way of determining whether or not a patient will respond to hydroxyurea following the treatment is administered would be helpful. An inverse linear relationship between total number of BFU-E colonies and Hb F levels in sickle cell patients treated with hydroxyurea has been demonstrated in our laboratory (Am J Hematol 46:259-263). We conducted a study to assess the relationship between the BFU-E colony growth in response to hydroxyurea in vitro and the fetal hemoglobin induction by hydroxyurea treatment in vivo in sickle cell patients. We performed in vitro cell cultures by using erythroid precursor cells isolated from sickle patients and incubated these cells with hydroxyurea in cell culture systems. Mononuclear cells from peripheral blood of each patient were incubated in both the absence and presence of hydroxyurea (100 uM) in multiwell tissue culture plates. BFU-E colonies were counted on day 14. The Hb F levels were determined by alkaline denaturation procedure followed by spectrophotometric analysis in cells harvested from culture plates on day 14. After the patients were started with hydroxyurea, blood samples were drawn every 2-4 weeks for BFU-E assay and Hb F levels. Seven patients with sickle cell anemia not previously treated with hydroxyurea were included in this study. The in vitro experimental results show that the number of BFU-E colonies is decreased from 173.5 ± 46.0 to 9.5 ± 10.8 with concomitant increase in Hb F levels from 4.0 ± 1.2 to 22.6 ± 9.2 percent. One patient completed in vivo study. The results show that the number of BFU-E colonies is decreased gradually from 180 to 2 where as Hb F levels are increased from 0{\%} to 18.5{\%} over a 12-week period. These data suggest that the change in the number of BFU-E colonies and Hb F levels after in vitro hydroxyurea treatment in cell culture systems may predict the in vivo response to hydroxyurea in sickle cell patients. Further studies that include more patients are underway.",
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