Quantitation of erythropoietin stimulatory activity using [3H]thymidine uptake by K562 cells.
A microassay for erythropoietin (Ep) activity in serum using [3H]thymidine uptake by K562 cells is presented. The method is similar to that of Krystal except that cells of the K562 human pluripotent leukemia cell line replace spleen cells from phenylhydrazine-treated anemic mice. Response to the hormone by K562 cells and spleen cells was colinear. Using the Krystal bioassay, 14 young hemoglobin S homozygotes had Ep activity levels of 17.9-113.8 mU/ml serum, whereas the new method with K562 cells gave a range of 19.2-115.3 mU/ml. The correlation coefficient between the two sets of data (r) was 0.999 (p less than 0.001). With the modified technique we have assayed 34 sickle cell patients, whose sera ranged from 19.2 to 1400 mU of Ep/ml with corresponding hemoglobin concentrations of 10.7 g % to 3.0 g %. Values for normal subjects were 22.1 +/- 2.1 mU/ml (n = 7). The stimulation of [3H]thymidine uptake is significantly inhibited by an anti-Ep antiserum. The assay permits quantification of stimulatory activities in a large number of samples with relative ease and is also suitable to explore the interactions of erythropoietic factors with their appropriate receptors on stem cells.
Lewis
J P
JP
Laboratory of Molecular Hematology, Veterans Administration Medical Center, Augusta, GA 30910.
McKie
V C
VC
Faguet
G B
GB
Felice
A E
AE
D000293:Adolescent; D000328:Adult; D000818:Animals; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D004921:Erythropoietin / Q000097:blood* / Q000276:immunology; D005260:Female; D006801:Humans; D007106:Immune Sera; D004915:Leukemia, Erythroblastic, Acute / Q000097:blood*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009500:Neutralization Tests; D013154:Spleen / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism*
D007106:Immune Sera; D004921:Erythropoietin; D013936:Thymidine