Assay for thrombopoietin: a new, more sensitive method based on measurement of the small acetylcholinesterase-positive cell.

Abstract

Abstract A sensitive assay for a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) that utilizes measurement of the small acetylcholinesterase-positive (SAChE+) cell in the marrow of mice is described. The results of this assay were compared with a previously published procedure that measures 35S incorporation into platelets of immuno-thrombocythemic mice. In the rebound-thrombocytotic mouse assay, TSF from kidney cell culture medium caused a dose-related increase in the amount of 35S incorporation into platelets; the minimum detectable dose of TSF was 15 mg of protein/mouse. For the SAChE+ cell assay, mice received single intraperitoneal injections of TSF or platelet-specific antiserum. Other mice were injected with plasma from thrombocytopenic donor mice. For controls, groups of mice were given saline or normal mouse plasma. Bone marrow from the mice (killed at 10 hr after injection) was taken for smears and stained for acetylcholinesterase. The results show that both TSF and rabbit anti-mouse platelet serum (RAMPS) caused a highly significant dose-dependent increase in the percentage of SAChE+ cells and that plasma from thrombocytopenic mice stimulated an increase (P < 0.0005) in the percentage of SAChE+ cells. The minimum detectable dose of TSF in the SAChE+ cell assay was 1.875 mg protein/mouse. Therefore, SAChE+ cells in the marrow of mice can detect smaller doses of TSF than the rebound-thrombocytotic mouse assay.