A BSTRA CT A method has been developed for measuring neutrophil cellularity in normal human bone marrow, in which the neutrophil-erythroid ratio was determined from marrow sections and marrow normoblasts were estimated by the erythron iron turnover. Neutrophil maturational categories, defined by morphologic criteria, were supported by autoradiographs of marrow flashed-labeled with 3H-thymidine. Correction for multiple counting error was empirically derived by counting serial sections through cells of each maturational category. The normal neutrophil-erythroid ratio in 13 normal human subjects was 1.5±0.07. The mean number of normoblasts in the same subjects was estimated to be 5.07+0.84 X 10' cells/kg. Total marrow neutrophils (X 10' cells/kg) were 7.70±+1.20, the postmitotic pool (metamyelocytes, bands, and segmented forms) was 5.59±0.90 and the mitotic pool (promyelocytes + myelocytes) was 2.11+0.36.Marrow neutrophil ("total") production has been determined from the number of neutrophils comprising the postmitotic marrow pool divided by their transit time. Transit time was derived from the appearance in circulating neutrophils of injected 3H-thymidine. The postmitotic pool comprised 5.59±0.90 X 10' neutrophils/ kg, and the transit time was 6.60±0.03 days. From these data marrow neutrophil production was calculated to be 0.85 X 10' cells/kg per day.Effective production, measured as the turnover of circulating neutrophils labeled with 3H-thymidine, was 0.87±0.13 X 10' cells/kg per day. This value correlated well with the calculation of marrow neutrophil production. A larger turnover of 1.62+0.46 X 10' cells/kg per day was obtained when diisopropylfluorophosphate-3P Dr. Dancey's present address is Division of Hematology, Montreal General Hospital, Montreal, Quebec, Canada.Dr. Deubelbeiss' present address is Division of Hematology, Inselspital, Bern, Switzerland.Received for publication 27 February 1975 and in revised form 24 May 1976. was used to label circulating neutrophils. Studies using isologous cells doubly labeled with 3H-thymidine and diisopropylfluorophosphate-UP demonstrated a lower recovery and shorter ti of the 'P label. INTRODUCTIONThe rate of neutrophil production can be measured as the turnover of neutrophils in either marrow or blood. Blood neutrophil turnover as determined with ["P1di-isopropylfluorophosphate (DFP) 1 cells has gained general acceptance: Cartwright et al.(1) in a study of 109 subjects, reported a mean turnover of 1.63 x 109 cells/ kg per day, and a similar figure of 1.36 X 109/kg per day was reported by Galbraith et al. (2) who studied 18 subjects. Such studies have provided an estimate of blood neutrophil turnover in normal man. Attempts to measure marrow neutrophil production have been indirect in the absence of reliable measurements of marrow cellularity. In this paper we describe a method for determining the number of marrow neutrophils and report measurements of basal rates of total production (marrow turnover) and effective production (blood turnover) of neutrophils in man.

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A B S T R A C T This paper describes a method for determining the number of marrow erythroid and neutrophil cells in which the cellularity of marrow sections was related to that of the total marrow by radioiron dilution. Tissue sections were prepared from methacrylateembedded dog marrow biopsies, and neutrophils were identified by staining of their primary granules. After correction of direct section counts for multiple counting error, accurate neutrophil-erythroid ratios were established with a coefficient of variation of less than 10% when 104 cells were examined. An average neutrophil-erythroid ratio of 1.2 was found in six normal dogs.The total number of nucleated red cells in the dog was 5.48±0.78 X 109/kg (+1 SD), and the corresponding erythron iron turnover was 0.90±0.11 mg Fe/100 ml whole blood/day. The total number of marrow neutrophils, derived from the neutrophil-erythroid ratio, was 6.6±0.59 X 109 cells/kg, of which 1.4 were promyelocytes and mvelocytes, 2.3 were metamyelocytes and bands, and 3.0 were segmented neutrophils. Leukopheresis studies were carried out in six dogs to confirm the accuracy of these cellular measurements. 1\Iarrow counts showed a mean decrease of 22.7 X 109 cells or 35% of the postmitotic neutrophil pool, and it was calculated that 10.2 x 109 additional cells had been taken from already circulating blood. This estimated deficit of 32.9 X 109 was almost identical to the 33 X

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SYNOPSIS Preparation of human marrow sections has been studied systematically in order to facilitate accurate identification of marrow cells. Both of the methods developed involve embedding marrow cores in methyl methacrylate. In one, acrolein fixation is followed by staining of deplasticized sections with eosine-y followed by azure II; in the other, neutrophilic forms are identified by their esterase-specific reactivity in marrow fixed with neutral-buffered formalin. These preparations are suitable for quantitative studies of marrow cellularity.The purpose of this paper is to report our experience in the preparation of sections of human marrow for light microscopy, and to describe two methods that permit reliable recognition of all normal cell types in marrow sections. Methods and resultsMarrow biopsies, taken from the posterior superior iliac spine with a Westerman Jensen (Ellis et al, 1964) or Jamshidi (Jamshidi and Swaim, 1971) needle, were placed immediately in fixative. FIXATIONThe following fixative techniques were evaluated: Zenker's fixative incubated for 24 hours at room temperature (Lillie, 1965); Zenker's fixative without acetic acid but with the same incubation conditions; glutaraldehyde at concentrations of 1 25%, 25%, 5-0y%, and 6-25% in 0-1 M phosphate buffer, pH 7-2, for 24 hours at room temperature, andat a5 50 % concentration in 0-065 and 0-15 M phosphate buffer, pH 7-2, for 24 hours at room temperature; glutaraldehyde-formol (glutaraldehyde 6'25 % in 0-1 M phosphate buffer, pH 7-2, with formalin 10% v/v) for 24 hours at room temperature; neutral-buffered formalin (10% formalin in 0-1 M phosphate buffer, '