Total Red Cell Volume in Healthy Young Males

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 8, No. 5 Copyright 1978, Institute for Clinical Science Total Red Cell Volume in Healthy Young Males BERNARD S. MORSE, M.D. Division of Hematology of the Department of Medicine, College of Medicine and Dentistry of New jersey, New Jersey Medical School, Newark, NJ and Internal Medicine Branch, Clinical Sciences Division, USAF School o f Aerospace Medicine, Brooks AFB, TX ABSTRACT Regression equations of total red cell volume on body weight, body surface area, lean body mass, etc., are provided for a series of 79 normal male subjects. The regression of total red cell volume on a combination of height and weight provided the smallest standard error of the estimate and the largest correlation coefficient. The coefficient of variation was 1.6 percent betw een the first and second red cell volume determination in 31 of these subjects. The popular expression of total red cell volume in ml per kg remains unsatisfactory. It is recom m ended that a comparison be made betw een a measured total red cell volume and a predicted normal range derived from a regression using body surface area as the independent variable. A simplified graph is provided which clearly delineates normal from abnormal and should prove adequate for m ost clinical situations. Introduction nevertheless recommends that for routine purposes, it is to be regarded adequate to The m easurem ent of total red cell volum e (TRCV) using 51 Cr labeled erythro Several large series of TRCV estimates in express blood volum e in those terms. cytes shows a large variation in normal normal subjects, reported prior to the publication of the ICSH report, indicate an individuals. T he International Com m ittee for Standardization in Hematology improved correlation with body surface (ICSH) published standard techniques for area (BSA) and combined height-weight the measurements of red cell and plasma rather than body weight alone.4,10,14 In volumes.7 Although it has been claimed clinical m edicine, the m easurem ent of that the popular m ethod of presenting TRCV is used routinely and primarily to blood volume estimates in ml per kg is establish the diagnosis of an absolute th eo retically unsatisfactory, th e IC SH erythrocytosis. Every effort should there /78/ $00.90 Institute for Clinical Science, Inc.

2 4 1 4 M ORSE fore be made to improve the prediction of normal to allow better recognition of the abnormal. The purpose of this paper is to present the m easured values and regression equations for TRCV derived from a sample of 79 normal male subjects using a technique similar to that proposed by the ICSH. A sim plified approach to the clinical assessm ent of the TRCV based on body surface area regression is presented. Materials and Methods Su b j e c t s Seventy-nine normal male Air Force subjects, ages 18 to 42, were used. These subjects gave inform ed consent to participate in one of several research protocols at the School of Aerospace M edicine. The m easurem ent of total red cell volume constituted one of several clinical tests that were performed during their stay at the School. All subjects passed a routine class III flight physical which included a chest x-ray and electrocardiogram. Blood donation w ith in six m onths c o n stitu te d grounds for exclusion. Fifty-six of the subjects com pleted basic training at least one month prior to their participation and the rem ainder were stationed perm anently at Brooks AFB, TX. The w eights ranged from 57.1 to kg and the surface areas ranged from 1.61 to 2.33 sq.m.* M e t h o d s Total red cell volume estimates were performed with 51 Cr labeled autologous red cells. Blood, collected at 0730 hours into 0.1 volume ACD solutionf was incubated with 15 /uc Na51C r 0 4for45m inutes at room tem perature. The chromated red * The complete tabulated data have been submitted to Crowell Collier MacMillan Information Sciences of the American Society for Information Science. f Special formula, Abbott Laboratories. cells were washed three times with sterile saline and w ere then resu sp en d ed in sterile saline for injection. The quantity reinjected, as well as an aliquot for the standard, was m easured gravimetrically to th e n e a re st 0.01 g on a M ettler balance. Subjects fasted overnight and rem ained supine for an hour prior to and during the test. The height and weight in the nude were recorded to the nearest 0.5 cm and 0.1 kg. The chromated cell suspension was injected intravenously at 1000 hours, and blood sam ples w ere collected in heparin from the opposite arm at 1015 and 1030 hours. Duplicate microhematocrits from each sample were centrifuged for five m inutes in a centrifuge. $ The height of the cell column to the top of the black line,1or if not visible to the beginning of the buffy coat, was measured, and a 0.98 correction was a p p lie d for tra p p e d plasm a.3 Aliquots of lysed blood and standards were counted to 0.5 percent counting error in an autogam ma scintillation detector. Replicate red cell volum e estim ates were performed in 31 of the subjects imm ediately after the 1030 hour blood sample was collected. Another aliquot o f51 Cr labeled red cells was injected and blood samples were collected at 1045 and 1100 hours. The radiochromium activity of the 1030 hour sample was subtracted from the 1045 and 1100 hour sample to correct for the circulating 51 Cr resulting from the 1000 hour injection. Body w ater was e stim a ted w ith deuterium oxide.11 Plasm a sam ples obtained three hours after the oral ingestion of 20 g of deuterium oxide (enriched) were quantitated for deuterium content by infrared spectrophotom etry.12 Lean body mass was derived with the Rathbun-Pace equation.9 Nomographic charts derived from the Dubois formula2 were used for the determ ination of surface area. } International Microhematocrit.

3 TO TAL RED CELL VOLUME IN HEA LTH Y YOUNG MALES 415 Results The summary of the m easured values is shown in table I. The TRCV was 26.6 ± 2.5 ml per kg and 1038 ± 94 ml per M2. Regression equations for the prediction of TRCV from body weight, height, BSA, lean body mass, etc., are listed in table II. The standard error of the estimate was smallest (± 150 ml) when a combination of height and w eight was used as the somatic reference standard. Regression of TRCV on total body water, lean body mass and body fat failed to improve the estimate. Replicate TRCV determinations in 31 subjects revealed a mean volume difference of + 9 ml betw een the first and second test. The standard deviation was ± 30 ml with a coefficient of variation of 1.6 percent. Discussion The usual custom of relating TRCV to body weight provides the clinician with what appears to be a simple interpretable parameter. Such a value is then compared with the average or normal value. The probability is implied that it differs from the normal. The use of an average TRCV value rather than a prediction equation based upon a regression analysis has serious drawbacks. W ith increasing body weight, for example, there is increasing TRCV, but it does not conform to an equivalent relationship (table III). If this were the case, then the TRCV in ml per kg would show the same value regardless of body weight. In table III, the TRCV in ml per kg has been calculated from the prediction equations provided for the present series as well as those reported in or calculated from the literature. In each case, the TRCV in ml per kg decreases w ith increasing body weight. These calculations clearly illustrate the pitfall of using an average norm al value w ith respect to a weight reference. In heavy but not obese TABLE I Summary o f Measured Values Mean S ta n d a r d D e v i a tio n Total red cell volume ml Weight Kg Body surface area sq. m Height cm Lean body mass Kg individuals, the confirmation of a diagnosis of erythrocytosis can readily be m issed since the TRCV is disproportionately low er in such individuals. Consider, for example, a 100 kg (2.20 BSA) male with a predicted red cell mass o f2381 ml (table II). An observed value of 3,000 ml would place this individual beyond the 99 percent confidence limits, th ereb y substantiating a diagnosis of erythrocytosis. I f th e TRCV was converted to ml per kg, then 30 ml per kg is compared to 26.6 ± 2.5. Since this value falls within two standard deviation units of the mean, it would be interpreted as a normal value. According to ICSH recom mendations, 30 ml per kg is the normal value for an adult male. Again, the use of an arbitrary or average value for diagnostic purposes or for inclusion of a patient into a treatm ent program will tend to TABLE I I Regression Analysis of Total Red Cell Volume on Somatic Standards P r e d i c tio n E q u a tio n T o ta l Red C e l l Volume Sx y* ml C o e f f i c i e n t o f V a r ia tio n P e r c e n t 16.6 x body weight x surface area x height x height x body weight 29.4 x body water x lean body mass x body fat *Standard error of the estimate. r

4 416 MORSE TABLE I I I Predicted Red Cell Volume Expressed as ml per kg at Specified Body Weights I n v e s t i g a t o r 50 Kg Body W eigh t 70 Kg 100 Kg Present series Huff and F eller^ Hyde and Jones Wennesland et al Sterling and Gray Frenkel et al Retzlaff et al^ Piomelli et al select against individuals at both ends of the spectrum of body size. T he regression equation using BSA derived from the present study compares favorably w ith those m ost recently reported by Frenkel et al4 and Retzlaff et al.10 It is not our intent to analyze methods for prediction of the red cell mass. The elegant study of Frenkel et al provides an in depth treatise in this area and advocates the usefulness of BSA as the independent variable. The series reported by Retzlaff et al10 and W ennesland et al14 also corroborate the usefulness of the BSA. In the present study, a combination of height and w eight provided the largest correlation coefficient (r) and the lowest standard error of the estimate (Sxy). The equation using BSA was a close second. In attempts to reduce further the variation, the relationship of TRCV to body water, lean body mass and body fat was m easu red by a n u m b er of in v estigators.5,6,8 In some reports, the relationship to total body water was exceptional whereas in other studies, including the present, the use of total body water or lean body mass failed to improve the prediction of the TRCV. In the present studies, there was more variation in lean body mass than body surface area. This may, in part, explain why the data correlate better with surface area than lean body mass. Even though the relationship of TRCV to com bined height-w eight or body surface area explains only 50 percent to 60 percent of the variation in red cell mass, these are the most convenient, inexpensive and readily obtainable in dependent variables. Clearly, the tradition of reporting red cell mass in ml per kg should be replaced by a m ethod which uses some form of regression analysis. Sample size, subject selection and technical differences all influence the prediction of the norm al value. Technical differences are listed in table IV and center mainly around the hem atocrit m ethod and correction for trapped plasm a. The m icrohem atocrit m ethod provides slightly lower values than the macro m ethod since a greater relative centrifugal force is applied.15 The read in g errors are sim ilar for both methods when the hematocrit values are in the normal range. Therefore, when a m icrohem atocrit m ethod is used, the TRCV value will tend to be lower and is exem plified by the present series. W ennesland, et al and Retzlaff, et al inclu d e d th e buffy coat in the m acrohematocrit reading. If such a practice is continued, then another variable is introduced and patients w ith m arkedly elevated leucocyte and/or platelet counts will tend to have larger values for the TRCV. E ven w hen the leucocyte and platelet counts are normal, the TRCV value will tend to be higher. Frenkel, et al, u sing th e m acrohem atocrit m ethod, applied a relative centrifugal force of 1223 x g, a value insufficient for maximal packing of erythrocytes.15 Even w ith a correction factor of 0.98, the TRCV values are expected to be larger. The inclusion of obese subjects in their series tends to reduce th e TRCV value and probably counter-balances any increase owing to their hem atocrit m ethod. In view of the methodologic differences that exist among the largest reported series, it is difficult to select a single reported regression as a laboratory standard. The object is to derive the narrowest possible range of normal so that the abnormal

5 TO TAL RED CELL VOLUM E IN HEA LTH Y YOUNG MALES 417 TABLE IV Selection and Technical Differences Among Series of Subjects with Apparent Normal Total Red Cell Volume Estimates I n v e s t i g a t o r S u b j e c t s Age C e l l s W ashed P r i o r t o I n j e c t i o n D e te r m in a tio n o f D ose I n j e c t e d * H e m a to c r it M eth odf C o r r e c ti o n f o r T ra p p e d P la sm a Present series Air Force Yes G Micro 0.98 Huff and Feller13 Normal Yes V W 0 Hyde and J o n e s 11 Patients Yes V w 0.96 Wennesland et al2 Prisoner Yes V W++ 0 Sterling and G r a y 1^ Medical Yes V 4 cc tubes 0 student Frenkel et al4 Hospital Yes V W 0.98 employees & patients Retzlaff et a l 3 Clinic NO V W personnel Piomelli et a l 12 Seminarian NO G w 0 *Gravimetric (G) or volumetric (V). tmicrohematocrit (Micro); Wintrobe tube (W); Buffy coat included in measurement (++). can be identified with confidence. The predicted 95 percent confidence limits for the present series, the series of Wennesland et a l14 and the tolerance limits reported by Frenkel et al4 are shown graphically in figure 1. It can readily be seen that the present data show the smallest range of total red cell volume. W hether this is due to su b ject selectio n or m ethodologic differences cannot be resolved; how ever, m ethodologic differences as previously m entioned do complicate the previous investigations.4,14 The patient whose observed value falls within the overlap of the upper 95 percent confidence and tolerance limits remains the greatest problem. He would be classified abnormal by the present criteria but norm al by th e o th er c rite ria.4,14 For laboratories that do not have an established reference standard, it is recom m ended that such an individual be subjected to repeat TRCV estimates at three to six month intervals until it can be decided w hether or not the TRCV value is stationary, increasing or normal. If the pa- SURFACE AREA (M2) F i g u r e 1. The shaded area encloses the normal range of TRCV between the upper and lower 95 percent confidence limits for the present series. The upper and lower 95 percent confidence and tolerance -. - limits for the data of Wennesland et al14 and Frenkel et al4 are included for comparison.

6 4 1 8 MORSE tient s body configuration is unusual, then perhaps the TRCV should be related to body water, exchangeable K+, etc. Until a universally acclaimed laboratory standard emerges, the following is recommended: (1) hem atocrit by micro-m ethod; (2) exclusion of buffy coat from the hematocrit reading; (3) ap p ro p riate correction employed for trapped plasma based on relative centrifugal force of the m icrohematocrit centrifuge; and (4) observed TRCV estimate reported in ml along with the normal range at the m easured value for body surface area (table II). Acknowledgments The technical assistance of A2C Richard Miles and MSgt. James Green is gratefully acknowledged. Thanks are also extended to Major M. Stansell for the deuterium measurements. References 1. Ba u m b e r g e r, J. P.: A note on the black line in hematocrit determinations. J. Lab. Clin. Med. 27: , DuBoiS, D. and DuBoiS, E. F.: A formula to estimate the approximate surface area of height and weight be known. Arch. Int. Med. 17: , E b a u g h, F. G., J r., L e v in e, P. and E m e r s o n, C. P.: The amount of trapped plasma in the red cell mass of the hematocrit tube. J. Lab. Clin. Med. 46: , F r e n k e l, E. P., M c C a l l, M. S., R e i s c h, J. S., ET AL: An analysis of methods for the prediction of normal erythrocyte mass. Amer. J. Clin. Path. 58: , Hyde, R. D. and Jones, N. F.: Red-cell volume and total body water. Brit. J. Haemat. 8: , H u f f, R. L. and F e l l e r, D. D.: Relation of circulating red cell volume to body density and obesity. J. Clin. Invest. 35:1-10, I nternational Com mittee f o r Standardiz a t io n in H e m a t o l o g y : Standard techniques for the measurement of red-cell and plasma volume. Brit. J. Haemat. 25: , Pio m e l l i, S., Nathan, D. C., Cum m ins, J. F., ET AL: The relationship of total red cell volume to total body water in octogenarian males. Blood 19:89-98, Ra thbun, E. N. and Pace, N.: Studies on body composition: 1. The determination of total body fat by means ofthe body specific gravity. J. Biol. Chem. 158: , Re t z l a f f, J. A., Tauxe, W. N., Kie l y, J. M., ET AL: Erythrocyte volume, plasma volume, and lean body mass in adult men and women. Blood 33: , Sc h lo e r b, P. R., Fr iis-hansen, B. J., E d e l- MAN, I. S., ET AL: The measurement of total body water in the human subject by deuterium oxide dilution. J. Clin. Invest. 29: , STANSELL, M. J. and M ojica, L., Jr.: Determination of body water content using trace levels of deuterium oxide and infrared spectrophotometry. Clin. Chem. 14: , Sterling-, K. and Gray, S. J.: Determination of the circulating red cell volume in man by radioactive chromium. J. Clin. Invest. 29: , W ennesland, R., Br o w n, E., H o o per, J., Jr., et AL: Red cell, plasma and blood volume in healthy men m easured by radiochromium (CR51) cell tagging and hematocrit: Influence of age, somatotype and habits of physical activity on the variance after regression of volumes to height and weight combined. J. Clin. Invest. 38: , W in t r o b e, M. M.: Clinical Hematology, Philadelphia, Lea and Febiger, 1974, pp