HEMOGLOBIN STANDARDS*

HEMOGLOBIN STANDARDS* RUSSELL L. HADEN Clevelnd Clinic, Clevelnd, Ohio Estimtions of hemoglobin often re unstisfctory to the lbortory worker nd the reports my be confusing to the clinicin. Unfortuntely, there is no unnimity of opinion concerning the technic of determintion or mnner of recording results. This confusion is due lrgely to the multiplicity of methods employed nd to the fct tht there is now on the mrket no hemoglobinometer reding in per cent hving ny scientific bsis for the selection of the stndrd used. The following clinicl methods for the estimtion of hemoglobin re in use in this country: (1) oxygen combining power (Vn Slyke); (2) iron content (Wong); (3) spectrophotometer (Sherd nd Snford); (4) comprison with colored stndrds: () tinted pper (Tlquist, (b) glss replcing hemoglobin (Dre, Von Fleischl- Miescher), (c) crbon monoxide-hemoglobin (Hldne, Plmer), (d) cid hemtin (originl Shli), (e) solutions to substitute for cid hemtin, in the Shli pprtus or in Duboscq colorimeter (Felton's pyrogllte nd Osgood nd Hskins' iron-chromium solutions), nd (f) glss to substitute for cid hemtin (Busch nd Lomb-Newcomer, Klett, new Shli, Hden-Husser). The oxygen cpcity method employing the Vn Slyke pprtus is ccurte when crried out by n experienced nd trined technicin but is too complicted to be used s routine clinicl lbortory procedure. The determintion of the quntity of iron by the new technic of Wong 18 is ccurte, quite simple, nd deserves much wider use. The spectrophotometer is expensive, must be clibrted with solutions of unstble crystllized hemoglobin nd cn be utilized only s mjor lbortory procedure * Red before the Eleventh Annul Convention of the Americn Society of Clinicl Pthologists, New Orlens, Louisin, My 6-9, 1932. 85 Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

86 RUSSELL L. HADEN even when the humn eye is replced by photoelectric cell s in the Sherd-Snford 12 hemoglobinometer. Of the colorimetric methods, the Tlquist is little better thn guessing; the Dre is difficult to red ccurtely, nd subject to wide errors; the Shli with n cid hemtin, yellow glss or correctly mde rtificil stndrd (Osgood-Hskms) is good clinicl method; the Duboscq colorimeter with colored glss stndrd s in the Newcomer nd Klett methods or with correct stndrd solutions is stisfctory for lbortory use; nd the Hden-Husser instrument hs its plce of usefulness. TABLE 1 HEMOGLOBIN STANDARDS IN DIFFERENT HEMOGLOBINOMETERS READING PER CENT IN INSTRUMENT Hldne Oliver Tlquist Dre (prior to 1926) Dre (1926 to 1931) Dre (1931 to ) Busch nd Lomb-Newcomer (originl) Shli (originl) Shli (present) Von Fleischl-Miescher Klett GBAMS EQUAL TO 100 PER CENT HEMOGLOBIN 13.80 15.00 15.80 13.80 16.90 16.00 16.90 17.20 14.00 15.80 15.36 The estimtion of oxygen cpcity by mens of the Vn Slyke pprtus is the method of choice for reserch work nd for determining nd checking stndrds. The iron method is the only possible substitute; comprtive estimtions indicte tht it gives results eqully s ccurte s the oxygen cpcity method (tble 1). Where n inexpensive lbortory instrument is required, the Shli with solid glss rod stndrd, or with the Osgood-Hskins 10 rtificil stndrd is stisfctory, if clibrted in grms. The Hden-Husser 4 hemoglobinometer is simple nd ccurte but unfortuntely more expensive. For use only in the lbortory the Busch nd Lomb-Newcomer nd Klett instru- Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

HEMOGLOBIN STANDARDS 87 ments with glss or liquid stndrds re very good. The ccurcy of ll the colorimetric methods cited, however, is lmost entirely dependent upon the clibrtion of the glss or solution used s stndrd, nd cn be depended upon only when this hs been done correctly. With the oxygen cpcity nd the iron methods the hemoglobin redings re directly in grms. Until recently most hemoglobinometers hve yielded redings in per cent with no two instruments in greement s to wht should be tken s equivlent to 100 per cent (tble 2). In lter models of the Von Fleischl-Miescher TABLE 2 HEMOGLOBIN IN GRAMS PER 100 cc. Averge NUMBER 1 2 3 4 5 6 7 8 9 10 OXYGEN CAPACITY METHOD (VAN SLYKE) grms 13.13 15.11 15.02 13.28 17.31 15.82 14.95 13.15 12.40 13.43 14.36 IRON CONTENT METHOD ("WONG) grm 13.46 14.68 14.68 12.95 17.53 16.25 14.63 12.98 12.50 13.32 14.30 nd the new Busch nd Lomb-Newcomer pprtus, the mount of hemoglobin is determined in grms only, lthough figures given in the tble re suggested for trnsposing the figures into per cent. The Klett nd the new Shli with solid glss rod stndrd give redings in grms s well s in per cent; the results with the Hden-Husser instrument re clculted only in grms. The Von Fleischl-Miescher instrument is clibrted by comprison with solution of unstble hemoglobin crystls; the new Busch nd Lomb-Newcomer by spectrophotometric determintions, nd the Hden-Husser by direct Vn Slyke redings; the method of clibrtion of the Klett nd Shli instruments is not known to me. Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

88 RUSSELL L. HADEN It is pprent tht with proper clibrtion ny ccurte colorimetric method cn be trnsposed directly into grms. Thus Osgood nd Hskins' 79 results in norml individuls reported in grms were obtined by clibrting their colorimetric procedure by direct comprison with redings on the Vn Slyke instrument. Wintrobe's figures 16 ' 17 were obtined with Newcomer glss stndrd checked by the oxygen cpcity method. It seems fir to require tht ll hemoglobinometers, reding either in per cent or in grms should hve s their bsis of clibrtion figures TABLE 3 COMPARISON OF HEMOGLOBIN READINGS WITH OXYGEN CAPACITY AND IRON CONTENT METHODS AND HADEN-HATTSSER HEMOGLOBINOMETER BLOOD NUMBER OXYGEN CAPACITY METHOD (VAN SLYKE) IBON METHOD (WONG) HADEN-HAU88ER HEMOGLOBINOMETER 1 2 3 4 5 6 7 8 9 10 grms* 12.1 12.4 10.6 10.4 9.4 7.6 14.4 6.0 8.0 11.2 grms* 11.8 12.1 10.3 10.7 9.4 7.8 14.4 6.2 8.4 11.8 grms* 12.0 12.0 10.5 10.5 9.5 7.8 14.5 6.0 8.0 11.5 Averge 10.23 10.29 10.23 * Grms per 100 cc. obtined by direct Vn Slyke redings or determintion of the iron content. Any hemoglobin determintion would thus be designted or understood in the lbortory: hemoglobin = per cent ( gms. by the direct Vn Slyke or the iron content method); or hemoglobin = gms. (direct Vn Slyke or iron content method) with the hemoglobinometer. In tble 3 is shown series of determintions with the Hden- Husser hemoglobinometer compred with both the oxygen cpcity nd iron methods. Such comprison should be vilble for every hemoglobinometer used. So fr s the technic of Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

HEMOGLOBIN STANDAKDS 89 hemoglobin estimtions is concerned, one my sy tht severl methods nd instruments re stisfctory, but method or instrument should only be employed which llows the trnsltion of the redings into grms of hemoglobin s determined by the direct Vn Slyke or the iron method. This point could esily be settled by common consent mong lbortory workers. The next problem concerns the mount of hemoglobin per hundred cubic centimeters of blood in norml individuls when determined by n ccurte method. Hldne, 6 on the bsis of very smll number of determintions found n verge of 13.8 grms of hemoglobin in helthy norml men in Englnd. He employed the oxygen cpcity method in the Hldne blood-gs pprtus. This so-clled Hldne stndrd (13.8 grms = 100 per cent) is frequently used in Americ, lthough every one hs relized tht norml men show reding well over 100 per cent when this stndrd is used. Recently, Price-Jones 12 hs found n verge hemoglobin content of 112 per cent in norml men in Boston with this stndrd, using colorimetric procedure clibrted by the gsometric method. The Hldne stndrd should never be used in this country, since with its use norml individuls will not hve color index of 1.00. The Willimson 14 stndrd should lso be discrded since the verge norml for men (16.92 grms) is much higher thn other workers hve found with other ccurte methods vilble. The spectrophotometer used by Willimson ws clibrted with hemoglobin crystls which re unstble nd difficult to prepre in pure form. Hence the results obtined with it cn not be compred with the most ccurte clinicl method (direct Vn Slyke) now is common use. Willimson's tble is of gret vlue, however, in showing the reltive hemoglobin content t different ges in the two sexes. Wht is the hemoglobin content per hundred cubic centimeters of norml individuls in this country by the direct Vn Slyke or iron content method? No lrge series of normls hs been reported with the iron method. Using the oxygen cpcity method I 8 found the verge in forty norml men severl yers go to be 15.4 grms. This series I hve recently incresed to seventy nd for this number the verge hemoglobin content is Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

90 RUSSELL L. HADEN 15.34 grms per hundred cubic centimeters (tble 4). Using the sme method, Dill 2 found the verge in forty men in Boston, 15.38 grms per hundred cubic centimeters nd Sopkey 13 in Indi, 15.30 grms in series of 121 men. These three series re the only ones reported in which ll the determintions were done by the oxygen cpcity method. Osgood nd Hskins (Portlnd, Oregon) using their inorgnic stndrd clibrted ginst direct Vn Slyke redings reported n verge hemoglobin vlue of 16 grms in 153 men. 6 Wintrobe nd Muller (New Orlens) first reported n verge of 15.85 in men using the Newcomer method, TABLE 4 A SUMMARY OF THREE SERIES OF SIMULTANEOUS ESTIMATIONS OF HEMOGLOBIN AND RED CELLS IN BLOOD DETERMI NATIONS MEAN BED CELL COUNTS HEMOGLOBIN OBSERVER AND DATE Osgood nd Hskins* (1926-27) Wintrobe* (1929-30) Hden (1922 nd 1932)... e 153 100 70 I 100 50 30 5.60 5.85 4.95 1 4.80 4.93 4.38 Men concentrtion o 16.0 17.0 15.34 1 13.69 13.76 13.37 g grnts^ 14.29 14.53 15.50 Coefficient 9 1 grm&\ 14.29 13.97 15.26 o» *B o grms\ 14.29 14.25 15.38 * Figures given in personl communiction from uthors. t Grms per 100 cc. clibrted by direct Vn Slyke redings. Lter Wintrobe 1 ' 6 revised his figures to 17 grms in men since mistke in clibrtion of his instrument hd been found. The figures for women re, of course, lower. Figures reported for other prts of the world vry little from those cited. The clinicin is interested in the bsolute vlues for hemoglobin in the blood, but is even more interested in the hemoglobin content reltive to norml nd in the mount reltive to the number of red cells present. Such reports re of gret vlue nd re ccurte, provided one cn trnslte them t will into grms, which is lwys possible if n bsolute figure is tken s 100 per Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

HEMOGLOBIN STANDARDS 91 cent. The men concentrtion of hemoglobin in norml men should be tken s 100 per cent. It is pprent tht different figure would hve to be tken for women. In studying the vrious types of nemi, the clinicin must know the reltion of the mount of hemoglobin to the red cell count, nmely, the color index, nd in clculting the per cent of cells, 5,000,000 red cells re tken s 100 per cent of cells. It is thus necessry to tke s, 100 per cent for the hemoglobin the number of grms of hemoglobin found per hundred cubic centimeters of blood per five million cells (the hemoglobin coefficient). In determining stndrd the erythrocyte count s well s the mount of hemoglobin must be known. Hldne nd Willimson did not record the number of red blood cells. The Hldne nd Willimson stndrds hve lredy been discussed s impossible of cceptnce on other grounds but this is n dded reson why they should not be used. Unfortuntely, lso, no red cell counts re reported for the hemoglobin estimtions of Dill. Only three lrge series of simultneous estimtions of hemoglobin nd of red cells hve been mde in this country, those of Osgood nd Hskins, (1926-1927), of Wintrobe (1929-1930) nd of Hden (1922 nd 1932). A summry of the findings in these three series is shown in tble 4. Osgood nd Hskins found the sme hemoglobin coefficient in men nd women (14.29 grms); Wintrobe found 14.53 grms in men nd 13.97 in women; my results show 15.50 grms in men nd 15.26 grms in women. The difference in hemoglobin per cell in men nd women is smll so it is unnecessry to use different stndrd for the two sexes. It is pprent from tble 4 tht the vrition in the hemoglobin coefficient is due lrgely to differences in the erythrocyte counts. If ll the figures re correct, prt from the vrition in hemoglobin, there is evident wide vrition in red cell count in different prts of the country. This might well be, since with the higher red cell counts reported for the Scndinvin countries, the hemoglobin is much the sme s in this county. I hve found the number of red cells in norml men in Detroit (5,030,000), in Knss City (4,880,000), nd in Clevelnd (5,030,000) pproximtely the sme. Likewise I found tht red cell counts done by medicl Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

92 RUSSELL L. HADEN students in Knss City on their own blood with Husser-Levy counting chmbers vried little from yer to yer s is indicted in tble 5. Sixteen femle students during this sme period showed n verge of 4,420,000, per cubic centimeters. Emerson 1 reported n verge of 5,000,000 mong 176 medicl students in Bltimore. No lrge series hve been reported from other centers. The hemoglobin stndrd which I suggested in 1922 s 100 per cent ws 15.6 grms (15.55 grms). I m now using 15.4 grms on the bsis of the lrger series (100 men nd women) reported here, lthough there is ctully difference of only 0.12 grm per hundred cubic centimeters in the stndrd for the two groups. TABLE 5 RED CELL COUNTS MADE BT MEDICAL STUDENTS IN KANSAS CITY YEAR 1924-1925 1925-1926 1926-1927 1927-1928 1928-1929 1929-1930 NUMBER OF STUDENTS 25 37 32 36 46 54 Totl: 230 AVERAGE COUNT 5.12 5.06 5.04 4.99 5.02 4.85 Averge: 4.997 My stndrd hs been found to give norml color indices for other workers in Phildelphi, Boston, nd Bltimore, nd for me in Detroit, Knss City nd Clevelnd. How is one to explin the differences in the three reported series, ech suggesting different hemoglobin coefficient? This cn not be done. Only further ccurte studies in different prts of this country will determine to wht extent the norml red cell count vries. Until some common figure is greed upon, the selection of single stndrd is impossible. Tbles such s Osgood 8 hs given for clculting percentges cn not be universlly used since they re correct only when the red cell counts nd the hemoglobin re the sme s he hs found them in Portlnd. Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

HEMOGLOBIN STANDARDS 93 It is necessry tht some method be mde vilble for reporting to clinicins the hemoglobin in per cent nd for clculting ccurtely the color nd sturtion indices. To meet this need I hve the following pln to offer. Only method or instrument for the determintion of hemoglobin should be used which reds in grms s clibrted by the direct Vn Slyke or iron content method nd ll redings should be recorded in the lbortory in grms. In ech lbortory, then, the men vlue for hemoglobin nd for the red cell count is determined for the plce in which the lbortory is locted. This is done esily nd quite ccurtely by counting the red cells in ten or more norml dults nd by determining simultneously the hemoglobin in the sme individuls with n instrument reding in grms per hundred cubic centimeters. The men vlue for erythrocytes nd for hemoglobin is thus found nd the men vlue for hemoglobin in grms per hundred cubic centimeters of blood per five million cells or per million cells is clculted. The number of grms of hemoglobin so determined is tken s norml or 100 per cent for the lbortory in which the clibrtion is done. When so determined the hemoglobin percentge in given blood will be the sme everywhere nd with every observer even though there my be wide vrition in bsolute figures, nd the color index in norml individuls lwys will be within norml limits (0.90 to 1.10). Using this pln, hemoglobin report would red: hemoglobin = per cent (100 per cent = hemoglobin content of norml dult with 5,000,000 erythrocytes). If, on further investigtion, it is found tht the hemoglobin coefficient is ctully everywhere the sme, bsolute figures cn then be used. Until such time, ech worker my use stndrd hemoglobin coefficient determined on the bsis of hemoglobin estimtions nd red cell counts mde in his own lbortory, nd my report the mount of hemoglobin in per cent of this stndrd. SUMMARY AND CONCLUSIONS 1. In mking hemoglobin determintions only n instrument or method should be employed in which the results re recorded in Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

94 EUSSELL L. HADEN grms s determined by the oxygen cpcity or iron content method. 2. For clinicl purposes the reports of hemoglobin re best given in per cent of norml of helthy dult with red cell count of 5,000,000 cells. 3. The Hldne nd Willimson stndrds cn not be so trnsposed since in determining the stndrds no red cell counts were done. 4. There is wide vrition in hemoglobin nd erythrocyte counts nd consequently in the hemoglobin coefficient in the three lrge series (Hden, Osgood nd Hskins, nd Wintrobe) reported for norml men nd women. 5. A stisfctory stndrd must give color index within norml limits (0.90 to 1.10) in norml individuls. 6. In the present stte of confusion, the hemoglobin coefficient should be determined for ech lbortory nd the hemoglobin should be reported only in per cent of this norml. 7. The hemoglobin percentge of given blood when determined by this method is necessrily the sme in ll lbortories within the limits of technicl error. REFERENCES (1) EMEESON, C. P.: Clinicl dignosis, fourth ed. Phildelphi, J. P. Lippincott Co. 513 pp. (2) DILL: Quoted by Price-Jones. (3) HADEN, R. L.: The norml hemoglobin stndrd. Jour. Am. Med. Assn., 79: 149&-1497. 1922. (4) HADEN, R. L.: A new hemoglobinometer. Jour. Lb. & Clin. Med., 16: 68-72. 1930. (5) HALDANE, JOHN, AND SMITH, J. L.: The mss nd oxygen cpcity of the blood in mn. Jour. Physiol., 25: 331-343. 1899. (6) OSGOOD, E. E.: Personl communiction. (7) OSGOOD, E. E.: Hemoglobin, color index, sturtion index nd volume index stndrds: redetermintion bsed on the findings in 137 helthy young men. Arch. Int. Med., 37: 685-706. 1926. (8) OSGOOD, E. E.: Tbles for clcultion of color index, volume index nd sturtion index bsed on recently determined stndrds. Jour. Lb. & Clin. Med., 12: 899-906. 1927. Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

HEMOGLOBIN STANDARDS 95 (9) OSGOOD, E. E., AND HASKINS, D. D.: Reltion between cell count, cell volume nd hemoglobin content of venous blood of norml young women. Arch. Int. Med., 39: 643-855. 1927. (10) OSGOOD, E. E., HASKINS, H, D., AND TEOTMAN, F. T.: A simplifiction of the Osgood-Hskins hemoglobin method. Jour. Lb. & Clin. Med., 16: 482-486. 1931. (11) PRICE-JONES, C: The concentrtion of hemoglobin in norml humn blood. Jour. Pth. & Bct., 34: 779-787. 1931. (12) SHEAED, C, AND SANFOED, A. H.: Photo-electrometer with one stge of mplifiction: s pplied to the determintion of hemoglobm. Jour. Am. Med. Assn., 93: 1951-1957. 1929. (13) SOPKEY: Quoted by Price-Jones. (14) WILLIAMSON, C. S.: Influence of ge nd sex on hemoglobin. Arch. Int. Med., 18: 505-528. 1916. (15) WINTEOBE, M. M.: Hemoglobin stndrds in norml men. Proc. Soc. Exp. Biol. & Med., 26: 848-851. 1929. (16) WINTEOBE, M. M.: Blood of norml young women residing in subtropicl climte. Arch. Int. Med., 45: 287-301. 1930. (17) WINTEOBE, M. M., AND MULLEB, M. W.: Norml blood determintions in the South. Arch. Int. Med., 43: 96-113. 1929. (18) WONG, S. Y.: Colorimetric determintion of iron nd hemoglobin in blood. II. Jour. Biol. Chem., 77: 409-412. 1928. Downloded from https://cdemic.oup.com/jcp/rticle-bstrct/3/1/85/1767892 on 11 Februry 2018

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