Quantitative determination of hemoglobin and cytochemical staining for peroxidase using 3,3′,5,5′-tetramethylbenzidine dihydrochloride, a safe substitute for benzidine

A&AI

YTICAI

BIOCHEkllSIR~

98, 388-393

(1979)

Quantitative Determination of Hemoglobin and Cytochemical for Peroxidase Using 3,3’,5,5’-Tetramethylbenzidine Dihydrochloride, A Safe Substitute for Benzidine H. H. LIEM,”

Staining

F. CARDENAS,$ M. TAVASSOLI,$ M. B. POH-FITZPATRICK,? AND U. MULLER-EBERHARD*$

Received

February

I. 1979

The quantitative determination of hemoglobin employing 3,3’,5.5’-tetramethylbenzidine dihydrochloride (TMB-dl is described. This agent can also be used for staining myeloperoxidase-containing granules in granulocytes. TMB-d is at least as sensitive a reagent as benzidine but. unlike benzidine and diaminobenzidine. it is not a carcinogen.

Benzidine is widely employed in the re- testing methods (5.6). but its solubility in action for measuring hemoglobin in the water is limited. urine and plasma (1). The chemical reaction We now report on the relative merit of (TMB-d) as a subleads to the formation of hematin which TMB-dihydrochloride stitute for benzidine in the quantitative catalyzes the conversion of hydrogen peroxide to water and oxygen. In this con- measurement of hemoglobin using the version benzidine is oxidized to a chromomethod of Crosby and Furth (I) and staining of hemoglobin-containing cells using the genic product; the intensity of the resulting method of Ralph (7). We have also comcolor is used as an index of hemoglobin conpared TMB-d with 3,3’-diaminobenzidine centration. Benzidine and some of its (DAB) which is the most commonly used derivatives have also been used as hydrogen donors in the reaction to identify and benzidine derivative for cytochemical staining localize the enzyme peroxidase. of myeloperoxidase (8) and of horseradish Since benzidine has been demonstrated to peroxidase employed in the immunoperoxibe carcinogenic and the safety of many other dase technique (2,3). substitutes such as diaminobenzidine, otolidine, and others has been questioned (4). MATERIALS AND METHODS the search for a safe compound has been initiated. A methylated derivative of benzidine, 1. Tetramethylbenzidine dihydrochlo3,5.3’ S’-tetramethylbenzidine (TMB) ,’ was ride is purchased from Polysciences, Inc. found not to be carcinogenic by current (Warrington, Pa.). We use solutions ranging from 50 to 250 mg in 100 ml of distilled water ’ Abbreviations used: TMB. 3.5.3’.5’-tetramethylor 10% acetic acid. bewidine; d, dihydrochloride: DAB. 3.3’.diamino2. Hydrogen peroxide is freshly prebrnzidine: PAP, horseradish peroxidase-antiperoxidase. pared. Four milliliters of a 30-~01% solution 0003.2697/79/140388-06$02.00/O Copyright All nghtr

8~ 1979 hy Acadrmtc Press. Inc. ut rrproduct,i,n I,, an, form re\erved.

388

QUANTITATIVE

DIETERMINATION

is added to 96 ml of distilled water. This solution can be used for several days if stored in the refrigerator. 3. Hemoglobin standards are fireshly prepared (the solutions range from 10 to 100 mgi100 ml water) from a reference stock solution (100 mgil00 ml). Methodological details are described in the section below on the preparation of hemoglobin standard. 4. Distilled water, 10%’ acetic acid, or sodium citrate buffer (pH 5) is usled as diluent.

Duplicate tubes are prepared for reagent blank and hemoglobin standards. One milliliter of TMB-d solution is added to each test tube, and 0.02 ml of the hemoglobin standard containing different concentrations is added to the tubes so labeled. No hemoglobin is added to the reagent blank tube. After 2 min, 1 ml of hydrogen pereoxide solution is added to each of the tubes which are mixed thoroughly by swirling. Ten milliliters of diluent (distilled water, citrate buffer, or acetic acid) is added to each tube which is then covered with Parafilm and mixed by inversion. The reaction product is monitored spectrophotometrically using a Beckman Model 25 spectrophotometer in the wavelength range 750 to 350 nm.

OF HEMOGLOBIN

389

This layer of hemoglobin may be decanted or aspirated. 5. Occasionally a slight turbidity remains in the hemoglobin solution. If an exceptionally clear hemoglobin solution is desired, shake with chloroform for a second time. Centrifuge and remove the supernatant hemoglobin, which is invariably transparent. 6. To prepare a hemoglobin solution which can be stored indefinitely at -2O”C, add an equal volume of glycerol to the solution. The hemoglobin content can be assayed by the cyanmethemoglobin method. 7. Dilutions are made from this stock solution for use as reference standards. Aliquots of a lOO-mg% hemoglobin solution are stored at -20°C in 30% glycerol in a quantity needed for several weeks. Before use, the aliquot is defrosted and further dilutions are made with distilled water or phosphate-buffered saline. pH 7.4. These solutions are stable in the refrigerator for several days.

Myeloperoxidase staining of neutrophilic granulocytes and hemoglobin staining of hemoglobin-containing cells (erythrocytes and its precursors) are performed on airdried smears of normal human blood and bone marrow aspirate. Smears are fixed for 30 s in phosphate-buffered formalPwparution c?f’Hrtnoglohirl Strrndcrrtt acetone solution. Stock Soll~tiotis For immunoperoxidase staining, the 1. Wash red cells three times with sandwich method of Sternberger et LII. (9) normal saline; centrifuge at 4OOgfor 5 min, utilizing the horseradish peroxidase-antibetween washes. peroxidase immune complex (PAP) is em2. After the last centrifugation, replace ployed. We have previously used this the saline with an equal volume of distilled method for demonstration of hemopexin and water and shake thoroughly. cytochrome h, in a variety of rat tissues (3). 3. Add 0.33 vol of chloroform and shake In the present study. we use goat anti-rat vigorously for 3 min. hemopexin to localize hemopexin in normal 4. Centrifuge at 14OOgfor 30 min. After rat liver. Methods for the preparation of centrifugation, the hemoglobin solution will the reagents have been described (IO) and form a clear layer on the top and is s:epa- will not be repeated here. Paraffin-embedded rated from the chloroform by a solid disk. pieces of rat liver are sectioned at about

390

LlEM

5 Frn, mounted on glass slides, and deparaffinized. The sections are then sequentially treated with goat anti-rat hemopexin, rabbit anti-goat IgG. and goat antiperoxidaseperoxidase complex. In control experiments, normal goat serum or phosphatebuffered saline replaces the anti-rat hemopexin. TMB-d, 37.5 mg/lOO ml, replaces benzidine, 300 mg/lOO ml. and DAB, 7.5 mg/lOO ml, according to the hemoglobin staining method of Ralph (7) and the myeloperoxidase staining method of Kaplow (8). Duplicate slides, including the appropriate control slides, are stained with benzidine or DAB in comparison with TMB-d followed by counterstaining with methyl green or neutral red. RESULTS

ET AL.

300

450

600 Wavelength

750

(nmi

FIG. I Effect oftime and temperature on absorption spectra of TMB-d dissolved in water. (. I freshly prepared, room temperature: ( - - - ) 1 day. room temperature: ( -.-.) I day. refrigerated.

AND DISCUSSION

globin reveals two absorption maxima. at 450 and 650 nm. The absorption maximum TMB-d is a white powder which is at 450 nm is dependent on the presence of stable in the vacuum seal of the manuacetic acid. When water or citrate buffer is facturer. On exposure to air, it begins to used as diluent. this maximum is not present. oxidize and within days attains a blue tint. The formation of the maximum at 4.50 nm Contrary to TMB which is not water soluble may, therefore. be related to a secondary ( 1 I), the dihydrochloride dissolves slowly oxidation product. A similar “secondary in water (or in citrate buffer) and the solution maximum absorption” has been reported remains colorless when stoppered and kept when ortlzo-tolidine was substituted for at 4°C. When the solution is exposed to benzidine (12). On the other hand, the air, oxidation of the agent produces a pale blue product. TMB-d also dissolves in 10% absorption at 650 nm is independent of the type of diiuent (Fig. 2) and appears to acetic acid in which a green oxidation correspond in a quantitative manner with product is formed (see below). The absorpthe “primary maximum absorption” when tion spectrum of TMB-d solution in water benzidine or ortho-tolidine is used (12). shows progressive oxidation at room Whereas the absorption at 450 nm is detemperature. Upon refrigeration, oxidation pendent on the reaction with hemoglobin occurs at a considerably slower rate (Fig. 1). (Fig. 2), the absorption at 650 nm can occur A positive correlation between oxidation upon self-oxidation (Fig. 1). Therefore. we and increase in temperature is found when suggest the use of the absorption maximum the solutions are exposed to room temperaat 450 nm for hemoglobin measurement. ture, 30°C. 37°C. and 45°C (not shown). Compared to cjrtlro-tolidine and benzidine, this “primary maximum absorption” is shifted to a longer wavelength. The maxiSpectrophotometric monitoring of this mum appears at 515 nm with benzidine and reaction using a standard solution of hemoat 630 nm with cjrhto-tolidine.

QUANTITATlVE

Wavelength

DETERMINATION

(nm)

FIG. 2. Effect of diluent on absorption spectra of hemoglobin reaction products using TMB-d. I___ ) diluent, IOR, acetic acid. final pH 2.5: ( - -- ) diluent. water, final pH 4.0: t -.-.) diluent. citrate buffer, final pH 4.5.

Trrrninntion

of tht> Reaction

To determine the endpoint of the rea’ction, the absorbances at 450 and 650 nm were measured at IO-min intervals after addition of diluent. It was found that the concen-

391

OF HEMOGLOBIN

tration of TMB-d affected the endpoint reading. At the high concentration (in the range of 250 mg/lOO ml) the reaction did not terminate for as long as 2 days. At lower concentrations of TMB-d, its tendency to become oxidized was reduced. A concentration of 50 mg/lOO ml was found to be optimal. At this concentration the readings (at 450 nm) were stable between 20 to 30 min after addition of the diluent. The absorbancy of the same sample at wavelength 650 nm was more variable due to the described effect of autooxidation. The intensity of the color development was, in accordance with Beer’s law, directly dependent upon the quantity of hemoglobin present (Fig. 3).

Myeloperoxidase-containing granules in granulocytes in blood smears stained readily with TMB-d with an intensity comparable to the benzidine reaction obtained by the method of Kaplow (8) (Fig. 4). The granules appeared blue in this reaction. As compared to those of the myeloperoxidase reaction of Kaplow. the reagents for the TMB-d re-

0.6 -

m g % hemoglobin

FIG. results

3. Optical density were obtained using

at 4.50 nm graphed as a function of hemoglobin the absorbance maximum at 650 nm (not shown).

concentration.

Similar

392

LiEM

action are easier to make but they must be freshly prepared. TMB-d does not compare favorably with benzidine in staining hemoglobin in erythrocytes employing the method described by Ralph (7) nor can it replace DAB in immunohistochemistry using the PAP method for localization of hemopexin in rat liver. TMB-d has a pronounced tendency to crystallize on the slide when the tissue is preincubated in phosphate-buffered saline. Because of the difficulty in completely removing this buffer, crystallization remains a problem, although it does not interfere significantly with the interpretation of the results. The alcohol-soluble TMB does not crystallize in the buffer, but its poor solubility in commonly used buffer systems limits its usage. Two other noncarcinogenic compounds have recently been shown to be satisfactory for demonstrating myeloperoxidase activity in blood and bone marrow cells. These are 3-amino9-ethylcarboxyl (13) and 4-chloro-1-naphthol (14). Although we have successfully used 3-amino-9-ethylcarboxyl as an indicator of myeloperoxidase. no systematic comparison was made in the present study. CONCLUSIONS Tetramethylbenzidine dihydrochloride is a suitable substitute for benzidine in the quantitative determination of hemoglobin in solutions. It also compares favorably with diaminobenzidine in the identification and localization of myeloperoxidase in cytochemical reaction. Since tetramethylbenzidine dihydrochloride readily crystallizes in commonly used buffer systems, its usage in histochemistry is limited. At present, tetramethylbenzidine dihydrochloride is expensive. However, utilization of a much smaller amount of the chemical than benzidine yields consistent results. The tendency of TMB-d to oxidize can be easily controlled by using freshly prepared solutions. Solutions and/or dilutions which are re-

ET AL.

FIG. 4. Myeloperoxidase-containing leukocytes stained with TMB-d.

granules

frigerated and tightly for at least 1 day.

are stable

stoppered

In

ACKNOWLEDGMENTS This work was supported by Research Grants AM18549(M.B.P.F.), AM-IX329and CA-17735 (U.M.E.). and AM-Z.510 (M.T.) from the National Institutes of Health, Bethesda, Maryland. M.T. is a recipient of RCDA AM-7055 I.

REFERENCES 1. Crosby, W. H., 11, 380-383. 2. Aurameas.

and Furth,

S. (1970)

[,I/. Kv\,.

F. W. (1956) (.!/(I/.

B/~I~x/

27, 349-385.

3. Tavassoli. M.. 0~01s. J.. Sugimoto. Cr.. Cox, K. H.. and Muller-Eberhard. U. (1976) Bioc,hcnr. Bic~/dy\ A’<,, Cc~r,~r,~on. 72. 281 -287.

QUANTITATIVE

DETERMINATION

4. Clayson. D. B.. and Garner. R. C. (1976) i/t Chemical Carcinogens (Searle. C. E., ed.). pp. 366-461. American Chemical Society. Washington. D. C. 5. Holland. V. R., Saunders, B. C.. Rose. F. L.. and Walpole. A. L. (1974) 7’c,trtrlrrdrr,/1 30, 329993302. 6. White, W. I. (1977)J. C‘hvon~trt~~jir. 138, 220-222. 7. Ralph, P. H. (1941) Strri/i 7~e~~/rn~1/. 16, 105. 8. Kaplow. L. S. (1965) B/or~d 26. 215-219. 9. Sternherger, L. A.. Hardy. P. H., Jr.. Cuculis,

393

OF HEMOGLOBIN

J. J., and Meyer, H. G. ( 1970) J. Histoc.l~e~~~. C‘~td~cttr. 18. 3 15-333. IO. Muller-Eberhard, U., Yam, L.. Tavaasoli. M., Cox. K.. and 0~01s. J. ( 1974)Biochem. Bi~jph~\. Rr.\.

II.

C‘omrtrrrt~.

61,

983-987.

Standefer. J. C.. and Vanderjagt, D. (1977) C‘lir~. Clrrrtf. 23, 749-75 1. 12. Lewis, G. P. (1965) J. Clrn. P&lrr~/. 18, 235-239. 13. Kaplow, L. S. (1975) Awwr. .I. (‘/it/. Pnrhl. 63, 45 I. 14. Ornstein, L., Ansley, H.. and Saunders, A. (1976) B/orM/ CC//.\ 2, 557-585.