Letters to the Editor Substitute for Benzidine in Myeloperoxidase Stains .02 M Acetate buffer, pH 5.0-5.2 0.3% Hydrogen peroxide
50 ml. 0.4 ml.
Final pH is 5.5. Wash gently in running tap water and counterstain with Mayer's hematoxylin for 8 minutes. Wash, dry, mount in glycerolgelatin and examine. Peroxidase activity is represented by red-brown granular deposits. The distribution of dye is identical to that seen with benzidine stains. This technic is suggested as an alternative to methods employing benzidine and its derivatives. L E O N A R D S. K A P L O W ,
M.D.
Chief, Laboratory Service Veterans Administration Hospital West Haven, Conn. 06516 and Associate Professor of Pathology and Laboratory Medicine Yale University School of Medicine New Haven, Conn. 06520
Method Fix thin blood smears in buffered formalin acetone 5 for 15 seconds at room temperature. Wash gently and incubate slides for 2'/2 minutes at room temperature in a filtered mixture containing: 3-Amino-9-ethylcarbazole* Dimethylsulfoxide
10 mg. 6 ml.
Received September 3, 1974; accepted for publication September 10, 1974. Address reprint requests to Dr. Kaplow. * Aldrich Chemical Co., Inc. Milwaukee, Wis. 53233.
References 1. Federal Register 39, # 2 0 pp 3756-3797 Jan. 29, 1974 2. Graham RC JR, Lundholm U, Karnovsky MJ: Cytochemical demonstration of peroxidase activity with 3-amino-9-ethylcarbazole. J HistochemCytochem 13:150-152, 1965 3. Kaplow LS: Simplified myeloperoxidase stain using benzidine dihydrochloride. Blood 26: 215-219, 1965 4. Schaefer HE, Fischer R: Peroxidase detection in smear preparations and tissue sections after decalcification and paraffin embedding. Klin Wochenschr 46:1228-1230, 1968 5. Yam LT, Li CY, Crosby WH: Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 55:283-290, 1971
NBT Slide Test To the Editor: — I read with interest the article of Okamura and associates (Am J Clin Pathol 62:27-31, 1974) concerning 451
an improved method for the so-called "NBT slide test" in human neutrophils. Among other aims they tried to improve
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
To the Editor: — Federal regulations 1 now prohibit the use of benzidine and its derivatives in concentrations above 0.1% unless rigorously defined safety precautions are taken. These safety requirements are too complex and demanding for clinical laboratories. Indeed, it is likely that manufacturing laboratories will also find it very difficult to comply with these regulations. It is thus more than a likelihood that benzidine and its derivatives will shortly become unavailable. These carcinogenic compounds are used in clinical laboratories primarily in procedures for detecting occult blood and in staining leukocytes for myeloperoxidase activity.3 Our laboratory has been investigating a number of alternative methods for peroxidase stains, using indicator compounds other than benzidine. A method modified from Graham and associates2 and Schaefer and Fischer 4 has yielded most consistent and satisfactory results. It is given below.
50 ml. 0.4 ml.
Final pH is 5.5. Wash gently in running tap water and counterstain with Mayer's hematoxylin for 8 minutes. Wash, dry, mount in glycerolgelatin and examine. Peroxidase activity is represented by red-brown granular deposits. The distribution of dye is identical to that seen with benzidine stains. This technic is suggested as an alternative to methods employing benzidine and its derivatives. L E O N A R D S. K A P L O W ,
M.D.
Chief, Laboratory Service Veterans Administration Hospital West Haven, Conn. 06516 and Associate Professor of Pathology and Laboratory Medicine Yale University School of Medicine New Haven, Conn. 06520
Method Fix thin blood smears in buffered formalin acetone 5 for 15 seconds at room temperature. Wash gently and incubate slides for 2'/2 minutes at room temperature in a filtered mixture containing: 3-Amino-9-ethylcarbazole* Dimethylsulfoxide
10 mg. 6 ml.
Received September 3, 1974; accepted for publication September 10, 1974. Address reprint requests to Dr. Kaplow. * Aldrich Chemical Co., Inc. Milwaukee, Wis. 53233.
References 1. Federal Register 39, # 2 0 pp 3756-3797 Jan. 29, 1974 2. Graham RC JR, Lundholm U, Karnovsky MJ: Cytochemical demonstration of peroxidase activity with 3-amino-9-ethylcarbazole. J HistochemCytochem 13:150-152, 1965 3. Kaplow LS: Simplified myeloperoxidase stain using benzidine dihydrochloride. Blood 26: 215-219, 1965 4. Schaefer HE, Fischer R: Peroxidase detection in smear preparations and tissue sections after decalcification and paraffin embedding. Klin Wochenschr 46:1228-1230, 1968 5. Yam LT, Li CY, Crosby WH: Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 55:283-290, 1971
NBT Slide Test To the Editor: — I read with interest the article of Okamura and associates (Am J Clin Pathol 62:27-31, 1974) concerning 451
an improved method for the so-called "NBT slide test" in human neutrophils. Among other aims they tried to improve
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 5, 2016
To the Editor: — Federal regulations 1 now prohibit the use of benzidine and its derivatives in concentrations above 0.1% unless rigorously defined safety precautions are taken. These safety requirements are too complex and demanding for clinical laboratories. Indeed, it is likely that manufacturing laboratories will also find it very difficult to comply with these regulations. It is thus more than a likelihood that benzidine and its derivatives will shortly become unavailable. These carcinogenic compounds are used in clinical laboratories primarily in procedures for detecting occult blood and in staining leukocytes for myeloperoxidase activity.3 Our laboratory has been investigating a number of alternative methods for peroxidase stains, using indicator compounds other than benzidine. A method modified from Graham and associates2 and Schaefer and Fischer 4 has yielded most consistent and satisfactory results. It is given below.
