INFECTIOUS DISEASES Original Article
Monoclonal Antibody to Pneumocystis carinii Comparison with Silver Stain in Bronchial Lavage Specimens KEVIN S. HOMER, M.D.,1 ELIZABETH L. WILEY, M.D.,1 ALICE L. SMITH, M.D.,1 LOLA McCOLLOUGH, M.T.(ASCP),1 DOROTHY CLARK, M.T.(ASCP),2 STEPHEN D. NIGHTINGALE, M.D.,1 AND FRANK VUITCH, M.D.1
39 had organisms detected using Grocott’s modified silver stain. Of 37 patients without clinically apparent Pneumocystis pneu› monia any time in their course, 4 had abundant organisms and 33 had negative stains with MAB-3F6. MAB-3F6 detected Pneumocystis organisms in 22 of 31 cases of Pneumocystis pneumonia that had no organisms identified using Grocott’s silver stain (X2 = 5.76, P = 0.016). MAB-3F6 immunochemical stain› ing is a more sensitive method than Grocott’s modified silver stain to detect Pneumocystis organisms. (Key words: Pneumo› cystis pneumonia; Grocott silver stain; Human immunodeficiency virus; Immunohistochemistry monoclonal antibody; Bronchial lavage) Am J Clin Pathol 1992; 97:619-624
Pneumocystis pneumonia is a common and frequently lethal infection in patients with acquired immune defi› ciency syndrome (AIDS). Because no effective means of culture exists, definitive diagnosis of Pneumocystis pneu› monia is made by recognizing organisms morphologically in cytologic or histologic preparations. A group of studies determined that the combination of Grocott’s modified Gomori methenamine silver stain1 and Papanicolaou stains on bronchoalveolar lavage specimens is the most sensitive, least invasive method to identify organisms.2"8 A number of studies using indirect fluorescent tagged monoclonal antibody, clone 2G2 (MAB-2G2), have shown a greater sensitivity than Grocott stains using bronchial washings and sputum samples. 69 "" However,
Braughman and associates9 found no difference between the detection rates of MAB-2G2 and Grocott on bronchial lavage specimens. A new monoclonal antibody, clone 3F6 (MAB-3F6) (DAKO, Carpinteria, CA), against Pneumo› cystis carinii antigen 82-kD, was shown to detect Pneu› mocystis organisms in formalin-fixed paraffin-embedded tissue.12 We began this study to determine whether this antibody can detect Pneumocystis organisms in bronchial lavage specimens and whether it is as specific and sensitive as Grocott’s silver stain in their identification. MATERIALS AND METHODS
The cytopathology laboratory at Parkland Memorial Hospital received 310 bronchial lavage specimens from patients infected with the human immunodeficiency virus between July 1, 1985 and July 1, 1989. According to a From the ’Department of Pathology and the Department of Internal standard protocol of the cytopathology service, the spec› Medicine, Parkland Memorial Hospital,2 University of Texas Southwestern Medical Center, Dallas, Texas; and the DAKO Corporation, Carpinteria, imen material was centrifuged, the supernatant removed, California. and the button resuspended in buffer (volume four times the button volume). Six to eight cytospin slides were pre› Received June 18, 1991; received revised manuscript and accepted for publication September 18, 1991. pared from each specimen. Four to six were stained with This study was funded by the DAK.O Corporation. Papanicolaou stain, one was stained with Grocott’s mod› Address reprint requests to Dr. Wiley: Division of Surgical Pathology, ification of the Gomori methenamine silver stain, and Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9072. one was stained for acid fast bacilli. The lavage material 619
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Monoclonal 3F6 anti-Pneumocystis carinii antibody (MAB-3F6) was used to stain cell blocks from 164 bronchial lavage specimens from patients with the acquired immune deficiency syndrome (AIDS) and AIDS-related complex and compared with slides stained with Grocott’s modification of the Gomori methenamine silver stain. Pneumocystis organisms were present in 83 of 164 cases using MAB-3F6 stain, whereas Grocott’s modified silver stain demonstrated Pneumocystis organisms in 48. MAB-3F6 demonstrated Pneumocystis organisms in 38 cases with negative silver stains, whereas silver stain identified Pneumocystis or› ganisms in only three MAB-3F6-negative cases. Of 70 patients with clinical Pneumocystis pneumonia at the time the specimen was obtained, 59 had MAB-3F6-positive specimens, whereas
620
INFECTIOUS DISEASES f Article
To preserve diagnostic bronchial lavage material on file, this study restricted itself to the use of archived cell block material for staining with the antibody MAB-3F6. Of the original 310 cases, 164 cell block specimens from 156 patients had enough tissue remaining in the archived material for further study. Additional 5-jum-thick sections were cut from the 164 specimens selected for study and were mounted on polylysine-coated slides, dried overnight at 37 C, deparaffinized, and hydrated. The sections were digested with 0.1% protease Type XXIV for 15 minutes and incubated with prediluted mouse monoclonal 3F6 anti-Pneumocystis antibody in a 25% solution of non› immune rabbit serum (DAKO). Sections were washed briefly in TRIS-buffered saline and incubated with pre› diluted biotinylated rabbit anti-mouse immunoglobulins (link antibody) followed by alkaline phosphatase-labeled streptavidin. The color was developed using a naphtol phosphate substrate and 0.5% solution of new fuchsin. The slides were washed, counter-stained with Mayer’s he› matoxylin, and mounted in glycerol. Slides stained using MAB-3F6 were reviewed blindly and independently by two investigators (KH, EW) and grouped according to the presence or absence of Pneumocystis organisms. Cases were considered positive if more than two small groups of cysts were present or if a single large cluster of organisms was present. Cases without positively stained organisms were considered negative. Discrepant cases were reviewed collectively to reach a consensus. The Parkland Memorial Hospital AIDS Clinic registry supplied clinical information on all patients with Pneu› mocystis pneumonia treated in the AIDS clinic. Clinical information from the registry and from the patients’ medical charts was correlated with dates of bronchoscopic specimens. The diagnosis of Pneumocystis pneumonia was
established independently by the Parkland Memorial Hospital AIDS service and preceded the date of this study by 1 to 3 years. The diagnosis was established by (1) the presence of a characteristic infiltrate on chest x-ray, low arterial percentage oxygen saturation, and response to anti-Pneumocystis treatment or respiratory death, (2) or› ganisms demonstrated on a bronchoscopy specimen (us› ing Gomori methenamine silver stain), or (3) in the case of three patients, identification of Pneumocystis organisms in autopsy material. Clinical information and slide data for each patient was recorded on a personal computer using PC-File Vfi, a dBase-compatible relational data base program (Button Ware Software Inc., Bellevue, WA). Statistical analyses of the data were performed using 2 X 2 matrices and calculating the Chi-square values. RESULTS According to the records available from the Parkland Memorial Hospital AIDS clinic registry for the 145 pa› tients studied with MAB-3F6, a diagnosis of Pneumocystis pneumonia made by the AIDS service preceded bron› choscopy by 1 to 7 days for 24 patients, by 8 to 14 days for 8 patients, and by 15 to 31 days for 12 patients. Bron› choscopy was performed on the date of diagnosis for 13 patients. Bronchoscopy preceded establishment of diag› nosis of Pneumocystis pneumonia by 1 to 11 days for 12 patients and by 21 days for 1 patient. If a clinical diagnosis of Pneumocystis pneumonia had been made within 31 days before or after bronchoscopy, a patient was consid› ered to have Pneumocystis pneumonia at the time of bronchoscopy. There were 38 patients who had a diagnosis of Pneumocystis pneumonia made more than 31 days before or 31 days after the studied bronchoscopic speci› men was obtained and are designated as patients with history/future of Pneumocystis pneumonia. Thirty-seven patients did not have a diagnosis of Pneumocystis pneu› monia at anytime during their clinical course and are considered to be clinically negative. The MAB-3F6 technique heavily outlined individual Pneumocystis organisms with the fuschia-colored dye product as irregular spheres with accentuation of the cyst outlines. The exudate associated with clumps of organisms stained bright pink (Fig. 1). Clumps of organisms present were associated with pulmonary material, but organisms also were present within clotted blood in specimens that contained scant pulmonary material (Fig. 2). In most cases scattered single organisms and granular debris also were present, usually near the larger clumps of organisms. Of the 310 bronchial lavage specimens, Pneumocystis organisms were originally identified in 114 and review identified 3 additional cases. Of these, 94 were from 139 specimens of patients with Pneumocystis pneumonia, two
A.J.C.P. May 1992
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
remaining after preparation of cytospin slides was centrifuged into a tight button, placed in one or two tissue bags, and fixed in 10% buffered formalin. The cell block tissue was processed on a Shandon Hypercenter 2 auto› matic tissue processor (Shandon Scientific Ltd., Cheshire, UK), embedded in paraffin, and sectioned at 5 nm. One slide with two levels from each block was stained with hematoxylin and eosin and one slide with two levels was stained using Grocott’s modified Gomori methenamine silver stain. Ail silver-stained slides, both cytospin and cell block preparations, of all 310 bronchial lavage spec› imens were reviewed blindly and independently by three investigators (KH, EW, AS) and assessed for the presence or absence of Pneumocystis organisms. Two to four slides stained with silver stain were examined in each case, at least one slide each of a cytospin and a cell block section. Cases in which organisms were detected on either silverstained cytospin slide or cell block section were considered positive for Pneumocystis. Discrepant cases were reviewed collectively to reach a consensus.
7 .^TT
*£fc**W. ’Iff H£- ^ t e t t e r 1 * -
Monoclonal Antibody to Pneumocystis carinii Comparison with Silver Stain in Bronchial Lavage Specimens KEVIN S. HOMER, M.D.,1 ELIZABETH L. WILEY, M.D.,1 ALICE L. SMITH, M.D.,1 LOLA McCOLLOUGH, M.T.(ASCP),1 DOROTHY CLARK, M.T.(ASCP),2 STEPHEN D. NIGHTINGALE, M.D.,1 AND FRANK VUITCH, M.D.1
39 had organisms detected using Grocott’s modified silver stain. Of 37 patients without clinically apparent Pneumocystis pneu› monia any time in their course, 4 had abundant organisms and 33 had negative stains with MAB-3F6. MAB-3F6 detected Pneumocystis organisms in 22 of 31 cases of Pneumocystis pneumonia that had no organisms identified using Grocott’s silver stain (X2 = 5.76, P = 0.016). MAB-3F6 immunochemical stain› ing is a more sensitive method than Grocott’s modified silver stain to detect Pneumocystis organisms. (Key words: Pneumo› cystis pneumonia; Grocott silver stain; Human immunodeficiency virus; Immunohistochemistry monoclonal antibody; Bronchial lavage) Am J Clin Pathol 1992; 97:619-624
Pneumocystis pneumonia is a common and frequently lethal infection in patients with acquired immune defi› ciency syndrome (AIDS). Because no effective means of culture exists, definitive diagnosis of Pneumocystis pneu› monia is made by recognizing organisms morphologically in cytologic or histologic preparations. A group of studies determined that the combination of Grocott’s modified Gomori methenamine silver stain1 and Papanicolaou stains on bronchoalveolar lavage specimens is the most sensitive, least invasive method to identify organisms.2"8 A number of studies using indirect fluorescent tagged monoclonal antibody, clone 2G2 (MAB-2G2), have shown a greater sensitivity than Grocott stains using bronchial washings and sputum samples. 69 "" However,
Braughman and associates9 found no difference between the detection rates of MAB-2G2 and Grocott on bronchial lavage specimens. A new monoclonal antibody, clone 3F6 (MAB-3F6) (DAKO, Carpinteria, CA), against Pneumo› cystis carinii antigen 82-kD, was shown to detect Pneu› mocystis organisms in formalin-fixed paraffin-embedded tissue.12 We began this study to determine whether this antibody can detect Pneumocystis organisms in bronchial lavage specimens and whether it is as specific and sensitive as Grocott’s silver stain in their identification. MATERIALS AND METHODS
The cytopathology laboratory at Parkland Memorial Hospital received 310 bronchial lavage specimens from patients infected with the human immunodeficiency virus between July 1, 1985 and July 1, 1989. According to a From the ’Department of Pathology and the Department of Internal standard protocol of the cytopathology service, the spec› Medicine, Parkland Memorial Hospital,2 University of Texas Southwestern Medical Center, Dallas, Texas; and the DAKO Corporation, Carpinteria, imen material was centrifuged, the supernatant removed, California. and the button resuspended in buffer (volume four times the button volume). Six to eight cytospin slides were pre› Received June 18, 1991; received revised manuscript and accepted for publication September 18, 1991. pared from each specimen. Four to six were stained with This study was funded by the DAK.O Corporation. Papanicolaou stain, one was stained with Grocott’s mod› Address reprint requests to Dr. Wiley: Division of Surgical Pathology, ification of the Gomori methenamine silver stain, and Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9072. one was stained for acid fast bacilli. The lavage material 619
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Monoclonal 3F6 anti-Pneumocystis carinii antibody (MAB-3F6) was used to stain cell blocks from 164 bronchial lavage specimens from patients with the acquired immune deficiency syndrome (AIDS) and AIDS-related complex and compared with slides stained with Grocott’s modification of the Gomori methenamine silver stain. Pneumocystis organisms were present in 83 of 164 cases using MAB-3F6 stain, whereas Grocott’s modified silver stain demonstrated Pneumocystis organisms in 48. MAB-3F6 demonstrated Pneumocystis organisms in 38 cases with negative silver stains, whereas silver stain identified Pneumocystis or› ganisms in only three MAB-3F6-negative cases. Of 70 patients with clinical Pneumocystis pneumonia at the time the specimen was obtained, 59 had MAB-3F6-positive specimens, whereas
620
INFECTIOUS DISEASES f Article
To preserve diagnostic bronchial lavage material on file, this study restricted itself to the use of archived cell block material for staining with the antibody MAB-3F6. Of the original 310 cases, 164 cell block specimens from 156 patients had enough tissue remaining in the archived material for further study. Additional 5-jum-thick sections were cut from the 164 specimens selected for study and were mounted on polylysine-coated slides, dried overnight at 37 C, deparaffinized, and hydrated. The sections were digested with 0.1% protease Type XXIV for 15 minutes and incubated with prediluted mouse monoclonal 3F6 anti-Pneumocystis antibody in a 25% solution of non› immune rabbit serum (DAKO). Sections were washed briefly in TRIS-buffered saline and incubated with pre› diluted biotinylated rabbit anti-mouse immunoglobulins (link antibody) followed by alkaline phosphatase-labeled streptavidin. The color was developed using a naphtol phosphate substrate and 0.5% solution of new fuchsin. The slides were washed, counter-stained with Mayer’s he› matoxylin, and mounted in glycerol. Slides stained using MAB-3F6 were reviewed blindly and independently by two investigators (KH, EW) and grouped according to the presence or absence of Pneumocystis organisms. Cases were considered positive if more than two small groups of cysts were present or if a single large cluster of organisms was present. Cases without positively stained organisms were considered negative. Discrepant cases were reviewed collectively to reach a consensus. The Parkland Memorial Hospital AIDS Clinic registry supplied clinical information on all patients with Pneu› mocystis pneumonia treated in the AIDS clinic. Clinical information from the registry and from the patients’ medical charts was correlated with dates of bronchoscopic specimens. The diagnosis of Pneumocystis pneumonia was
established independently by the Parkland Memorial Hospital AIDS service and preceded the date of this study by 1 to 3 years. The diagnosis was established by (1) the presence of a characteristic infiltrate on chest x-ray, low arterial percentage oxygen saturation, and response to anti-Pneumocystis treatment or respiratory death, (2) or› ganisms demonstrated on a bronchoscopy specimen (us› ing Gomori methenamine silver stain), or (3) in the case of three patients, identification of Pneumocystis organisms in autopsy material. Clinical information and slide data for each patient was recorded on a personal computer using PC-File Vfi, a dBase-compatible relational data base program (Button Ware Software Inc., Bellevue, WA). Statistical analyses of the data were performed using 2 X 2 matrices and calculating the Chi-square values. RESULTS According to the records available from the Parkland Memorial Hospital AIDS clinic registry for the 145 pa› tients studied with MAB-3F6, a diagnosis of Pneumocystis pneumonia made by the AIDS service preceded bron› choscopy by 1 to 7 days for 24 patients, by 8 to 14 days for 8 patients, and by 15 to 31 days for 12 patients. Bron› choscopy was performed on the date of diagnosis for 13 patients. Bronchoscopy preceded establishment of diag› nosis of Pneumocystis pneumonia by 1 to 11 days for 12 patients and by 21 days for 1 patient. If a clinical diagnosis of Pneumocystis pneumonia had been made within 31 days before or after bronchoscopy, a patient was consid› ered to have Pneumocystis pneumonia at the time of bronchoscopy. There were 38 patients who had a diagnosis of Pneumocystis pneumonia made more than 31 days before or 31 days after the studied bronchoscopic speci› men was obtained and are designated as patients with history/future of Pneumocystis pneumonia. Thirty-seven patients did not have a diagnosis of Pneumocystis pneu› monia at anytime during their clinical course and are considered to be clinically negative. The MAB-3F6 technique heavily outlined individual Pneumocystis organisms with the fuschia-colored dye product as irregular spheres with accentuation of the cyst outlines. The exudate associated with clumps of organisms stained bright pink (Fig. 1). Clumps of organisms present were associated with pulmonary material, but organisms also were present within clotted blood in specimens that contained scant pulmonary material (Fig. 2). In most cases scattered single organisms and granular debris also were present, usually near the larger clumps of organisms. Of the 310 bronchial lavage specimens, Pneumocystis organisms were originally identified in 114 and review identified 3 additional cases. Of these, 94 were from 139 specimens of patients with Pneumocystis pneumonia, two
A.J.C.P. May 1992
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
remaining after preparation of cytospin slides was centrifuged into a tight button, placed in one or two tissue bags, and fixed in 10% buffered formalin. The cell block tissue was processed on a Shandon Hypercenter 2 auto› matic tissue processor (Shandon Scientific Ltd., Cheshire, UK), embedded in paraffin, and sectioned at 5 nm. One slide with two levels from each block was stained with hematoxylin and eosin and one slide with two levels was stained using Grocott’s modified Gomori methenamine silver stain. Ail silver-stained slides, both cytospin and cell block preparations, of all 310 bronchial lavage spec› imens were reviewed blindly and independently by three investigators (KH, EW, AS) and assessed for the presence or absence of Pneumocystis organisms. Two to four slides stained with silver stain were examined in each case, at least one slide each of a cytospin and a cell block section. Cases in which organisms were detected on either silverstained cytospin slide or cell block section were considered positive for Pneumocystis. Discrepant cases were reviewed collectively to reach a consensus.
7 .^TT
*£fc**W. ’Iff H£- ^ t e t t e r 1 * -
