Comparison of formalin, buffered formalin, and Bouin's fixation on the detection of human papillomavirus deoxyribonucleic acid from genital lesions

Detection of nucleic acid sequences homologous to human papillomavirus (HPV) relies primarily on their extraction from unfixed tissue. We detected HPV sequences in DNA extracted from paraffin-embedded tissue fixed in formalin (buffered and unbuffered) and Bouin's solution by dot blot hybridization. A detectable hybridization signal was noted in 32% of these fixed tissues which were chosen from cases where HPV DNA was detected in the unfixed tissue. When using a homologous 32P-labeled probe and a high stringency wash, the hybridization signal was lost if DNA was extracted after Bouin's fixation and diminished after formalin fixation, more so with unbuffered formalin. Similar differences in the hybridization signals among the different fixatives after high stringency wash were noted with in situ hybridization. Southern blot analysis showed that DNA extracted from tissues fixed in Bouin's was degraded and ranged in size from 100 to 500 base pairs as compared with 100 to 900 base pairs for DNA extracted from tissue fixed with unbuffered formalin. In contrast, no degradation was noted after fixation with buffered formalin. These results demonstrate that HPV sequences can be identified in DNA extracted from paraffin-embedded, fixed tissue. However, use of some fixatives may preclude identification of HPV type, by either dot blot or in situ hybridization.     

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.     

The effect of fixation and trypsinization on the immunohistochemical demonstration of intracellular immunoglobulin in paraffin embedded material

Using an indirect labelled immunoperoxidase technique the influence of fixation time on the antigenicity of intracellular immunoglobulin in lymphoid tissue fixed in buffered formalin has been investigated. Within a fixation period of 96 hours a decrease of 15% of stainable immunoglobulin containing cells was found, for every 24 hours the fixation time was prolonged. By comparing sections from tissue fixed in buffered formalin and selected fixatives (Lillie's AAF, Bouin's fluid, Clarke's fluid and 96% ethanol 1% acetic acid (E--A) processed at 4 degrees C and at 25 degrees C) an increased number of stained immunoglobulin containing cells was found in tissue fixed in Lillie's AAF, Bouin's fluid, Clarke's fluid and E--A processed at 4 degrees C. No difference was found between tissue fixed in buffered formalin and E--A processed at 25 degrees C. In addition the effect of pretreatment of the sections with trypsin on the number of stainable immunoglobulin containing cells was investigated. Trypsinization of sections from formalin fixed material increased the number of stainable cells substantially. No essential effect was seen on tissue fixed in Lillie's AAF and Bouin's fluid. In contrast trypsin treatment of sections from tissue fixed in Clarke's fluid and E--A completely destroyed the tissue. No differences were observed between different immunoglobulin classes examined as regards the effect of fixation time, fixatives and trypsinization.     

Effects of several commonly used fixatives on DNA and total nuclear protein analysis by flow cytometry

Five commonly used fixatives (AZF, B-5, Bouin's, formalin, and Zenker's) were evaluated for their effect on the flow cytometric analysis of DNA and total nuclear protein (TNP) in solid tumors. Data were obtained with the use of colonic adenocarcinoma, squamous carcinoma of the lung, mammary adenocarcinoma, and spleen with a plasma cell leukemic infiltrate. The parameters examined were G0-G1 DNA staining intensity, %G0-G1, percent coefficient of variation (%CV), percent debris, and TNP staining intensity. The results showed that variations in the fixation of solid tumor significantly affected flow cytometric-derived parameters. In this study, paraffin-embedded tissue (PET) fixed in 10% (v/v) neutral buffered formalin (NBF) produced the best results, with a %CV below 4.7, whereas fixatives such as Zenker's and B-5 produced poor %CVs (above 6.0) or uninterpretable TNP and light scatter data. These data suggest that a portion of all tissue samples be fixed in NBF to allow for subsequent analysis by fixative-sensitive assays such as DNA in situ hybridization and flow cytometry.     

Deleterious effects of formalin/acetic acid/alcohol (FAA) fixation on the detection of HPV DNA by in situ hybridization and the polymerase chain reaction.

As part of a study of archival cervical cancer specimens (1920s to 1980s) to determine whether changes have occurred in the prevalence of human papillomavirus (HPV) DNAs, investigations were performed on tissues which had been fixed in either 10% buffered formalin (NBF) or formalin-acetic acid-alcohol (FAA). HPV DNA was detected by in situ hybridization (ISH) using HPV 6, 11, 16 and 18 32P-labelled DNA probes under conditions of high stringency; and by the polymerase chain reaction (PCR) using 20-mer oligonucleotide primers to amplify 109 bases of the E6 region of HPV 16. In some instances results obtained from Southern blot hybridizations, which had been carried out on specimens of fresh cancer tissue, were available for comparison. When tissues had been fixed in NBF, HPV DNA sequences were detected in 53% of specimens by ISH and in 72% by PCR. In comparison, the rates of detection of HPV by ISH and PCR in tissues fixed in FAA were 17% and 21% respectively. These results indicate that FAA is clearly inferior to NBF for the preservation of detectable HPV DNA sequences in tissue sections.     

PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time.

The polymerase chain reaction (PCR) DNA amplification method is a powerful new tool for the retrospective analysis of paraffin-embedded tissue (PET). The technique has afforded the sensitive and specific detection of nucleic acid sequences associated with genetic and infectious diseases. However, PET processing conditions vary in their suitability for amplification. The authors have examined the effects of 11 fixatives at three fixation times. The effect of fixation was measured by the ability of the DNA in a treated tissue to serve as a template for the amplification of DNA fragments that ranged from 110 to 1,327 base pairs in length. Specimens fixed in acetone or 10% buffered neutral formalin were found to be best suited for subsequent analysis by PCR. A second group of fixatives, including Zamboni's, Clarke's, paraformaldehyde, formalin-alcohol-acetic acid, and methacarn, compromised amplification efficiency. Tissues treated with Carnoy's, Zenker's, or Bouin's, respectively, were even less desirable for amplification analysis.     

Immersion fixation methods for glycol methacrylate-embedded testes

Routine processing of testicular tissue through 10% neutral buffered formalin (NBF) into paraffin produces severe cellular shrinkage which obscures most of the morphologic detail. The following studies were performed to compare different combinations of immersion fixatives and embedding media for optimal cellular detail in the final histologic sections. We examined sections of testes from rats, mice, and rabbits fixed in either NBF, Bouin's, Zenker's, or Helly's fixatives, and embedded in either standard paraffin or glycol methacrylate. The results were similar in all 3 species. In paraffin, Bouin's or Helly's fixatives produced the fewest artifacts, while in glycol methacrylate, NBF-fixed tissue showed the greatest intracellular detail and preservation. The merits and limitations of each method are discussed for the rat, with exceptions for the other species noted where appropriate. The use of glycol methacrylate as the support medium for sectioning makes high-quality tissue sections available from formalin-fixed testes.     

Variability in beta-globin and HPV DNA amplification by PCR from fixed tissues.

Paraffin-embedded tissues from a variety of sources and treated with different fixatives were tested for beta-globin and HPV amplification by polymerase chain reaction (PCR). In tests of tissues collected in the previous 2 to 3 yr, excellent rates (87% to 93%) for beta-globin amplification were obtained for specimens fixed in buffered formalin, Bouin's, and Hartmann's solutions. In contrast, the rate of beta-globin amplification was low for tissues fixed in Hollande's solution (7%) and in Hartmann's solution with eosin (33%). The results of beta-globin amplification from archival tissues stored for variable time periods showed no decrease in the amplification rate with longer periods of storage. Human papillomaviruses (HPVs) were identified in 17% of globin negative and in 43% of globin positive tissues. HPV-16 amplification was more efficient when the targeted DNA sequence was small. Variability in amplification depends not only on the type of fixative used, but also on other ill-defined factors. Therefore, conditions for optimal amplification should be determined before undertaking studies of archival material.     

The effect of fixation on detection of B-cell clonality by polymerase chain reaction.

It has been suggested that neutral buffered formalin (NBF)-fixed, paraffin-embedded, or fresh specimens might provide satisfactory DNA templates for polymerase chain reaction (PCR) assays used in establishing the clonality and presumptive B-cell lineage of lymphoma. The suitability of other fixatives used by hematopathologists, such as B5, is still undetermined. Thirty cases were identified from the files of the Cleveland Clinic Foundation, Cleveland Ohio, that showed abnormal immunoglobulin heavy chain (IgH) rearrangement by Southern blot analysis (SBA). Corresponding paraffin-embedded tissue samples fixed in NBF (21 cases), B5 (18 cases), Hollande's fixative (17 cases), zinc formalin (ZF) (5 cases), and Bouin's fixative (3 cases) were studied. With use of consensus primers against the framework 3 (FR3) and FR2 regions of the VH gene, paired against JH primer(s), PCR analysis was performed. bcl-2/IgH translocation was also studied. Ten reactive lymphoid samples were used as controls, and 40 cases were evaluated. Successful amplification of a clonal proliferation was manifested as one or two discrete narrow bands in the appropriate size range. The sensitivity of detecting clonality was 95, 94, 67, 80, and 0% for NBF, Hollande's fixative, B5, ZF, and Bouin's fixative, respectively. Although NBF and Hollande's fixative were 100% specific, consistent false-positive results were a major problem with B5-fixed tissue. Paraffin-embedded tissue, fixed in NBF, Hollande's fixative, and ZF solutions, may be used for DNA extraction and PCR assays for establishing B-cell clonality. The precipitating fixative B5 and Bouin's solution should not be used for this purpose until the issue of false-positive results is resolved.     

Successful application of indirect in-situ polymerase chain reaction to tissues fixed in Bouin''s solution

AIMS: To evaluate the value of polymerase chain reaction-in situ hybridization (PCR-ISH) for the detection of human papillomaviruses (HPV) in paraffin sections of cervical biopsies fixed either in 10% formalin or in Bouin's solution. METHODS AND RESULTS: We analysed 40 biopsies from Italian women infected with the human immunodeficiency virus type 1 (HIV 1). In-situ hybridization techniques were performed with commercial biotinylated probes. The PCR-ISH was carried out by the 'hot start modification'. Cervical intraepithelial neoplasia (CIN) was found in 23 of 40 patients (57. 5%); eight cases showed condylomatous features. Human papillomavirus was detected in 42.5% by ISH and in 65% by PCR-ISH. Sixty-nine per cent of positive biopsies contained HPV 16, 18, 31 and 33. HPV 6 and 11 were found only in condylomata acuminata samples. CONCLUSIONS: The results point to a high incidence of HPV infection as well as of CIN in HIV-positive patients. Human papillomavirus type 16 appears to be most frequently associated with CIN. Polymerase chain reaction-ISH is more sensitive than ISH in the detection and typing of HPV DNA both in clinical and in 'latent' infections. The two techniques yielded the same results with either formalin- or Bouin's-fixed material.     

In situ demonstration of tissue proliferative activity using anti-bromo-deoxyuridine monoclonal antibody.

Immunohistochemical staining with anti-bromo-deoxyuridine (BrdU) monoclonal antibody was performed on a variety of human tissues following in vitro incubation with BrdU. The effect of different fixatives and DNA denaturation techniques on the reactivity with anti-BrdU was investigated. Optimal preservation of the antigenicity of BrdU incorporated into the DNA of proliferating cells was seen in tissues fixed in Bouin's fluid, while samples which had been fixed with cross-linking reagents, such as formalin, were usually unreactive. Positivity for BrdU was restored in formalin fixed tissues after digestion with pepsin, but this was usually associated with loss of morphological details. Acid and thermal DNA denaturation techniques gave similar results. It is concluded that Bouin fixation followed by acid or thermal denaturation of DNA is the method of choice for the in situ detection of cells in S-phase using anti-BrdU monoclonal antibody.     

Immunohistochemical assays in prostatic biopsies processed in Bouin's fixative

AIMS: To investigate the problems involved in undertaking immunohistochemistry (IHC) and nuclear morphometry using Bouin's fixed prostate biopsies. METHODS: Archival Bouin's fixed and formalin fixed, paraffin wax embedded prostatic biopsies were immunostained for three nuclear biomarkers (minichromosome maintenance protein 2 (MCM-2), p27, and Ki-67), one membrane localised biomarker (C-erb-B2), CD34, and alpha methylacyl-CoA racemase (AMACR). The quality of IHC staining was compared between tissues prepared separately in both fixatives. Feulgen staining was also performed on Bouin's fixed tissues to check its suitability for nuclear morphometry. RESULTS: MCM-2 staining was completely negative in Bouin's fixed tissues, whereas p27 showed more background and excess cytoplasmic staining in Bouin's fixed versus formalin fixed tissues. C-erb-B2 showed non-specific, strong luminal cell staining in the Bouin's fixed tissue. Feulgen staining was also very weak in Bouin's fixed tissue. However, Ki-67, AMACR, and CD34 worked equally well in Bouin's and formalin fixed tissues. CONCLUSIONS: Bouin's fixed tissues may be unsuitable when subsequent IHC and morphometry are contemplated. An awareness of which antibodies are suitable for use in Bouin's fixed biopsies is essential.     

Efficiency of in situ hybridization as a function of probe size and fixation technique

In an attempt to improve fixation technique for viral RNA detection by in situ hybridization, we have quantitatively compared the hybridization signal obtained when measles virus or visna virus infected cell cultures were fixed with eight different fixatives and hybridized with 35S-labeled virus-complementary DNA probes of several size ranges. Small probes (mean length, 70 bases) gave higher signals than larger probes (mean lengths 140, 350, and 780 bases) with all fixatives. This increase in signal was minimal with acetic ethanol or formalin, but was dramatic with fixatives containing glutaraldehyde; with these fixatives the signals with small probes were 6.5- to 22-fold greater than with large probes. The highest signals were obtained with periodate-lysine-paraformaldehyde-glutaraldehyde (PLPG) fixed cells hybridized with small probes, and were 1.5- to 6.7-fold greater than those obtained with the commonly used fixative acetic ethanol. PLPG and other glutaraldehyde based fixatives also greatly improved the preservation of cellular morphology compared to acetic ethanol.     

Influence of fixation on human papillomavirus DNA detection in frozen and embedded paraffin lesions by in situ hybridization with biotinylated probes.

Series of frozen or paraffin-embedded tissues from various body sites, taken from non-immunosuppressed or immunosuppressed patients with persistent papilloma lesions were examined for the presence of group specific antigen from human papillomavirus (HPV) by indirect immunofluorescence or HPV DNA by in situ hybridization with biotinylated probes. We have shown that it is possible to detect HPV DNA after fixation of tissues in neutral formalin, Bouin's or Baker's solution. However, the sensitivity was reduced as compared to frozen tissues. The HPV DNA was detected in nuclei of heavily infected epithelial cells such as plantar or hand warts or in dispersed cells containing high copy numbers of HPV DNA from lesions such as squamous cell carcinomas or keratoacanthomas. In premalignant or malignant lesions of both immunosuppressed or non-immunosuppressed patients, HPV DNA was rarely detected after fixation. HPV types commonly described for skin and genital samples were identified in non-immunosuppressed patients, whereas in transplant recipients oncogenic HPV type 16 was identified in benign warts as well as in premalignant or malignant lesions. Positive reactions with several HPV types were more frequent in lesions from grafted patients than from the normal population. Virus antigen was detectable more frequently in frozen sections than in fixed tissues. Our findings indicate that in situ hybridization is an appropriate and rapid technique to study the presence of HPV infection. However, numerous controls are needed to avoid misinterpretations.     

Effects of fixation on polymerase chain reaction detection of Mycobacterium leprae.

The effects of standard fixatives (10% neutral buffered formalin, ethanol and mercury based) on the detection of Mycobacterium leprae DNA by the polymerase chain reaction (PCR) were studied. Mercury-based fixatives (Zenker's and Carnoy-Lebrun's fluids) strongly inhibited PCR amplification of M. leprae DNA. Ten percent neutral buffered formalin was inhibitory, but significant inhibition was observed only when fixation times exceeded 24 h. Ethanol-based fixatives provided the best medium for holding specimens for subsequent PCR with both free bacilli and skin biopsy specimens containing M. leprae. The M. leprae-specific, 360-bp region of the 18-kDa protein gene could be amplified from paraffin-embedded sections of formalin-fixed skin biopsy specimens from patients with either multibacillary or paucibacillary infections when proper fixation conditions were used. Results of the study demonstrate that tissues properly fixed with two standard fixatives (10% neutral buffered formalin and 50 or 70% ethanol) can be analyzed by PCR for the presence of M. leprae with no loss in specificity and only minimal diminution in sensitivity compared with the specificities and sensitivities obtained by use of freshly prepared, unfixed specimens.     

Detection of multiple types of human papillomavirus in a giant condyloma from a grafted patient. Analysis by immunohistochemistry, in situ hybridisation, Southern blot and polymerase chain reaction.

Immunosuppressed patients such as transplant recipients are known to develop multiple lesions suggestive of human papillomavirus (HPV) infection. A giant anal condyloma was obtained from a transplant patient; several fragments taken from different areas were examined for the presence of HPV DNA using in situ hybridisation, polymerase chain reaction (PCR) and Southern blot. Typical koilocytes were seen in routinely stained tissue sections, suggesting an HPV infection; furthermore, group specific HPV antigen was detected in one of four frozen fragments. Different results were obtained by in situ hybridisation according to the fragment tested. HPV types 6/11 were detected in each of the five fragments, frozen or fixed in Bouin's or formalin solutions. However, the number of HPV DNA positive cells and the intensity of the reaction greatly varied with the specimen. HPV 16 and 18 probes also reacted positively with the sample fixed in formalin; a stronger signal was observed with HPV 18 in one large focus than with HPV 16. HPV type 5 was detected in a few isolated cells of two frozen fragments. With the Southern blot technique, the profile of an HPV 6/11 was seen only in one of two frozen fragments; in this case, the bands were intense. A slight positive reaction was also obtained in one frozen fragment with HPV 16 probe. Four frozen fragments were analyzed with PCR: HPV 6/11 was detected in each fragment; HPV 18 was detected in the four samples but with different intensities; HPV types 5 and 16 did not show any positive signal. In conclusion, the lesion is an example of infection with several HPV types, demonstrated by three different techniques. This suggests the need for careful dermatological or colposcopic follow-up of transplant recipients, in order to prevent possible malignant transformation of anogenital lesions.     

Detection of HPV DNA in archival specimens of cervical cancer using in situ hybridisation and the polymerase chain reaction.

An archival survey of 98 cervical cancer specimens dating from the 1920s to the 1980s was undertaken to determine whether changes had occurred in the prevalence of human papilloma-virus (HPV) DNA. HPV DNA was detected in paraffin sections of cancers fixed in 10% formalin by in situ hybridisation (ISH) using HPV 6, 11, 16, and 18 32P-labelled DNA probes under conditions of high stringency; and by the polymerase chain reaction (PCR) using 20-mer oligonucleotide primers to amplify 109 bases of the E6 region of HPV 16. In 30 instances results obtained from Southern blot hybridisations which had been carried out on specimens of fresh tissue from the same cancers collected during the 1980s were available for comparison. The rates of HPV DNA detection in cervical cancers ranged from 83% (by Southern or PCR) and 70% (by ISH) on specimens from the 1980s, to 50% and 63% (by ISH and PCR, respectively) on specimens from the 1920s. HPV 16 was by far the most common type, being identified by Southern or ISH in approximately 92% of HPV DNA-positive specimens. No significant change in the prevalence of HPV DNA, or of HPV types, in cervical cancers was found over the 65 year period examined.     

Effects of fixative type and fixation time on the detection of Maedi Visna virus by PCR and immunohistochemistry in paraffin-embedded ovine lung samples

In doubtful cases, the histopathological diagnosis of lesions induced by Maedi Visna virus (MVV), a chronic multisystemic lentiviral disease of sheep, needs to be confirmed by the demonstration of MVV in the tissues. The influence of fixatives and the duration of fixation on the detection of MVV by immunohistochemistry (IHC) and PCR in paraffin-embedded tissues was assessed in lung samples with lesions in different degree, from five sheep serologically positive. Samples were fixed in 10% neutral buffered formalin (NBF), Bouin's solution (BS) and a zinc salts-based fixative (ZSF), for different periods of time between 24 h and 30 days. The three fixatives preserved the morphology of the tissues, although in ZSF-fixed samples an increase in the number of desquamated cells was seen in the alveoli. Tissues showed a similar degree of immunolabelling, irrespective of the duration of fixation using ZSF and NBF fixatives. MVV nucleic acids could be detected in samples fixed up to 14 days in NBF and 30 days in ZSF. However, in BS fixed tissues, immunostaining was weak and non-specific signals were observed after 4 days of fixation. Amplification of proviral DNA could not be obtained by PCR in these samples. IHC detected viral antigens in all sheep whereas one sheep with mild lesions was always negative by PCR.     

Morphology of the fetal rat testis preserved in different fixatives

Histopathological examination of the testes of exposed fetuses and neonates is important in assessing the developmental effects of environmental toxins, including sex hormone modulators. Modified Davidson's fluid (mDF) has been suggested as a superior substitute for Bouin's fluid for fixation of adult animal testes. We compared the morphology of fetal rat testes stained with hematoxylin and eosin (H&E) or immunochemically after fixation in 10% neutral buffered formalin (NBF), Bouin's fluid, or mDF. Fixation in mDF resulted in more sharply defined nuclear detail and better preservation of cellular cytoplasm on H&E-stained sections of rat testes on gestation day 19. Use of Bouin's fluid did not allow satisfactory detection of apoptotic cells by fluorescent terminal deoxynucleotide transferase-mediated deoxy-UTP nick labeling. Staining with the immunoperoxidase system and the conventional chromogen diaminobenzidine tetrahydrochloride to visualize 5-bromo-2-deoxyuridine-positive cells demonstrated that the number of positive nuclei and intensity of staining were similar with all 3 fixatives. Immunostaining for cytoskeletal protein vimentin was more intense and provided better details of the Sertoli cell cytoplasm with formalin fixation than with mDF. Our study demonstrates that fixation in mDF provided better morphologic detail in the fetal rat testis compared with 10% NBF and Bouin's fluid and illustrates the importance of establishing the correct fixation conditions for each immunostaining protocol.     

Human intestinal mucosal mast cells: evaluation of fixation and staining techniques

The staining properties of tissue mast cells are influenced by the method of fixation. Differences in fixation and staining techniques may explain the contradictory results in the published reports on the number of human mucosal mast cells (MMC) in the gastrointestinal mucosa in health and disease. We have examined the influence of fixatives on the staining properties of human MMC in operative biopsy specimens of human jejunum. Specimens were divided into pieces, each of which was fixed in one of the following fixatives: Carnoy's, basic lead acetate (BLA), Baker's, Bouin's, isotonic formol-acetic-acid (IFAA), 10% neutral buffered formalin, formol sublimate, and formol saline. Thereafter, tissues were paraffin-embedded and 5 micron sections were cut and stained with either astra-blue/safranin pH 0.3, or toluidine blue pH 0.5. Counts of the number of MMC/mm2 were obtained for each fixation method. The results show a critical influence of the fixative on the number of mast cells identified after staining. For example with astra-blue/safranin the mean MMC/mm2 count was 40 in formol-saline-fixed specimens, and 268 in Carnoy's-fixed specimens. In biopsies fixed with formalin-based fixatives, mast cells were more readily stained with toluidine blue. It is recommended that Carnoy's or BLA be used as the fixative for any light microscopic study of human MMC.