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PubMed1989

Differential staining of ocular goblet cells.

Millipore filters were used to obtain sheets of cells from the ocular surface. Using Periodic Acid Schiff-haematoxylin the intracellular neutral mucus of the goblet cells stains a brilliant, bright pink and the cell nuclei dark blue making it possible to observe the epithelial cells and the goblet cell population. In certain ocular surface diseases the size of the PAS-haematoxylin staining goblet cell population is reduced, returning towards normal as ocular health improves. These observations had previously been interpreted as showing degeneration followed by regeneration of the goblet cells. However, Alcian blue stains an acidic mucus, which does not stain with PAS-haematoxylin. Acidic mucus containing goblet cells have been revealed using Alcian blue staining when added to the PAS stained cells in conditions that have previously been shown to have reduced goblet cell population when assessed by PAS-haematoxylin staining. This suggests that the staining properties of some of the goblet cells have changed. The extra goblet cells revealed by Alcian Blue staining makes the total of goblet cells present close to normal in numbers. Improved clearing methods and cytological preservation has revealed that there is also a population of goblet cells that do not stain with either technique. These observations must cast doubts on previous claims of reduced goblet cell population associated with some extraocular diseases.

Conjunctiva↗

PubMed1999

Changes in NADPH-d staining in the paraventricular and supraoptic nuclei during pregnancy and lactation in rats: role of ovarian steroids and oxytocin.

Staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a histochemical marker for nitric oxide synthase (NOS), is increased in the supraoptic (SON) and paraventricular (PVN) nuclei in late pregnant rats. To determine whether increases in staining were evident at other times during pregnancy and lactation the number of cells that stained for NADPH-d in the SON and PVN in rats on days 4, 12, 16, and 22 of pregnancy and on days 4, 12, and 20 of lactation was compared to that in virgin females. In a second experiment the influence of ovarian hormones on NADPH-d staining was assessed by comparing staining in the SON and PVN among ovariectomized animals exposed to either a steroid hormone replacement schedule that mimics late pregnancy (oestrogen and progesterone with progesterone removal), oestrogen alone, oestrogen and progesterone, or cholesterol alone. In the last experiment of this series staining was compared among ovariectomized animals given either oestrogen or cholesterol priming accompanied by oxytocin (OT) or vehicle infusion into the third ventricle for 7 days. The number of cells showing dense staining for NADPH-d in both the SON and PVN increased on days 12 and 22 of pregnancy and 4 and 12 of lactation compared to that observed in virgins. NADPH-d staining in these areas was also increased by both the steroid treatment that mimicked late pregnancy and chronic central OT infusion in oestrogen-primed animals. These data suggest that NADPH-d staining in the SON and PVN is increased at times when oxytocinergic cells are known to be active and that the hormonal state associated with late pregnancy is sufficient to increase NADPH-d staining.

Animals↗

PubMed2001

Methylene blue staining of dysplastic and nondysplastic Barrett's esophagus: an in vivo and ex vivo study.

BACKGROUND AND STUDY AIMS: Methylene blue selectively stains specialized columnar epithelium in Barrett's esophagus with high accuracy. We prospectively evaluated the methylene blue staining properties of dysplastic and nondysplastic Barrett's esophagus and the association of these properties with the risk for dysplasia and cancer. PATIENTS AND METHODS: In a ex vivo study, we mapped, photographed, and sampled esophagectomy specimens with high grade dysplasia and/or early adenocarcinoma before and after methylene blue staining. In a concurrent in vivo study, we performed methylene blue staining and characterized methylene blue stain characteristics. Pathologists estimated the proportion of specialized columnar epithelium in each specimen and graded dysplasia. RESULTS: We examined 551 biopsies from 47 patients with biopsy-proven Barrett's esophagus and 48 sections from five surgical specimens with Barrett's esophagus and dysplasia and early adenocarcinoma. The accuracy of ex vivo and in vivo methylene blue staining for specialized columnar epithelium was 87% and 90%, respectively. It was influenced by the length of Barrett's esophagus, biopsy location, and the presence of esophagitis and/or dysplasia. Light to absent staining (p = 0.01) and moderate to marked heterogeneity (p = 0.01) were significantly associated with high grade dysplasia or cancer in the univariate analysis and in a multivariate model that adjusted for the length of Barrett's esophagus and the presence of a lesion. These staining characteristics were present in all patients with severe dysplasia and/or adenocarcinoma. CONCLUSIONS: Highly dysplastic or malignant Barrett's esophagus stains differently with methylene blue. Increased heterogeneity and decreased methylene blue stain intensity are significant independent predictors of high grade dysplasia and/or cancer. These features may help to direct biopsies in patients without a lesion.

Adenocarcinoma↗

PubMed1982

Automated staining of bone marrow and peripheral blood by a modified Wright's technic.

A Wright's stain provided for the automatic staining of routine peripheral blood smears is recommended for use in the Hematrak instrument. This stain at a pH range of 6.5-7.15 shows a dull and bluish coloration of erythrocytes, a finding not readily acceptable for visual microscopy of cell morphology or for photomicroscopy. In order to achieve a desirable erythrocyte color, but still retain the staining intensity of leukocyte chromatin, a two-stage staining method was devised. This involves the use of a mixture of Eosin y and a buffered Wright's solution to secondarily stain Wright-stained smears. This modification of Wright's technic offers a stain of high quality for both bone marrow and peripheral blood films with the advantage of little or no day-to-day variability. In comparison to the frequently advocated dual staining methods, it is cheap and easy to automate. The total staining time for peripheral blood films is about 12 minutes and about 20 minutes for bone marrow smears.

Automation↗

PubMed1992

Detection of Pneumocystis carinii. Comparative study of monoclonal antibody and silver staining.

The sensitivity of Pneumocystis carinii detection using silver stain (Grocott method) was compared to that using the avidin-biotin-complex immunoperoxidase (IP) staining method with anti-P. carinii monoclonal antibody. Silver stain detected only cyst wall, whereas IP stained both cyst wall and trophozoites. Serial sections of formalin-fixed, paraffin-embedded autopsy lung specimens from 41 acquired immune deficiency syndrome patients in three disease categories were stained: I--premortem or autopsy diagnosis of P. carinii pneumonia (13 cases); II--history of treated P. carinii pneumonia but no P. carinii detected in autopsy tissue specimens (15 cases); and III--no clinical or autopsy evidence of P. carinii pneumonia (13 cases). Smears from 7 bronchoalveolar lavages (3 positive) and 11 induced sputa (1 positive) also were stained. All cases of P. carinii in category I were detected with equal sensitivity. P. carinii undetected by silver stain in category II and III cases and in bronchoalveolar lavages and induced sputa were not revealed by IP. Detection of trophozoites by IP did not improve sensitivity because the staining pattern was amorphous or focally granular, and thus easily confused with nonspecific staining of mucin or intracellular or free particulate material. Reliable identification of trophozoites could be made only with coexisting cyst structures. Silver staining was more advantageous because it also identified fungal infections and was faster and more cost effective than IP.

Antibodies, Monoclonal↗

PubMed2001

Decreased cochlear DNA receptor staining in MRL.MpJ-Fas(lpr) autoimmune mice with hearing loss.

OBJECTIVES: Previous studies of decreased cochlear DNA binding in autoimmune mice suggested that antibodies against a cochlear cell surface DNA receptor cause autoimmune hearing loss. However, the presence of a cochlear DNA receptor has not been determined. Therefore, immunohistochemistry with an anti-DNA receptor antibody was performed on MRL.MpJ-Fas(lpr) (MRL/lpr) autoimmune mice to determine 1) which inner ear structures contain DNA receptors and 2) whether the receptor staining pattern changes as autoimmune disease progresses and hearing thresholds increase. STUDY DESIGN: A prospective study of the progression of hearing loss in autoimmune mice and correlated alterations in immunostaining for the inner ear DNA receptor. METHODS: One group of MRL/lpr mice (n = 10) was allowed to develop autoimmune disease, and auditory brainstem response (ABR) audiometry was performed at 4, 6, and 9 months of age to measure the progression of hearing loss. A second group (n = 5) was tested for ABR thresholds at 2 months of age and immediately killed to assess receptor staining before the onset of autoimmune disease and hearing loss. The inner ears from all mice were immunohistochemically stained with an anti-DNA receptor antibody, and a qualitative analysis of the staining of cochlear structures was performed. RESULTS: Auditory brainstem response audiometry revealed a significant 20- to 30-dB elevation of thresholds as systemic disease progressed. Anti-DNA receptor staining was heaviest in the spiral ligament and less intense in the spiral ganglion and cochlear nerve. Both groups showed a similar pattern of staining in these structures. The stria vascularis and hair cells also stained in both groups. However, the stria cells of normal-hearing mice showed diffuse intracellular immunoreactivity, whereas older mice displayed less staining that was confined to the cell membranes. CONCLUSIONS: The inner ears of MRL/lpr mice contain DNA receptors. Autoimmune hearing loss was correlated with weaker overall intracellular staining in the stria vascularis and hair cells but increased staining of the cell membranes. This suggested DNA receptors have impaired endocytosis and more receptors remain on the cell membrane, possibly as a result of binding by circulating autoantibodies.

Age Factors↗

PubMed2000

An update on "special stain" histochemistry with emphasis on automation.

For nearly 100 years, pathologists have utilized "special histochemical stains" to assist in tissue-based diagnosis. As illustrated in Figures 1 and 2, histochemical stains have been used to identify infectious microorganisms (e.g., Mycobacterium tuberculosis with acid-fast bacillus (AFB) stain), to detail inflammatory stromal or structural alterations (e.g., fibrosis in liver cirrhosis with Masson trichrome), to identify microanatomic sites of disease (e.g., basement membrane in glomerulonephritis with Jones methenamine silver), to identify abnormal chemical deposits (e.g., iron in hemochromatosis with Prussian blue stain), or abnormal immune deposits (e.g., amyloid via Congo red stain). The current surgical pathology laboratory may employ a repertoire of 20 to 25 "special stains" to ensure the full diagnostic complement. While the diagnostic repertoire and the biochemical recipes for the stains are now a well-established, codified part of surgical pathology, there is an ever-moving, leading edge of new developments including new reagents, applications, and methods. This review seeks to update the reader on some of the new applications including both new reagents and methods. Particular emphasis will be placed on the recent technologic advance of automating special stains in kinetic-mode (1-4). The authors consider in turn: 1. In brief, the "news" (recent literature review) of new staining applications; 2. In greater detail, two new applications for detection of Microsporidia and Helicobacer pylori; 3. The new technologic advancement of kinetic mode automation of special stains.

Automation↗

PubMed1975

An electron microscopic study of the location of teichoic acid and its contribution to staining reactions in walls of Streptococcus faecalis 8191.

The location of the glucosylated teichoic acid in whole cells and isolated walls of Streptococcus faecalis 8191 has been investigated using ruthenium red, gold-labelled concanavalin A and concanavalin A-peroxidase-diaminobenzidine. Dense laminae were revealed in sections of osmium-fixed walls stained with ruthenium red which corresponded to similar regions stained by uranyl and lead. Such regions were not seen after teichoic acid had been extracted, suggesting that the uptake of stain was by teichoic acid. However, these regions were not labelled on exposure to gold concanavalin A or concanavalin A-peroxidase-diaminobenzidine; these stains indicated that teichoic acid was situated between the dense laminae, although the distribution of stain could have been due to the inability of the concanavalin A stains to penetrate deeply. Chemical binding studies showed that the teichoic acid was the major uranyl binding component in isolated walls, from which it might be inferred that teichoic acid was located in the densely staining regions. However, since osmification significantly increased the binding of uranyl (and lead stains) to non-teichoic acid material, such an inference was not necessarily valid. It is concluded that the presence of teichoic acid can be demonstrated in certain regions of the wall by concanavalin A, but its presence in densely staining regions has not been established. These experiments therefore suggest that teichoic acid may not be intimately associated with the mechanisms that generate contrast patterns in stained sections of cell walls of Streptococcus faecalis.

3,3'-Diaminobenzidine↗

PubMed2006

Routine elastic staining assists detection of vascular invasion in colorectal cancer.

AIM: To determine the value of routine staining of colorectal cancer (CRC) specimens with elastic stains. METHODS AND RESULTS: Four hundred and ninety-eight cases of CRC were included. In 208 cases, vascular invasion (VI) was assessed using elastic stains [Elastic van Gieson or Elastin haematoxylin and eosin (H&E)] that were introduced as routine staining in CRC in our hospital. As a control, 290 cases in which VI was assessed solely on H&E staining were included. The site, stage and presence of vascular invasion were determined in both groups. The cost, time and workload resulting from the addition of these stains were also taken into account. The sensitivity of detection of VI in CRC was significantly improved after the introduction of these elastic stains in our routine practice (46.2% compared with 35.5% in the control group; P = 0.014). Particular improvement was noted in Dukes' stage A and B. We also noted that the net benefits gained from the introduction of these special stains outweighed the small extra cost and effort and, in addition, saved time for the reporting pathologists. CONCLUSION: Routine elastic stains are very useful and practical in evaluation of VI status in CRC and we recommend implementing these stains in routine pathological practice to improve patient care.

Adenocarcinoma↗

PubMed1995

Acid mucin and high molecular weight cytokeratin in prostatic lesions: evaluation of a combined histochemical and immunohistochemical stain.

OBJECTIVE: To evaluate the presence of acid mucins and high molecular weight cytokeratin (KER) in prostatic lesions using a combined histochemical and immunohistochemical stain consisting of Alcian blue at pH 2.5(AB) with a strept-avidin-biotin complex (SAB) staining for KER (SAB-KER). MATERIALS AND METHODS: Sections were obtained from archival paraffin blocks which included 20 cases of prostatic carcinoma, 30 cases of benign hyperplasia, and five cases of basal cell hyperplasia. Sections were stained for mucosubstances using the AB stain, for KER using SAB-KER and by both AB and SAB-KER, the combined stain (CS). RESULTS: With the CS stain KER, which is present in the prostatic basal cells, was not detected in malignant glands and in 60% of these cases intraluminal blue-stained acidic mucin was seen. On the other hand, all benign hyperplastic prostatic glands were devoid of intraluminal acidic mucin and showed staining for KER of their basal cells. Areas of basal cell hyperplasia were strongly positive for KER and intraluminal acidic mucin was seen in one case. Each of the stains separately gave similar results to the CS method but the contrast between the areas of carcinoma and hyperplasia was accentuated by the CS, and small foci of carcinoma were easily detected. CONCLUSION: The combined AB+SAB-KER stain is quicker to perform and allows the simultaneous appraisal of acid mucin and KER.

Avidin↗

PubMed1980

Electron dense staining affinities of mouse oxytalan and elastic fibers.

The staining affinity of electron dense stains to mouse palatal elastic fibers and periodontal oxytalan fibers was assessed and compared. Tissues were stained routinely with uranyl acetate and lead citrate or with elastic tissue stains including: (1) silver tetraphenylporphine sulfonate, (2) tannic acid technique, and (3) a high iron diamine sequence. Staining results were assessed with an electron microscope. Palatal elastic fibers demonstrated staining affinity to all elastic stain techniques. Oxytalan fibers did not demonstrate affinity to elastic fiber stains used. Based on electron dense stains, elastic and oxytalan fibers were dissimilar, in contrast to results reported utilizing light microscopic stains.

Animals↗

PubMed2003

Comparison of tracheal aspirates and bronchoalveolar lavage in racehorses. 1. Evaluation of cytological stains and the percentage of mast cells and eosinophils.

OBJECTIVE: To compare a fast Romanowsky cytological stain (Diff-Quik) and Leishman's stain for the detection of mast cells in samples from the lower airways of racehorses, and to compare the proportion of mast cells and eosinophils in the total inflammatory cells in tracheal aspirate (TA) with those in paired bronchoalveolar lavage (BAL) samples. DESIGN: Retrospective case series of 48 young Thoroughbred and Standardbred racehorses. PROCEDURE: Fifty-one paired TA and BAL samples were collected after treadmill exercise from 48 horses with poor racing performance. Two slides were prepared from each sample; one was stained with Diff-Quik stain and the other with Leishman's stain. Differential cell counts of eosinophils and mast cells were recorded from each slide. Comparison of the suitability of the stains for the detection of mast cells, and comparisons of eosinophil and mast cell percentages in TA and BAL samples were analysed using the non-parametric Wilcoxon matched pairs test. RESULTS: Percentages of mast cells were significantly higher in Leishman than in Diff-Quik stained slides in both TA (P = 0.03) and BAL samples (P < 0.0001). Mast cell percentages were significantly higher in BAL than in TA samples using Leishman's stain (P < 0.0001). There was no significant difference in eosinophil percentages between TA and BAL samples (P = 0.07). CONCLUSIONS: Fast Romanowsky type stains (for example Diff-Quik) are not appropriate for the detection of mast cells in samples from the equine lower respiratory tract. Therefore, a metachromatic stain that reliably identifies mast cells (for example Leishman's) should be used if evaluation of mast cells in lower respiratory tract is undertaken. Mast cells are predominantly found in the distal small airways and alveoli sampled with a BAL. In contrast, eosinophils appear to be evenly distributed in the lower respiratory tract. However, high percentages of eosinophils are occasionally found only in TA samples. We recommend that both a TA and BAL be used for the evaluation of eosinophils and mast cells within the equine lower respiratory tract.

Animals↗

PubMed1996

Detection of infection or infectious agents by use of cytologic and histologic stains.

A wide variety of stains are useful for detection of different organisms or, for viruses, the cytopathologic changes they induce, in smears prepared directly from clinical specimens and in tissue sections. Other types of stains, such as hematoxylin and eosin, are used routinely to stain tissue sections and are most valuable for assessing the immunologic response of the host to the invading pathogen. In many cases, the pattern of inflammation provides important clues to diagnosis and helps to guide the selection of additional "special" stains used predominantly for diagnosis of infectious diseases. A stain may be nonspecific, allowing detection of a spectrum of organisms, as do the Papanicolaou stain and silver impregnation methods, or detection of only a limited group of organisms, as do the different acid-fast techniques. Some nonspecific stains, such as the Gram stain, are differential and provide valuable preliminary information concerning identification. Immunohistochemical stains, on the other hand, are specific for a particular organism, although in some cases cross-reactions with other organisms occur. Despite the wealth of information that can be gleaned from a stained smear or section of tissue, however, the specific etiology of an infection often cannot be determined on the basis of only the morphology of the organisms seen; culture data are essential and must be considered in the final diagnosis.

Animals↗

PubMed1981

Comparison of acridine orange and Gram stains for detection of microorganisms in cerebrospinal fluid and other clinical specimens.

Acridine orange, a fluorochrome strain, is potentially superior to the Gram stain in the direct microscopic examination of clinical specimens because it gives striking differential staining between bacteria and background cells and debris. Its value in clinical laboratories was evaluated by testing 209 cerebrospinal fluids and 288 other body fluids, tissues, and exudates by both techniques. Smears were made in duplicate, fixed with methanol, stained, and examined without knowledge of the result of the companion smear or culture. Overall, acridine orange was slightly more sensitive than the Gram stain (acridine orange, 59.9%; Gram stain, 55.8%) and equally specific in detecting microorganisms. One smear was falsely positive by the Gram stain; none was falsely positive by the acridine orange stain. We conclude that acridine orange staining is a sensitive method for screening clinical specimens and reviewing selected specimens that are purulent, but negative by the Gram stain. Bloody fluids, thick exudates, and other normally difficult-to-read specimens were easily and quickly examined. We recommend, however, that positive smears be reexamined with the Gram stain to confirm the result and determine the Gram reaction of the microorganisms.

Acridine Orange↗

PubMed1994

Detection of acid-fast bacilli in concentrated primary specimen smears stained with rhodamine-auramine at room temperature and at 37 degrees C.

Many laboratory workers prefer the rhodamine-auramine method of staining acid-fast bacilli (AFB) in primary specimen smears rather than carbol fuchsin stains because the stain is more readily interpreted and yields greater sensitivity. The increasing incidence of AFB infections serves as an impetus to optimize the rhodamine-auramine stain. A total of 782 primary smears were evaluated blindly by the rhodamine-auramine method at both room temperature and 37 degrees C. Thirty-five smears (4.5%) were positive for AFB, 30 were positive by both methods, and 5 were positive at 37 degrees C only. Room temperature staining detected only 85.7% of the positive primary smears. Of the 30 smears positive by both methods, 13 (43.3%) had equal numbers of AFB on both smears, 13 (43.3%) had more AFB on the smear stained at 37 degrees C, and 4 (13.3%) had greater numbers of AFB on the smear stained at room temperature. No smears were positive only when stained at room temperature. The increasing diagnostic emphasis placed on the primary smear underscores the importance of optimizing AFB smear methods, and rhodamine-auramine staining at 37 degrees C enhances the detection of AFB compared with conventional staining at room temperature.

Benzophenoneidum↗

PubMed1997

Uniform staining of Cyclospora oocysts in fecal smears by a modified safranin technique with microwave heating.

Cyclospora, a coccidian protist, is increasingly being identified as an important, newly emerging parasite that causes diarrhea, flatulence, fatigue, and abdominal pain leading to weight loss in immunocompetent persons with or without a recent travel history as well as in patients with AIDS. Modified Kinyoun's acid-fast stain is the most commonly used stain to identify the oocyst of this parasite in fecal smears. Oocysts of Cyclospora stain variably by the modified acid-fast procedure, resulting in the possible misidentification of this parasite. We examined fecal smears stained by six different procedures that included Giemsa, trichrome, chromotrope, Gram-chromotrope, acid-fast, and safranin stains. We report on safranin-based stain that uniformly stains oocysts of Cyclospora a brilliant reddish orange, provided that the fecal smears are heated in a microwave oven prior to staining. This staining procedure, besides being superior to acid-fast staining, is fast, reliable, and easy to perform in most clinical laboratories.

AIDS-Related Opportunistic Infections↗

PubMed2001

Preservation of calcium pyrophosphate dihydrate crystals: effect of Mayer's haematoxylin staining period.

OBJECTIVE: To clarify the deleterious effects of Mayer's haematoxylin staining procedure which result in a decrease in, or complete loss of, the number of calcium pyrophosphate dihydrate (CPPD) crystals, and to determine the proper staining period for preserving the crystals in a histological paraffin section of articular tissues. METHODS: Paraffin sections of CPPD crystal-bearing articular tissues of six patients were stained with Mayer's haematoxylin for 3, 8, or 15 minutes, and subsequently with eosin for one minute. The specimens were examined with an Olympus BHS polarised light microscope. The pH of Mayer's haematoxylin solution was measured with a TOA pH meter. RESULTS: Positive birefringent CPPD crystals were seen clearly in all specimens stained with Mayer's haematoxylin for three minutes. The specimens stained for eight minutes showed a reduced number of crystals. No crystals were seen in the specimens stained for 15 minutes. Ordinary light microscopy showed no notable differences in the stainability of nucleus, cell membrane, and their surrounding tissues among specimens when stained with Mayer's haematoxylin for either 3, 8, or 15 minutes. The pH of Mayer's haematoxylin solution was 2.31. CONCLUSIONS: To find CPPD crystals in the paraffin sections of articular tissues, the staining period with Mayer's haematoxylin should be limited to three minutes. The longer the staining period, the greater the reduction in the number of crystals owing to the strong acidity of the haematoxylin solution. A staining period of 15 minutes causes a complete loss of CPPD crystals.

Aged↗

PubMed1987

Secretion from corticotropes after avidin-fluorescein stains for biotinylated ligands (CRF or AVP).

Viable pituitary target cells were identified with biotin-labeled corticotropin-releasing factor (1-Bio-CRF) or arginine vasopressin (Bio-AVP) and avidin-fluorescein (cell sorter grade). Stain was reduced from 8-10% of pituitary cells to less than 3% if nonbiotinylated ligands were added to compete with their respective biotinylated ligand for specific binding sites. However, AVP did not decrease the stain for 1-Bio-CRF nor did CRF decrease the Bio-AVP stain. Media adrenocorticotropin (ACTH) levels increased 4-18 times when 1-Bio-CRF or Bio-AVP were added during the staining process. One hour after staining, the cells secreted ACTH at levels identical to prestain basal levels. When challenged with CRF or AVP after this hour, the cells responded again with increases not different from those seen during staining. Tests of stained cell populations also showed that the cells were fully responsive 24 h after staining. The reverse hemolytic plaque assay for ACTH, which was used to study secretion from individual stained cells, showed that approximately 80% of plaques contained a stained cell and that plaque numbers and areas were like those of unstained populations. These data show that corticotropes are not chronically desensitized during exposure to low doses of biotinylated ligands (50-500 pM).

Adrenocorticotropic Hormone↗