Assessment of immune phagocytosis of Plasmodium falciparum infected red blood cells by human monocytes and polymorphonuclear leukocytes. A method for visualizing infected red blood cells ingested by phagocytes

A method is described for the visualization of red blood cells infected with Plasmodium falciparum ingested by monocytes or polymorphonuclear leukocytes (PMN) after in vitro incubation. Smears were stained with peroxidase followed by 4,6-diamino-2-phenylindole (DAPI) staining specific for DNA. Monocytes or PMN were identified under normal illumination by the peroxidase stain and the nuclei of these cells as well as the parasites were identified by means of the DAPI stain with ultraviolet light. Using this method we found that monocytes and PMN from normal blood donors preferentially phagocytose plasmodium falciparum infected red blood cells in the presence of sera from subjects living in areas endemic for malaria.     

Combined staining of elastic and collagen fibers for histopathological and differential diagnosis in aorta pathologies

Histochemical techniques are important for diagnosis of connective tissue fibers involved in different aorta pathologies. The aim of this study was to develop a histochemical staining method that identifies both elastic and collagen fibers simultaneously in the same section of aortic wall. Fragments of aorta from deceased dogs were processed according to standard histological technique for paraffin embedding. Sections were stained with orcein with counterstained with either light green or single step Gomori trichrome, and also orcein stain and picro-sirius red. Orcein stain combined with picro-sirius red allowed visualization of both elastic fibers (brown) and collagen fibers (red) with light microscopy using bright field illumination. Moreover, under polarized light microscopy only collagen fibers were detected, appearing as reddish birefringent fibers, confirming that picro-sirius red-stained fibers were collagen fibers. The combination staining by orcein and picro-sirius solutions provided visualization of both collagen and elastic fibers in the same section, facilitating identification of these connective tissue fibers in the aortic wall.     

Oligonucleotide probe for the visualization of Escherichia coli/Escherichia fergusonii cells by in situ hybridization: specificity and potential applications

There are several occasions when enumeration of Escherichia coli cells is needed. These include examination of urine specimens and water or food samples. Present methods rely on growth in more or less selective media (colony-forming units on agar or the most probable number method using liquid media). Unfortunately, no really selective medium with 100% efficiency of plating is available for E. coli. A 24-mer oligonucleotide probe (Colinsitu), complementary to a piece of 16S ribosomal ribonucleic acid, has been tested for specifically visualizing E. coli cells by in situ hybridization and epifluorescence microscopy. The fluorescent dye-labeled probe was able to stain cells of E. coli, Shigella spp. and E. fergusonii. Shigella spp. are known to belong to the E. coli genomospecies and E. fergusonii is the nomenspecies closest to E. coli by DNA-DNA hybridization. The probe did not stain any strain of 169 other genomospecies of the family Enterobacteriaceae or of a few other species frequently encountered in the environment. Revivification without cell division allowed the visualization of E. coli cells in contaminated water. In situ hybridization using the Colinsitu probe is a potential tool for the confirmation of (atypical) E. coli in reference centers and the rapid (3-6 h) detection and enumeration of E. coli in urine specimens, contaminated water and food. More work is needed to include in situ hybridization in laboratory routine.     

Pneumocystis carinii in sputum. Comparable efficacy of screening stains and determination of cyst density

To determine whether accurate quantification of Pneumocystis carinii cysts in sputum is possible using currently available means and to determine the optimal stain for screening sputum for P carinii, we undertook a comparative evaluation of readily available stains: (1) Diff-Quik, a stain that provides tinctorial detail comparable with Giemsa, (2) toluidine blue, and (3) silver methenamine on simultaneously thawed sputum samples containing P carinii. We also studied the Diff-Quik stain in combination with (4) toluidine blue and (5) silver methenamine. The three stains were comparable for screening purposes. Each disclosed similar numbers of P carinii aggregates on similarly prepared slides. Cysts were difficult to recognize on Diff-Quik due to the negatively staining character of the cyst wall with this stain. The cyst-wall stains, toluidine blue and silver methenamine, clearly stained the cyst wall. The combination of a cyst-wall stain with Diff-Quik allowed visualization of cysts, highlighted by the cyst-wall stain, and aggregate area, delineated by the Diff-Quik, allowing expression of number of cysts per aggregate area, a measure termed cyst density.     

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.     

Polarized incident light microscopical enhancement of immunogold and immunogold-silver preparations: its role in immunohistology

Polarized incident light microscopy (PILM) is a recognized method of visualization of bound colloidal gold in cytological immunocytochemical preparations. This study investigates its role in the assessment of histological sections using both indirect immunogold and immunogold-silver staining methods. With dark field or bright field illumination this technique was found to be advantageous and allowed easy detection staining at low magnification and accurate detection of low levels of stain product. We advocate this technique as a valuable microscopical enhancement method for immunogold immunohistology.     

Combined Fontana-Masson-mucin staining of Cryptococcus neoformans.

Cryptococci react positively with various histochemical stains, including the Fontana-Masson (FM), which stains the cell wall, and mucin stains, such as alcian blue and mucicarmine, which stain the capsule. Combinations of the FM stain with both the alcian blue and mucicarmine stains were performed on paraffin-embedded tissue specimens that were obtained from 15 patients who had culture-proved cryptococcosis. Combined FM-mucicarmine and FM-alcian blue stains were compared with other individual fungal stains. The FM stain, followed by either the mucicarmine or alcian blue stain, distinctively demonstrated both the cell wall and capsule of most organisms. More organisms were recognized in the combined stains than with either stain done individually. No interference between the stains was noted. Combining the FM stain with either of these two mucin stains appears to be helpful for identifying cryptococci.     

Enhancement of the Paraldehyde-Fuchsin Staining

Abstract

We have modified the aldehyde-fuchsin (PAF) stain to enhance its ability to simultaneous demonstration of different tissue compounds. We have substituted the light green dye of Hallni mixture with fast green dye and extended the reduction time by oxalic acid. The resulting stain provides better visualization of several tissue elements, such as the distinction between the nucleus and secretion products of goblet cells and between elastic and collagen fibers and others tissue elements. (The J Histotechnol 21:147, 1998)     

Proteoglycans and their relationship with the other components of the rabbit aorta wall observed in two different experimental conditions.

An electron microscopic study of the aorta wall of rabbit was carried out employing electron microscopic histochemical methods aimed at examining the distribution of proteoglycans and their correlation with the other structural components of the aorta wall. The ultrastructural evidence of the proteoglycans has been obtained by treating the arterial wall with alcian blue stain and supporting this staining procedure with control enzyme treatments. Moreover, in order to attain a better interpretation of the ultrastructural findings, the arterial wall has been studied under 2 different experimental conditions, the 1st one being represented by an accentuated contraction of the vase's wall, following immersion in the fixator, the other one by an extreme distension of the wall, reached prior to fixation, subjecting the tissue to its tension limit of 700 dyn/mm2. This type of electron microscopic visualization of the proteoglycans following alcian blue treatment leads on to think that at least 2 distinct macromolecular entities of proteoglycans exist, each one having an unusual relationship with the other components of the arterial wall. Different site and types of interaction are to be noted for each of the 2 distinct ultrastructural proteoglycans entities.     

Recombinant human single-chain MHC-peptide complexes made from E. coli By in vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens

Soluble recombinant MHC-peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single-chain human MHC (scMHC)-peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA-A2 in the presence of tumor-associated or viral peptides. The scMHC molecule was composed of beta2-microglobulin connected to the first three domains of the HLA-A2 heavy chain through a 15-amino acid flexible linker. Highly purified scMHC-peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART-1 or a viral peptide derived from HTLV-1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA-A2-restricted peptides. We also generated scMHC-peptide tetramers, which were biologically functional; they induced a peptide-specific CTL clone to be activated and secrete IFN-gamma, and were able to stain specifically CTL lines. Such recombinant soluble scMHC-peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen-specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.     

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.     

Evaluation of an indirect fluorescent-antibody stain for detection of Pneumocystis carinii in respiratory specimens.

Two prospective studies were undertaken to evaluate a commercial indirect fluorescent-antibody (IFA) stain for the detection of Pneumocystis carinii in respiratory specimens from individuals at risk for or with the acquired immunodeficiency syndrome. The first study compared IFA with Diff-Quik (DQ; a rapid Giemsa-like stain) for detecting P. carinii in 95 induced sputa obtained from 77 asymptomatic patients who had survived one previous episode of P. carinii pneumonia and who were being treated prophylactically with aerosolized pentamidine. Only one induced sputum specimen was found to contain P. carinii; organisms were detected by both stains. The second study compared the performance of the IFA stain versus DQ, modified toluidine blue O, and Gomori methenamine silver stains for detecting P. carinii in symptomatic individuals at risk for or with acquired immunodeficiency syndrome. Of 182 specimens examined, P. carinii was detected in 105 by one or more stains; the DQ stain detected 73 (70%), the modified toluidine blue O stain detected 75 (71%), the Gomori methenamine silver stain detected 76 (72%), and the IFA stain detected 95 (90%). The IFA stain was more sensitive (P less than 0.01) than the other traditional stains for detecting P. carinii; however, a subsequent clinical evaluation revealed that a subset of IFA-positive-only specimens were from patients whose clinical symptoms resolved without specific anti-P. carinii therapy.     

3D approach visualizing cellular networks in human lymph nodes

Lymph node diagnostics are essentially based on cutting thin sections of formalin fixed tissues. After hematoxylin and eosin stain, Giemsa stain and immunohistochemical staining of these tissues, the lymph node diagnosis is done using a light microscope, looking at two-dimensional pictures. Three-dimensional visualizations of lymph node tissue have not been used in lymphoma diagnostics yet. This article describes three-dimensional visualization of lymphoid tissue, using thick paraffin sections, immunostained with monoclonal antibodies, confocal laser scanning and data processing with appropriate software and the 3D printing process itself. The advantages and disadvantages of different printing techniques are discussed as well as the application of 3D models in diagnostics, teaching and research of lymph nodes.     

Immunoreactivity for calretinin and other calcium-binding proteins in cerebellum

Two calcium-binding proteins, calbindin and parvalbumin, have been reported to be abundant in Purkinje cells and other cell types in the cerebellum. Immunoreactivity for a related protein, calretinin, is now reported in cerebellum of chick and rat. In the chick, antibodies against calretinin stain mossy fibres throughout, and climbing fibres in a distinct group of folia. They also stain several cell types in the molecular layer. As there is no detectable calretinin mRNA in the cerebellar cortex, this cellular staining may be due to cross-reaction with an unknown antigen. In the rat, antibodies against calretinin stain the Lugaro cells, and some granule cells in lobe X; they also give weak staining of all the granule cells in the other lobes. Thus almost all the neuronal cell types in the cerebellum show immunoreactivity for at least one of the calcium-binding proteins in one or both species.     

Efficacy of different methods for detection of lowCryptosporidium parvum oocyst numbers or antigen concentrations in stool specimens

The detection ofCryptosporidium parvum oocysts in stool specimens by acid-fast (AF) stains or immunofluorescence assays (IFA) requires the presence of large numbers of oocysts. To determine whether new commercially available enzyme immunoassays (EIAs) are more sensitive alternatives, three EIAs, a direct IFA, and the modified cold Kinyoun AF stain were compared, particularly with respect to detection of low oocyst numbers or antigen concentrations. Thirty-one negative and 31 calf stool-enriched human stool specimens were tested. One EIA method detected only nine positive specimens, demonstrating a sensitivity significantly less (p<0.0001) than that of the IFA, the AF stain, and the other two EIAs. No differences could be found with respect to specificity. In addition, serial dilutions of 28 patients' stool samples containing cryptosporidian oocysts were prepared and examined using two EIAs, IFA, and the AF stain. One EIA yielded significantly inferior results (p<0.0001), whereas the other one and the two microscopic methods did not differ significantly in either part of the study. The results indicate that the new EIAs do not exhibit higher sensitivities for detection ofCryptosporidium parvum than the two routinely used microscopic methods. Thus, for most laboratories, the IFA or AF stain may still represent the preferred method for the diagnosis of cryptosporidiosis.     

Comparison of five elastin histochemical stains to identify pulmonary small vasculature*

Optimization of an elastic tissue histochemical stain to enable clear, crisp visualization and quantification of pulmonary small vasculature is central to the histomorphologic quantitation of pulmonary vasculature wall thickness. To accomplish elastic tissue histochemical stain optimization, five histochemical elastin stains were compared to identify the internal and external elastic laminae of small arteries (50–100 μm in external diameter) to very small intra-acinar vessels (10–50 μm in external diameter) in rat lung tissue sections. The five elastin stains included: a modified Verhoeff’s elastin stain, Miller’s elastic van Gieson, and three modifications of the Miller’s stain. The Miller elastin stain is a progressive procedure that does not require a differentiation step, thus enabling consistency and reliability of staining from slide to slide. A modified Miller’s histochemical staining methodology successfully highlighted the pulmonary small caliber vasculature wall thickness. The modified method was technically easier and less time consuming to perform than regressive methods. To improve elastin-to-background contrast, modifications to the Miller’s stain included bypassing the nuclear staining and using a neutral red counterstain in place of the van Gieson counterstain, both of which greatly facilitated observer-assisted pulmonary vascular structure identification for histomorphometric quantitation.     

A modification of the cell culture agar diffusion test using fluoresceindiacetate staining

In order to reduce the uncertainties involved in the morphologic evaluation of the cell culture agar diffusion test, the originally recommended neutral red stain was replaced by fluoresceindiacetate (FDA). This stain proved to be nontoxic in the concentrations used in this study (0.002%). Toxicity tests with phenol, formalin, and methylmethacrylate monomer revealed results corresponding to literature evaluations by other methods. Since FDA makes cell metabolism visible and easily accessible to automated evaluation techniques, it presents certain advantages over the morphologic evaluation using the neutral red stain.     

Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen‐specific T cells

Fluorochrome‐conjugated peptide–major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen‐specific T cells. The most common multimers, streptavidin–biotin‐based ‘tetramers’, can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen‐specific T cells within a sample, an issue that is particularly problematic when staining tumour‐specific, autoimmune or MHC class II‐restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran‐based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR–pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co‐receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state‐of‐the‐art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology.     

Giemsa staining for cysts and trophozoites of Pneumocystis carinii.

Although Giemsa staining has been routinely used for the detection of trophozoites and intracystic bodies in smears of bronchoalveolar lavage fluid (BAL) from patients with Pneumocystis carinii pneumonia, it does not normally stain the cyst wall. For detection of the cysts other stains such as toluidine Blue 'O' and methenamine silver must be used as well. Sulphation of smears before staining with Giemsa allows cysts to be visualised, thus enabling a single stain to be used to show all the stages of BAL or sputum, which is particularly useful, considering the increase in the prevalence of P carinii pneumonia in conjunction with the spread of AIDS.     

Transfer of proteins from acrylamide gels to nitrocellulose paper after silver stain detection

A method is described for the silver staining of polyacrylamide gels and transfer to nitrocellulose sheets which couples the advantages of silver stain detection sensitivity with efficient transfer to nitrocellulose sheets. Using this procedure, a detection sensitivity of 10 ng was achieved while retaining a transfer efficiency for most proteins that is equivalent or better than methodology that does not permit prior visualization of protein. When coupled with the use of an acrylamide template, this procedure permits protein detection, simultaneous transfer of a large number of cored protein products from many one- or two-dimensional polyacrylamide gels to a single sheet of nitrocellulose, and retention of immunoreactivity. Prior detection, simultaneous transfer, and retention of immunoreactivity provide a substantial number of biochemical and technical advantages.