Histological Staining Methods Research Articles

The Ultrastructure of the Human Spinal Nerve Root Cuff in the Lumbar Spine

Spinal nerve root cuffs may be relevant in selective nerve root and epidural blockade. We examined the ultrastructural aspects of spinal nerve root cuffs, such as their cellular and fibrillar components, using special histological staining methods, transmission and scanning electron microscopy, from six human cadavers. The morphology of the spinal nerve root cuff resembles that of the spinal subdural compartment. Cells gather together in compact layers due to specialized junctions. The thickness of its cellular layers is 5 to 8 microns; cells appear oriented parallel to the direction of their own nerve roots. The fibrillar component, made largely of collagen fibers, is found in the outer part of the spinal nerve root cuff and measures 100 to 150 microns. Numerous adipocytes separate dural laminas in concentric layers, extending from the dural sac to the spinal nerve root ganglia. However, adipocytes are not found within the thickness of the dural sac. The presence of few capillaries and the short distance between fat and axons may affect the passage of epidurally injected substances towards nerve root axons.

Muscle Biopsy in Diagnosis of Neuromuscular Disorders: The Technical Aspects, Clinical Utility, and Recent Advances

Skeletal muscle biopsies present unique preparatory challenges to the histotechnologist and unique diagnostic challenges to the clinician and pathologist. A muscle biopsy sample does not proceed through conventional fixation, nor is it processed via routine surgical pathology paraffin methods. A muscle biopsy must be dealt with as fresh tissue whose subsequent processing is a low-temperature freezing method that is ideally accomplished in a timely manner after surgical excision for optimal staining results. A panel of histological (nonenzyme) and enzymatic staining methods should be used on every muscle biopsy for full and complete clinical diagnostic results. Electron microscopy and immunohistochemistry may add valuable information in some cases. Many of these special procedures are not undertaken in the standard histology laboratory and are sent to specialized referral laboratories for testing and interpretation.The aim of this review is to reveal and summarize the unique aspects of muscle biopsies to ensure that any laboratory faced with handling a muscle biopsy will be prepared to take the correct action for optimal processing results. We also introduce the reader to muscle pathology and discuss the clinical utility of muscle biopsy in diagnosis of patients with neuromuscular disorders. (The J Histotechnol 30:257, 2007)Submitted September 24, 2007; accepted with modifications October 8, 2007

Muscle Biopsy in Diagnosis of Neuromuscular Disorders: The Technical Aspects, Clinical Utility, and Recent Advances

Skeletal muscle biopsies present unique preparatory challenges to the histotechnologist and unique diagnostic challenges to the clinician and pathologist. A muscle biopsy sample does not proceed through conventional fixation, nor is it processed via routine surgical pathology paraffin methods. A muscle biopsy must be dealt with as fresh tissue whose subsequent processing is a low-temperature freezing method that is ideally accomplished in a timely manner after surgical excision for optimal staining results. A panel of histological (nonenzyme) and enzymatic staining methods should be used on every muscle biopsy for full and complete clinical diagnostic results. Electron microscopy and immunohistochemistry may add valuable information in some cases. Many of these special procedures are not undertaken in the standard histology laboratory and are sent to specialized referral laboratories for testing and interpretation.The aim of this review is to reveal and summarize the unique aspects of muscle biopsies to ensure that any laboratory faced with handling a muscle biopsy will be prepared to take the correct action for optimal processing results. We also introduce the reader to muscle pathology and discuss the clinical utility of muscle biopsy in diagnosis of patients with neuromuscular disorders. (The J Histotechnol 30:257, 2007)Submitted September 24, 2007; accepted with modifications October 8, 2007

Cytomegalovirus-induced pneumonia and myocarditis in three cases of suspected sudden infant death syndrome (SIDS): Diagnosis by immunohistochemical techniques and molecularpathologic methods

Immunohistochemical and molecularpathologic techniques have improved the diagnosis of myocarditis as compared with conventional histologic staining methods done according to the Dallas criteria. Additionally, immunohistochemistry and in situ-hybridization are able to demonstrate viral infection, e.g. cytomegaloviruses in salivary glands and lungs, locations both known to be involved in cytomegalovirusinfection. However, in many cases of proved cytomegalovirusinfection the cause of death remains unclear. We report on three children younger than 1-year of age, who died suddenly without prodromal symptoms. Their deaths were attributed to SIDS (sudden infant death syndrome). In situ-hybridization, immunohistochemical (LCA, CD45R0, CD68, MHC-class-II-molecules, E-selectine) and molecularpathologic investigations (PCR), however, suggested that death was caused by a cytomegalovirus-induced pneumonia or myocarditis. In the future, these methods should be used for investigating cases with suspicion of SIDS.

A sensitive stain for aluminum in undecalcified cancellous bone

Aluminum is known to accumulate with age in bone and other tissues of humans, even in the absence of renal disease. Our study aimed to develop a histological staining method sufficiently sensitive to detect aluminum in plastic sections of undecalcified bone biopsies from healthy volunteers as well as from patients with renal and non-renal bone diseases. We used quantitative histomorphometry to measure the percentage of trabecular surface stained by aluminum and found that our new method was approximately 50% more sensitive for detecting aluminum than the Acid Solochrome Azurine (ASA) method which in turn was significantly more sensitive than the Aluminon method. Aluminon is widely used in pathology laboratories for diagnostic purposes despite concerns in the literature about Aluminon’s limited sensitivity for aluminum. Our histomorphometric results showed that the newly developed method stained approximately 10% of the trabecular surface in bone sections from healthy controls, 38% from renal patients, 26% from patients with vitamin D deficiency, and 29% from patients with osteoporosis. Histomorphometric measurements of aluminum-stained trabecular surfaces in sections stained with ASA were consistent with those obtained in Walton-stained sections but proportionately lower. Moreover, the Walton and ASA methods stained aluminum at similar locations in adjacent bone sections. As the ASA and Walton methods are considerably more sensitive for bone aluminum than the Aluminon method, we recommend that either of them should be used in place of the Aluminon method for routine diagnostic purposes.

The contribution of pathologic diagnosis and research of the faculty members engaged in teaching histology and embryology to undergraduate medical studies

The theoretical and practical aspects of teaching are generally defined by the curriculum, but professional experience plays an important role as well. The teaching experience of the faculty members is developed through health services, education and research. THE DEPARTMENT OFHISTOLOGY AND EMBRIOLOGY: This paper reviews the activities of the teaching staff of the Department of Histology and Embryology, pointing to the importance of pathologic diagnostics and research. It underlines the necessity of using some non-standard histologic staining methods on some tissues, organs and pathogens, as well as the results of experimental studies of the endocrine system. The experimental tumor models are used to demonstrate the interdependence of the endocrine and immune systems. Modalities and possibilities of regeneration of the digestive tract and connective tissue mucosa are explained This paper also deals with histologic criteria used for assessment of fetal age and in the diagnosis of developmental malformations. Linear and stereologic methods, used in quantification of normal morphology, the degree of pathologic changes, and regeneration of normal structures in the courses of and after therapy, are given. The authors particularly emphasized the importance of above-mentioned activities in the teaching of Histology and Embryology to undergraduate students.

Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls

Histological staining methods commonly used for detecting cellulose and lignin in cell walls were combined with epifluorescence microscopy to visualize differences in lignification between and within cellular elements. We tested our approach on sections of one-year-old branches of Fraxinus ornus L., Myrtus communis L., Olea europaea L., Pistacia lentiscus L. and Rhamnus alaternus L., containing both normal and tension wood. Sections were subjected to various staining techniques, viz. safranin O, safranin O/fast green FCF, and alcoholic solutions of safranin O/astra blue, according to the commonly accepted protocols. Stained and unstained sections were compared using both light and epifluorescence microscopy. Safranin O with or without counterstaining hid the strong fluorescence of vessel walls, cell corners and middle lamellae allowing the secondary wall fibers to fluoresce more clearly. Epifluorescence microscopy applied to stained sections showed more cell wall details than autofluorescence of unstained sections or white light microscopy of counterstained sections. This simple approach proved reliable and valuable for detecting differences in lignification in thick sections without the need for costly equipment.

Morphological features of cartilage observed during mandibular distraction in rabbits

Ossification during distraction osteogenesis can be classified as intramembranous or endochondral. It is not known whether cartilage in the distraction gap is transformed into new bone. The aim of this study was to investigate the morphological features of ossification in the transition of cartilage to bone during mandibular distraction osteogenesis in a rabbit model. A cortical osteotomy was performed and custom-made devices were applied. Immediately after surgery, the devices were lengthened by 0.25 mm every 12 h for up 10 days, during which time four rabbits were killed at 0, 5 and 10 days and examined using histological staining and immunohistochemical methods. Apoptotic cells were identified by an in-situ detection assay for nuclear DNA fragmentation using a modified TUNEL procedure, with several sections analyzed using software for histomorphometric analysis. The results showed that the amount of cartilage in the distraction gap was significantly decreased. The cartilage had ossified in two ways, termed endochondral ossification and transchondroid bone formation.

Mammary Gland Secretory Concretions Contain Non‐Collagenous Bone Matrix Proteins

Secretory concretions in mammary gland alveoli are commonly of microscopical size. However, some concretions reach clinically palpable dimensions and may occlude teat canals and obstruct milk flow. We studied secretory concretions in sheep, goat and cow mammary glands, using routine histological staining methods, conventional histochemistry and electron microscopy. As concretions frequently mineralize, immunostaining for keratan sulphate and calcium-binding non-collagenous bone matrix proteins (bone sialoprotein, osteocalcin, osteonectin and osteopontin) was performed. Concretions consisted of organic matrix (condensed secretions) with calcium precipitates. Mineralized deposits mostly show concentric organization, bound haematoxylin, and were readily identified in H&E-stained sections. Mineral components of concretions reacted for calcium carbonate and phosphate, organic matrix was found to contain sialoglycan material. Immunohistochemistry revealed bone sialoprotein, osteonectin and keratan sulphate in cow and goat concretions. Osteocalcin was detected in sheep, cow and goat concretions, whilst osteopontin was not identified in any of the specimens studied. Our results indicate the presence of non-collagenous bone matrix proteins (except osteopontin) in mammary gland concretions. These glycoproteins are commonly thought to govern mineralization of organic matrix and are assumed also to promote mineral deposition in mammary gland secretory concretions. Besides caseins, these particular glycoproteins have to be considered as calcium-binding milk proteins.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA

BackgroundGene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays.ResultsThe MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases.ConclusionRNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Senile systemic amyloidosis, cerebral amyloid angiopathy, and dementia in a very old Finnish population

Senile systemic amyloidosis (SSA) and cerebral amyloid angiopathy (CAA) are amyloid disorders, which typically manifest with old age. The aim of our study was to examine the possible association of these disorders in very old Finns. We performed a prospective, population-based post mortem study and used histological and immunohistochemical staining methods to verify the presence of these types of amyloid. All 63 subjects (59% of the 107 individuals 95 years of age or more, who died during the 10-year follow-up study), 53 women and 10 men), had been neurologically examined. The prevalence of SSA and its association with CAA, dementia, and neuropathologically verified AD was analyzed. Overall SSA occurred in 23 (37%) and CAA in 28 (44%) of the 63 subjects. At clinical examination 41 individuals (65%) were demented; 24 (38%) had Alzheimer's disease. SSA showed no association with the presence of CAA (P = 0.45), clinical dementia (P = 0.09), or Alzheimer's disease (P = 0.21), or sex (P = 0.53). Our prospective population based study shows that SSA and CAA are frequent in very old Finns, but they do not associate.

Cerebral amyloid angiopathy in a 95+ cohort: complement activation and apolipoprotein E (ApoE) genotype

There is growing evidence that in Alzheimer's disease (AD) amyloid beta-protein (Abeta) triggers a chronic inflammatory reaction in cerebral amyloid plaques, including complement proteins. Abeta also accumulates cerebrovascularly in age- and AD-associated cerebral amyloid angiopathy (CAA). We investigated complement proteins in CAA in a population-based series using histological and immunohistochemical staining methods. The 74 subjects, aged 95 years or more, had undergone clinical neurological examination and apolipoprotein E (ApoE) genotyping. The brains had been studied for AD post-mortem, allowing us to relate the histopathological findings to clinical and genetic conditions. CAA with congophilic amyloid was found in 36/74 individuals (48.6%). The vascular amyloid deposits immunoreacted with antibodies to Abeta and complements 3d (C3d) and 9 (C9). The positivity in complement stains increased with growing severity of CAA (P = 0.001). The presence of CAA associated with ApoE epsilon4 (P = 0.0005) and overrepresentation of epsilon4 among those with moderate or severe vs. mild CAA (P = 0.03) was demonstrated. The presence of CAA associated with dementia (P = 0.01), which was contributed by both epsilon4+ (P = 0.02) and epsilon4- (P = 0.06) subjects. Our study shows that complement proteins are deposited in the affected vessels in Abeta-associated CAA. They may solely represent the cerebral Abeta- burden associated to inflammatory stimuli, or signal a contribution in the clearance of cerebral Abeta, thereby contributing to the events associated with evolution of clinical dementia. Our results demonstrate a strong association between CAA and ApoE epsilon4 as well as dementia and suggest that the contribution of CAA to dementia is largely independent of ApoE epsilon4.

Single sperm cell isolation by laser microdissection

In 1996, Van Oorschot and Jones firstly reported in scientific correspondence that short tandem repeat (STR) profiles could be obtained from cells left on different objects. Since then, forensic scientists have focused their efforts in isolating single cells as it can be extremely helpful in solving caseworks where sexual violence was concerned. Laser microdissection is a micromanipulation procedure allowing to cut off precisely the cells of interest from tissue samples or smears by a laser beam fitted with an optical microscope. We have harvested single sperm cells by laser microdissection using a Leica AS LMD (Leica Microsystems, Germany); laser setting, pulse laser intensity and laser alignment as well as recovery of the specimen have been properly fitted to the samples we were dealing with. Different tissue preservation, fixation, histological staining (Papanicolau, Nuclear Fast Red—Picroindigocarmine) methods and number of harvested cells for each sample have been evaluated as well. Finally, the genotype of sperm cells has been determined by STR typing, evaluating the sensibility of this forensic technique according to instrumental and biological abovementioned variables.

Development of Neural Stem/Progenitor Cells from Human Brain by Transplantation into the Brains of Adult Rats

The aim of the present work was to study human neural stem/progenitor cells (SPC) cultured in vitro and their potential to survive, migrate, and differentiate after transplantation into adult rat brain. SPC were extracted from the brains of nine-week human embryos and were cultured in selective medium for three weeks. Transplantation was with suspensions of cells or whole neurospheres; these were studied four weeks after transplantation into the hippocampus, striatum, and lateral ventricles of adult rats. Analysis of transplanted cells was based on various histological and immunohistological staining methods: bisbenzimide, bromodeoxyuridine, and antibodies to human nuclei, vimentin, beta-tubulin, neurofilaments, and glial fibrillar acidic protein, which allowed us to make independent assessments of their state and differentiation. Transplanted SPC from human brains survived well for one month in all areas of adult rat brain without immunosuppression. Cells from suspension transplants migrated intensely and differentiated into neurons and gliocytes. At the same time, transplants of whole neurospheres showed limited or no migration because of the development of a glial barrier.

Onco-surgical significance of anatomomorhological specificity of glottic region of the larynx

Glottis is a medium level of the larynx, involving the vocal cords, vocal process of arytenoid cartilage, and the anterior and posterior commissures. This region of heterogeneous histomorphological structure has specific characteristics: the internal striated muscles, the outer and inner perichondrium, atypical intra-cartilaginous areas of ossification, and unusual muscular insertion to cartilage. Microtomy of 3 thick successive sections was performed at the level of the upper surface of vocal cords. Standard histological staining methods were used. Microphotographs were taken by light microscope under different magnification along with histomorphometric measurements. Cancer spread is partially restricted by anatomic barriers: vocal ligament and tendon of the anterior commissure, and subsequently by the elastic cone. Easier tumor extension may be due to absence of the internal perichondrium and ossification of thyroid cartilage. Microvascularization of the anterior commissure is significant for tumor spread in glottis and paraglottic space. The role of commissural lymph network in local spread of the cancer is completely vague. It is certain that there are causes, still unrecognized, which have an effect on the pathways and direction of malignant tumor spread.

Two-dimensional mapping of copper and zinc in liver sections by laser ablation-inductively coupled plasma mass spectrometry.

Metals are not homogeneously distributed in organ tissues. Although most mapping techniques, such as histologic staining methods, have been developed for element imaging on a subcellular level, many suffer from either low precision or poor detection limits. Therefore, small variations in elemental distribution cannot be identified. We developed a method for two-dimensional mapping of trace elements to identify the influence of metabolic zonation by the liver on trace element distribution. A prepared homogeneous Certified Reference Material (CRM; LGC 7112, pig liver) was used to characterize the laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-MS) in terms of precision. Different isotopes for copper and zinc were monitored, and the use of carbon as an internal standard was investigated to correct for differences in ablation efficiency to identify the most precise mapping technique for liver samples. For the homogeneous CRM, the reproducibility of the copper and zinc signals was approximately 3-24% depending on spot size and number of pulses. When carbon was used as an internal standard, the reproducibility was improved significantly. Line scan signals over a length of 1.5 mm were more precise [relative SD (RSD), 1.6-6.1% for copper ((63)Cu, (65)Cu) and zinc ((64)Zn, (66)Zn) depending on the spot size, the scanning speed, and the element]. Thin section of sheep liver achieved precisions of 27-59% (raster scan) and 9-47% (line scan) RSD for copper, whereas the precision for zinc was significantly better: 8-18% (raster scan) and 4-21% (line scan) RSD. Long line scans and two-dimensional element maps of the thin sections revealed the zonation of copper in sheep liver containing extremely low copper concentrations. Elemental mapping of trace elements generated by LA-ICP-MS can be very precise so that small changes in the elemental concentration in the tissue can be detected and nonuniform spatial distribution of the elements in tissues can be established.

In situ hybridization analysis of chemokines and chemokine receptors in the central nervous system

The expression of a number of chemokines and chemokine receptors by cells resident in normal and pathological central nervous system (CNS) tissue has been characterized by in situ hybridization techniques. As a result, our understanding of the role of this cytokine family in neurobiology has been enhanced greatly. Specific methods for detecting chemokine and chemokine receptor mRNAs in situ vary with the number of these genes that have been characterized and encompass approaches widely utilized by other investigators characterizing cell-specific gene expression patterns. We describe methods that our laboratory has used successfully in characterizing chemokine and chemokine receptor expression in the CNS, focusing on protocols that utilize radiolabeled in vitro-transcribed riboprobes for detecting these transcripts. Because general dye-based histological staining methods do not readily differentiate astrocytes and microglia, specific immunohistochemical protocols are required for definitive localization of gene expression to these glial cell types. As such, methods that are compatible with the in situ hybridization procedure are included for staining astrocytes and microglia.

Histological Findings After Colocystoplasty And Gastrocystoplasty

We conducted a prospective, long-term assessment of the histological changes that can occur following bladder augmentation with colon or stomach. Histological evaluations of biopsies from 44 consecutive patients undergoing augmentation (colocystoplasty in 26, gastrocystoplasty in 18) were performed. Patients underwent endoscopic assessment and tissue sampling at 2 or 4-year intervals following the initial augmentation procedure. Patients with less than 2 years of followup were excluded from the analysis. Specimens were taken from the native bladder, the augment segment (large bowel or stomach) and the anastomotic line. Sections (4 mu.) were examined using standard histological staining methods (hematoxylin and eosin and periodic acid-Schiff) and immunohistochemistry was performed for different markers of neoplasia, cellular proliferation and blood group antigens. Histological findings were correlated with the incidence of stone formation and urinary tract infection. Group 1 consisted of 20 patients undergoing colocystoplasty who met the criteria for study inclusion. Of the patients 10 (50%) had stones, 19 (95%) had a positive urine culture and 6 had no histological changes. While no cases of malignancy were identified, other forms of pathological change were noted in 14 of the 20 patients (70%). Group 2 included 15 patients undergoing gastrocystoplasty who met the criteria for study inclusion. No stones or malignancy were identified in this group. Positive urine cultures were recorded in 2 patients (13%), no histological changes were found in 6 and 9 (60%) had pathological changes. Periodic prospective biopsy evaluation of children who have undergone either colocystoplasty or gastrocystoplasty failed to reveal any histological evidence of malignancy after 10-year followup. However, histological evidence of a premalignant lesion 13 years after followup suggests that screening for premalignant lesions should be initiated no later than 6 to 10 years following enterocystoplasty.

Coxsackie B3 myocarditis in 4 cases of suspected sudden infant death syndrome: diagnosis by immunohistochemical and molecular-pathologic investigations.

Immunohistochemical and molecular-pathologic techniques have improved the diagnosis of myocarditis as compared with conventional histologic staining methods done according to the Dallas criteria. Most investigations were carried out on adults, and only a few authors investigating childhood deaths applied these modern methods, used for diagnosing myocarditis. We report on four children under one year of age, who suddenly died without prodromal symptoms. Their deaths were attributed to SIDS (sudden infant death syndrome). Immunohistochemical (LCA, CD68, CD45R0, MHC-class-II-molecules, VP1-capsid-protein of enteroviruses) and molecular-pathologic (RT-PCR) investigations, however, suggested that death was caused by a coxsackie-B3-myocarditis. In the future, these methods should be used for investigating cases with suspicion of SIDS.

Estudo comparativo entre citopatologia e histopatologia no diagnóstico de neoplasias caninas

Foi realizado um estudo comparativo entre os diagnósticos citológico e histopatológico em diversas neoplasias de 150 cães, pelas colorações de Wright, May-Grünwald-Giemsa, Novo Azul de Metileno e Papanicolau. Colorações histológicas como Hematoxilina-Eosina, van Gieson, Sudan, Azul de Toluidina e Ácido Periódico de Schiff também foram empregadas. Os dados revelaram uma eficácia de ordem de 85,3% no diagnóstico citopatológico, considerando-se os resultados histopatológicos como corretos. Em 4,0% dos casos somente a origem embrionária das neoplasias foi estabelecida. Em 1,3% das neoplasias apenas o prognóstico foi determinado; o diagnóstico citológico diferiu da histopatologia em 8,1% dos casos. Em dois casos (1,3%) o diagnóstico citológico diferiu do histológico, mas um reexame determinou que o primeiro estava correto, o que elevou a sua eficácia para 86,6%. Entre as técnicas utilizadas, a punção aspirativa por agulha fina foi o melhor método para obter amostras. A citologia não foi adequada para o diagnóstico de neoplasias mamárias, dadas às variações morfológicas em diferentes áreas. A impressão em lâmina não é recomendada para análise de tumores mesenquimais e deve ser substituída pela citologia esfoliativa. O Wright revelou-se o método de coloração mais eficiente. As colorações adaptadas da histopatologia, van Gieson em leiomiomas e leiomiossarcomas, Sudan em lipomas e lipossarcomas, e Ácido Periódico de Schiff e Azul de Toluidina em mastocitomas, foram empregadas com sucesso fornecendo assim maior clareza de detalhes para as diversas neoformações de origem epitelial e mesenquimal.