Microwave histoprocessing of bone marrow trephine biopsies

Summary In recent years, the microwave oven has been increasingly used in the pathology laboratory for processing of tissue for diagnostic purposes with a remarkable reduction in processing time and also reports of excellent morphology and immunohistochemistry. We evaluated some of these processes on post mortem bone marrow trephine biopsies and describe a novel way of processing these biopsies in the microwave oven.     

AgarCyto: a novel cell-processing method for multiple molecular diagnostic analyses of the uterine cervix.

In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.     

Role of bone marrow imprints in haematological diagnosis: a detailed study of 3781 cases

X. Gong, X. Lu, X. Wu, R. Xu, Q. Tang, G. Xu, L. Wang, X. Zhang and X. Zhao Role of bone marrow imprints in haematological diagnosis: a detailed study of 3781 cases Objectives: To explore the role of imprints in routine bone marrow (BM) diagnosis. Methods: The cellularity and diagnostic accuracy of BM imprints, aspirate smears and trephine biopsy sections from 3781 patients were assessed using routine cytochemical staining. Seventy‐nine cases of lymphoma and 114 cases of plasma cell myeloma (PCM) were selected for correlation analysis of tumour cell infiltration patterns. Another 21 cases of lymphoma were selected to detect t(14;18)(q32;q21) and t(11;14)(q13;q32) by fluorescent in situ hybridization (FISH) on BM imprints, and the G‐banding technique was performed for comparison. Results: BM imprints were better than smears for evaluating cellularity. In the BM imprint group, diagnostic accuracy for metastatic carcinoma, myeloproliferative neoplasm, myelodysplastic/myeloproliferative neoplasm and PCM was better than in the smear group, while accuracy for megaloblastic anaemia, acute myeloid leukaemia, refractory cytopenia with unilineage or multilineage dysplasia, refractory anaemia with excess blasts and lymphoplasmacytic lymphoma was higher than in the section group, but not statistically different from the smear group. Good correlation of infiltration patterns of lymphoma and myeloma cells was found between BM imprints and sections (r = 0.90 and 0.78, respectively). Detection of t(11;14)(q13;q32) by FISH on imprints was higher than G‐banding analysis. Conclusions: BM imprints show features of both smears and trephine sections. Imprints are superior to smears for evaluation of cellularity, and are also better than sections for analysis of cytological changes. In addition, FISH on BM imprints markedly improves the identification of chromosomal abnormalities.     

Diagnosis of soil-transmitted helminths in the era of preventive chemotherapy: effect of multiple stool sampling and use of different diagnostic techniques

Background

Soil-transmitted helminth infections are common throughout the tropics and subtropics and they disproportionately affect the poorest of the poor. In view of a growing global commitment to control soil-transmitted helminthiasis, there is a need to elucidate the effect of repeated stool sampling and the use of different diagnostic methods in areas targeted for preventive chemotherapy that are characterized by low-infection intensities. In this study, we focused on schoolchildren on Unguja Island, Zanzibar, an area where anthelminthic drugs have been repeatedly administered over the past decade.

Methodology/Principal Findings

Three serial stool samples from each of 342 schoolchildren were examined using the Kato-Katz (K-K), Koga agar plate (KAP), and Baermann (BM) techniques. These methods were used individually or in combination for the diagnosis of Ascaris lumbricoides (K-K), Trichuris trichiura (K-K), hookworm (K-K and KAP), and Strongyloides stercoralis (KAP and BM). The examination of multiple stool samples instead of a single one resulted in an increase of the observed prevalence; e.g., an increase of 161% for hookworm using the K-K method. The diagnostic sensitivity of single stool sampling ranged between 20.7% for BM to detect S. stercoralis and 84.2% for K-K to diagnose A. lumbricoides. Highest sensitivities were observed when different diagnostic approaches were combined. The observed prevalences for T. trichiura, hookworm, A. lumbricoides, and S. stercoralis were 47.9%, 22.5%, 16.5%, and 10.8% after examining 3 stool samples. These values are close to the ‘true’ prevalences predicted by a mathematical model.

Conclusion/Significance

Rigorous epidemiologic surveillance of soil-transmitted helminthiasis in the era of preventive chemotherapy is facilitated by multiple stool sampling bolstered by different diagnostic techniques.     

The use of the anti-factor VIII method on trephine biopsies of the bone marrow for the identification of immature and atypical megakaryocytes in myeloproliferative diseases and allied disorders. A morphometric study

A morphometric analysis was performed on trephine biopsies of the bone marrow to identify atypical megakaryocyte proliferation following PAS staining and the immunohistological demonstration of factor VIII. This study includes nine patients with a megakaryoblastic crisis in chronic myeloid leukemia (CML), four with acute megakaryoblastic leukemia (AM) and three with myeloid dysplasia later evolving into overt acute leukemia. Comparison and statistical evaluation of the PAS reaction with anti-factor VIII staining reveals that the latter technique not only facilitates the recognition of immature and abnormal megakaryocytes, but leads to a significantly increased count for all megakaryocytic elements in the bone marrow. Thus our retrospective investigation of routinely processed and paraffin-embedded trephine biopsies shows that the diagnosis of a megakaryoblastic crisis in CML as well as AM may be easily established with the aid of the anti-factor VIII method. In all cases of megakaryoblastic proliferation in CML and AM, the appearance of blasts was associated with moderate to pronounced myelofibrosis which could be also determined by morphometry.     

The determinants of the molecular substitution process in turtles

Neutral rates of molecular evolution vary across species, and this variation has been shown to be related to biological traits. One of the first patterns to be observed in vertebrates has been an inverse relationship between body mass (BM) and substitution rates. The effects of three major life‐history traits (LHT) that covary with BM – metabolic rate, generation time and longevity (LON) – have been invoked to explain this relationship. However, most of the theoretical and empirical evidence supporting this relationship comes from endothermic vertebrates, that is, mammals and birds, in which the environmental conditions, especially temperature, do not have a direct impact on cellular and molecular biology. We analysed the variations in mitochondrial and nuclear rates of synonymous substitution across 224 turtle species and examined their correlation with two LHT (LON and BM) and two environmental variables [latitude (LAT) and habitat]. Our analyses indicate that in turtles, neutral rates of molecular evolution are hardly correlated with LON or BM. Rather, both the mitochondrial and nuclear substitution rates are significantly correlated with LAT – faster evolution in the tropics – and especially so for aquatic species. These results question the generality of the relationships reported in mammals and birds and suggest that environmental factors might be the strongest determinants of the mutation rate in ectotherms.     

Metastatic anaplastic oligodendroglioma simulating acute leukemia. A case report

BACKGROUND: Anaplastic oligodendroglioma (OG) is an uncommon tumor that rarely metastasizes outside the central nervous system. Spread to the bone marrow (BM) is so rare that when it occurs in the course of follow-up of a case of OG, a disseminated second primary tumor may be a more likely possibility unless BM examination provides evidence to the contrary. Potentially misleading cytologic features of metastatic anaplastic OG can be seen in a BM touch preparation. CASE: A 50-year-old man had undergone left frontal lobectomy in September 1999 for anaplastic OG and presented seven months later with evidence, on BM scan, of focal abnormal uptake at multiple sites. Bone marrow biopsy confirmed OG secondaries, which, on the touch preparation, appeared not only in clusters but also as single cells, simulating acute leukemia. CONCLUSION: The morphology of anaplastic OG metastatic to BM simulates acute leukemia, as seen on the BM touch preparation. This is relevant particularly in the context of anaplastic OG on follow-up. This diagnostic pitfall can be heightened if a BM aspirate rather than biopsy is performed. Metastatic OG can be added to the list of tumors that metastasize to BM as single cells.     

Diagnostic methodology is critical for accurately determining the prevalence of Ichthyophonus infections in wild fish populations

Several different techniques have been employed to detect and identify Ichthyophonus spp. in infected fish hosts; these include macroscopic observation, microscopic examination of tissue squashes, histological evaluation, in vitro culture, and molecular techniques. Examination of the peer-reviewed literature revealed that when more than 1 diagnostic method is used, they often result in significantly different results; for example, when in vitro culture was used to identify infected trout in an experimentally exposed population, 98.7% of infected trout were detected, but when standard histology was used to confirm known infected tissues from wild salmon, it detected ~50% of low-intensity infections and ~85% of high-intensity infections. Other studies on different species reported similar differences. When we examined a possible mechanism to explain the disparity between different diagnostic techniques, we observed non-random distribution of the parasite in 3-dimensionally visualized tissue sections from infected hosts, thus providing a possible explanation for the different sensitivities of commonly used diagnostic techniques. Based on experimental evidence and a review of the peer-reviewed literature, we have concluded that in vitro culture is currently the most accurate diagnostic technique for determining infection prevalence of Ichthyophonus , particularly when the exposure history of the population is not known.     

Parasitoids, predators and PCR: the use of diagnostic molecular markers in biological control of Arthropods

Abstract:  The polymerase chain reaction (PCR) revolutionized the field of diagnostics, and today it has routine applications in medical, veterinary, forensic and botanical sciences. The fields of biological control and insect pest management have generally been slow to adopt PCR-based diagnostics in comparison with other fields of science. However, there has been increasing interest in the use of molecular diagnostic tools in arthropod biological control. In applied entomology, molecular techniques have generally been used for insect identification and systematics; however, PCR-based techniques are increasingly becoming recognized as valuable tools in ecological studies. Here, we review research that has used PCR-based techniques for parasitoid and predator/prey identification and detection, and place these studies in the context of their contributions to biological control of arthropods. The status and future directions of diagnostic molecular markers in applied entomology and insect pest management are also discussed.     

The histochemical evaluation of the glycogen storage diseases. A review of techniques and their limitations

Synopsis Histochemical techniques have been applied to biopsies of liver and muscle in cases of glycogen storage disease in order to demonstrate the presence of glycogen and the various enzymes which are involved in the degradation and synthesis of glycogen. This review of techniques is based on the examination of tissues from a group of patients with glycogen storage disease, and the results have been correlated with biochemical assays on the same tissues. It is possible to differentiate between the various forms of glycogen storage disease with the exception of Types III and VI which, due to the vagaries of the phosphorylase reaction, have very similar staining characteristics. The techniques have also been applied successfully to blood films from which considerable information can be obtained.     

Critical evaluation of the use of cell cultures for inclusion in clinical trials of patients affected by collagen VI myopathies

Collagen VI myopathies (Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM), and myosclerosis myopathy) share a common pathogenesis, that is, mitochondrial dysfunction due to deregulation of the permeability transition pore (PTP). This effect was first identified in the Col6a1(-/-) mouse model and then in muscle cell cultures from UCMD and BM patients; the normalizing effect of cyclosporin A (CsA) confirmed the pathogenic role of PTP opening. In order to determine whether mitochondrial performance can be used as a criterion for inclusion in clinical trials and as an outcome measure of the patient response to therapy, it is mandatory to establish whether mitochondrial dysfunction is conserved in primary cell cultures from UCMD and BM patients. In this study we report evidence that mitochondrial dysfunction and the consequent increase of apoptotic rate can be detected not only, as previously reported, in muscle, but also in fibroblast cell cultures established from muscle biopsies of collagen VI-related myopathic patients. However, the mitochondrial phenotype is no longer maintained after nine passages in culture. These data demonstrate that the dire consequences of mitochondrial dysfunction are not limited to myogenic cells, and that this parameter can be used as a suitable diagnostic criterion, provided that the cell culture conditions are carefully established.     

DREAMing: a simple and ultrasensitive method for assessing intratumor epigenetic heterogeneity directly from liquid biopsies

Many cancers comprise heterogeneous populations of cells at primary and metastatic sites throughout the body. The presence or emergence of distinct subclones with drug-resistant genetic and epigenetic phenotypes within these populations can greatly complicate therapeutic intervention. Liquid biopsies of peripheral blood from cancer patients have been suggested as an ideal means of sampling intratumor genetic and epigenetic heterogeneity for diagnostics, monitoring and therapeutic guidance. However, current molecular diagnostic and sequencing methods are not well suited to the routine assessment of epigenetic heterogeneity in difficult samples such as liquid biopsies that contain intrinsically low fractional concentrations of circulating tumor DNA (ctDNA) and rare epigenetic subclonal populations. Here we report an alternative approach, deemed DREAMing (Discrimination of Rare EpiAlleles by Melt), which uses semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of epiallelic species at single-CpG-site resolution in fractions as low as 0.005%, providing facile and inexpensive ultrasensitive assessment of locus-specific epigenetic heterogeneity directly from liquid biopsies. The technique is demonstrated here for the evaluation of epigenetic heterogeneity at p14^ARF and BRCA1 gene-promoter loci in liquid biopsies obtained from patients in association with non-small cell lung cancer (NSCLC) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), respectively.     

Role of molecular markers in diagnosis and prognosis of renal cell carcinoma

It has been demonstrated that different renal cell neoplasms have characteristic morphologic and genetic features. Histologic subtyping of renal epithelial neoplasms has been shown to be of prognostic value; therefore they must be correctly classified. Although adequate sampling and a good understanding of the morphologic features usually minimize diagnostic errors, the use of immunohistochemical and chromosomal analysis on formalin-fixed, paraffin-embedded tissues can be necessary. These techniques can facilitate diagnosis on small biopsies, which are increasingly obtained from renal masses. An immunohistochemical panel including CD10, parvalbumin, AMACR, CK7 and S100A1 seems the most promising; fluorescence in situ hybridization analysis using centromeric probes to evaluate the gains and losses of the chromosomes can be helpful in selected cases. A wide variety of molecular markers have been examined, but further research is required to prove their value as prognostic tools.     

Fièvres prolongées d’origine inconnue

Prolonged fever of unknown origin (FUO) identifies a pattern of fever that defined in 1961. The identification of the cause of FUO is a challenge in clinical practice despite recent advances in diagnostic techniques. No standardized diagnostic strategy could be determined. The diagnostic process should be guided by the potential diagnostic clues (PDCs) emerging from the history, meticulous physical examination and baseline tests. A standardized flow chart can be applied only in absence of PDCs or when the PDCs are contradictory. In the absence of clues, a staged diagnostic protocol was used to search elements contributing to the diagnosis (CT scan, scintigraphies, endoscopies and systematic biopsies). When diagnosis was not established and patient deteriorated, empiric therapeutic trial were started to presumptive diagnoses. Recently, the role of 18-FDG-PET scan as been intensively evaluated as a second-step investigation technique, as a part of structured diagnostic protocol, early after the initial clinical work-up and baseline biology, radiology and ultrasonography. This approach is based on the fact that reaching a diagnosis is extremely difficult in patients with FUO and that this tracer accumulates in infectious, neoplastic and non-infectious inflammatory disorders.     

Biochemical-markers for the diagnosis of bone metastasis: a clinical review

The preferential metastasis of cancer cells to skeleton not only disrupts the process of bone remodeling and influences the therapeutic decision, but also results in severe complications. Although the current diagnosis of bone metastases (BM) relies on bone imaging techniques, they are not sensitive enough for early detection as well as they are invasive and expensive to use. Since factors derived from bone metabolism are potentially useful to diagnose metastatic bone disease in cancer patients, a number of clinical trials have been carried out on this area. Results suggest that higher levels of bone biomarkers are associated with an increased risk of BM. As a result, biochemical-markers are showing prospects in early diagnosis of BM. This review summarizes the available evidence on the clinical use of biochemical-markers in the diagnosis of various cancers with high incidence of BM including breast, prostate and lung.     

The clinical diagnosis of splenomegaly.

Assessing for the presence of splenomegaly is an important component of the physical examination. Although several methods of palpation and percussion of the spleen have been described, until recently they have not been validated by noninvasive imaging techniques such as ultrasonography, radionuclide scanning, and computed tomography that offer objective means to assess splenomegaly. We review the literature comparing various physical examination techniques with noninvasive imaging modalities and conclude that palpation and percussion of the spleen are complementary but frequently insensitive and that further studies are needed to evaluate the efficacy of specific diagnostic methods.     

Trephine lung biopsy

Forty-five percutaneous trephine lung biopsies using the Steel apparatus were performed on 38 patients. Tissue was obtained on 42 occasions (94%) leading to histological or culture diagnosis in 33 patients (87%). Pneumothoraces (12 patients), bleeding into the airways or pleural space (4 patients), and tumour seeding along the needle track (1 patient) occurred in 38% of biopsy attempts (45% of patients). In contrast to the Vim-Silverman technique, the Steel trephine appears to produce a higher tissue yield and superior specimens for histological study. Trephine lung biopsy is comparable to open lung biopsy in providing positive diagnoses. With anticipation and expeditious management of complications, trephine lung biopsy is both safe and useful in the diagnosis of pulmonary disease.     

Distribution, ultrastructural localization, and ontogeny of the core protein of a heparan sulfate proteoglycan in human skin and other basement membranes

A variety of heparan sulfate proteoglycans (HSPG) have been identified on cell surfaces and in basement membrane (BM). To more fully characterize HSPG in human skin BM, we used two monoclonal antibodies (MAb) directed against epitopes of the core protein of a high molecular weight HSPG isolated from murine EHS tumor. Indirect immunofluorescence revealed linear distribution of HSPG within all skin BM, and within BM of all other human organs investigated. In a study of the ontogeny of HSPG in human skin BM, HSPG was detectable as early as 54 gestational days, comparable with other ubiquitous BM components, such as laminin and type IV collagen. Immunoelectron microscopy on adult skin and neonatal foreskin showed staining primarily within the lamina densa (LD) and sub-lamina densa regions of the dermoepidermal junction (DEJ) and vascular BM. In neonatal foreskin, additional staining was noted of basilar cytoplasmic membranes of keratinocytes, endothelial cells, and pericytes. We conclude that the core protein of a high molecular weight HSPG is ubiquitous in human BM, appears in fetal skin on or before 54 days, and is present primarily in the regions of the LD and sub-LD.