Development of new and accurate measurement devices (TruSlice and TruSlice Digital) for use in histological dissection: an attempt to improve specimen dissection precision.

Histological dissection of human tissue has relied on conventional procedures, which have largely remained unchanged for decades. Practices to determine measurement parameters employed in these procedures have largely relied on the use of rulers and weighing scales. It is well documented in the scientific literature that both fixation and processing of tissue can significantly affect the viability of the of tissue sections both for tinctorial and immunocytochemical investigations. Both of these factors can be compounded in their negative effects by inappropriate sampling of tissue at histological cut up. There are five key factors to ensure good surgical grossing technique, flat uniformly perpendicular specimen cutting face, appropriate immobilisation of the tissue specimen during grossing, good visualisation of the cutting tissue face, sharp cutting knives and the grossing knife action. Meeting these factors implies the devices are fit for purpose. Here we describe an innovative approach to designing cut up devices to improve accuracy and precision, which take these five key requirements into consideration. The devices showed accuracy and precision, enabling tissue slices to be produced in a uniformly perpendicular fashion to within 2 mm in thickness and to enable consistency and reproducibility of performance across a series of tissue types. The application of a digital rule on one of these devices ensures accuracy and also enables quality control issues to be clearly assessed. As cellular pathology laboratories conform to ever increasing standards of compliance and performance in practice, the advent of assured precision and accuracy at cut up is awaited. Recommendations from accreditation bodies such as the United Kingdom Accreditation Service (UKAS) continue to push for improvements in this area of histological investigation. These newly designed devices may give the answers to these requirements and provide the impetus for a new generation of innovative equipment for histological dissection.

Towards the lean lab: the industry challenge.

In anatomic pathology, the current state encompassing the pre-analytic processes of tissue collection, handling, examination, preparation, processing, and storage are largely uncontrolled, inconsistently performed, and/or not standardized according to the sound scientific data. Pre-analytic defects result in nearly three-quarters of the problems in laboratory diagnostics. This is evident in quality surveys from well-respected institutions that document high miss rates in the required basics of information related to patient and tissue identity, let alone parameters documenting quality aspects related to the surgical specimen and its preservation. This talk will describe the historical approach to tissue processing and identify gaps from worldwide observations in current laboratory practices. It will also offer potential methodological and technological solutions and process improvements that laboratories may consider in serving the ultimate users of pathology information: the clinician and the patient. It illustrates the need for scientifically validated specimen guidelines and a performance based, standardized and documented "chain of custody" of the pre-analytical steps from the patient's body through fixation. For thought leaders and professional standard setters, opportunities for optimizing molecular studies exist in specimen collection, transfer, grossing, fixation, and decalcification protocols. In this evolving era of molecular profiling and personalized therapeutic decision-making, a well-reasoned and coordinated focus on pre-analytic processes that optimizes specimens for subsequent testing will result in: Improved specimen quality for molecular testing Improved accuracy of diagnostic and molecular test results Reduced Turnaroundtimes for same-day diagnosis Enhanced satisfaction of clinicians and patients.

Internal calibrants allow high accuracy peptide matching between MALDI imaging MS and LC-MS/MS.

One of the important challenges for MALDI imaging mass spectrometry (MALDI-IMS) is the unambiguous identification of measured analytes. One way to do this is to match tryptic peptide MALDI-IMS m/z values with LC-MS/MS identified m/z values. Matching using current MALDI-TOF/TOF MS instruments is difficult due to the variability of in situ time-of-flight (TOF) m/z measurements. This variability is currently addressed using external calibration, which limits achievable mass accuracy for MALDI-IMS and makes it difficult to match these data to downstream LC-MS/MS results. To overcome this challenge, the work presented here details a method for internally calibrating data sets generated from tryptic peptide MALDI-IMS on formalin-fixed paraffin-embedded sections of ovarian cancer. By calibrating all spectra to internal peak features the m/z error for matches made between MALDI-IMS m/z values and LC-MS/MS identified peptide m/z values was significantly reduced. This improvement was confirmed by follow up matching of LC-MS/MS spectra to in situ MS/MS spectra from the same m/z peak features. The sum of the data presented here indicates that internal calibrants should be a standard component of tryptic peptide MALDI-IMS experiments.

Robust automated tumour segmentation on histological and immunohistochemical tissue images.

Tissue microarray (TMA) is a high throughput analysis tool to identify new diagnostic and prognostic markers in human cancers. However, standard automated method in tumour detection on both routine histochemical and immunohistochemistry (IHC) images is under developed. This paper presents a robust automated tumour cell segmentation model which can be applied to both routine histochemical tissue slides and IHC slides and deal with finer pixel-based segmentation in comparison with blob or area based segmentation by existing approaches. The presented technique greatly improves the process of TMA construction and plays an important role in automated IHC quantification in biomarker analysis where excluding stroma areas is critical. With the finest pixel-based evaluation (instead of area-based or object-based), the experimental results show that the proposed method is able to achieve 80% accuracy and 78% accuracy in two different types of pathological virtual slides, i.e., routine histochemical H&E and IHC images, respectively. The presented technique greatly reduces labor-intensive workloads for pathologists and highly speeds up the process of TMA construction and provides a possibility for fully automated IHC quantification.

Flow cytometric immunphenotyping of epithelial cancer cells in effusions--technical considerations and pitfalls.

BACKGROUND: Data regarding the role of flow cytometry (FCM) in the characterization of malignant effusions are limited to date. In the present study, we optimized the conditions for FCM immunphenotyping of effusions using a four-color analysis and investigated aspects related to the advantages and limitations of this method in this setting. METHODS: FCM analysis optimization for the study of epithelial cells was undertaken using five carcinoma cell lines, and subsequently applied to malignant pleural and peritoneal effusions using antibodies against epithelial and mesothelial markers (Ber-EP4 and EMA), CD138, and integrin subunits. FCM of frozen versus fresh specimens and the performance of FCM compared to immunhistochemistry were evaluated. RESULTS: FCM optimization was achieved and applied to clinical specimens, with resulting detection of epithelial markers and adhesion molecules on cancer cells. Frozen clinical specimens and cell lines showed reduced CD138 expression compared to fresh specimens, with conservation of the remaining epitopes. FCM generally showed comparable performance to immunhistochemistry. CONCLUSIONS: FCM is an effective method for characterization of cancer cells in clinical effusion specimens in both the diagnostic and research setting, and is comparable to immunhistochemistry in terms of sensitivity and specificity, with the additional advantage of providing quantitative data. The majority of epitopes are conserved in frozen cells, but a minority may be lost, suggesting that the thorough testing of each antibody in both conditions is mandatory.

Two distinct events, section compression and loss of particles ("lost caps"), contribute to z-axis distortion and bias in optical disector counting.

Deformation of tissue sections in the z-axis can bias optical disector counting. When samples of particle densities are not representative for the entire tissue section, significant bias of estimated numbers can result. To assess the occurrence, prevalence, extent, sequence of events, and causes of z-axis distortion, the distribution of neuronal nucleoli in thick paraffin and vibratome sections was determined in chicken, rodent, and human brain tissues. When positions of neuronal nucleoli were measured in the z-axis, nucleoli were more frequent at the surfaces (bottom and top) of tissue sections than in the core. This nonlinear z-axis distribution was not lab-, equipment-, or investigator-specific, and was independent of age, fixation quality, coverslipping medium, or paraffin melting temperature, but in paraffin sections, was highly correlated with the tilt of the knife (cutting) angle. Manipulation of subsequent tissue processing steps revealed that two events contribute to z-axis distortion. Initially, a higher density of particles results at surfaces after sectioning, apparently due to section compression. Subsequently, particles can be lost to varying degrees from surfaces during floating or staining and dehydration, resulting in "lost caps." These results may explain different degrees of z-axis distortion between different types of sections and different labs, and reinforce the importance of checking z-axis distributions as a "quality control" prior to selection of guard zones in optical disector counting. Indirect approaches to assess section quality, such as resectioning in a perpendicular plane, yield additional artifacts, and should be replaced by a direct quantitative measurement of z-axis distribution of particles.

Effect of thin-section diffusion-weighted MR imaging on stroke diagnosis.

BACKGROUND AND PURPOSE: Diffusion-weighted (DW) imaging has limited spatial resolution, especially in the z direction. We decreased the section thickness of DW imaging to 3 mm to determine if this change improves the depiction of small infarcts and if it affects stroke diagnosis. METHODS: We studied conventional (5-mm section thickness, 1-mm intersection gap) and thin-section (3-mm section thickness, no intersection gap) DW imaging data in 49 patients with symptoms of acute cerebral ischemia. Two radiologists who were not aware of the clinical findings reviewed all images and diagnosed the stroke subtype according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) method. Accuracies of stroke diagnosis with an experienced neuroradiologist and with a second-year radiology resident were compared. To quantify lesion conspicuity, contrast-to-noise ratios (CNRs) were measured. The CNR of thin-section DW imaging was then divided by the CNR of conventional DW imaging to yield the relative CNR (rCNR). RESULTS: The experienced neuroradiologist made the correct final diagnoses in 78% of cases with conventional DW imaging, improving to 100% with thin-section DW imaging. The resident made the correct diagnoses in 71% of cases with conventional DW imaging, improving to 94% with thin-section DW imaging. Lesion conspicuity was improved on thin-section DW imaging (rCNR = 1.47 +/- 0.63), especially for supratentorial lesions (rCNR = 1.51 +/- 0.63). CONCLUSION: Compared with conventional DW imaging, thin-section DW imaging permitted better lesion conspicuity and more precise stroke diagnosis.

The role of liquid-based cytology in the investigation of breast lesions using fine-needle aspiration: a cytohistopathological evaluation.

INTRODUCTION: Liquid-based cytology processing of breast fine-needle aspiration (FNA) specimens has been used in our institution since 1999. The aim of this study was to investigate the role of liquid-based cytology (Thin-Prep(R)) in the evaluation of breast FNA specimens. PATIENTS AND METHODS: This study was carried out on 352 cases corresponding to equal number of women (134 malignant and 218 benign) examined from September 1999 to June 2003. A direct to vial study was planned. Aspirated material was directly immersed to cytolytR vial (Cytyc Corporation, Boxborough, MA). The specimens were transported to the laboratory non-refrigerated within 24 hr. Thin-Prep slides were made using the Thin-Prep 2000 processor. The remaining material was embedded in paraffin. RESULTS: Altogether, 352 cases were examined and unsatisfactory smears were observed in 14 (3.9 %) out of all cases. The histologic examination verified that 128 out of 131 total malignant cases and 203 out of 207 total benign cases were correctly diagnosed with the cytological technique. False positives were found in four cases and false negative in three cases. Liquid-based technique presented a sensitivity of 97.7%, a specificity of 98%, a positive predictive value (PPV) of 96.9%, a PNV of 98.5%, and an over all accuracy (OA) of 97.9%. CONCLUSIONS: The processing of FNA breast specimens using liquid-based cytology offers an accurate diagnostic tool. Liquid-based cytology could reduce the uncertainty based in the cytological evaluation of FNA, because it offers the possibility to embed material in paraffin and take tissue sections with histological appearance identical to those observed in routine histological sections. Moreover it offers the potential advantage of retraining extra material for the purpose of ploidy analysis and immunohistochemical study in order to offer information concerning the expression of prognostic markers such as p53, MIB-1, steroid hormone receptors, and EGFR.

A standardized method for brain-cutting suitable for both stereology and MRI-brain co-registration.

We have developed an agar-embedding method for brain-slicing that minimizes the geometrical distortions which arise from handling and slicing the fixed postmortem brain. To facilitate postmortem brain-magnetic resonance imaging (MRI) co-registration, each hemisphere is processed separately. We embed the fixed brain hemisphere with reference markers in agar. The block containing the brain and markers is sliced at a fixed interval using a rotary slicer. Each slice is photographed with a high-resolution digital camera. The digital images are realigned as a 3-dimensional volume via a control point-based registration method for multi-slice registration. The realigned multiple slices of the reconstructed postmortem hemisphere are then co-registered to corresponding slices of an in vivo reference MRI-volume. We illustrate these postmortem MRI-brain co-registration methods to correlate in vivo T2-weighted MRI hyperintensities in gray and white matter with underlying pathology. For design-based stereology, the volume of interest (VOI) is defined using reproducible anatomical boundaries. This method is suitable for stereologic measures of structures ranging from defined nuclei to whole brain.

Quantitative histopathological analysis of cervical intra-epithelial neoplasia sections: methodological issues.

OBJECTIVES: As part a Program Project to evaluate emerging optical technologies for cervical neoplasia, our group is performing quantitative histopathological analysis of biopsies from 1,800 patients. Several methodological issues have arisen with respect to this analysis: (1) Finding the most efficient way to compensate for staining intensity variation with out losing diagnostic information; (2) Assessing the inter- and intra-observer variability of the semi-interactive data collection; and (3) the use of non-overlapping cells from the intermediate layer only. METHODS: Non-overlapping quantitatively stained nuclei were selected from 280 samples with histopathological characteristics of normal (199), koilocytosis (37), CIN 1 (18), CIN 2 (10) and CIN 3 (16). Linear discriminant analysis was used to assess the diagnostic information in three different feature sets to evaluate and compare staining intensity normalization methods. Selected feature values and summary scores were used to evaluate intra- and inter-observer variability. RESULTS: The features normalized by the internal subset of the imaged cells had the same discriminatory power as those normalized by the control cells and by both normalization methods seem to have additional discriminatory power over the set of features which do not require normalization. The use of the internal subset decreased the image acquisition time by approximately 50% at each center, respectively. The intra- and inter-observer variability was of a similar size. Good performance was obtained by measuring the intermediate layer only. CONCLUSION: The use of intensity normalization from a subset of the imaged non-overlapping intermediate layer cells works as well as or better than any of the other methods tested and provides a significant timesaving. Our intra- and inter-observer variability do not seem to affect the diagnostic power of the data. Although this must be tested in a larger data set, the use of intermediate layer cells only may be acceptable when using quantitative histopathology.

Ideal cooling process for paraffin-embedded tissues.

Back in the seventies everybody was convinced that it was no longer necessary to cool paraffinembedded tissues, because of the new advances in the production of paraffin. The reason for this assumption was the addition of plastic polymers and dimethyl sulfoxide. The quality of tissue sectioning improved because of these additives. The daily routine in the histology laboratories shows that it is impossible to produce good quality cutting without cooling. Cooling the paraffin by means of cooling plates or ice dishes creates drastic improvement; the cutting quality improves and it is much easier. Both improvements speed up the process and save time for the technicians and the physician. Long-term study indicates that not only the temperature but also the methodology of cooling and the cooling rate are playing a decisive role in the cutting quality.

Revised guides for organ sampling and trimming in rats and mice--part 1.

This is the first part of a series of three articles on trimming instructions of rat and mouse protocol organs and tissues in regulatory type toxicity studies. It is based on the experience made in the European RITA and American NACAD working groups and is an extended revision of trimming guides published in 1995 (Bahnemann et al.). The optimum localization for tissue preparation, the sample size, the direction of sectioning and the number of sections to be prepared is described organ by organ. These descriptions are illustrated for each organ by a schematic drawing and a macro-photograph showing the plane of section as well as a low power view of the H&E stained slide demonstrating the optimum "end-product". This revision will improve the quality and efficiency of routine procedures and facilitate daily work in the histotechnical lab. It will promote intra- and inter-study reproducibility and comparability and thus lead to a further coherence within each study and improvement of the validity of historical control data.

Differential tissue shrinkage and compression in the z-axis: implications for optical disector counting in vibratome-, plastic- and cryosections.

The optical disector is among the most efficient cell counting methods, but its accuracy depends on an undistorted particle distribution in the z-axis of tissue sections. Because the optical disector samples particle densities exclusively in the center of sections, it is essential for unbiased estimates of particle numbers that differential shrinkage or compression (and resulting differences in particle densities along the z-axis) are known and corrected. Here we examined, quantified, and compared differential shrinkage and compression of vibratome-, celloidin- and cryosections. Vibratome sections showed a significant z-axis distortion, while celloidin- and cryosections were minimally distorted. Results were directly compared with previous data obtained from paraffin and methacrylate sections. We conclude that z-axis distortion varies significantly between embedding and sectioning methods, and that vibratome-, methacrylate- and paraffin sections can result in grossly biased estimates. We describe a simple method for assessing differential z-axis shrinkage or compression, as well as simple strategies to minimize the bias of the optical disector. Minimal bias can be achieved by either adjusting the placement and extent of counting boxes and guard spaces for sampling, or by applying a correction factor in cases when guard spaces are deemed essential for particle recognition.

Electron tomographic analysis of frozen-hydrated tissue sections.

Electron tomography of frozen-hydrated tissue sections enables analysis of the 3-D structure of cell organelles in situ and in a near-native state. In this study, 160-200-nm-thick sections were cut from high-pressure frozen rat liver, and improved methods were used for handling and mounting the sections. Automated data collection facilitated tilt-series recording at low electron dose (approximately 4000 e(-)/nm(2) at 400 keV). Higher doses (up to 10,000 e(-)/nm(2)) were found to increase contrast and smooth out surface defects, but caused section distortion and movement, with likely loss of high-resolution information. Tomographic reconstruction showed that knife marks were 10-40 nm deep and located on the "knife face" of the section, while crevices were 20-50 nm deep and found on the "block face." The interior of the section was normally free of defects, except for compression, and contained useful structural information. For example, the topology of mitochondrial membranes in tissue was found to be very similar to that in frozen-hydrated whole mounts of isolated mitochondria. In rare cases, a 15-nm banding pattern perpendicular to the cutting direction was observed in the interior of the section, most evident in the uniformly dense, protein-rich material of the mitochondrial matrix.

Counting particles in tissue sections: choices of methods and importance of calibration to minimize biases.

Investigators must choose between counting methods to quantify microscopic particles in tissues. The conventional profile-based ("model-based" or "2D-") counting methods have been criticized for their potential biases due to assumptions about shapes, sizes, and orientation of particles when converting profile counts into cell numbers. New stereological methods ("design-based" or "3D-") methods such as the optical disector or physical disector were initially introduced as being inherently unbiased. Recent calibration analyses and comparisons of results from different investigators have revealed the potential for significant biases in the most efficient and most frequently used design-based method, the optical disector. This review aims to objectively assess the strengths and limitations of current profile- and disector-based cell counting methods by examination of studies in which these methods have been calibrated against the "gold-standard", counts obtained by 3-dimensional reconstruction of serial sections. Advantages and disadvantages of each counting method and the associated embedding and sectioning techniques are compared and frequent mistakes and pitfalls of each technique are discussed. The importance of a calibration step for each technique is emphasized, and a protocol is provided for a quick and simple calibration by a "sampling" 3-D reconstruction of limited serial sections. Trends in the usage of counting methods are analyzed in four major journals. It is hoped that this review will be helpful, for both investigators and manuscript reviewers, in clarifying some of the contentious issues in the choice and implementation of appropriate methods for particle counting in tissue sections.

Effectiveness of thin-layer preparations vs. conventional Pap smears in a blinded, split-sample study. Extended cytologic evaluation.

OBJECTIVE: To further evaluate the effectiveness of the AutoCyte PREP thin-layer slide preparation (TriPath Imaging, Inc., Burlington, North Carolina) as compared to conventional Pap smears. STUDY DESIGN: A split-sample, blinded evaluation of matched thin-layer preparations and conventional smears from 2,438 patients was performed. This material was enriched by including 260 cases of high grade squamous intraepithelial lesions (HSILs) and cancer cases from an earlier study. Many of these cases were difficult to diagnose, containing very few abnormal cells on one or both matching slides. The preparations were evaluated multiple times by both thin-layer-inexperienced and -experienced cytology professionals to better compare performance related to preparation quality alone. RESULTS: The initial evaluations of the slides by personnel with only brief training in thin-layer interpretation demonstrated equivalent performance for the two preparations. The reevaluation study by cytology professionals with several months of thin-layer experience demonstrated a statistically significant improvement in detection of both LSIL and HSIL lesions using AutoCyte PREP slides. There was also a statistically significant improvement in the number of satisfactory samples using the AutoCyte PREP method. CONCLUSION: The study demonstrated that the AutoCyte PREP thin-layer slide preparation is at least equivalent to conventional Pap smears in the detection of LSIL and HSIL, even when evaluated by cytology professionals who have been newly trained in the thin-layer method and that, with increased experience, the thin-layer AutoCyte PREP slide preparation method showed a statistically significant improvement in disease detection.

Whiplash injury determination with conventional spine imaging and cryomicrotomy.

STUDY DESIGN: Soft tissue-related injuries to the cervical spine structures were produced by use of intact entire human cadavers undergoing rear-end impacts. Radiography, computed tomography, and cryomicrotomy techniques were used to evaluate the injury. OBJECTIVES: To replicate soft tissue injuries resulting from single input of whiplash acceleration to whole human cadavers simulating vehicular rear impacts, and to assess the ability of different modes of imaging to visualize soft tissue cervical lesions. SUMMARY OF BACKGROUND DATA: Whiplash-associated disorders such as headache and neck pain are implicated with soft tissue abnormalities to structures of the cervical spine. To the authors' best knowledge, no previous studies have been conducted to determine whether single cycle whiplash acceleration input to intact entire human cadavers can result in these soft tissue alterations. There is also a scarcity of data on the efficacy of radiography and computed tomography in assessing these injuries. METHODS: Four intact entire human cadavers underwent single whiplash acceleration (3.3 g or 4.5 g) loading by use of a whole-body sled. Pretest and posttest radiographs, computed tomography images, and sequential anatomic sections using a cryomicrotome were obtained to determine the extent of trauma to the cervical spine structures. RESULTS: Routine radiography identified the least number of lesions (one lesion in two specimens). Although computed tomography was more effective (three lesions in two specimens), trauma was not readily apparent to all soft tissues of the cervical spine. Cryomicrotome sections identified structural alterations in all four specimens to lower cervical spine components that included stretch and tear of the ligamentum flavum, anulus disruption, anterior longitudinal ligament rupture, and zygopophysial joint compromise with tear of the capsular ligaments. CONCLUSIONS: These results clearly indicate that a single application of whiplash acceleration pulse can induce soft tissue-related and ligament-related alterations to cervical spine structures. The pathologic changes identified in this study support previous observations from human volunteers observations with regard to the location of whiplash injury and may assist in the explanation of pain arising from this injury. Although computed tomography is a better imaging modality than radiography, subtle but clinically relevant injuries may be left undiagnosed with this technique. The cryomicrotome technique offers a unique procedure to understand and compare soft tissue-related injuries to the cervical anatomy caused by whiplash loading. Recognition of these injuries may advance the general knowledge of the whiplash disorder.

A valid quantitative histochemical technique for assaying cytochrome c oxidase in single cells.

A quantitative histochemical method for assaying cytochrome c oxidase (COX) has been validated with two new findings concerning the optimal tissue thickness and a suitable substrate. The kinetics of a COX-catalysed reaction coupled to the oxidation of diaminobenzidine (DAB) were followed at 37 degrees C in single muscle fibres in unfixed sections of mouse gastrocnemius using a real-time image analysis system. The optimum composition of the substrate medium for the reaction was 0.1 mM reduced cytochrome c, 4 mM DAB, 2% dimethylsulphoxide, 2% polyvinyl alcohol and 0.1 mM HEPES buffer, final pH 7.5. The absorbances at 451 nm of the final reaction products, DAB polymer oxides, deposited in the intermyofibrillar mitochondria increased linearly as a function of incubation time for at least 80 s after the start of incubation. The initial velocities (v(i)) of the COX reaction calculated from the gradients of the linear regression best fits for times between 40 and 60 s were reproducible. The v(i) determined in single muscle fibres at a saturated concentration of cytochrome c (0.1 mM) were proportional to section thickness for thicknesses less than 3 microns, but they decreased exponentially when the thickness was greater than 4 microns. Thus, for the quantitative assay, unfixed sections 3 microns thick must be used. The Michaelis constants (Km) determined for commercial cytochrome c in the range of 20-26 microM for COX in three types of skeletal muscle fibres of mouse gastrocnemius were higher than the corresponding in situ Km (12-13 microM) for reduced cytochrome c. However, the Km values for commercial cytochrome c were in good agreement with the value previously determined with homogenates of rat hind limb muscle. Therefore, reduced cytochrome c is a more suitable substrate for the kinetic study and assay of COX in situ.