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.

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.

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.

Intraoperative pathologic consultation. An audit of 1,000 recent consecutive cases.

In order to assess the accuracy of the intraoperative consultation, or "frozen section," a retrospective review of 1,000 consecutive intraoperative consultations was performed. We present these data as a quality assurance assessment and as an update to a body of older literature examining the accuracy of the frozen section. The indications for intraoperative consultation and types of specimen submitted have changed in recent years, and these changes are related to the results in the present study. The increasingly important role of intraoperative cytology as an adjunct to and, in many cases, a replacement for frozen section is also emphasized.

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.

Guidelines for practical utilization of intraoperative frozen sections.

We reviewed 4057 intraoperative frozen sections from 1980 through 1984 to assess the accuracy, strengths, and weaknesses of this technique. Breast, lymph node, and skin comprised half of the sites evaluated. Frozen-section and final diagnoses agreed in 91.5% and disagreed in 6.8% of the cases; 1.7% of the cases were deferred. False-negative frozen-section diagnoses were due to pathologist sampling or judgment errors and surgeon sampling errors. There were eight (0.15%) false-positive diagnoses, none of which altered patient treatment. We recommend that lymph nodes for lymphoproliferative disorders and breast tissue for which a malignant diagnosis will not result in an immediate mastectomy not be submitted for frozen-section diagnosis. Appropriate studies of these tissues can be carried out without an intraoperative diagnosis; such a policy will increase the cost-effectiveness of frozen sections without compromising patient care.

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.

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.

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.

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.

A determination of thickness and surface relief in reembedded sections of an epoxy- and a melamine-resin containing ferritin as size standard.

Chemical and physical data of two electron microscopic embedding media (the non-polar epoxy resin Epon 812 and the polar melamine resin Nanoplast FB 101) suggest that less kinetic energy must be applied for cutting a section from a Nanoplast block than from an Epon block of the same hardness and that, consequently, the cutting qualities of Nanoplast are better. To test this hypothesis, normal and extremely thin sections of Epon- and Nanoplast-embedded horse spleen ferritin micropellets were reembedded and resectioned for a determination of thickness and surface roughness. The ease with which extremely thin sections can be cut from the Nanoplast resin (8 nm versus 15 nm in Epon) and the smooth surface of these sections support the hypothesis that the cutting quality of an embedding material is determined primarily by its energy balance, i.e. by the kinetic energy which must be introduced for sectioning and the bonding energy which is released exothermically from a polymer while being sectioned.

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.

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.

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.

RITA--registry of industrial toxicology animal-data--guides for organ sampling and trimming procedures in rats.

RITA (Registry of Industrial Toxicology Animal-data) is a pathology data base for the collection of validated histopathological data on tumours and potentially pre-neoplastic lesions observed in laboratory rodents. To enable a better comparison of information, standardized techniques for the preparation of histological slides have been established for all organs. The current paper describes the guidelines for organ sampling and trimming procedures applied in the RITA project, i.e. the number of sections to be taken, the direction in which an organ should be cut, the localization (anatomical site) from which a sample should be taken, and the size of an organ (or part of an organ) to be placed in cassettes for processing. Schematical illustrations and additional explanations are provided to support the proposed standardized procedures in histology laboratories.

Standardization of fixation, processing and staining methods for the central nervous system of vertebrates.

This paper reports the standardization of methods used for processing and embedding various vertebrate brains of different size in paraffin. Other technical details developed for avoiding frequent difficulties arising during laboratory routine are also reported. Some modifications of the Nissl and Klüver-Barrera staining methods are proposed. These modifications include: 1) a Nissl stain solution with a rapid and efficient action with easier differentiation; 2) the use of a cheap microwave oven for the Klüver-Barrera stain. These procedures have the advantage of permitting Nissl and Klüver-Barrera staining of nervous tissue in about five and fifteen minutes respectively. The proposed procedures have been tested in brains obtained from fish, amphibians, reptiles and mammals of different body sizes. They are the result of our long experience in preparing slides for comparative studies. Serial sections of excellent quality were regularly obtained in all the specimens studied. These standardized methods, being simple and quick, are recommended for routine use in neurobiological laboratories.

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.