Validation of a method to directly and specifically measure nitrite in biological matrices.

The bioactivity of nitric oxide (NO) is influenced by chemical species generated through reactions with proteins, lipids, metals, and its conversion to nitrite and nitrate. A better understanding of the functions played by each of these species could be achieved by developing selective assays able of distinguishing nitrite from other NO species. Nagababu and Rifkind developed a method using acetic and ascorbic acids to measure nitrite-derived NO in plasma. Here, we adapted, optimized, and validated this method to assay nitrite in tissues. The method yielded linear measurements over 1-300 pmol of nitrite and was validated for tissue preserved in a nitrite stabilization solution composed of potassium ferricyanide, N-ethylmaleimide and NP-40. When samples were processed with chloroform, but not with methanol, ethanol, acetic acid or acetonitrile, reliable and reproducible nitrite measurements in up to 20 sample replicates were obtained. The method's accuracy in tissue was ≈ 90% and in plasma 99.9%. In mice, during basal conditions, brain, heart, lung, liver, spleen and kidney cortex had similar nitrite levels. In addition, nitrite tissue levels were similar regardless of when organs were processed: immediately upon collection, kept in stabilization solution for later analysis or frozen and later processed. After ip nitrite injections, rapidly changing nitrite concentrations in tissue and plasma could be measured and were shown to change in significantly distinct patterns. This validated method could be valuable for investigations of nitrite biology in conditions such as sickle cell disease, cardiovascular disease, and diabetes, where nitrite is thought to play a role.

Subsite awareness in neuropathology evaluation of National Toxicology Program (NTP) studies: a review of select neuroanatomical structures with their functional significance in rodents.

This review article is designed to serve as an introductory guide in neuroanatomy for toxicologic pathologists evaluating general toxicity studies. The article provides an overview of approximately 50 neuroanatomical subsites and their functional significance across 7 transverse sections of the brain. Also reviewed are 3 sections of the spinal cord, cranial and peripheral nerves (trigeminal and sciatic, respectively), and intestinal autonomic ganglia. The review is limited to the evaluation of hematoxylin and eosin-stained tissue sections, as light microscopic evaluation of these sections is an integral part of the first-tier toxicity screening of environmental chemicals, drugs, and other agents. Prominent neuroanatomical sites associated with major neurological disorders are noted. This guide, when used in conjunction with detailed neuroanatomic atlases, may aid in an understanding of the significance of functional neuroanatomy, thereby improving the characterization of neurotoxicity in general toxicity and safety evaluation studies.

Onset of puberty and normal histological appearances of the reproductive organs in peripubertal female Göttingen minipigs.

In preclinical studies, it is important to know whether the animals used are sexually mature or not. Precise data have not yet been published, however, about the histological features of the female reproductive organs during the peripubertal period or about the age of acquisition of sexual maturity in the minipig. The histological characteristics of the genital organs of female control minipigs from toxicology studies were described and, based on the presence of ovarian corpora lutea, used to assess the age at which maturity was reached. Only 50% of females can be considered mature at about 6.5 months old (a body weight of 11.8 kg), and 100% were not mature until about 7.5 months old (13.1 kg), although it is said that females reach sexual maturity at the age of approximately 5 months, by the time the body weight is about 10 to 12 kg. The uterine weights of mature females were higher than 94.4 g, whereas the maximum weight reached in the immature females was 55.2 g. In contrast, the differences between immature and mature ovarian weights were not significant. The histological appearance of the mature vagina in the various stages of the estrous cycle is also described.

Design and validation of the biliary atresia research consortium histologic assessment system for cholestasis in infancy.

BACKGROUND & AIMS: Pathologists participating in the National Institutes of Health-sponsored Biliary Atresia Research Consortium (BARC) developed and then evaluated a standardized system for histologic reporting of liver biopsies from infants with cholestasis. METHODS: A set of 97 anonymous liver biopsy samples was sent to 10 pathologists at BARC centers. A semiquantitative scoring system that had 16 histologic features was developed and then used by the pathologists, who had no knowledge of clinical history, imaging results, or laboratory data. Interobserver agreement was evaluated statistically. Agreement on scoring of each feature and on the pathologists' diagnosis, compared with the final clinical diagnosis, was evaluated by using weighted kappa statistics. RESULTS: There was moderate to substantial interobserver agreement in identification of bile plugs in ducts, giant-cell transformation, extramedullary hematopoiesis, and bile duct proliferation. The pathologists' diagnosis of obstruction in clinically proven cases of biliary atresia (BA) ranged from 79%-98%, with a positive predictive value of 90.7%. Histologic features that best predicted BA, on the basis of logistic regression, included bile duct proliferation, portal fibrosis, and absence of sinusoidal fibrosis (each P<.0001). CONCLUSIONS: The BARC histologic assessment system identified features of liver biopsies from cholestatic infants, with good interobserver agreement, that might be used in diagnosis and determination of prognosis. The system diagnosed BA with a high level of sensitivity and identified infants with biliary obstruction with reasonable interobserver agreement. However, distinguishing between BA and disorders such as total parenteral nutrition-associated liver disease and alpha(1)-antitrypsin deficiency is not possible without adequate clinical information.

A method for utilizing automated machine learning for histopathological classification of testis based on Johnsen scores.

We examined whether a tool for determining Johnsen scores automatically using artificial intelligence (AI) could be used in place of traditional Johnsen scoring to support pathologists' evaluations. Average precision, precision, and recall were assessed by the Google Cloud AutoML Vision platform. We obtained testicular tissues for 275 patients and were able to use haematoxylin and eosin (H&E)-stained glass microscope slides from 264 patients. In addition, we cut out of parts of the histopathology images (5.0 × 5.0 cm) for expansion of Johnsen's characteristic areas with seminiferous tubules. We defined four labels: Johnsen score 1-3, 4-5, 6-7, and 8-10 to distinguish Johnsen scores in clinical practice. All images were uploaded to the Google Cloud AutoML Vision platform. We obtained a dataset of 7155 images at magnification 400× and a dataset of 9822 expansion images for the 5.0 × 5.0 cm cutouts. For the 400× magnification image dataset, the average precision (positive predictive value) of the algorithm was 82.6%, precision was 80.31%, and recall was 60.96%. For the expansion image dataset (5.0 × 5.0 cm), the average precision was 99.5%, precision was 96.29%, and recall was 96.23%. This is the first report of an AI-based algorithm for predicting Johnsen scores.

Artificial intelligence for cellular phenotyping diagnosis of nasal polyps by whole-slide imaging.

BACKGROUND: artificial intelligence (AI) for cellular phenotyping diagnosis of nasal polyps by whole-slide imaging (WSI) is lacking. We aim to establish an AI chronic rhinosinusitis evaluation platform 2.0 (AICEP 2.0) to obtain the proportion of inflammatory cells for cellular phenotyping diagnosis of nasal polyps and to explore the clinical significance of different phenotypes of nasal polyps on the WSI. METHODS: a total of 453 patients were enrolled in our study. For the development of AICEP 2.0, 179 patients (WSIs) were obtained from the Third Affiliated Hospital of Sun Yat-Sen University (3HSYSU) from January 2008 to December 2018. A total of 24,625 patches were automatically extracted from the regions of interest under a 400× HPF by Openslide and the number of inflammatory cells in these patches was counted by two pathologists. For the application of AICEP 2.0 in a prospective cohort, 158 patients aged 14-70 years old with chronic rhinosinusitis with nasal polyps (CRSwNP) who had undergone endoscopic sinus surgery at 3HSYSU from June 2020 to December 2020 were included for preoperative demographic characteristics. For the application of AICEP 2.0 in a retrospective cohort, 116 patients with CRSwNP who had undergone endoscopic sinus surgery from May 2016 to June 2017 were enrolled for the recurrence rate. The proportion of inflammatory cells of these patients on WSI was calculated by our AICEP 2.0. FINDINGS: for AICEP 2.0, the mean absolute errors of the ratios of eosinophils, lymphocytes, neutrophils, and plasma cells were 1.64%, 2.13%, 1.06%, and 1.22%, respectively. The four phenotypes of nasal polyps were significantly different in clinical characteristics (including asthma, itching, sneezing, total IgE, peripheral eosinophils%, tissue eosinophils%, tissue neutrophils%, tissue lymphocytes%, tissue plasma cells%, and recurrence rate; P <0.05), but there were no significant differences in age distribution, onset time, total VAS score, Lund-Kennedy score, or Lund-Mackay score. The percentage of peripheral eosinophils was positively correlated with the percentage of tissue eosinophils (r = 0.560, P <0.001) and negatively correlated with tissue lymphocytes% (r = -0.489, P <0.001), tissue neutrophils% (r = -0.225, P = 0.005), and tissue plasma cells% (r = -0.266, P = 0.001) in WSIs.

Greater interobserver agreement by endoscopic mucosal resection than biopsy samples in Barrett's dysplasia.

BACKGROUND & AIMS: Endoscopic mucosal resection (EMR) is an important diagnostic, staging, and therapeutic tool for patients with Barrett's esophagus (BE)-associated neoplasia. We analyzed the histopathologic characteristics of specimens collected during EMR compared with biopsy specimens from patients with BE and assessed interobserver variability in pathologists' assessment of EMR and biopsy specimens. METHODS: We evaluated EMR (n = 251) and biopsy (n = 269) specimens collected from patients with BE at 2 tertiary referral centers. A detailed histologic analysis was performed for each EMR and biopsy specimen to determine the grade of dysplasia, depth of the specimen, proportion of specimen with dysplasia, and quality of samples. Interobserver agreement for both biopsy and EMR specimens (among 4 experienced pathologists) was calculated by using kappa statistics. RESULTS: Histologic analysis showed that submucosa was present in the majority of EMRs, compared with biopsy specimens (88% vs 1%, P < .0001). Almost all biopsy specimens (99%) included lamina propria. However, the muscularis mucosa was observed in only 58% of biopsy specimens. For both EMR and biopsy specimens, the highest grade of dysplasia comprised < or =25% of the total area in >50% of the specimens. Interobserver agreement on the diagnosis of dysplasia was significantly greater for EMR specimens than biopsy specimens (low-grade dysplasia, 0.33 vs 0.22, P < .001; high-grade dysplasia, 0.43 vs 0.35, P = .018). CONCLUSIONS: Submucosa can be examined in most samples collected from EMR; the distribution of neoplasia is focal within biopsy and EMR specimens. There is more interobserver agreement among pathologists in the analysis of EMR samples than biopsy specimens for the diagnosis of dysplasia.

Diagnostic efficacy of midnight cortisol and midnight ACTH in the diagnosis and localisation of Cushing's syndrome.

Classical tests for diagnosis of Cushing's syndrome (CS) like urine free cortisol and dexamethasone suppression tests have limitations in various clinical settings. This study evaluated the usefulness of sleeping midnight serum cortisol (SMNC) as a diagnostic test for hypercortisolemia. A simultaneously done midnight plasma ACTH level was used to classify the disease as ACTH dependent or independent. Standard biochemical tests, SMNC, midnight plasma ACTH and appropriate imaging evaluated patients with a clinical suspicion of Cushing's syndrome. We evaluated 43 patients with CS comprising of 34 patients with Cushing's disease (CD), 2 patients with thymic carcinoid producing ectopic CS, 5 patients with adrenal carcinoma and 2 with adrenal adenoma. Thirteen patients with clinical suspicion were also evaluated with the above tests and CS was ruled out. SMNC, midnight plasma ACTH and dexamethasone suppressed cortisol was collected from patients with a suspicion of CS. SMNC was evaluated against histopathology as the gold standard. SMNC achieved 100% sensitivity in the diagnosis of endogenous CS at cut offs of 138 nmol/l and below. Raising the cut off to 207 nmol/l resulted in a test sensitivity of 90.5%. At a cut off of 1.65 pmol/l, midnight plasma ACTH could distinguish ACTH independent causes of CS with 100% sensitivity. We concluded that a single midnight collection could identify all patients with CS and classify the ACTH status at the proposed cut offs.

Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor end plates of a vertebrate twitch muscle.

Tritiated diisopropylfluorophosphate (DFP) was used to phosphorylate acetylcholinesterase (AChase) in the motor end plate of mouse sternomastoid muscle, and its distribution within the end plate was evaluated quantitatively by electron microscope radioautography. With the use of emulsion layers whose sensitivity to tritium had been calibrated, the density of AChase in different components of the end plate was calculated. The AChase was primarily localized (85%) in the junctional fold region. The concentration of AChase there was more than 20,000 active sites per cubic micron of tissue. The resolution of the technique was not sufficient to determine whether there was some AChase in the nerve end bulb; however, if there is any there, the concentration must be less than 10% of that at the junctional fold region.

[Values of cerebrospinal fluid cytochemistry in pediatric patients with ventriculoperitoneal shunt]. / Valores del análisis citoquímico de líquido cefalorraquídeo en pacientes con derivación ventrículo-peritoneal.

BACKGROUND: The ventriculo-peritoneal shunt (VPS) is the treatment for hydrocephalus, the cerebrospinal fluid (CSF) is evaluated for the management of its complications; however, information on the values of the cytochemistry in this population is insufficient. AIM: To describe the characteristics of the CSF cytochemistry of children in VPS management. METHODS: Descriptive observational study, developed in Bogotá (Colombia), from 2008 to 2016. VPS and related procedures records were reviewed. Patients between 6 months and 18 years were included. RESULTS: A total of 285 records were reviewed, 31 samples were entered. The CSF values were, respectively, for the median and 90% percentile: total leukocytes: 0 and 7 cells/mm3, neutrophils: 0 and 6.8 cells/mm3, lymphocytes: 0 and 2 cells/mm3, proteins: 13.4 and 67.2 mg/dL, glucose: 59 and 27.4 mg/dL. DISCUSSION: Glucose values evinced a normal rank towards the widest inferior limit with protein values exceeding the values expected. Cellularity is the variable with the lowest variation. CONCLUSIONS: The values of the CSF cytochemistry in patients with VPS are not comparable to those of the healthy population and should be interpreted according to the characteristics of this population.

Do pathologists agree with each other on the histological assessment of pT1b oesophageal adenocarcinoma?

Background: In early (T1) oesophageal adenocarcinoma (OAC), the histological profile of an endoscopic resection specimen plays a pivotal role in the prediction of lymph node metastasis and the potential need for oesophagectomy with lymphadenectomy. Objective: To evaluate the inter-observer agreement of the histological assessment of submucosal (pT1b) OAC. Methods: Surgical and endoscopic resection specimens with pT1b OAC were independently reviewed by three gastrointestinal pathologists. Agreement was determined by intraclass correlation coefficient for continuous variables, and Fleiss' kappa (κ) for categorical variables. Bland-Altman plots of the submucosal invasion depth were made. Results: Eighty-five resection specimens with pT1b OAC were evaluated. The agreement was good for differentiation grade (κ=0.77, 95% confidence interval (CI) 0.68-0.87), excellent for lymphovascular invasion (κ=0.88, 95% CI 0.76-1.00) and moderate for submucosal invasion depth using the Paris and Pragmatic classifications (κ=0.60, 95% CI 0.49-0.72 and κ=0.42, 95% CI 0.33-0.51, respectively). Systematic mean differences between pathologists were detected for the measurement of submucosal invasion depth, ranging from 297 µm to 602 µm. Conclusions: A substantial discordance was found between pathologists for the measurement of submucosal invasion depth in pT1b OAC. Differences may lead to an over- or underestimation of the lymph node metastasis risk, with grave implications for the treatment strategy. Review by a second gastrointestinal pathologist is recommended to improve differentiating between a favourable and an unfavourable histological profile.

Factors Affecting Ultrastructural Quality in the Prefrontal Cortex of the Postmortem Human Brain.

Electron microscopy (EM) studies of the postmortem human brain provide a level of resolution essential for understanding brain function in both normal and disease states. However, processes associated with death can impair the cellular and organelle ultrastructural preservation required for quantitative EM studies. Although postmortem interval (PMI), the time between death and preservation of tissue, is thought to be the most influential factor of ultrastructural quality, numerous other factors may also influence tissue preservation. The goal of the present study was to assess the effects of pre- and postmortem factors on multiple components of ultrastructure in the postmortem human prefrontal cortex. Tissue samples from 30 subjects were processed using standard EM histochemistry. The primary dependent measure was number of identifiable neuronal profiles, and secondary measures included presence and/or integrity of synapses, mitochondria, and myelinated axonal fibers. Number of identifiable neuronal profiles was most strongly affected by the interaction of PMI and pH, such that short PMIs and neutral pH values predicted the best preservation. Secondary measures were largely unaffected by pre- and postmortem factors. Together, these data indicate that distinct components of the neuropil are differentially affected by PMI and pH in postmortem human brain.

Complex I deficiency in Parkinson's disease frontal cortex.

A study of complex I (NADH:ubiquinone oxidoreductase) activity in Parkinson's disease (PD) brain has identified loss of activity only in substantia nigra although loss of activity of this enzyme has been identified in a number of non-brain tissues. We investigated this paradox by studying complex I and other complexes of the mitochondrial electron transport chain in frontal cortex from PD and aged control brain using a variety of assay conditions and tissue preparations. We found increasingly significant losses of complex I activity in PD frontal cortex as increasingly pure mitochondria were studied. Complexes II, III, and IV were comparable in PD and controls. Inclusion of bovine serum albumin in the assay increased enzyme activity but lessened discrimination between PD and controls. Complex I deficiency in PD brain is not confined to substantia nigra. Methodological issues are critical in demonstrating this loss of activity.

[Absolute and relative errors of individual determination of cytochemical indices in clinical practice].

The study assesses absolute and relative errors in the calculation of cytochemical indices in a precise patient. At present, the semiquantitative index (the mean cytochemical coefficient (MCC) after G. Astaldi and L. Verg) is widely used to evaluate the cytochemical activity of leukocytes. At the same time other indices, such as differentiating cytochemical coefficient (DCC) after I. G. Bondarenko, occasionally find application. Analysis of the results of simulation of an array containing 500 leukograms has demonstrated that the individual relative detection error for MCC ranges from 13 to 21% and that for DCC 20 to 51%. The results of the study enable an adequate estimation of the ways of expressing the cytochemical findings, which are proposed by different authors.

Differential diagnosis of well-differentiated squamous cell carcinoma from non-neoplastic oral mucosal lesions: New cytopathologic evaluation method dependent on keratinization-related parameters but not nuclear atypism.

BACKGROUND: The cytology of oral squamous cell carcinoma (SCC) is challenging because oral SCC cells tend to be well differentiated and lack nuclear atypia, often resulting in a false negative diagnosis. The purpose of this study was to establish practical cytological parameters specific to oral SCCs. METHODS: We reviewed 123 cases of malignancy and 53 of non-neoplastic lesions of the oral mucosa, which had been diagnosed using both cytology and histopathology specimens. From those, we selected 12 SCC and 4 CIS cases that had initially been categorized as NILM to ASC-H with the Bethesda system, as well as 4 non-neoplastic samples categorized as LSIL or ASC-H as controls, and compared their characteristic findings. After careful examinations, we highlighted five cytological parameters, as described in Results. Those 20 cytology samples were then reevaluated by 4 independent examiners using the Bethesda system as well as the 5 parameters. RESULTS: Five cytological features, (i) concentric arrangement of orangeophilic cells (indicating keratin pearls), (ii) large number of orangeophilic cells, (iii) bizarre-shaped orangeophilic cells without nuclear atypia, (iv) keratoglobules, and (v) uneven filamentous cytoplasm, were found to be significant parameters. All malignant cases contained at least one of those parameters, while none were observed in the four non-neoplastic cases with nuclear atypia. In reevaluations, the Bethesda system did not help the screeners distinguish oral SCCs from non-neoplastic lesions, while use of the five parameters enabled them to make a diagnosis of SCC. CONCLUSION: Recognition of the present five parameters is useful for oral SCC cytology. Diagn. Cytopathol. 2017;45:406-417. © 2017 Wiley Periodicals, Inc.

Biologically Relevant Heterogeneity: Metrics and Practical Insights.

Heterogeneity is a fundamental property of biological systems at all scales that must be addressed in a wide range of biomedical applications, including basic biomedical research, drug discovery, diagnostics, and the implementation of precision medicine. There are a number of published approaches to characterizing heterogeneity in cells in vitro and in tissue sections. However, there are no generally accepted approaches for the detection and quantitation of heterogeneity that can be applied in a relatively high-throughput workflow. This review and perspective emphasizes the experimental methods that capture multiplexed cell-level data, as well as the need for standard metrics of the spatial, temporal, and population components of heterogeneity. A recommendation is made for the adoption of a set of three heterogeneity indices that can be implemented in any high-throughput workflow to optimize the decision-making process. In addition, a pairwise mutual information method is suggested as an approach to characterizing the spatial features of heterogeneity, especially in tissue-based imaging. Furthermore, metrics for temporal heterogeneity are in the early stages of development. Example studies indicate that the analysis of functional phenotypic heterogeneity can be exploited to guide decisions in the interpretation of biomedical experiments, drug discovery, diagnostics, and the design of optimal therapeutic strategies for individual patients.

The relevance of tissue thiol histochemistry to diagnostic hematopathology.

Expression analyses suggest that alterations of the antioxidant state of some diffuse large B-cell lymphomas can assist prognosis; reversibly oxidized thiols may serve as a surrogate marker for identifying such cases. Little is known about the distribution of free thiols and reversibly oxidized thiols in human tissues. We developed a staining technique that enables visualization of tissue thiols in situ using bright field microscopy and validated it using gastrointestinal tissue specimens. We used our thiol staining technique to assess benign tonsillectomy and diffuse large B-cell lymphoma specimens. The gastrointestinal series revealed the presence of free thiols within epithelial cells and cells of the lamina propria. Staining for reversibly oxidized thiols was robust in gastric foveolar cells, intestinal goblet cells and the mucus they produce. Tonsillectomy specimens exhibited diffuse presence of free thiols. Staining for reversibly oxidized thiols was confined to germinal center macrophages and sinus histiocytes. Among the diffuse large B-cell lymphoma specimens, we observed strong staining for free thiols within malignant cells. By contrast to benign B-cells, the malignant cells demonstrated pronounced and diffuse staining for reversibly oxidized thiols. We demonstrated intrinsic differences between benign and malignant cells.

News from the Biological Stain Commission, no. 16.

In the 16(th) issue of News from the Biological Stain Commission (BSC) under the heading of Regulatory affairs, the Biological Stain Commission's International Affairs Committee presents information from the 28(th) meeting of CEN/TC 140 In vitro diagnostic medical devices held on October 23, 2013 in Berlin, Germany. Information is also presented from the 19(th) meeting of ISO/TC 212 Clinical laboratory testing and in vitro diagnostic test systems held on October 19 - 21, 2013 in Singapore.

External quality assessment for acid fast bacilli smear microscopy in eastern part of Ethiopia.

BACKGROUND: External quality assessment (EQA) of sputum smear microscopy is essential and indispensable component of any tuberculosis program. This study assessed the EQA of acid fast bacilli (AFB) smear microscopy through onsite evaluation, blinded rechecking and panel test. A one year study was conducted on eight health institution laboratories from December 2011 to December 2012. Onsite evaluation, blinded rechecking and panel tests were used to collect data. Data were analyzed using SPSS version 16. Sensitivity, specificity, predictive values, and proportions of false readings were calculated. The level of agreement was measured using Kappa (κ) value. RESULTS: Problems observed during onsite evaluation include shortages of materials, disinfectant, and poor storage and working condition. A total of 578 slides were collected for blinded rechecking, of which 102 (17.6%) were reported as positive by peripheral laboratories. The panel test revealed an overall error of 17 (25.25%) of which 14 (17.5%) were minor errors [low false negative 6 (7.5%) and low false positive 8 (10%)], and 3 (3.75%) were major errors (high false positive). The sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) of the peripheral laboratories were 83.5, 97.8, 91.7, and 95.7, respectively. The false readings at the peripheral laboratories were 32 (5.5%). Agreement on reading the slides was observed on 546 (94.5%) slides (K = 0.84, SE = 0.054). CONCLUSIONS: Lack of reagents, supplies, favorable working environment and AFB related technical problems were identified in the peripheral laboratories. High false negative error was found to be the predominant major error. A continuous and strong EQA scheme should be implemented to avoid reporting errors and produce quality sputum results.

Guidelines for the cytopathologic diagnosis of epithelioid and mixed-type malignant mesothelioma: Complementary Statement from the International Mesothelioma Interest Group, Also Endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology.

OBJECTIVE: To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma. DATA SOURCES: Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks. RATIONALE: This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.