A Large Postmortem Database of COVID-19 Patients Can Inform Disease Research and Public Policy Decision Making.

CONTEXT.­: Autopsies performed on COVID-19 patients have provided critical information about SARS-CoV-2's tropism, mechanisms of tissue injury, and the spectrum of disease. OBJECTIVE.­: To provide an updated database of postmortem disease in COVID-19 patients, assess relationships among clinical and pathologic variables, evaluate the accuracy of death certification, and correlate disease variables to causes of death. DESIGN.­: The 272 postmortem examinations reported in this paper were submitted by 14 pathologists from 9 medical or forensic institutions across the United States. The study spans the eras of the 3 principal COVID-19 strains and incorporates surveyed demographic, clinical, and postmortem data from decedents infected with SARS-CoV-2, including primary and contributing causes of death. It is the largest database of its kind to date. RESULTS.­: Demographics of the decedents reported here correspond well to national statistics. Primary causes of death as determined by autopsy and official death certificates were significantly correlated. When specifically cited disease conditions found at autopsy were correlated with COVID-19 versus non-COVID-19 death, only lung findings characteristic of SARS-CoV-2 infection or the absence of lung findings were significantly associated. CONCLUSIONS.­: Changes in hospitalization and disease likely stem from longer lifespans after COVID-19 diagnosis and alteration in treatment approaches. Although Omicron variants preferentially replicate in the upper airways, autopsied patients who died of COVID-19 in that time period showed the same lung damage as earlier decedents. Most importantly, findings suggest that there are still unelucidated risk factors for death from COVID-19 including possibly genetic susceptibility.

Undiagnosed Malignancy and Therapeutic Complications in Oncology Patients.

CONTEXT.­: Despite technologic and medical advancements, autopsies are essential to uncover clinically unsuspected diagnoses, to advance our understanding of disease processes, and to help reduce medical errors. OBJECTIVE.­: To investigate the percentage of malignancy clinically diagnosed and undiagnosed in a series of hospital autopsies. Secondarily, to explore the therapeutic complications directly contributing to death in cancer patients. DESIGN.­: A 10-year retrospective study (2008-2018). All nonforensic autopsies performed at the University of Vermont Medical Center during this period were reviewed by 2 pathologists, and data, including antemortem diagnoses of malignancy, and autopsy findings, including therapeutic complications, were collected. RESULTS.­: A total of 246 cases documented a diagnosis of malignancy. In 34.5% (85 of 246) of cases a tissue diagnosis of malignancy was first documented following postmortem examination. In 41.2% (35 of 85) of cases there was clinical antemortem suspicion of malignancy, whereas in 58.8% (50 of 85) clinically unsuspected malignancy was first diagnosed after postmortem examination. In 16.0% (8 of 50) of cases the undiagnosed malignancy was the primary cause of death. The overall rate of therapeutic complication related to the treatment of oncologic disease in patients that resulted in death was 21.7% (35 of 161). CONCLUSIONS.­: Our study shows the percentage of clinically unsuspected malignancies revealed by postmortem examination to be 5% (50 of 1003) of all autopsy cases. In 16% (8 of 50) of cases, the cause of death was due to the clinically undiagnosed malignancy, and hence not to an incidental finding. Despite advances in medical therapy in the management of oncologic disease, in up to 21.7% (35 of 161) of cases therapeutic complications directly contributed to death.

A Postmortem Portrait of the Coronavirus Disease 2019 (COVID-19) Pandemic: A Large Multi-institutional Autopsy Survey Study.

CONTEXT.­: This study represents the largest compilation to date of clinical and postmortem data from decedents with coronavirus disease 2019 (COVID-19). It will augment previously published small series of autopsy case reports, refine clinicopathologic considerations, and improve the accuracy of future vital statistical reporting. OBJECTIVE.­: To accurately reflect the preexisting diseases and pathologic conditions of decedents with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection through autopsy. DESIGN.­: Comprehensive data from 135 autopsy evaluations of COVID-19-positive decedents is presented, including histologic assessment. Postmortem examinations were performed by 36 pathologists at 19 medical centers or forensic institutions in the United States and Brazil. Data from each autopsy were collected through the online submission of multiple-choice and open-ended survey responses. RESULTS.­: Patients dying of or with COVID-19 had an average of 8.89 pathologic conditions documented at autopsy, spanning a combination of prior chronic disease and acute conditions acquired during hospitalization. Virtually all decedents were cited as having more than 1 preexisting condition, encompassing an average of 2.88 such diseases each. Clinical conditions during terminal hospitalization were cited 395 times for the 135 autopsied decedents and predominantly encompassed acute failure of multiple organ systems and/or impaired coagulation. Myocarditis was rarely cited. CONCLUSIONS.­: Cause-of-death statements in both autopsy reports and death certificates may not encompass the severity or spectrum of comorbid conditions in those dying of or with COVID-19. If supported by additional research, this finding may have implications for public health decisions and reporting moving forward through the pandemic.

Insights into pathogenesis of fatal COVID-19 pneumonia from histopathology with immunohistochemical and viral RNA studies.

INTRODUCTION: We describe post-mortem pulmonary histopathologic findings of COVID-19 pneumonia in patients with a spectrum of disease course, from rapid demise to prolonged hospitalisation. METHODS AND RESULTS: Histopathologic findings in post-mortem lung tissue from eight patients who died from COVID-19 pneumonia were reviewed. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were performed to detect virus. Diffuse alveolar damage (DAD) was seen in all cases with a spectrum of acute phase and/or organising phase. IHC with monoclonal antibodies against SARS-CoV-2 viral nucleoprotein and spike protein detected virus in areas of acute but not organising DAD, with intracellular viral antigen and RNA expression seen predominantly in patients with duration of illness less than 10 days. Major vascular findings included thrombi in medium- and large-calibre vessels, platelet microthrombi detected by CD61 IHC and fibrin microthrombi. CONCLUSIONS: Presence of SARS-CoV-2 viral RNA by NGS early in the disease course and expression of viral antigen by IHC exclusively in the acute, but not in the organising phase of DAD, suggests that the virus may play a major role in initiating the acute lung injury of DAD, but when DAD progresses to the organising phase the virus may have been cleared from the lung by the patient's immune response. These findings suggest the possibility of a major change during the disease course of COVID-19 pneumonia that may have therapeutic implications. Frequent thrombi and microthrombi may also present potential targets for therapeutic intervention.

Autopsy Service Death Certificate Review.

CONTEXT.­: Despite the importance of accurate death statistics for epidemiologic studies and public health initiatives, there remains a high frequency of errors in death certification. This deficiency can be addressed by the hospital autopsy service. OBJECTIVES.­: To improve the quality and accuracy of death certificates issued in the hospital and improve resident and clinician education by initiating a death certificate review process, performed by pathology residents while on their hospital autopsy rotation. DESIGN.­: A resident reviewed all death certificates issued in the hospital daily through the state electronic death certificate filing system and correlated with the decedent's medical record. When errors were found, the resident filed an amended death certificate with the state. If applicable, the Office of the Medical Examiner was contacted to investigate. The original certifying physician was then contacted via email with an explanation for the amendment. RESULTS.­: In 12 months, 590 death certificates were issued by the hospital. Eighty-eight of 590 (15%) were amended. Of those 88 amended, 41 (47%) were missing an underlying cause of death, 7 (8%) had an inaccurate cause of death, 41 (47%) failed to include relevant contributory causes of death, and 17 (19%) had major typographic errors. Of 88, 24 (27%) fell under the Office of the Medical Examiner's jurisdiction and were reported with a subsequent change in the manner of death in 23 of 88 cases (26%). CONCLUSIONS.­: Death certificate review by the autopsy service improves the accuracy of death certification, impacts resident and clinician education, and serves as quality assurance for both the hospital and the state.

Langerhans cell histiocytosis limited to the female genital tract: A review of literature with three additional cases.

•LCH of the female reproductive tract has four patterns of involvement.•A comprehensive literature review revealed 35 cases of pure genital LCH.•We report two new cases of pure LCH lesions of the vulva and one of the cervix.•Treatment of LCH varies and there is no standard for pure genital involvement.•Prognosis of LCH confined to the gynecologic tract appears to be favorable.

Double trouble: para-neoplastic anti-PCA-2 and CRMP-5-mediated small fibre neuropathy followed by chorea associated with small cell lung cancer and evolving radiological features.

Patients with Purkinje cell cytoplasmic autoantibody type 2 (PCA-2) and collapsin response-mediator protein-5 (CRMP-5) autoantibody can present with multifocal elements of encephalomyeloneuropathy. Except for an anecdotal report, case descriptions of paraneoplastic small fibre neuropathy are lacking. We report paraneoplastic small fibre neuropathy followed by chorea associated with small cell lung cancer. A man aged 57 years with a 35 pack-year smoking history presented with painless subacute paresthesia and weight fluctuation. A non-length-dependent small fibre neuropathy was confirmed by skin biopsy. Further testing revealed positive serum PCA-2 and CRMP-5 autoantibodies, which after positron emission tomography-CT led to histological confirmation of a small cell lung cancer. Initially, abnormal MRI and cerebrospinal fluid studies suggested central nervous system (CNS) involvement which was subclinical; however, 6 months later during antitumour therapy, the patient became symptomatic with choreoathetosis. After combined chemoradiation as well as immunosuppressive and symptomatic therapies, the clinical course stabilised, although residual neurological deficits remained at follow-up a year later. Coexistent PCA-2 and CRMP-5 autoantibodies may occur in the setting of small fibre peripheral neuropathy and choreoathetosis and predict cancer type. Two paraneoplastic syndromes can present successively over months; subclinical CNS involvement with evolving basal ganglia abnormalities can be a paraneoplastic manifestation. In the appropriate clinical setting, paraneoplastic testing should be considered in patients presenting with small fibre neuropathy.

Primary vaginal mucinous adenocarcinoma of intestinal type, associated with intestinal metaplasia of Skene ducts in a diethylstilbestrol-exposed woman.

OBJECTIVES: Primary mucinous vaginal adenocarcinoma of intestinal type is an extremely rare malignancy of uncertain histogenesis, which makes for a diagnostic challenge. We report a case and describe the histopathologic features and the unusual immunoprofile of this rare entity. METHODS: We report a case of vaginal mucinous adenocarcinoma of intestinal type in a diethylstilbestrol-exposed woman in which intestinal metaplasia of the Skene duct was found at the time of recurrence. RESULTS: As the histogenesis of primary vaginal intestinaltype adenocarcinomas remains uncertain, the finding of Skene duct metaplasia in association with invasive adenocarcinoma lends support to the origin of vaginal mucinous adenocarcinomas of intestinal type to be metaplasia, at least in some cases. Such an origin accounts for the unusual immunohistochemical profile, which raises concern for a metastatic adenocarcinoma of gastrointestinal origin. CONCLUSIONS: Recognition of this rare entity is important, particularly to avoid the pitfall of misdiagnosing metastatic disease.

Increased utilization, verification, and clinical implications of immunocytochemistry: Experience in a northern New England hospital.

BACKGROUND: We report our experience in utilization, verification, and clinical implications of antibodies for use in diagnostic immunocytochemistry (ICC). METHODS: A computer search identified cytology cases utilizing ICC and corresponding surgical pathology material. Alcohol-fixed liquid based cytology (LBC) specimens were generated from surgical pathology bench specimens. ICC on LBC and immunohistochemistry on formalin fixed paraffin embedded tissue (FFPE) were performed in parallel for 71 commonly used antibodies. Cytology and corresponding surgical pathology reports were reviewed for all cases in which antibodies failed verification studies but had been used in the four years prior to implementation of our verification process. RESULTS: From 2007 to 2011, the number of cytology cases in which ICC was performed increased from 98 (or 5% of all non-Pap test/nonurine cytology cases in our laboratory) to 306 (or 15%). Verification studies revealed calretinin, CD5, c-kit/CD117, inhibin, napsin A, OCT 3/4, and PAX-5 to be nonreliable in LBC despite consistent immunoreactivity in concurrent IHC on surgical specimens. No antibodies were found to be immunoreactive on LBC but nonreactive on FFPE. No adverse clinical outcomes resulted from the use of nonverified antibodies. CONCLUSIONS: Utilization of ICC at our institution has increased dramatically in recent years. Our verification process confirmed reliability in the majority of antibodies, but did identify several inconsistent antibodies. Although, in our series, no adverse clinical outcomes resulted from preverification use of these inconsistent antibodies, we encourage other institutions to confirm reliability of antibodies prior to use for diagnostic ICC.

The utility of human papillomavirus testing in young women with atypical glandular cells on pap test.

OBJECTIVE: This study aimed to gain information regarding the follow-up diagnoses and human papillomavirus (HPV) status of women younger than 35 years diagnosed with atypical glandular cells (AGCs) on Pap test. MATERIALS AND METHODS: This is a retrospective observational study in which the cytopathology files at Fletcher Allen Health Care were reviewed from 2000 to 2013 for the diagnoses of AGC in women younger than 35 years. Subsequent pathology reports and HPV testing results were obtained. Significant lesions were defined as cervical intraepithelial neoplasia (CIN) 2 or 3, invasive squamous cell carcinoma, adenocarcinoma in situ, or adenocarcinoma. RESULTS: One hundred six women younger than 35 years with an AGC Pap diagnosis and subsequent follow-up were identified. Significant lesions were diagnosed in 44.3% of the women (47); the majority (55.3%, 26 patients) of which were classified as CIN 2 or 3. Adenocarcinoma in situ was diagnosed in 27.7% of the cases (13). A diagnosis of both CIN 2 or 3 and adenocarcinoma in situ was made in 14.9% of the cases (7). One patient (2.1%) was diagnosed with endometrial adenocarcinoma. The HPV status was identified in 36.8% of the women (39): 69.2% (27) was HPV positive, and 30.8% (12) was HPV negative. Fifty-five percent of HPV-positive women were diagnosed with a significant lesion upon follow-up. No known HPV-negative women were diagnosed with a significant lesion. CONCLUSIONS: Human papillomavirus testing may be useful in risk stratifying young women with AGC on Pap test because they are at risk of having an HPV-positive cervical lesion.

Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer.

BACKGROUND: Ovarian tumors create a dynamic microenvironment that promotes angiogenesis and reduces immune responses. Our research has revealed that threonyl-tRNA synthetase (TARS) has an extracellular angiogenic activity separate from its function in protein synthesis. The objective of this study was to test the hypothesis that TARS expression in clinical samples correlates with angiogenic markers and ovarian cancer progression. METHODS: Protein and mRNA databases were explored to correlate TARS expression with ovarian cancer. Serial sections of paraffin embedded ovarian tissues from 70 patients diagnosed with epithelial ovarian cancer and 12 control patients were assessed for expression of TARS, vascular endothelial growth factor (VEGF) and PECAM using immunohistochemistry. TARS secretion from SK-OV-3 human ovarian cancer cells was measured. Serum samples from 31 tissue-matched patients were analyzed by ELISA for TARS, CA-125, and tumor necrosis factor-α (TNF-α). RESULTS: There was a strong association between the tumor expression of TARS and advancing stage of epithelial ovarian cancer (p < 0.001). TARS expression and localization were also correlated with VEGF (p < 0.001). A significant proportion of samples included heavy TARS staining of infiltrating leukocytes which also correlated with stage (p = 0.017). TARS was secreted by ovarian cancer cells, and patient serum TARS was related to tumor TARS and angiogenic markers, but did not achieve significance with respect to stage. Multivariate Cox proportional hazard models revealed a surprising inverse relationship between TARS expression and mortality risk in late stage disease (p = 0.062). CONCLUSIONS: TARS expression is increased in epithelial ovarian cancer and correlates with markers of angiogenic progression. These findings and the association of TARS with disease survival provide clinical validation that TARS is associated with angiogenesis in ovarian cancer. These results encourage further study of TARS as a regulator of the tumor microenvironment and possible target for diagnosis and/or treatment in ovarian cancer.

CT-guided transthoracic needle aspiration biopsy of subsolid lung lesions.

PURPOSE: To assess the diagnostic performance of computed tomography (CT)-guided transthoracic needle aspiration biopsy (TNAB) in the evaluation of persistent subsolid lung lesions. MATERIALS AND METHODS: A retrospective review of all CT-guided TNABs performed at a single institution from January 2002 to November 2012 was conducted to identify patients with persistent subsolid lung lesions. The diagnostic performance of CT-guided TNAB was assessed through comparison of cytologic diagnoses with core needle biopsy, surgical resection, or imaging and clinical follow-up. The cytologic, histologic, and imaging features of each lesion were characterized, and CT-guided TNAB complications were recorded. RESULTS: In 32 patients, a diagnosis of benign or malignant disease was identified through evaluation of pathologic or follow-up data. There were 18 men and 14 women, with a mean age of 67.1 years ± 9.6 (range, 52-86 y). The mean lesion diameter was 21 mm ± 11 (range, 8-62 mm). A final diagnosis of malignancy was made in 28 cases (87.5%); four benign lesions were also diagnosed. The overall sensitivity of CT-guided TNAB in the evaluation of these lesions was 89.2%, and the specificity and positive predictive value were 100%. Two pneumothoraces (6.3%) were identified. CONCLUSIONS: Among patients with subsolid lung lesions, CT-guided TNAB is safe and shows high sensitivity. The high specificity and positive predictive value of the procedure allow for definitive treatment decisions to be made for most patients.

Testing of integrated human papillomavirus mRNA decreases colposcopy referrals: could a change in human papillomavirus detection methodology lead to more cost-effective patient care?

OBJECTIVES: This study investigates potential colposcopy referral rates, as per the latest American Society for Colposcopy and Cervical Pathology recommendations, following the change in high-risk human papillomavirus (HR-HPV) detection methodology from Hybrid Capture 2 (HC2) to APTIMA at our institution. STUDY DESIGN: Rates of colposcopy referral were compared between two cohorts, each comprising all Pap samples with a diagnosis of atypical squamous cells of undetermined significance (ASCUS) tested for HR-HPV in our laboratory during a 12-month period. Cohorts I and II included Pap samples tested with HC2 (n = 1,856) and APTIMA (n = 1,651), respectively. The rates of quantity not sufficient (QNS) results were determined for all Pap samples during the same time periods. RESULTS: The proportion of HR-HPV-positive Pap samples with an ASCUS diagnosis was significantly lower with APTIMA (42%) than with HC2 (53%; p < 0.0001). APTIMA also resulted in a significantly lower QNS rate among all Pap samples (0.42 vs. 4.3% with HC2; p < 0.0001). CONCLUSION: The change in HR-HPV detection methodology from HC2 to APTIMA has led to a 21% reduction in colposcopy referrals and a 90% decrease in QNS rates at our institution. The new methodology has resulted in more cost-effective patient care and fewer insufficient samples requiring repeat HR-HPV testing.

Recurrent pregnancy loss in a woman with NLRP7 mutation: not all molar pregnancies can be easily classified as either "partial" or "complete" hydatidiform moles.

Recurrent hydatidiform moles is an uncommon occurrence. Over the past decade, genetic studies of women with multiple recurrent molar pregnancies have revealed that maternal mutations in two different genes, NLRP7 and C6orf221, result in recurrent moles. We report a 23 year old woman, born of unrelated parents, who has experienced three molar pregnancies in succession. Whilst the first pregnancy was classified as a complete hydatidiform mole, the second and third moles defied classification as complete or partial mole using conventional histology, p57 nuclear staining pattern and ploidy studies. Molecular and cytogenetic studies proved that all three molar pregnancies were diploid and biparental in origin. Gene sequencing analysis showed that the patient is homozygous for a previously described mutation in NLRP7. A SNP microarray ruled out the presence of deletion of the NLRP7 locus. This case draws attention to the fact that recurrent molar pregnancies may be the result of specific, identifiable gene mutations, even in patients from non-consanguineous backgrounds. When pathologists encounter patients with molar pregnancies that are diploid and p57 negative and yet have fetal elements such as nucleated red blood cells or immature fetal tissues, it should heighten their suspicion of a possible genetic basis and appropriate molecular genetic workup performed with counseling offered.

Müllerian and mesothelial lymph node involvement in patients with borderline ovarian tumors.

OBJECTIVE: To determine the true incidence of Müllerian and mesothelial lymph node involvement in serous and mucinous borderline ovarian tumors (BLOT) with serial sectioning and immunohistochemistry. STUDY DESIGN: Formalinfixed, paraffin-embedded lymph node blocks from patients with serous (N = 21) and mucinous (N = 5) BLOT who underwent lymphadenectomy between 1995 and 2002 were serially sectioned at 5 microm levels with 3 consecutive sections taken at surface, 125 microm and 475 microm. One slide from each level was stained with hematoxylin-eosin (H-E), cytokeratin (AE1-AE3, DAKO) and calretinin (DAKO). Lymph node involvement was defined as epithelioid cells recognized by H-E and confirmed by immunoreaction with keratin (Müllerian) and calretinin (mesothelial) or identified by immunohistochemistry alone. The results obtained by serial sectioning and immunohistochemistry were compared with those obtained by routine histologic examination at the time of the original surgery. RESULTS: A total of 240 lymph nodes (215 from patients with serous and 25 from patients with mucinous BLOT) were examined. Original pathologic examination identified lymph node involvement in 29/215 lymph nodes from 21 patients with serous BLOT. Twelve of the 21 patients with serous BLOT (57%) and none of the 5 patients with mucinous BLOT (0%) demonstrated Müllerian lymph node involvement. Serial sectioning and keratin immunostaining identified Müllerian involvement in 4 (1.6%) and 10 (4.2%) additional nodes not diagnosed in original sections, respectively. However, no additional node-positive patients were identified. Mesothelial involvement was identified in 2 patients (2/26, 7.6%). CONCLUSION: Patients with serous BLOT have a high incidence of Müllerian lymph node involvement. Distinction between Müllerian and mesothelial differentiation may require immunohistochemical study. Compared with routine histologic examination, serial sectioning and immunohistochemical examination yield a higher number of involved lymph nodes.

Topical testosterone for breast cancer patients with vaginal atrophy related to aromatase inhibitors: a phase I/II study.

PURPOSE: Controversy exists about whether vaginal estrogens interfere with the efficacy of aromatase inhibitors (AIs) in breast cancer patients. With the greater incidence of vaginal atrophy in patients on AIs, a safe and effective nonestrogen therapy is necessary. We hypothesized that vaginal testosterone cream could safely treat vaginal atrophy in women on AIs. METHODS: Twenty-one postmenopausal breast cancer patients on AIs with symptoms of vaginal atrophy were treated with testosterone cream applied to the vaginal epithelium daily for 28 days. Ten women received a dose of 300 µg, 10 received 150 µg, and one was not evaluable. Estradiol levels, testosterone levels, symptoms of vaginal atrophy, and gynecologic examinations with pH and vaginal cytology were compared before and after therapy. RESULTS: Estradiol levels remained suppressed after treatment to <8 pg/mL. Mean total symptom scores improved from 2.0 to 0.7 after treatment (p < .001) and remained improved 1 month thereafter (p = .003). Dyspareunia (p = .0014) and vaginal dryness (p <.001) improved. The median vaginal pH decreased from 5.5 to 5.0 (p = .028). The median maturation index rose from 20% to 40% (p < .001). Although improvement in total symptom score was similar for both doses (-1.3 for 300 µg, -0.8 for 150 µg; p = .37), only the 300-µg dose was associated with improved pH and maturation values. CONCLUSIONS: A 4-week course of vaginal testosterone was associated with improved signs and symptoms of vaginal atrophy related to AI therapy without increasing estradiol or testosterone levels. Longer-term trials are warranted.

Documentation of immunocytochemistry controls in the cytopathologic literature: a meta-analysis of 100 journal articles.

Although a detailed description of the procedure and tissue used as controls is considered a necessary component in surgical pathology articles in which immunohistochemistry is utilized, such documentation seems less stringent in the cytopathologic literature. A comprehensive literature search was done for articles published in English within the last 15 years on nine of the most widely used antibodies in cytopathology. Individual case reports were excluded. Of the 100 articles reviewed, 13 articles were review articles or commentaries and hence not included in the analysis. Only 11 (13%) of the remaining 87 articles described positive and negative controls run on identically prepared samples. Forty-seven articles (54%) either did not mention controls or did not run controls as separate specimens. Sixteen articles (18%) included a vague statement about controls. Twelve (14%) commented only on the negative control, included only histology tissue controls, or included cell block controls, but the study also included other types of preparations, such as cytospins. One article (1%) did not include controls because of insufficient material. The College of American Pathologists recognizes the impracticality of maintaining separate positive control samples for every possible combination of fixation, processing, and specimen type. However, more stringent documentation of procedure and use of controls in the cytopathologic literature will ensure that immunocytochemistry results in diagnostic cytopathology as well as in research are valid and reproducible.