Gerard van Swieten, the Dutch personal physician of Empress Maria Theresia (1700-1780).

Born as orthodox catholic in 1700 in Leyden, Gerard van Swieten was orphaned as a child in 1712. He studied medicine under Herman Boerhaave in Leyden from 1720, recording the lectures of his mentor and publishing them after his death. Following his graduation in 1715, van Swieten practiced medicine and pharmacy in Leyden, giving private lectures to students in both fields. Van Swieten became known as a brilliant doctor, and it was expected that he might succeed to Boerhaave's position after his death in 1738; however, his catholic faith was an obstacle for the protestant State University. These very beliefs, however, contributed to his instatement as the personal physician of the Austrian Empress Maria Theresia (1717-1780) in October 1744. In the summer of 1745 he was appointed physician to Maria Theresa in Vienna by Franz I. and at the same time appointed prefect of the court library. In addition to taking care of the library, other tasks he received from Maria Theresia included reformation of the medical faculty, improving the quality of Vienna's clinics and promoting healthcare in the empire. Van Swieten is seen as one of the founders of the so-called First Wiener Medical School (Erste Wiener Medizinische Schule) in 1745, and was at the founding of the first modern clinic in 1754. Van Swieten died June 18, 1772.

An 18th century description of endometriosis : The autopsy of the Countess von Reitzenstein.

Descriptions of endometriosis in 18th century monographs and manuscripts are rare and the recorded macroscopic features of endometriosis seldom support this attribution to the described cases. Recently, we became aware of an anonymous German autopsy report from the 18th century. After transcription, the manuscript was assessed by pathologists with historical expertise. This revealed that the patient died because of a malignant tumor, most probably of a gynecological origin. Furthermore, the described ovarian pathologic findings strongly support the diagnosis endometriotic ovarian cyst. Like Giovanni Battista Morgagni (1668-1772) in his landmark publication De Sedibus et Causis Morborum per Anatomen Indagatis (1761), the author correlated the pathological findings at autopsy with the symptoms of the patient. The identity of the patient could, with high probability, be established as being the Countess of Reitzenstein, the wife of a Prussian general major in the army of Friedrich the Great: Karl Erdmann von Reitzenstein (1722-1789).

The estimation of tumor cell percentage for molecular testing by pathologists is not accurate.

Molecular pathology is becoming more and more important in present day pathology. A major challenge for any molecular test is its ability to reliably detect mutations in samples consisting of mixtures of tumor cells and normal cells, especially when the tumor content is low. The minimum percentage of tumor cells required to detect genetic abnormalities is a major variable. Information on tumor cell percentage is essential for a correct interpretation of the result. In daily practice, the percentage of tumor cells is estimated by pathologists on hematoxylin and eosin (H&E)-stained slides, the reliability of which has been questioned. This study aimed to determine the reliability of estimated tumor cell percentages in tissue samples by pathologists. On 47 H&E-stained slides of lung tumors a tumor area was marked. The percentage of tumor cells within this area was estimated independently by nine pathologists, using categories of 0-5%, 6-10%, 11-20%, 21-30%, and so on, until 91-100%. As gold standard, the percentage of tumor cells was counted manually. On average, the range between the lowest and the highest estimate per sample was 6.3 categories. In 33% of estimates, the deviation from the gold standard was at least three categories. The mean absolute deviation was 2.0 categories (range between observers 1.5-3.1 categories). There was a significant difference between the observers (P<0.001). If 20% of tumor cells were considered the lower limit to detect a mutation, samples with an insufficient tumor cell percentage (<20%) would have been estimated to contain enough tumor cells in 27/72 (38%) observations, possibly causing false negative results. In conclusion, estimates of tumor cell percentages on H&E-stained slides are not accurate, which could result in misinterpretation of test results. Reliability could possibly be improved by using a training set with feedback.

Diagnosing ocular surface squamous neoplasia in East Africa: case-control study of clinical and in vivo confocal microscopy assessment.

OBJECTIVE: To examine the reliability of clinical examination and in vivo confocal microscopy (IVCM) in distinguishing ocular surface squamous neoplasia (OSSN) from benign conjunctival lesions. DESIGN: Case-control study. PARTICIPANTS: Sixty individuals with conjunctival lesions (OSSN and benign) and 60 age-matched controls with normal conjunctiva presenting to Kilimanjaro Christian Medical Centre, Moshi, Tanzania. METHODS: Participants were examined and photographed, and IVCM was performed. Patients with conjunctival lesions were offered excisional biopsy with histopathology and a human immunodeficiency virus (HIV) test. The IVCM images were read masked to the clinical appearance and pathology results. Images were graded for several specific features and given an overall categorization (normal, benign, or malignant). A group of 8 ophthalmologists were shown photographs of conjunctival lesions and asked to independently classify as OSSN or benign. MAIN OUTCOME MEASURES: Comparison of the histopathology diagnosis with the clinical and IVCM diagnosis. RESULTS: Fifty-two cases underwent excisional biopsy with histopathology; 34 were on the OSSN spectrum, 17 were benign, and 1 was lymphoma. The cases and controls had comparable demographic profiles. Human immunodeficiency syndrome infection was more common in OSSN compared with benign cases (58.8% vs. 5.6%; odds ratio, 24.3, 95% confidence interval [CI], 2.8-204; P = 0.003). Clinically, OSSN lesions more frequently exhibited feeder vessels and tended to have more leukoplakia and a gelatinous appearance. Overall, the ophthalmologists showed moderate agreement with the histology result (average kappa = 0.51; 95% CI, 0.36-0.64). The masked grading of IVCM images reliably distinguished normal conjunctiva. However, IVCM was unable to reliably distinguish between benign lesions and OSSN because of an overlap in their appearance (kappa = 0.44; 95% CI, 0.32-0.57). No single feature was significantly more frequent in OSSN compared with benign lesions. The sensitivity and specificity of IVCM for distinguishing OSSN from benign conjunctival lesions were 38.5% and 66.7%, respectively. CONCLUSIONS: In East Africa, conjunctival pathology is relatively common and can present significant diagnostic challenges for the clinician. In this study, neither clinical examination nor IVCM was found to reliably distinguish OSSN from benign conjunctival pathology because of an overlap in the features of these groups. Therefore, IVCM cannot currently replace histopathology, and management decisions should continue to rely on careful clinical assessment supported by histopathology as indicated.

The pathology of bone marrow failure.

An important indication for bone marrow investigation is the presence of bone marrow failure, which manifests itself as (pan)cytopenia. The causes of cytopenia are varied and differ considerably between childhood and adulthood. In the paediatric age group inherited bone marrow failure syndromes are important causes of bone marrow failure, but they play only a minor role in later life. This review gives a comprehensive overview of bone marrow failure disorders in children and adults. We classified the causes of bone marrow failure according to the main presenting haematological abnormality, i.e. anaemia, neutropenia, thrombocytopenia or pancytopenia. The following red cell disorders are discussed: red cell aplasia, sideroblastic anaemia, congenital dyserythropoietic anaemia, haemolytic anaemia, paroxysmal nocturnal haemoglobinuria, iron deficiency anaemia, anaemia of chronic disease and megaloblastic anaemia. The neutropenias occur in the context of Shwachman-Diamond syndrome (SDS), severe congenital neutropenia, cyclic neutropenia, immune-related neutropenia and non-immune neutropenia. In addition, the following causes of thrombocytopenia are discussed: congenital amegakaryocytic thrombocytopenia, thrombocytopenia with absent radii, immune-related thrombocytopenia and non-immune thrombocytopenia. Finally, we pay attention to the following pancytopenic disorders: Fanconi anaemia, dyskeratosis congenita, aplastic anaemia, myelodysplastic syndromes and human immunodeficiency virus (HIV) infection.

Unison or cacophony: postgraduate training in pathology in Europe.

With the free movement of people in the European Union, medical mobility has increased significantly. This is notably the case for disciplines for which shortage of well-trained staff has occurred. Pathology is among those specialties and effectively the discipline is confronted with a striking increase in mobility among trainees and qualified specialists. The presumption underlying unlimited mobility is that the competencies of the medical specialists in the European countries are more or less equal, including significant similarities in the postgraduate training programs. In order to assess whether reality corresponds with this presumption, we conducted a survey of the content and practice requirements of the curricula in the EU and affiliated countries. The results indicate a striking heterogeneity in the training program content and practice requirements. To name a few elements: duration of the training program varied between 4 and 6 years; the number of autopsies required varied between none at all and 300; the number of biopsies required varied between none at all and 15,000. We conclude that harmonization of training outcomes in Europe is a goal that needs to be pursued. This will be difficult to reach through harmonization of training programs, as these are co-determined by political, cultural, and administrative factors, difficult to influence. Harmonization might be attained by defining the general and specific competencies at the end of training and subsequent testing them through a test to which all trainees in Europe are subjected.

Activating KIRs exert a crucial role on relapse and overall survival after HLA-identical sibling transplantation.

Recognition of HLA-C molecules by killer cell immunoglobulin-like receptors (KIRs) is an important mechanism in the regulation of natural killer (NK) cell activity. Eradication of residual leukaemic cells by alloreactive donor NK cells after haematopoietic stem cell transplantation (HSCT) fulfils a crucial role in the control of relapse. This retrospective study evaluates 83 patients and their related donors. All individuals were typed at low-resolution level to determine their HLA repertoire. KIR genotyping data were obtained by the use of sequence-specific oligonucleotide (SSO) analysis. All data were combined with patient and donor characteristics and post-transplant clinical data. A higher overall survival was seen when KIR2DS1 in the donor was mismatched with the HLA-C group 2 ligand in the patient (p=0.03). The number of activating KIRs either in the patient or in the donor was significantly correlated with the occurrence of relapse (p=0.003 and p=0.02, respectively). In addition, the presence of KIR2DS5 in the patient alone or in both the patient and donor was significantly correlated with the occurrence of relapse (p=0.004 and p=0.005, respectively). In conclusion, significant correlations were found for activating KIRs with overall survival and relapse.

Patients benefit from the addition of KIR repertoire data to the donor selection procedure for unrelated haematopoietic stem cell transplantation.

Killer cell immunoglobulin-like receptors (KIRs) expressed on donor natural killer (NK) cells are important for induction of NK cell alloreactivity in haematopoietic stem cell transplantation (HSCT). Current criteria in the selection procedure of an unrelated donor do not account for this potential NK alloresponse. In this study the KIR gene repertoire of 21 HSCT patients and all their potential, unrelated donors (N=64) has been identified by the sequence-specific priming (SSP) procedure. KIR genotype characteristics are correlated with HLA and clinical data. These data show that for 16 cases an HLA compatible alternative donor was available. Among those 16 were 8 donors with a favourable predicted NK alloreactivity directed against the leukaemic cells. In conclusion, it is feasible and clinically relevant to add the KIR repertoire to the unrelated donor selection procedure.

Type IV collagen degradation in the myocardial basement membrane after unloading of the failing heart by a left ventricular assist device.

After left ventricular assist device (LVAD) support in patients with end-stage cardiomyopathy, cardiomyocytes decrease in size. We hypothesized that during this process, known as reverse remodeling, the basement membrane (BM), which is closely connected to, and forms the interface between the cardiomyocytes and the extracellular matrix, will be severely affected. Therefore, the changes in the myocardial BM in patients with end-stage heart failure before and after LVAD support were studied. The role of MMP-2 in this process was also investigated. Transmission electron microscopy showed that the BM thickness decreased post-LVAD compared to pre-LVAD. Immunohistochemistry indicated a reduced immunoreactivity for type IV collagen in the BM after LVAD support. Quantitative PCR showed a similar mRNA expression for type IV collagen pre- and post-LVAD. MMP-2 mRNA almost doubled post-LVAD (P<0.01). In addition, active MMP-2 protein as identified by gelatin zymography and confirmed by Western blot analysis was detected after LVAD support and in controls, but not before LVAD support. Active MMP was localized in the BM of the cardiomyocyte, as detected by type IV collagen in situ zymography. Furthermore, in situ hybridization/immunohistochemical double staining showed that MMP-2 mRNA was expressed in cardiomyocytes, macrophages, T-cells and endothelial cells. Taken together, these findings show reduced type IV collagen content in the BM of cardiomyocytes after LVAD support. This reduction is at least in part the result of increased MMP-2 activity and not due to reduced synthesis of type IV collagen.

Long-term prognosis of young breast cancer patients (≤40 years) who did not receive adjuvant systemic treatment: protocol for the PARADIGM initiative cohort study.

INTRODUCTION: Currently used tools for breast cancer prognostication and prediction may not adequately reflect a young patient's prognosis or likely treatment benefit because they were not adequately validated in young patients. Since breast cancers diagnosed at a young age are considered prognostically unfavourable, many treatment guidelines recommend adjuvant systemic treatment for all young patients. Patients cured by locoregional treatment alone are, therefore, overtreated. Lack of prognosticators for young breast cancer patients represents an unmet medical need and has led to the initiation of the PAtients with bReAst cancer DIaGnosed preMenopausally (PARADIGM) initiative. Our aim is to reduce overtreatment of women diagnosed with breast cancer aged ≤40 years. METHODS AND ANALYSIS: All young, adjuvant systemic treatment naive breast cancer patients, who had no prior malignancy and were diagnosed between 1989 and 2000, were identified using the population based Netherlands Cancer Registry (n=3525). Archival tumour tissues were retrieved through linkage with the Dutch nationwide pathology registry. Tissue slides will be digitalised and placed on an online image database platform for clinicopathological revision by an international team of breast pathologists. Immunohistochemical subtype will be assessed using tissue microarrays. Tumour RNA will be isolated and subjected to next-generation sequencing. Differences in gene expression found between patients with a favourable and those with a less favourable prognosis will be used to establish a prognostic classifier, using the triple negative patients as proof of principle. ETHICS AND DISSEMINATION: Observational data from the Netherlands Cancer Registry and left over archival patient material are used. Therefore, the Dutch law on Research Involving Human Subjects Act (WMO) is not applicable. The PARADIGM study received a 'non-WMO' declaration from the Medical Ethics Committee of the Netherlands Cancer Institute - Antoni van Leeuwenhoek hospital, waiving individual patient consent. All data and material used are stored in a coded way. Study results will be presented at international (breast cancer) conferences and published in peer-reviewed, open-access journals.

The medical autopsy as quality assurance tool in clinical medicine: dreams and realities.

The purpose of medical autopsy has changed to issues of quality assurance today. In addition, autopsies are considered valuable in medical education, e.g., delivering cases for problem-based learning for students. Many studies underscore the need for autopsies also in the era of technical progress emphasizing the continuing discrepancies between antemortem and post mortem diagnoses. Despite these important tasks, we face a decline of autopsy for several reasons with complex interactions. The role of all persons involved in this decline is evaluated and suggestions for changes are proposed. Last but not least, the future of the autopsy is in the hands of pathology itself.

Identification of HLA-A*0111N: a synonymous substitution, introducing an alternative splice site in exon 3, silenced the expression of an HLA-A allele.

A new variant of the HLA-A*010101 allele designated as HLA-A*0111N, previously known as HLA-A*010101var, was identified in a patient requiring a stem-cell transplantation. The patient was typed by serologic methods as HLA-A2 homozygous and by sequence-based typing (SBT) as A*010101,020601. Flow-cytometric (FCM) analysis with 11 human monoclonal antibodies (mAbs) for the A1 molecule confirmed lack of any cell membrane expression of the A*0111N allele. One-dimensional isoelectric focusing (1D-IEF) of total cell lysate from the patient's cells revealed no cell surface and cytoplasmic A1 protein expression, whereas the HLA-A2 molecule was identified by both FCM analysis and 1D-IEF. DNA sequence analysis showed the presence of a synonymous substitution from G to T at position 597 in codon 175. RNA SBT revealed a deletion of 24 bp in exon 3, position 596 through 619, encoding codons 175 through 182 of the HLA-A*0111N allele. The synonymous substitution introduced a new splice site, resulting in an efficient splicing, because no classical A1 protein could be detected in the patient. This alternative splicing prevented the translation into a correct and stable class I molecule expression on the cell surface.

Similar left and right ventricular sarcomere structure after support with a left ventricular assist device suggests the utility of right ventricular biopsies to monitor left ventricular reverse remodeling.

BACKGROUND: To evaluate whether the morphology of the contractile filaments in cardiomyocytes of patients with end-stage heart failure, treated with a left ventricular assist device (LVAD), is identical in the left- and right ventricle (LV, RV) and in the interventricular septum (IVS) and can be monitored by biopsies taken with a bioptome. The application of an LVAD as a bridge to recovery of cardiac function requires monitoring of myocyte recovery. The use of RV biopsies for this purpose might be feasible, if morphologic findings in the RV coincide with those in the LV. METHODS AND RESULTS: At the time of heart transplantation, myocardial biopsies of LV, RV and IVS from 13 patients after LVAD support were compared using immunohistochemistry with monoclonal antibodies against contractile proteins. Additionally, in five of these patients, small biopsies obtained with a diagnostic bioptome were compared with large transmural biopsies of the same region. Hemodynamic monitoring was performed when the patients were fully recovered from the implantation, to rule out persistent RV failure. The staining pattern of actin, myosin, tropomyosin, troponin T and C was identical in the biopsies of LV, RV and IVS. Small biopsies taken with a bioptome appeared to be representative for the larger biopsies. Hemodynamic monitoring showed absence of RV failure in our study group. CONCLUSION: In the absence of RV failure, morphology of the contractile myofilaments after LVAD support for 215+/-143 days is identical in LV, RV and IVS. This may allow monitoring of the possible occurrence of LV reverse remodeling by RV biopsies.

Donor interleukin-4 promoter gene polymorphism influences allograft rejection after heart transplantation.

BACKGROUND: The cytokine interleukin-4 (IL-4) is secreted mainly by activated T lymphocytes and characterizes the T-helper 2 (Th2) sub-type. In transplantation Th2 cells are believed to induce graft tolerance. Previous studies revealed that patients with a relatively high frequency of IL-4 producing helper T lymphocytes (HTL) before heart transplantation (HTX) had no or less rejection episodes compared with patients with a low frequency of IL-4 producing HTL. Three single nucleotide polymorphisms (SNPs) have been identified in the promoter region of the IL-4 gene, which influence promoter strength. We investigated whether there was a correlation between SNP genotypes in the IL-4 promoter and heart failure, and rejection after HTX. METHODS: Seventy HTX patients, 61 donors, and 36 controls were genotyped for the 3 SNPs by sequencing. RESULTS: Of the SNPs at -285 and -81, only the C and A alleles, respectively, were found in this study. Both alleles were found for the -590 SNP. No relation between patient genotype of the SNP at -590 and heart failure and rejection was found. However, incidence of rejection was significantly lower in patients that received a donor heart with the T-positive genotype compared with patients that received a heart from a T-negative donor. Patients who had the T-negative genotype and received a heart from a T-positive donor, suffered significantly less from rejection than T-negative patients that received a T-negative donor heart. This was not significant in the T-positive patient group. CONCLUSIONS: This indicates that IL-4 production within the donor heart and by cells from the donor is important for reducing incidence of episodes of rejection.

Cardiomyocyte death in patients with end-stage heart failure before and after support with a left ventricular assist device: low incidence of apoptosis despite ubiquitous mediators.

BACKGROUND: Left ventricular assist device (LVAD) implantation in patients with end-stage heart failure results in impressive hemodynamic improvement. The effects on myocardial apoptosis and its mediators are unknown. METHODS: Myocardial biopsies from 17 patients at the time of LVAD implantation and after explantation, at the time of heart transplantation (HTx), were examined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) reaction and with antibodies against Fas ligand (FasL), Fas, tumor necrosis factor (TNF)-alpha receptor 1 (TNF-R1), TNF-alpha receptor 2 (TNF-R2), TNF-alpha, TNF-alpha-converting enzyme (TACE), poly(ADP-ribose) polymerase (PARP), poly(ADP-ribose) (PAR), caspase-3 and FLICE inhibitory protein (FLIP). RESULTS: Apoptosis incidence was low: 0.8% (range 0% to 3%) positive cardiomyocytes nuclei before support, and 0.1% (range 0% to 0.6%) after support (p < 0.01). This was accompanied by low expression of caspase-3 and high expression of the DNA repair enzyme, PARP. Its product, PAR, increased after support. Mediators and receptors inducing apoptosis as well as FLIP were widely present before and after support. CONCLUSIONS: Despite the abundant presence of mediators and receptors inducing apoptosis, the incidence of apoptosis itself was low before and after mechanical support. The abundant expression of FLIP may suggest an important role for this protein in the inhibition of cardiomyocyte death.

[Tracheal rupture as a cause of unanticipated perioperative mortality]. / Trachearuptuur als oorzaak van onverwacht peroperatief overlijden.

Here we report two cases in which healthy young patients died during surgery because ventilation was impossible by a clinical picture of massive subcutaneous emphysema. The probable diagnosis was tracheal rupture. This diagnosis was not confirmed during coroner's autopsy, but there had been no systematic search for a puncture in the trachea or the main bronchial tubes. Immediate recognition of this situation, and implementation of ventilation of one lung by pushing a narrower endobronchial tube beyond the tracheal rupture, is potentially life-saving.

The use of virtual slides in the EUROPALS examination.

BACKGROUND: The only realistic way to improve harmonisation of European pathology training is to define the generally accepted competencies and to test them periodically during the training programme (progress test). The European Association of Pathology Chairs and Program Directors therefore decided to implement an annual on-line test using virtual slides in addition to static jpeg images and theoretical MCQ's. The EU supported this endeavour as EUROPALS (EUROpean Pathology Assessement & Learning System). METHODS: To address the challenges of large scale digital testing EUROPALS teamed up with i-Path Diagnostics Ltd, a company specialising in utilisation of virtual slides in histology/pathology education and examination. Specific examination software was used in the test system. RESULTS: In the first 2 years we provided at five occasions progress tests, including 2 proctored tests, attracting hundreds of participants. The accessibility varied from suboptimal to good and improved with each subsequent test. It was influenced both by the hosting server capacity and the internet bandwidth at the user's location. CONCLUSION: On-line testing using virtual slides is possible but requires a good collaboration between the provider and the user. Both should be aware of the requirements and threads of large scale testing with hundreds of simultaneous users.

[The rise and fall of pathology techniques]. / Opkomst en teloorgang van technieken in de pathologie.

For the past 150 years the most constant factor in the pathologist's histopathological diagnostic work-up has been haematoxylin staining. This technique, in combination with later additional staining techniques, determined knowledge on a cellular level for a long time. The invention of the transmission electron microscope added an ultrastructural dimension, and for many decennia in the middle of the twentieth century this was an important diagnostic tool. Enzyme histochemistry and morphometry came next, but these techniques never really became important as they were largely overtaken by immunohistochemistry and molecular diagnostics. These, in their turn, will face competition from proteomics and other forms of genomics. It seems likely that the trusty light microscope will lose out to digital microscopy, which is developing rapidly and offers the possibility to make a diagnosis at a distance. Pathology will continue to be a specialty on the move.