DeepFocus: fast focus and astigmatism correction for electron microscopy.

High-throughput 2D and 3D scanning electron microscopy, which relies on automation and dependable control algorithms, requires high image quality with minimal human intervention. Classical focus and astigmatism correction algorithms attempt to explicitly model image formation and subsequently aberration correction. Such models often require parameter adjustments by experts when deployed to new microscopes, challenging samples, or imaging conditions to prevent unstable convergence, making them hard to use in practice or unreliable. Here, we introduce DeepFocus, a purely data-driven method for aberration correction in scanning electron microscopy. DeepFocus works under very low signal-to-noise ratio conditions, reduces processing times by more than an order of magnitude compared to the state-of-the-art method, rapidly converges within a large aberration range, and is easily recalibrated to different microscopes or challenging samples.

A comparison of invasive lobular carcinoma with other invasive breast cancers at Tygerberg Academic Hospital.

BACKGROUND: The second most common histological subtype of invasive breast carcinoma is invasive lobular carcinoma (ILC) occuring with a frequency 10-15% in Western countries and approximately 5%, in Africa, the Middle East and Asia (AMA). Combined hormone replacement therapy (CHRT) is a risk factor for the development of ILC which is infrequently diagnosed at our centre.This study aimed to investigate the incidence and clinicopathological characteristics of ILC as compared to invasive breast carcinoma of no special type (IBC-NST). METHODS: Clinical and pathological data on breast carcinoma patients attending the breast and endocrine unit at Tygerberg Academic Hospital since 2017 have been recorded on a Stellenbosch University REDCap® database. RESULTS: IBC-NST was the most frequent subtype diagnosed (83.9%) and ILC the second most common subtype (5.2%). Most ILCs were of luminal B intrinsic subtype, and the median size was slightly smaller than IBC-NST. There were significantly more grade 2 ILCs than IBC-NSTs (81.5% vs 50.9%). There was no statistical difference between stage and histological subtype. CONCLUSION: ILC has clinicopathological differences when compared to IBC-NST, although these were less pronounced in this study. The prevalence of ILC was similar to numbers reported in AMA. We hypothesise that there may be a discrepancy in the prevalence of ILC between public and private healthcare systems in South Africa, and that it may be due to differing trends in prescribing CHRT.

The initial impact of the COVID-19 pandemic on the diagnosis of new cancers at a large pathology laboratory in the public health sector, Western Cape Province, South Africa.

BACKGROUND: The COVID-19 pandemic has disrupted cancer diagnostic services. A decline in the number of new cancers being diagnosed over a relatively short term implies a delay in diagnosis and subsequent treatment. This delay is expected to have a negative effect on cancerrelated morbidity and mortality. The impact of the pandemic on the number of new cancer diagnoses in our setting is unknown. OBJECTIVES: To assess the impact of COVID-19 on the number of new cancers diagnosed at our institution in the first 3 months following the implementation of lockdown restrictions, by focusing on common non-cutaneous cancers. METHODS: A retrospective laboratory-based audit was performed at a large anatomical pathology laboratory in Western Cape Province, South Africa. The numbers of new diagnoses for six common cancers (breast, prostate, cervix, large bowel, oesophagus and stomach) from 1 April 2020 to 30 June 2020 were compared with the corresponding period in 2019. RESULTS: Histopathological diagnoses for the six cancers combined decreased by 192 (-36.2%), from 531 new cases in the 2019 study period to 339 in the corresponding period in 2020. Substantial declines were seen for prostate (-58.2%), oesophageal (-44.1%), breast (-32.9%), gastric (-32.6%) and colorectal cancer (-29.2%). The smallest decline was seen in cervical cancer (-7%). New breast cancers diagnosed by cytopathology declined by 61.1%. CONCLUSIONS: The first wave of the COVID-19 pandemic and the associated response resulted in a substantial decline in the number of new cancer diagnoses, implying a delay in diagnosis. Cancer-related morbidity and mortality is expected to rise as a result, with the greatest increase in mortality expected from breast and colorectal cancer.

Postmortem lung biopsies from four patients with COVID-19 at a tertiary hospital in Cape Town, South Africa.

BACKGROUND: An outbreak of a novel coronavirus in China in late 2019 has resulted in a global pandemic. The virus (SARS-CoV-2) causes a severe acute respiratory syndrome and had been responsible for >14 000 deaths in South Africa (SA) at the time of writing, 30 August 2020. Autopsies in our setting have not been prioritised owing to the infective risks for staff, resulting in a lack of information on the histopathology of the disease in the SA setting. Postmortem biopsies are relatively quick and easy to perform and reduce the infective risk posed by full autopsies. OBJECTIVES: To determine whether postmortem biopsies of lung tissue could be used to determine cause of death in lieu of full autopsies in patients dying from COVID-19. METHODS: We performed postmortem biopsies of lung tissue on 4 patients with SARS-CoV-2 confirmed by reverse transcriptase polymerase chain reaction who died in the Tygerberg Hospital (Cape Town, SA) intensive care unit (ICU) in June - July 2020, in order to determine their cause of death. The biopsies were performed in the ICU with the necessary personal protective equipment within 2 hours after death. Clinical information was obtained from the hospital records and the histopathology was reviewed by two consultant histopathologists. Microbiology and electron microscopy were also performed on this tissue. RESULTS: All 4 patients were aged >50 years and had multiple comorbidities. Pulmonary pathology was present in only 3 cases, and the findings were surprisingly heterogeneous. One case demonstrated several findings including diffuse alveolar damage, extensive fibrin thrombi in pulmonary arteries with pulmonary infarction, organising pneumonia and bronchopneumonia. Other findings included type 2 pneumocyte hyperplasia, intra-alveolar macrophages and squamous metaplasia. An organising pneumonia was present in 2 other cases, although these findings were not deemed to be severe enough to be the cause of death. Fibrin thrombi were present in pulmonary arteries of 3 cases. One case showed no significant acute pulmonary pathology. The cause of death could only be determined in 1 case. CONCLUSIONS: The pulmonary findings we observed are in keeping with those described in the international literature. However, the pathology was surprisingly heterogeneous between cases, and was only deemed severe enough to be the cause of death in 1 of 4 cases. While lung-targeted, standardised postmortem biopsies may be safe, easy to perform and provide useful insights into the disease, they are not suitable to replace full autopsies in determining cause of death.

The current aetiology of malignant pleural effusion in the Western Cape Province, South Africa.

BACKGROUND: Malignant pleural effusion (MPE) represents a very common cause of pleural exudates, and is one of the most challenging pleural disorders to manage. This could be attributed to the paucity of high-quality experimental evidence, and inconsistent practice worldwide. South Africa (SA) currently has no data regarding the aetiology of MPE. OBJECTIVES: To identify the most common malignancies causing MPE in a population served by a large tertiary hospital in SA, and specifically the relative contribution of mesothelioma. A secondary objective was to evaluate the efficacy of chemical pleurodesis in a subset of patients. METHODS: We retrospectively included all known cases of MPE evaluated at our institution over a 3-year period with a tissue diagnosis of MPE. RESULTS: The most common causes of MPE in a total of 274 patients were lung cancer (n=174, 63.5%), breast cancer (n=32, 11.7%), unknown primary (n=22, 11.7%) and mesothelioma (n=27, 9.9%). Talc pleurodesis was performed in 81 of 194 patients (41.8%) referred to our division, and was radiologically successful in 22 of 25 (88.0%) followed up to 3 months. CONCLUSIONS: The main cause of MPE in our setting was lung cancer, followed by breast cancer, unknown primary and mesothelioma. Chemical pleurodesis was a viable palliative measure for MPE in this population.

Diagnosing diffuse lung disease in children in a middle-income country: the role of open lung biopsy.

SETTING: A tertiary care hospital situated in a middle-income country with a high burden of tuberculosis (TB) and human immunodeficiency virus (HIV) infection. OBJECTIVE: To determine the diagnostic yield of open lung biopsy (OLB) in children with diffuse lung disease (DLD), comparing findings in HIV-infected and non-HIV-infected children. DESIGN: This 9-year retrospective study included 51 children with DLD (oxygen-dependent or on artificial ventilation), who required an OLB where the diagnosis remained uncertain after extensive investigations. RESULTS: The median age was 7 months, median body weight was 6.6 kg (61% were severely malnourished) and 30% were HIV-infected (62% on antiretroviral treatment). The diagnostic yield of the OLB was 86% (n = 44) and was significantly higher in HIV-infected (77%) than in non-HIV-infected (48%) children (P = 0.01). Pneumonia was the most common diagnosis (n = 25, 57%), with common agents being cytomegalovirus (CMV), viruses other than CMV, Pneumocystis jiroveci pneumonia and previously undiagnosed TB (10%). Mycobacterium tuberculosis as a cause of DLD was not suspected before the OLB, as all investigations for TB were negative. Non-infectious causes of DLD were established in 10% of cases. CONCLUSION: The OLB is a useful diagnostic tool to diagnose idiopathic DLD, including TB, in young children.

Early γ-irradiation and subsequent storage of red cells in SAG-M additive solution potentiate energy imbalance, microvesiculation and susceptibility to stress-induced apoptotic cell death.

γ-Irradiation of red blood cell (RBC) concentrates prevents transfusion-associated graft-versus-host disease but may diminish RBC quality. Herein, we show that early γ-irradiation (25 Gy) of RBC units and their subsequent storage in SAG-M additive solution altered membrane microvesiculation, supernatant haemoglobin and cytosolic ATP. γ-Irradiation did not influence phosphatidylserine externalization, a marker of erythrocyte apoptotic cell death (eryptosis), in RBC stored for 42 days. However, shorter periods (4-21 days) of storage accentuated eryptosis in γ-irradiated RBC versus untreated RBCs following energy depletion, suggesting that γ-irradiated RBC is primed for stress-induced eryptosis during storage.

Efficiency of riboflavin and ultraviolet light treatment against high levels of biofilm-derived Staphylococcus epidermidis in buffy coat platelet concentrates.

BACKGROUND AND OBJECTIVES: Staphylococcus epidermidis forms surface-attached aggregates (biofilms) in platelet concentrates (PCs), which are linked to missed detection during PC screening. This study was aimed at evaluating the efficacy of riboflavin-UV treatment to inactivate S. epidermidis biofilms in buffy coat (BC) PCs. MATERIALS AND METHODS: Biofilm and non-biofilm cells from S. epidermidis ST-10002 and S. epidermidis AZ-66 were individually inoculated into whole blood (WB) units (~106 colony-forming units (CFU)/ml) (N = 4-5). One spiked and three unspiked WB units were processed to produce a BC-PC pool. Riboflavin was added to the pool which was then split into two bags: one for UV treatment and the second was untreated. Bacterial counts were determined before and after treatment. In vitro PC quality was assessed by flow cytometry and dynamic light scattering. RESULTS: Bacterial counts were reduced during BC-PC production from ~106 CFU/ml in WB to 103 -104 CFU/ml in PCs (P < 0·0001). Riboflavin-UV treatment resulted in significantly higher reduction of S. epidermidis AZ-66 than strain ST-10002 (≥3·5 log reduction and 2·6-2·8 log reduction, respectively, P < 0·0001). Remaining bacteria post-treatment were able to proliferate in PCs. No differences in S. epidermidis inactivation were observed in PCs produced from WB inoculated with biofilm or non-biofilm cells (P > 0·05). Platelet activation was enhanced in PCs produced with WB inoculated with biofilms compared to non-biofilm cells (P < 0·05). CONCLUSION: Riboflavin-UV treatment was similarly efficacious in PCs produced from WB inoculated with S. epidermidis biofilm or non-biofilm cells. Levels of biofilm-derived S. epidermidis ≥103 CFU/ml were not completely inactivated; however, further testing is necessary with lower (real-life) bacterial levels.

Bacterial survival and distribution during buffy coat platelet production.

BACKGROUND AND OBJECTIVES: At Canadian Blood Services, buffy coat (BC) platelet concentrates (BC-PCs) show a generally lower bacterial contamination rate than apheresis PCs. This study investigated whether the PC production method contributes to this observation. MATERIALS AND METHODS: Whole blood (WB) inoculated with eight bacterial strains was processed using the BC method. Bacteria were enumerated throughout BC-PC production and subsequent PC storage. Endotoxin production and bacterial adhesion to PC bags were evaluated during PC storage. PC quality was monitored by CD62P expression (flow cytometry) and changes in dynamic light scattering (ThromboLUX® ). RESULTS: During overnight WB hold, Staphylococcus epidermidis titres remained unchanged, commercial Escherichia coli and Klebsiella pneumoniae were eliminated and the remaining organisms proliferated to high concentrations. Through BC-PC production, bacteria segregated preferentially towards the cellular fractions compared to plasma (P < 0·05). During PC storage, most bacteria adhered to the PC bags and Gram negatives produced clinically significant endotoxin levels. Changes in CD62P expression or ThromboLUX scoring did not consistently reflect bacterial contamination in BC-PCs. CONCLUSION: WB hold during BC-PC production does not have a broad-spectrum bactericidal effect, and therefore, other factors contribute to low rates of contamination in BC-PCs.

Direct comparison of liquid-based and smear-based cytology with and without rapid on-site evaluation for fine needle aspirates of thoracic tumors.

BACKGROUND: Liquid-based cytology (LBC) and rapid on-site evaluation (ROSE) are proposed to improve the quality of fine needle aspirates (FNA) and their diagnostic yield compared with conventional smear cytology (CSC). This prospective study directly compared outcomes of sonar-guided FNA of thoracic tumors supported by LBC, CSC, or CSC with ROSE. METHODS: Three aspirates each for both LBC and CSC with separate 22G spinal needles in a randomized, alternating sequence during 64 transthoracic FNA of thoracic tumors were collected. Smears were prepared by cytology staff on site but evaluated with ROSE only when all six samples had been collected. If no diagnostic material was found on the first three CSC additional needle passes guided by ROSE were performed. RESULTS: Final diagnoses were non-small cell lung cancer in 50 (78.1%), small cell lung cancer in 11 (17.2%), mesothelioma in 1 (1.6%), and inflammation in 2 cases (3.1%), respectively. LBC and CSC were diagnostic in 42 (65.6%) and 49 (76.6%) cases, respectively (P = 0.039), with both methods diagnostic in 41 cases (64.1%). Fifteen cases (23.4%) remained undiagnosed following three passes for CSC but 9 (14.1%) of these were diagnosed using FNA and ROSE with a total yield of 58 cases (90.6%; P < 0.001). CONCLUSION: The diagnostic yield of transthoracic FNA submitted for LBC is significantly lower than with CSC when slides are prepared professionally. ROSE significantly increases the yield of transthoracic FNA.

The real value of endobronchial ultrasound.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has become a standard procedure worldwide, used in conjunction with bronchoscopy to obtain biopsies for mediastinal disorders. A 67-year-old man with a 40 pack-year smoking history presented with a 2-year history of hoarseness and weight loss. He also had a history of asbestos exposure. On examination under anaesthesia a lesion of the right false vocal fold was found and histology showed a moderately differentiated infiltrating keratinising squamous carcinoma. The question posed was whether this mass could be ascribed to metastatic supraglottic carcinoma or if it was indeed a metachronous primary bronchus carcinoma, as the treatment of these two malignancies differs significantly. Traditional bronchoscopy with TBNA is the least invasive procedure to obtain a cytological diagnosis, but the proximity of the aorta and pulmonary arteries and the mass being 14 mm from the bronchus would have made sampling by means of this procedure near impossible. We used EBUS to localise the mass and noted the position of the major vessels on Doppler ultrasound. Real-time ultrasound guidance allowed us to bridge the tissue plane between the mass and bronchial lumen using the longer EBUS needle and to obtain a fine-needle aspirate of the mass, which proved to be a keratinising squamous carcinoma. We describe this case in which EBUS-TBNA was pivotal in reducing the number of invasive procedures in a patient with metastatic supraglottic carcinoma.

Impact of routine sputum cytology in a population at high risk for bronchial carcinoma.

SETTING: Sub-Saharan Africa carries a high burden of lung cancer, with limited access to specialised health care. OBJECTIVE: To investigate the diagnostic value of sputum cytology and its potential in reducing the need for invasive diagnostic procedures in a high-risk population. DESIGN: We collected spontaneously expectorated sputum from 108 patients referred for a diagnostic procedure for suspected lung cancer between June 2010 and June 2012, and examined the diagnostic yield of sputum cytology for malignant cells as well as factors predicting a positive result. RESULTS: Bronchial carcinoma was diagnosed in 90 patients (83.3%), of whom 35 (38.9%) had sputum cytology positive for malignant cells with 100% diagnostic accuracy. Positive sputum cytology was significantly associated with endobronchial tumour and obstruction seen during bronchoscopy (OR 4.69 and OR 8.89, respectively), and with a histology of squamous cell carcinoma (OR 1.9). All but one patient with positive sputum were inoperable (97.1%), and we estimated that up to a third of all invasive procedures could be avoided if sputum cytology was used for triage. CONCLUSION: Sputum cytology had a high yield and accuracy in this high-risk group. Its routine use in selected patients is likely to result in reduced costs and less patient risk and discomfort.

Increased leukocyte survival and accelerated onset of lymphoma in the absence of MCL-1 S159-phosphorylation.

The antiapoptotic BCL-2 protein MCL-1, which opposes mitochondrial outer membrane permeabilization, was shown to have a crucial role in the survival of hematopoietic cells. We have previously shown that, upon loss of phosphatidylinositol 3-kinase signaling, S159 of MCL-1 is phosphorylated by glycogen synthase kinase-3 (GSK-3), earmarking MCL-1 for enhanced ubiquitylation and degradation. In this study, we introduced MCL-1(wt) or the phosphorylation-deficient mutant MCL-1(S159A) in mouse BM cells, followed by adoptive transfer to recipient mice. Mice expressing MCL-1(S159A) exhibited significantly elevated white blood cell and lymphocyte counts, whereas no effect was observed on the distribution of T and B lymphocyte subsets or the numbers of monocytes, red blood cells or platelets. Expression of MCL-1(S159A) in Eµ-Myc transgenic bone marrow significantly accelerated the onset of disease, and these mice displayed increased spleen weights compared with Eµ-Myc/MCL-1(wt) mice. Our data demonstrate that the absence of MCL-1 S159 phosphorylation provides a survival advantage for hematopoietic cells in vivo and facilitates oncogenesis.

Hypoxia induces the expression of transketolase-like 1 in human colorectal cancer.

BACKGROUND AND AIMS: Transketolase-like (TKTL) 1 is one of the key enzymes for anaerobic sugar degradation even in the presence of oxygen (aerobic glycolysis). Transketolase-dependent reactions supply malignant tumors with ribose and NADPH. Therefore, TKTL1 activity could be crucial for tumor proliferation and survival. The aim of the study was to evaluate the expression of TKTL1 in colorectal cancer (CRC) and its regulation under hypoxic conditions. METHODS: We studied TKTL1 mRNA and protein expression in CRC cell lines and human CRC biopsies by quantitative real-time PCR, Western blotting and immunohistochemistry. Regulation of TKTL1 under oxygen depletion was analyzed by cultivating cells either in a three-dimensional spheroid model or in a hypoxia incubator chamber. RESULTS: TKTL1 mRNA was heterogeneously expressed in monolayers of cells with high levels in HT-29 and SW480. TKTL1 protein was also clearly detectable in HT-29 and SW480. Hypoxia-inducible factor (HIF)-1α protein expression correlated with TKTL1 protein expression in SW480 spheroids over time. On the one hand, induction of hypoxia in T84 spheroids did not induce TKTL1; on the other hand, hypoxia by incubation at 1% O2 in a hypoxia incubator chamber clearly showed an upregulation of TKTL1. In 50% of CRC patients, TKTL1 protein expression was upregulated in tumor compared to non-tumor tissue. The immunohistochemical staining of TKTL1 in CRC patient samples resulted in 14 positive and 30 negative samples. CONCLUSIONS: TKTL1 expression correlated with HIF-1α protein expression and was induced upon hypoxic conditions which could facilitate energy supply to tumors under these circumstances.

Effect of altered surfaces on postural sway characteristics in elderly subjects.

Mobility is essentially based on successful balance control. The evaluation of functional strategies for postural stability is requisite for effective balance rehabilitation and fall prevention in elderly subjects. Our objective was to clarify control mechanisms of different standing positions reflecting challenges of typical everyday life situations. For this purpose, elderly subjects stood on different surfaces resulting in a change of the biomechanical constraints. Sway parameters out of time and frequency domain were calculated from center-of-pressure (COP) excursions. Besides the classic quantification of the amount of sway variability, we investigated the temporal organization of postural sway by means of nonlinear time series analysis. Limb load symmetry was quantified via foot pressure insoles. We found task dependent motor outputs: (1) asymmetrical loading in all conditions; (2) altered amount and structure of COP movements with dissimilar changes in medio-lateral and anterior-posterior direction; (3) changes of the motor output affect several time scales especially when standing on a balance board or with one foot on a step. Our results indicate that elderly subjects preferred forcefully one limb which supports a step-initiation strategy. Modifications of the postural sway structure refer to the interaction of multiple control mechanisms to cope with the altered demands. The identification of postural strategies employed in daily activities augments the ecological validity of postural control studies.

Segmental aganglionosis (zonal aganglionosis or "skip" lesions) in Hirschsprungs disease: a report of 2 unusual cases.

INTRODUCTION: There is accumulating evidence that "skip" lesions or zonal aganglionosis do occur in HSCR disease, albeit rarely. They are of interest because it may cause confusion in interpreting surgical margins as well as understanding the pathophysiology of HSCR disease. Normally described as "a skip area" of normally ganglionated bowel, surrounded proximally and distally by aganglionosis with variations may occur. CASE REPORTS: We report two cases of infants with unusual types of "skip lesions", identified within the last 5 years. RESULTS: One patient had an area of zonal aganglionosis in the transverse colon and recto-sigmoid, bordered by areas of normally enervated bowel in the right and descending colon. In the second patient, the terminal ileum, transverse, descending and sigmoid colons and rectum were histologically aganglionic, but focal patches of ganglion cells were identified in 21 cm of the right ascending colon and the appendix, suggesting some ENS plasticity and possible incomplete apoptosis. CONCLUSION: These cases illustrate the point that the presence of ganglion cells at the resection line is not sufficient to guarantee postoperative function and "skip" lesions may uncommonly confuse the picture. In addition, they raise questions as to its pathophysiology and favor an alternate hypothesis of local changes promoting neuroblast apoptosis as the possible cause.

Transbronchial fine needle aspiration biopsy and rapid on-site evaluation in the setting of superior vena cava syndrome.

There is a paucity of prospective data on flexible bronchoscopy with rapid on-site evaluation (ROSE) in the setting of superior vena cava (SVC) syndrome. The aims of this prospective study were to assess the diagnostic yield and safety of these investigations and specifically to evaluate the role of ROSE in limiting the need for tissue biopsies. Over a 5-year period 48 patients (57.4 ± 9.7 years) with SVC syndrome secondary to intrathoracic tumors underwent flexible bronchoscopy with TBNA and ROSE. Endobronchial Forceps biopsy was reserved for visible endobronchial tumors with no on-site confirmation of diagnostic material. ROSE confirmed diagnostic material in 41 cases (85.4%), and in only one of the remaining cases did the addition of a forceps biopsy increase the diagnostic yield (overall diagnostic yield of 87.5%). No serious complications were noted. The final diagnoses made included nonsmall lung cancer (n = 27), small cell lung cancer (n = 16), and metastatic carcinoma (n = 3). Two undiagnosed cases died of suspected advanced neoplasms (unknown primary tumors). We conclude that TBNA has a high diagnostic yield and is safe in the setting of SVC syndrome. With the addition of ROSE, tissue biopsy is required in the minority of cases.