Making sense of a haemolysis monitoring and reporting system: a nationwide longitudinal multimethod study of 68 Australian laboratory participant organisations.

BACKGROUND: The key incident monitoring and management systems (KIMMS) quality assurance program monitors incidents in the pre- and postanalytical phases of testing in medical laboratories. Haemolysed specimens have been found to be the most frequent preanalytical error and have major implications for patient care. The aims of this study were to assess the suitability of KIMMS for quality reporting of haemolysis and to devise a meaningful method for reporting and monitoring haemolysis. METHODS: A structured survey of 68 Australian KIMMS laboratory participant organisations was undertaken. Quarterly haemolysis reports (2011-2014) were analysed. RESULTS: Among 110 million accessions reported, haemolysis rates varied according to the reporting methods that participants used for assigning accessions (16% of participants reported haemolysis by specimen and 83% reported by episode) and counting haemolysis rejections (61% by specimen, 35% by episode and 3% by test). More than half of the participants (56%) assigned accessions by episode and counted rejections by specimen. For this group, the average haemolysis rate per 100,000 episodes was 177 rejected specimens with the average rate varying from 100 to 233 over time. The majority of participants (91%) determined rejections using the haemolysis index. Two thirds of participants (66%) recorded the haemolysis manually in laboratory information systems. CONCLUSIONS: KIMMS maintains the largest longitudinal haemolysis database in the world. However, as a means of advancing improvements in the quality of the preanalytical laboratory process, there is a need to standardise reporting methods to enable robust comparison of haemolysis rejection rates across participant laboratories.

Key factors influencing the incidence of hemolysis: A critical appraisal of current evidence.

Hemolysis is a leading cause of pre-analytical laboratory errors. The identification of contributing factors is an important step towards the development of effective practices to reduce and prevent hemolysis. We performed a review of PUBMED, Embase, Medline and CINAHL to identify articles published between January 2000 and August 2016 that identified factors influencing in vitro hemolysis rates. The 40 studies included in this review provide excellent evidence that hemolysis rates are higher in Emergency Departments (EDs), for non-antecubital draws, for specimens drawn using an intravenous catheter compared to venipuncture and for samples transported by pneumatic tube compared to by hand. There is also good evidence that hemolysis rates are higher when specimens are not collected by professional phlebotomists, larger volume specimen tubes are used, specimen tubes are filled less than halfway and tourniquet time is greater than one minute. The results of this review suggest that hospitals and clinical laboratories should consider deploying phlebotomists in EDs, drawing all blood through a venipuncture, using the antecubital region as the optimum blood collection site and transporting specimens by laboratory assistant/other personnel, or if this in not practical, ensuring that pneumatic transport systems are validated, maintained and monitored. Studies also recommend making hemolysis a hospital-wide issue and ensuring high-quality staff training and adherence to standard operating procedures to reduce hemolysis rates. Awareness of the factors that influence hemolysis rates, and adoption of strategies to mitigate these risk factors, is an important step towards creating quality practices to reduce hemolysis rates and improve the quality of patient care.

Decreased expression of key tumour suppressor microRNAs is associated with lymph node metastases in triple negative breast cancer.

BACKGROUND: Breast cancer is the most common malignancy that develops in women, responsible for the highest cancer-associated death rates. Triple negative breast cancers represent an important subtype that have an aggressive clinical phenotype, are associated with a higher likelihood of metastasis and are not responsive to current targeted therapies. miRNAs have emerged as an attractive candidate for molecular biomarkers and treatment targets in breast cancer, but their role in the progression of triple negative breast cancer remains largely unexplored. METHODS: This study has investigated miRNA expression profiles in 31 primary triple negative breast cancer cases and in 13 matched lymph node metastases compared with 23 matched normal breast tissues to determine miRNAs associated with the initiation of this disease subtype and those associated with its metastasis. RESULTS: 71 miRNAs were differentially expressed in triple negative breast cancer, the majority of which have previously been associated with breast cancer, including members of the miR-200 family and the miR-17-92 oncogenic cluster, suggesting that the majority of miRNAs involved in the initiation of triple negative breast cancer are not subtype specific. However, the repertoire of miRNAs expressed in lymph node negative and lymph node positive triple negative breast cancers were largely distinct from one another. In particular, miRNA profiles associated with lymph node negative disease tended to be up-regulated, while those associated with lymph node positive disease were down-regulated and largely overlapped with the profiles of their matched lymph node metastases. From this, 27 miRNAs were identified that are associated with metastatic capability in the triple negative breast cancer subtype. CONCLUSIONS: These results provide novel insight into the repertoire of miRNAs that contribute to the initiation of and progression to lymph node metastasis in triple negative breast cancer and have important implications for the treatment of this breast cancer subtype.

The expression of Dicer and Drosha in matched normal tissues, tumours and lymph node metastases in triple negative breast cancer.

BACKGROUND: Breast cancer is the most common malignancy in women world-wide. Triple negative breast cancer (TNBC) is a highly aggressive subtype that lacks expression of hormone receptors for estrogen, progesterone and human epidermal growth factor 2; and is associated with a high propensity for metastatic spread. Several studies have identified critical roles for microRNAs in breast cancer, but the role of two critical enzymes involved in microRNA biogenesis, Dicer and Drosha, is not well understood, particularly with respect to metastatic progression in this subtype. METHODS: We examined the expression of Dicer and Drosha in a series of invasive 35 TNBCs with matched normal adjacent tissues (n = 18) and lymph node metastases (n = 15) using semi-quantitative real time RT-PCR. The relationship of their expression with clinical features including age at diagnosis, lymph node positivity and tumour size was analysed. RESULTS: We report that Dicer was significantly decreased while Drosha was significantly increased in tumours when compared to normal adjacent tissues. While there was no difference in Drosha expression in lymph node metastases when compared to the primary tumour, Dicer was significantly increased. There was no correlation between the expression of either Dicer or Drosha to age at diagnosis, lymph node positivity and tumour size. CONCLUSIONS: In conclusion, Dicer and Drosha are dysregulated in TNBC and matched lymph node metastases however, the clinical relevance of this is still not known. The altered expression of Dicer and Drosha may serve as markers for disrupted miRNA biogenesis in TNBC.

The accuracy of the 'triple test' in the diagnosis of papillary lesions of the breast.

BACKGROUND AND OBJECTIVE: The literature on fine-needle aspiration (FNA) cytology for papillary lesions presents a very mixed picture. Many authors advocate mandatory excision of these lesions. This recommendation is largely based on the 'atypical' nature of the FNA report. The aim of this work is to see if breast papillomas can be treated conservatively. STUDY DESIGN: We report a retrospective study of outcomes for patients with a provisional diagnosis of a 'papillary breast lesion' based on assessment by palpation (no clinically suspicious features), sonography (benign or probably benign according to the Breast Imaging Reporting and Data System 'BI-RADS®'), and FNA (benign cytological category with a papillary architecture) findings from one integrated breast service. RESULTS: Thirty-six cases were identified over a period of 6 years. Thirty-four of the patients had surgical excision. All of the 34 surgical cases were confirmed to be benign in nature on histopathology (intraduct papilloma). The remaining 2 cases were stable on follow-up. CONCLUSION: We believe that a policy of mandatory excision of papillary lesions of the breast is unnecessarily cautious.

Building Research Support Capacity across Human Health Biobanks during the COVID-19 Pandemic.

Human health biobanks are forms of research infrastructure that supply biospecimens and associated data to researchers, and therefore juxtapose the activities of clinical care and biomedical research. The discipline of biobanking has existed for over 20 years and is supported by several international professional societies and dedicated academic journals. However, despite both rising research demand for human biospecimens, and the growth of biobanking as an academic discipline, many individual biobanks continue to experience sustainability challenges. This commentary will summarize how the COVID-19 pandemic is creating new challenges and opportunities for both the health biobanking sector and the supporting discipline of biobanking. While the challenges for biobanks may be numerous and acute, there are opportunities for both individual biobanks and the discipline of biobanking to embrace change such that biobanks can continue to support and drive biomedical research. We will therefore describe numerous practical steps that individual biobanks and/or the discipline of biobanking can take to survive and possibly thrive in response to the COVID-19 pandemic.

Adjuvant letrozole versus tamoxifen according to centrally-assessed ERBB2 status for postmenopausal women with endocrine-responsive early breast cancer: supplementary results from the BIG 1-98 randomised trial.

BACKGROUND: The Breast International Group (BIG) 1-98 trial (a randomised double-blind phase III trial) has shown that letrozole significantly improves disease-free survival (DFS) compared with tamoxifen in postmenopausal women with endocrine-responsive early breast cancer. Our aim was to establish whether the benefit of letrozole versus tamoxifen differs according to the ERBB2 status of tumours. METHODS: The BIG 1-98 trial consists of four treatment groups that compare 5 years of monotherapy with letrozole or tamoxifen, and sequential administration of one drug for 2 years followed by the other drug for 3 years. Our study includes data from the 4922 patients randomly assigned to the two monotherapy treatment groups (letrozole or tamoxifen for 5 years; 51 months median follow-up [range <1 to 90 months]). A central assessment of oestrogen receptor (ER), progesterone receptor (PgR) and ERBB2 status using paraffin-embedded primary tumour material was possible for 3650 (74%) patients. ER, PgR, and ERBB2 expression were measured by immunohistochemistry (IHC) and ERBB2-positivity was confirmed by fluorescence in-situ hybridisation (FISH). Positive staining in at least 1% of cells was considered to show presence of ER or PgR expression. Tumours were deemed ERBB2-positive if amplified by FISH, or, for the few tumours with unassessable or unavailable FISH results, if they were IHC 3+. Hazard ratios (HR) estimated by Cox modelling were used to compare letrozole with tamoxifen for DFS, which was the primary endpoint, and to assess treatment-by-covariate interactions. The BIG 1-98 trial is registered on the clinical trials site of the US National Cancer Institute website http://www.clinicaltrials.gov/ct/show/NCT00004205. FINDINGS: By central assessment 7% (257 of 3650) of tumours were classified as ERBB2-positive. In 3533 patients with tumours confirmed to express ER, DFS was poorer in patients with ERBB2-positive tumours (n=239) than in those with ERBB2-negative tumours (n=3294; HR 2.09 [95% CI 1.59-2.76]; p<0.0001). There was no statistical evidence of heterogeneity in the treatment effect according to ERBB2 status of the tumour (p=0.60 for interaction), thus, letrozole improves DFS compared with tamoxifen regardless of ERBB2 status. The observed HRs were 0.62 (95% CI 0.37-1.03) for ERBB2-positive tumours and 0.72 (0.59-0.87) for ERBB2-negative tumours. INTERPRETATION: A benefit of letrozole over tamoxifen was noted, irrespective of ERBB2 status of the tumour, and, therefore, ERBB2 status does not seem to be a selection criterion for treatment with letrozole versus tamoxifen in postmenopausal women with endocrine-responsive early breast cancer.

Staphylococcal scalded skin syndrome in an extremely premature neonate: a case report with a brief review of literature.

Staphylococcus aureus can cause a spectrum of exfoliative skin conditions including staphylococcal scalded skin syndrome (SSSS) which can present as a severe and life threatening illness in extremely premature neonates. We describe a case of an extremely premature neonate with SSSS and discuss relevant pathology and issues in clinical management.

Diagnostic 'errors' in anatomical pathology: relevance to Australian laboratories.

Failure to recognise that anatomical pathology diagnosis is a process of cognitive interpretation of the morphological features present in a small tissue sample has led to the public misperception that the process is infallible. The absence of a universally accepted definition of diagnostic error makes comparison of error rates impossible and one large study of laboratories in the United States shows a significant error rate of about 5%, most of which have no major impact on patient management. A recent review of the work of one pathologist in New South Wales confirms a lack of appreciation in medical administration that variable diagnostic thresholds result in an inherent fallibility of anatomical pathology diagnoses. The outcome of the review emphasises the need to educate both public and non-pathology colleagues of the nature of our work and brings into consideration the requirement to establish baseline error rates for Australian laboratories and the role of the Royal College of Pathologists of Australasia (RCPA) in developing fair and unbiased protocols for review of diagnostic errors. The responsibility of ensuring that diagnostic error rates are kept to the minimum is a shared one. Area health services must play their part by seeking to ensure that pathologists in any laboratory are not overworked and have adequate support and back-up from pathologists with expertise in specialised areas. It has been clearly enunciated by the Royal College of Pathologists in the United Kingdom that it is not safe for any histopathology service to be operated single-handedly by one histopathologist. Service managers and clinicians have to understand that country pathologists cannot provide the full range and depth of pathology expertise in the many clinical subspecialty areas that are often practised in non-metropolitan areas. Attending clinicians share the responsibility of accepting proffered pathology diagnoses only if it conforms to the clinical context. Pathology laboratories must continue to develop and maintain best-practice protocols and conduct periodic reviews of diagnosis, cytology-histology concordance, frozen section/permanent section correlations, conference reviews, intra and interdepartmental consultations, participate in external quality assurance programs and maintain ongoing education for all laboratory staff.

DNA methylation profile of triple negative breast cancer-specific genes comparing lymph node positive patients to lymph node negative patients.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with no targeted treatment available. Our previous study identified 38 TNBC-specific genes with altered expression comparing tumour to normal samples. This study aimed to establish whether DNA methylation contributed to these expression changes in the same cohort as well as disease progression from primary breast tumour to lymph node metastasis associated with changes in the epigenome. We obtained DNA from 23 primary TNBC samples, 12 matched lymph node metastases, and 11 matched normal adjacent tissues and assayed for differential methylation profiles using Illumina HumanMethylation450 BeadChips. The results were validated in an independent cohort of 70 primary TNBC samples. The expression of 16/38 TNBC-specific genes was associated with alteration in DNA methylation. Novel methylation changes between primary tumours and lymph node metastases, as well as those associated with survival were identified. Altered methylation of 18 genes associated with lymph node metastasis were identified and validated. This study reveals the important role DNA methylation plays in altered gene expression of TNBC-specific genes and lymph node metastases. The novel insights into progression of TNBC to secondary disease may provide potential prognostic indicators for this hard-to-treat breast cancer subtype.

Current Methods of Haemolysis Detection and Reporting as a Source of Risk to Patient Safety: a Narrative Review.

AIM: Haemolysis has a major impact on patient safety as the need for a replacement specimen increases the risk of injury and infection, delays test results and extends the duration of hospital stays. Consistency of haemolysis detection and reporting can facilitate the generation of benchmark data used to develop quality practices to monitor and reduce this leading cause of pre-analytical laboratory error. This review aims to investigate current methods of haemolysis detection and reporting. METHOD: Due to known heterogeneity and immaturity of the research field, a scoping search was conducted using PUBMED, Embase, Medline and CINAHL. Articles published between 2000 and 2014 that reported haemolysis rates in specimens from the general population were included. RESULTS: Of the 50 studies that met the inclusion criteria, 20 detected haemolysis using the Haemolysis Index (HI), 19 by visual inspection and 13 by undefined methods. There was large intra-study variation in the plasma free haemoglobin level used to establish haemolysis (HI: mean±SD 846±795 mg/L, range 150-3000 mg/L; Visual: 850±436 mg/L, 500-3000 mg/L). Sixteen studies reported the analyte of interest, with only three studies reporting a haemoglobin level at which the specimen would be rejected. CONCLUSION: Despite haemolysis being a frequent and costly problem with a negative impact on patient care, there is poor consistency in haemolysis detection and reporting between studies. Improved consistency would facilitate the generation of benchmark data used to create quality practices to monitor and reduce this leading cause of pre-analytical laboratory error.

Repair of UVB-induced DNA damage is reduced in melanoma due to low XPC and global genome repair.

UVB exposure leads to DNA damage, which when unrepaired induces C>T transitions. These mutations are found throughout the melanoma genome, particularly in non-transcribed regions. The global genome repair (GGR) branch of nucleotide excision repair (NER) is responsible for repairing UV-induced DNA damage across non-transcribed and silent regions of the genome. This study aimed to examine the relationship between UVB and GGR in melanoma. DNA repair capacity and relative expression of NER in melanocytes and melanoma cell lines before and after treatment with UVB was quantified. Transcript expression from 196 melanomas was compared to clinical parameters including solar elastosis and whole transcriptome data collected. Melanoma cell lines showed significantly reduced DNA repair when compared to melanocytes, most significantly in the S phase of the cell cycle. Expression of GGR components XPC, DDB1 and DDB2 was significantly lower in melanoma after UVB. In the melanoma tumours, XPC expression correlated with age of diagnosis and low XPC conferred significantly poorer survival. The same trend was seen in the TCGA melanoma dataset. Reduced GGR in melanoma may contribute to the UV mutation spectrum of the melanoma genome and adds further to the growing evidence of the link between UV, NER and melanoma.

The Conundrum of Papillary Breast Lesions within the C3 Category.

OBJECTIVES: To identify features that could define papillary ductal cell proliferation within the C3 category and to subcategorise papillary lesions into benign papillomas which can be managed conservatively and atypical/malignant papillary neoplasms which require surgical intervention. STUDY DESIGN: A blind microscopic rescreen of all C3 cases was conducted. The corresponding histological outcome was compared with the cytology. Statistical analysis was performed using papillary versus non-papillary outcomes and benign versus atypical/malignant papillary lesions. In addition, macropapillary lesions (papilloma and encysted papillary carcinoma) were plotted against micropapillary ductal carcinoma in situ. RESULTS: Two hundred thirty FNA cases reported as C3 included 72 papillary neoplasms (52 benign papillomas and 20 atypical/malignant papillary lesions). Features specific to papillary lesions within C3 include macropapillary fragments, complex sheets, palisading strips, cystic background, cohesion and a decreased nuclear-to-cytoplasmic ratio. Features favouring atypical/malignant papillary lesions include decreased numbers of bare bipolar nuclei, discohesion and a non-cystic background. These features are common to most breast malignancies; however, identification of papillary features often results in a downgraded diagnosis from C5. CONCLUSIONS: This study supports the ability to reliably identify papillary ductal cell proliferation within C3. Certain features can distinguish papillary lesions from other C3 pathologies. This separation is likely to be clinically useful as papillary lesions may require a different management approach.

Novel genes associated with lymph node metastasis in triple negative breast cancer.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and no targeted treatments. TNBC patients are more likely to develop metastases and relapse than patients with other breast cancer subtypes. We aimed to identify TNBC-specific genes and genes associated with lymph node metastasis, one of the first signs of metastatic spread. A total of 33 TNBCs were used; 17 of which had matched normal adjacent tissues available, and 15 with matched lymph node metastases. Gene expression microarray analysis was used to reveal genes that were differentially expressed between these groups. We identified and validated 66 genes that are significantly altered when comparing tumours to normal adjacent samples. Further, we identified 83 genes that are associated with lymph node metastasis and correlated these with miRNA-expression. Pathway analysis revealed their involvement in DNA repair, recombination and cell death, chromosomal instability and other known cancer-related pathways. Finally, four genes were identified that were specific for TNBC, of which one was associated with overall survival. This study has identified novel genes involved in LN metastases in TNBC and genes that are TNBC specific that may be used as treatment targets or prognostic indicators in the future.

The microscopic complexities of C3 in breast cytology.

BACKGROUND: Fine-needle aspiration (FNA) of difficult breast lesions often results in an atypical (C3) report. The assortment of outcomes generated by C3 reports varies widely, and this has given rise to different clinical management pathways. OBJECTIVE: To identify and objectively assess microscopic features associated with atypical/C3 breast FNA cases. MATERIALS AND METHODS: A total of 230 atypical breast FNAs were subjected to a blind microscopic rescreen using a range of robust qualitative and quantitative cytological criteria including cellularity, architectural qualities, cytomorphology and background features. A logistic regression with a receiver operating characteristic (ROC) curve and the resultant forward stepwise analysis were conducted to assess the results. This statistical testing was measured against malignant, benign proliferative and benign non-proliferative outcomes. RESULTS: The malignant and benign proliferative outcomes showed a mixture of opposing protective and predictive individual cytological criteria. The stepwise analysis produced models demonstrating the best combination of individual cytological criteria for malignancy, proliferative and benign non-proliferative entities. In the malignancy model, discohesion, nuclear crowding within sheets, diminished numbers of bare bipolar nuclei and myoepithelial cells, the presence of tubules or necrosis and the absence of a cystic background were important features. The benign proliferative model suggested the same criteria but with the opposite implication and with the addition of several others, such as the presence of apocrine metaplasia, retained polarity and a speckled or coarse chromatin pattern. Age was a significant factor in malignant and proliferative outcomes. The benign non-proliferative stepwise analysis produced a model with fewer criteria (complex sheets, bare bipolar nuclei and a cystic background) limiting clinical application. CONCLUSION: Atypical/C3 breast cytology remains a legitimate reporting category. However, it is associated with a number of different histological outcomes. The incidence of the C3 category can be significantly reduced by controlling extrinsic factors and understanding the associated microscopic features.

Regulators of global genome repair do not respond to DNA damaging therapy but correlate with survival in melanoma.

Nucleotide excision repair (NER) orchestrates the repair of helix distorting DNA damage, induced by both ultraviolet radiation (UVR) and cisplatin. There is evidence that the global genome repair (GGR) arm of NER is dysfunctional in melanoma and it is known to have limited induction in melanoma cell lines after cisplatin treatment. The aims of this study were to examine mRNA transcript levels of regulators of GGR and to investigate the downstream effect on global transcript expression in melanoma cell lines after cisplatin treatment and in melanoma tumours. The GGR regulators, BRCA1 and PCNA, were induced in melanocytes after cisplatin, but not in melanoma cell lines. Transcripts associated with BRCA1, BRCA2, ATM and CHEK2 showed altered expression in melanoma cell lines after cisplatin treatment. In melanoma tumour tissue BRCA1 transcript expression correlated with poor survival and XPB expression correlated with solar elastosis levels. Taken together, these findings provide evidence of the mechanisms underlying NER deficiency in melanoma.