bluuebunny/crossref_metadata_2025 huggingface dataset:
An algorithmic approach to the use of rapid molecular diagnostics for SARS-CoV-2, influenza viruses, and other respiratory pathogens during an unprecedented respiratory season
Authors: Maris A S , Tao L , Stratton C W , Humphries R M , Schmitz J E
Published: October 2021

Introduction/Objective

The COVID-19 pandemic exacerbated deficiencies of testing personnel, reagents, supplies and disposables, instruments, and automation in many clinical laboratories. Upon entering respiratory season, a strategy was warranted to optimize laboratory resources when supplies were already limited and expected respiratory season test volume was unknown. An algorithm was devised to prioritize test ordering and TAT based on patient clinical scenario.

Methods/Case Report

The institutional respiratory season SARS-CoV-2 algorithm was constructed by a multidisciplinary team including infectious disease, infection prevention, laboratory, and IT/LIS leadership. CDC guidance on influenza testing was incorporated. Antigen-based testing was discontinued; only molecular amplification- based platforms with FDA EUA were utilized. Platforms had a range of TAT (20 minutes to 8 hours) and included fully- automated high throughput, rapid random access, point-of-care, and CDC SARS-CoV-2 assays. Test bundles included SARS-CoV-2 (monoplex), or SARS-CoV-2 + fluA&B (triplex), or SARS-CoV-2 + respiratory pathogen panel (multiplex RPP; includes 22 targets, including flu A&B).

Results (if a Case Study enter NA)

Key factors in the algorithm included whether the patient was outpatient or inpatient, hospital employee or not, symptomatic or not, immunocompetent or immunocompromised, and whether a concurrent order for other respiratory pathogens was included or not. Clinician responses for these factors determined the type of swab collected (wet swab in VTM or dry swab) and how quickly the TAT was indicated for a given patient using a colored-dot sticker system. Priority TAT in decreasing order was symptomatic inpatients, asymptomatic pre- procedure patients, asymptomatic admissions, symptomatic employees, and symptomatic outpatients.

Conclusion

An algorithm for respiratory pathogen testing during an unprecedented respiratory season prioritizes result TAT to an individual patient’s clinical situation while maximizing laboratory stewardship by eliminating redundant influenza testing and requiring ‘all upfront’ orders to avoid add-on orders that require ‘dumpster diving’ for samples. Limitations include inherent differences in sensitivity, LOD, and specificity when multiple different platforms are utilized to detect the same analytes.
Blood Groups of Recipients of COVID-19 Convalescent plasma
Authors: Petersen J M , Jhala D
Published: October 2021

Introduction/Objective

COVID -19 Convalescent plasma therapy (CCP) is under an FDA Emergency Use Authorization to treat hospitalized patients with COVID-19. However, being ill enough to require hospitalization for COVID-19 is a negative outcome. There is also contradictory literature on whether ABO blood group is associated with worse outcomes with COVID-19 disease. Therefore, we share a regional Veterans Administration Medical Center (VAMC) experience comparing the blood groups of patients intended to receive CCP to a control group of patients positive for SARS-CoV-2.

Methods/Case Report

A retrospective review of all patients who had CCP ordered in the year 2020 was performed to identify the blood group of these patients, which was compared to a control population of positive patients early in the pandemic (March 17th, 2020 to May 20th, 2020).

Results (if a Case Study enter NA)

A total of 15 patients had CCP ordered as part of their care with an age range of 56-85 (average 69.7) years of age, entirely male composition, and a racial breakdown of 13 African Americans (86.7%), 1 Caucasian American (6.7%), and 1 Asian American (6.7%). The blood group distribution amongst these 15 patients for CCP was 1 AB+ (6.7%), 5 A+ (33.3%), 4 B+ (26.7%), and 5 O+ (33.3%).

The unrelated control population consisted of 81 SARS-CoV-2 positive patients whose blood groups were distributed as 3 group AB (3.7%), 21 group A (25.9%), 15 group B (18.5%), and 42 group O (51.8%). A Chi squared test did not show a statistically significant difference between the two groups in ABO composition.

Conclusion

The ABO proportions of patients for whom CCP was ordered compared to the control group was not statistically significant. This provides support to the literature arguing that ABO may not be related to worse outcomes such as hospitalization or need for CCP transfusion.
Comparison of virus concentration methods for the reverse-transcriptase quantitative PCR (RT-qPCR)-based detection of bacteriophage Φ6, an enveloped virus surrogate for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, from untreated wastewater at St. George’s University (SGU), Grenada
Authors: Farmer-Diaz K , Matthew-Bernard M , Ramos-Nino M , Fitzpatrick D , Cheetham S
Published: October 2021

Introduction/Objective

With the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the consequent opening of borders, it is of paramount importance to monitor communities for the presence of potential COVID-19 clusters. Wastewater-based epidemiology (WBE) is an effective way to monitor community spread of pathogens and has been implemented in many countries to identify potential outbreaks. Most standardized methodologies used in WBE for the detection of viruses have been validated for non-enveloped viruses; therefore, there is a need for the creation of a standardized method for the detection of enveloped viruses, such as SARS-CoV-2.

Methods/Case Report

Wastewater seeded with a Pseudomonas syringae bacteriophage (Φ6) was used to test the efficiency of five wastewater virus concentration methods. Two methods were based on AlCl3 flocculation with different pH adjustments. The other methods utilized membrane filtration followed by beef elution: one added a pretreatment with HCl; another added a pretreatment with MgCl2; and finally, an untreated control. A known amount of Bacteriophage Φ6 was used for all methods, and detection was quantified by reverse-transcription quantitative polymerase chain reaction.

Results (if a Case Study enter NA)

The Cq values for each method, ranging from 24.38–38.6, were compared. The highest recovery of Φ6 was obtained with the AlCl3 flocculation method with an adjusted pH of 6, followed in descending order by MgCl2-pretreated sample with beef elution; HCl-pretreated sample with beef elution, the untreated sample, and AlCl3 flocculation method with an adjusted pH of 3.5.

Conclusion

The results suggest that AlCl3 flocculation with an adjusted pH of 6 may be a quick and cost-effective methodology for the detection of enveloped viruses in WBE. This can facilitate the implementation of WBE at SGU, Grenada, as a useful tool to conduct active surveillance and track outbreaks of enveloped viral diseases, such as SARS-CoV-2.
Practical Risk Scoring System for Predicting Severity of COVID-19 Disease
Authors: Petersen J M , Jhala D
Published: October 2021

Introduction/Objective

SARS-CoV-2 has become an international pandemic with numerous casualties. The severity of the COVID-19 disease course depends on several clinical, laboratory, and radiological factors. This has led to risk scoring systems in various populations such as in China, but similar risk scoring systems developed based on the American veteran population are sparse. As a risk scoring system (RSS) would be very useful for future reference in similar pandemics, we share a simple Jhala Risk Scoring System (JRSS) developed early in the pandemic to assess the severity of disease risk.

Methods/Case Report

A retrospective review of all SARS-CoV-2 reverse transcriptase-polymerase chain reaction (RT-PCR) tests collected and performed at the regional Veterans Administration Medical Center (VAMC) serving the Philadelphia and surrounding areas from March 17th, 2020 to May 20th, 2020. Data was collected and analyzed in the same year. These tests were reviewed within the computerized medical record system for demographic, medical history, laboratory test history, and clinical course. Information from the medical records were then scored based on the criteria of the JRSS.

Results (if a Case Study enter NA)

The JRSS, based on age, ethnicity, presence of any lung disease, presence of cardiovascular disease, smoking history, and diabetes history with laboratory parameters correlated and predicted (with statistical significance) which patients would be hospitalized.

Conclusion

The JRSS reached statistical significance in its predictions on informing risk stratification for COVID-19 positive patients. Similar risk scoring systems may play a role in the rapid development of risk scoring in future pandemics of similar nature and thus provide a useful reference point. A simple RSS based on clinical parameters is a highly practical, cost effective and simple system to evaluate need for hospitalization, which is critical for operations in the intensive care unit and simultaneously the use of ventilators.
Necrotizing Fasciitis and Fatal Septic Shock Associated with Streptococcus constellatus
Authors: Rajack F , Naab T J
Published: October 2021

Introduction/Objective

Streptococcus anginosus group consists of three species: S. anginosus, S. intermedius, and S. constellatus (SC), normal flora in the human oral cavity, gastrointestinal and urogenital tracts. What makes them unique is their ability to form abscesses.

Methods/Case Report

We report a case of a 43 year old morbidly obese bipolar male presenting with general malaise and diarrhea. He had a history of uncontrolled diabetes mellitus Type 2 and persistent right thigh wound. Admitting labs revealed diabetic ketoacidosis. Physical examination revealed a right medial thigh ulcer with necrotic scrotal skin, after which he underwent urgent surgical debridement. Wound culture grew SC. Debrided tissue measured 44 x 17 x 4 cm. Histology revealed abscesses next to zones of pale gray necrotic tissue having a granular appearance without associated neutrophils. Gram stain of these pale gray zones revealed sheets of Gram positive cocci, consistent with Stage III necrotizing fasciitis (NF), which has 47% mortality. He died shortly after surgery with death attributed to septic shock. Toxic shock syndrome is most often associated with Group A Streptococcus. However, other rare pathogenic streptococci, including SC, may be associated with a similar syndrome. Gram stain is essential in confirming NF, characterized by pale gray necrosis with a granular appearance.

Results (if a Case Study enter NA)

NA

Conclusion

Our case highlights the risk of fatal NF due to SC. Identification of SC in a deep wound warrants early surgical intervention for adequate source control and administration of appropriate antibiotic. Poor diabetic control and ulcerated chronic thigh wound predisposed our patient to fatal necrotizing fasciitis due to SC, confirmed by Gram stain.
Comparison of longitudinal SARS-CoV-2 nasopharyngeal specimens reveals the transcriptomic COVIDome
Authors: Twa D D , Kuchinski K , Nikiforuk A , Krajden M , Prystajecky N , Jassem A , Sekirov I
Published: October 2021

Introduction/Objective

SARS-Cov-2 is well established to introduce a cytokine-like storm among select individuals that results in multisystem failure and death. Comorbidities, age, oxygen status, and real-time appraisal of inflammatory markers in the blood have been used to risk stratify patients, however, these clinical markers do not comprehensively characterize the at-risk population or disease course. To understand the molecular underpinnings of the primary site of SARS-CoV-2 infection, here, we interrogated the transcriptomic profile of the nasopharyngeal tissue among paired SARS-CoV-2 specimens.

Methods/Case Report

We performed ribosomal depletion RNAseq on 24 primary samples, including 16 paired samples from 8 unique patients who converted between SARS-CoV-2 negative and positive status via clinical diagnostic qRT-PCR. Additional targeted qRT-PCR was performed for ACE2 and TMPRSS2 in an extension sample of 54 paired specimens from 27 unique patients who converted in their SARS-CoV-2 status on the basis of the qRT- PCR test. Differential gene expression, differential correlative expression with ACE2, and correlative expression with viral load was used to identify genes, which were integral to SARS-CoV-2 pathogenesis, so termed the COVIDome. Gene ontologies, pathways, and reactive infiltrate was assessed between specimens and compared with measures of clinical outcome using regression with appropriate correction for multiple hypotheses.

Results (if a Case Study enter NA)

We observed significant enrichment for ontologies of lymphocyte activation, specifically interferon gamma signaling; (P<1E-20) and platelet activation (P<1E-5). Genes specifically enriched across all three modules included: ADAMDEC1, EPSTI1, GRIP2, IRF7, KLHDC7B, OAS3, OASL, PIK3R4, RSAD2, and XAF1. Using CIBERSORT to approximate immune cell populations from bulk RNA, we observed and enrichment for CD4 immune cells, which was associated with viral status (P<0.01) while high-risk gene signatures were associated with measures of clinical outcome (P<0.05).

Conclusion

We characterized the pathogenesis of SARS-CoV-2 in longitudinal nasopharyngeal samples of COVID- 19 patients and related these molecular manifestations with measures of clinical outcome. As proof of principal, our findings suggest additional study in a large, longitudinal extension sample is warranted to validate and assess molecular features of clinical outcome associated with SARS-CoV-2 infection.
Validation and Implementation of molecular RT-PCR COVID-19 Testing Platforms
Authors: Patel S , Jhala D
Published: October 2021

Introduction/Objective

COVID-19 is caused by the SARS-CoV-2 coronavirus that has led to a worldwide pandemic with an unprecedented need for fast and accurate testing under FDA Emergency Use Authorizations (EUA). Despite the promise of the Alinity-m as an upgrade from the Abbott m2000 for optimization of high throughput testing and random access for laboratory workflow, validation studies comparing Alinity-m to already used Abbott m2000 are sparse in the literature, particularly for the veteran population.

Methods/Case Report

The validation study for the Alinity m (Chicago IL) was performed in three parts 1) Method to method sample/patient correlation study, 2) precision study (at 250 virus copies/mL, 500 virus copies/mL and 1000 virus copies/mL versus 4 negatives), and 3) verification and confirmation of the accuracy of the assay at the lower limit of detection (LOD). For the validation study. The patient specimens were used side by side for both assays. The results from the Abbott m2000 (Chicago IL), which was already established in our lab, were used as a reference for validation.

Results (if a Case Study enter NA)

The validation with a method to method correlation included 157 valid specimens from which concordant results were obtained for all 157 specimens on both the Alinity-m and Abbott m2000. The precision or reproducibility of the Alinity-m was verified at all concentrations. The limit of detection verification on diluted samples determined the limit of detection to be 20 virus copies/mL (>95% of dilution samples agreed with positive results at this level), which confirmed even below the manufacturer provided LOD of 100 virus copies/mL.

Conclusion

Alinity m was validated for clinical use with study demonstrating that it is 1) equivalent in the method to method correlation, precision, and LOD determination. 2)The validation of Alinity m, holds great promise with its optimized throughput for testing of large numbers of specimens with less technician handling and random access, is a valuable addition to the literature of test validations under the FDA EUA. 3)The validation of tests under the FDA EUA is unprecedented and provides an important way to improve patient care during this extraordinary pandemic.
Molecular Profiling in Thyroid cancer- Using next generation sequencing to differentiate rare cases of papillary thyroid hyperplasia from papillary thyroid carcinoma
Authors: Lazim A , Luceno S , Arriola A , Seth A , Jhala N
Published: October 2021

Introduction/Objective

Differentiating papillary thyroid hyperplasia from papillary thyroid carcinoma is made primarily on differences in key histologic and cytomorphologic features. These include architectural features such as invasion and nuclear changes such as chromatin pallor, nuclear grooves, and intranuclear pseudoinclusions. Distinguishing between these two diagnoses is not always straightforward in a minority of cases, as papillary thyroid hyperplasia nodules can be misinterpreted as papillary thyroid carcinoma and vice versa. This is because the nuclear changes can be subtle and be induced artificially by poor or inadequate formalin fixation. Immunostains such as cytokeratin 19 (CK19), galectin-3, and HBME1 may be used to distinguish between papillary thyroid hyperplastic nodules and papillary thyroid carcinoma; however, as immunostaining results are not always definitive, other more definitive ancillary tests are sometimes utilized such as molecular testing.

Methods/Case Report

Thyroid cases with indeterminate diagnoses (n=6) were obtained from the archives. Such cases included papillary thyroid hyperplasia and cases with descriptive diagnoses such as well-differentiated encapsulated and low-grade papillary carcinoma. Five cases of definitive papillary thyroid carcinoma and one case of follicular thyroid carcinoma (n=6) were used as a comparison group. Next generation sequencing (NGS) assays were performed using a custom SLIMamp targeted DNA solid tumor panel (Pillar Biosciences) and a targeted multi gene RNA fusion panel (Invitae).

Results (if a Case Study enter NA)

All of the definitive cases of papillary thyroid carcinoma showed characteristic mutations such as BRAF V600E as well as RNA fusions such as RET/PTC while all of the indeterminate cases did not show any detectable DNA mutations or RNA fusions, confirming their benign nature. A separate case that was signed out as non-invasive follicular thyroid neoplasm with papillary-like nuclear features showed an NRAS Q61R mutation and a novel ETV1-ERG fusion.

Conclusion

This study demonstrates the utility of molecular testing as an adjunct to histopathological evaluation in definitive diagnosis and differentiation of papillary thyroid hyperplasia from papillary carcinoma. Next generation sequencing with targeted DNA and RNA panels will be beneficial for both known and novel variant or fusion detection in suspect cases.
EGFR testing patterns and detection of EGFR exon 20 insertions among patients with NSCLC in the US
Authors: Lin H M , Yin Y , Curran E , Crossland V , Wu Y , Ou S
Published: October 2021

Introduction/Objective

Epidermal growth factor receptor (EGFR) mutations are common in non-small cell lung cancer (NSCLC). EGFR exon 20 insertions (EGFRex20ins), a rare subset of EGFR mutations, are refractory to tyrosine kinase inhibitors. With the development of targeted therapies for EGFRex20ins, such as mobocertinib, molecular testing is required to optimize treatment. A better understanding of real-world EGFR detection patterns is needed to maximize patient outcomes.

Methods/Case Report

This retrospective study describes EGFR testing and EGFRex20ins detection patterns in patients with NSCLC in the United States. The Flatiron Health electronic health record database was used to identify patients ≥18 years, with advanced NSCLC, and with ≥2 clinic visits between 01/01/2011 and 12/31/2020. Baseline demographics, clinical characteristics, EGFR testing, and EGFRex20ins detection rates by sex, race, and smoking history were summarized.

Results (if a Case Study enter NA)

A total of 67,281 patients with NSCLC were identified. EGFR testing increased from 44% in 2011 to 77% in 2020. Of all patients, 44,926 (66.8%) were tested: 50.8% female; 3.3% Asian; 16.0% never-smokers. Of all patients, 22,355 (33.2%) were not tested: 41.4% female; 1.2% Asian; 7.5% never-smokers. Of those tested, 6,245 (13.9%) patients had EGFR mutations: 65.9% female; 11.8% Asian; 48.4% never-smokers.

EGFRex20ins detection rates changed from 0.6% in 2011 to 1.0% in 2019 and 0.7% in 2020. Of those tested, 304 patients had EGFRex20ins: 58.2% female; 8.2% Asian; 50.3% never-smokers. EGFR testing was higher in females (71.2%) than males (62.8%), never-smokers (84.5%) than those with a smoking history (64.6%), and Asian patients (84.2%) than White (66.6%), Black (65.4%), or other patients (69.5%). Of those tested, EGFRex20ins mutations were detected in 0.8% of females (males: 0.6%), 2.2% of never-smokers (with smoking history: 0.4%), and 1.7% of Asians (White: 0.6%, Black: 0.6%, other patients: 0.7%) had EGFRex20ins. A similar trend was observed for EGFR mutations with higher proportions of females, never-smokers, and Asian patients affected.

Conclusion

EGFR testing and EGFRex20ins detection rates have increased. However, not all patient subgroups were tested at the same rate and undertesting occurred in all subgroups. Further education of specialists diagnosing NSCLC is warranted to ensure all patients receive biomarker testing and benefit from emerging EGFRex20ins- targeted therapies.
Complete response of a colonic high-grade neuroendocrine carcinoma to platinum-based therapy: Insights from comprehensive genomic profiling
Authors: Wood R , Arnason T , DeCoste R , Gaston D , Carter M D , Rayson D
Published: October 2021

Introduction/Objective

Comprehensive genomic profiling (CGP) is an essential tool in precision medicine, providing diagnostic, prognostic, and predictive (therapeutic) information to enable personalized and optimized care for cancer patients. We present the case of a patient with a metastatic high-grade tumor of the colon who showed an impressive response to systemic therapy and discuss the role of CGP in cancer management.

Methods/Case Report

A 54-year-old woman with was diagnosed with stage IV large-cell neuroendocrine carcinoma (LCNEC) of the colon with large volume liver and nodal metastases with imminent hepatic failure. CGP was performed on the resected tumor (TSO500 panel, 523 cancer-related genes, Illumina), showing pathogenic mutations in multiple oncogenes and tumor suppressor genes, including BRCA1, BAP1, and BRAF. Additionally, global parameters revealed a very high tumor mutation burden (TMB, 351 / Mb), and high-degree microsatellite instability (MSI-H). Treatment of the patient’s metastases with platinum-based systemic therapy resulted in a complete radiographic response, with no evidence of disease recurrence after 6.5 years. This type of complete response to therapy is rarely reported in colonic LCNEC. Assessment by Medical Genetics did not identify a germline mutation suggestive of hereditary breast/ovarian cancer or Lynch syndrome.

Results (if a Case Study enter NA)

NA

Conclusion

The patient’s extraordinary response to therapy is likely due to loss of BRCA1 and/or BAP1 function, as deleterious mutations in both genes predict sensitivity to platinum-based therapy through exploitation of deficient homologous recombination repair (HRR). The high TMB and MSI-H status suggest the immune system may have contributed to tumor clearance through neoantigen activation of T-cells. The information provided by CGP also suggested potential tumor sensitivity to poly(ADP-Ribose) polymerase inhibitors (PARPi), immunotherapy (IT), and BRAF/MEK inhibitor therapy, should the tumor recur. This case highlights the value of CGP in guiding management of rare and aggressive tumors.
The optimization of an ultra-sensitive single molecule assay for the multiplexed detection of SARS-CoV-2 antigens
Authors: Senussi Y , Swank Z N , Walt D R
Published: October 2021

Introduction/Objective

SARS-CoV-2 antigens, including the nucleocapsid (N) protein, spike protein, and its S1 subunit have served as key biomarkers for research and diagnostic purposes. We previously developed quantitative single molecule array (Simoa) assays to measure the concentration of spike, S1 subunit and N protein in plasma samples with femtomolar limits of detection. We aimed to test antibodies that were not available early in the pandemic, reduce assay cross-reactivity, develop a multiplexed assay for spike, S1, and N protein in order to minimize the sample volume needed.

Methods/Case Report

Using the Simoa platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 17 S1 subunit and spike antibodies for a total of 130 antibody-pair combinations, we performed dilution linearity experiments to determine the ideal dilution factor, spike and recovery experiments, tested the assay using S1 subunit from other human coronavirus HKV1, NL63, and 229E, pre-pandemic plasma samples from patients that were sick with viral or bacterial respiratory infections. We then used the best antibody pairs to measure S1 and spike in plasma samples collected from patients with severe SARS-CoV-2. Lastly, we conjugated the best-performing capture antibodies for spike, S1 and N to beads labeled with different fluorophores to test if the assay for all three antigens could be multiplexed.

Results (if a Case Study enter NA)

We observed no cross-reactivity with S1 from other coronavirus strains, no detection of S1 or spike in a cohort of 30 pre-pandemic samples and successfully developed a multiplexed assay for the detection of spike, S1, and N protein, enabling us to use 50% less sample volume.

Conclusion

Reduction of necessary sample volume is important for studies involving multisystem inflammatory syndrome in children (MIS-C), and possible adverse effects of SARS-CoV-2 vaccinations on children and young adults. An improved assay with minimal cross-reactivity will also be useful to study individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).
Bioinformatics and wet laboratory analysis of rs1800562 to predict genetic aetiology of iron overload in Nigeria
Authors: Bolarinwa A B , Onawoga F
Published: October 2021

Introduction/Objective

The most reported single nucleotide polymorphism (SNP) of the HFE gene is rs1800562, representing the substitution of Adenine for Guanine at position 847 of the HFE gene. This has been widely implicated in hereditary haemochromatosis and other conditions like altered cholesterol balance, Alzheimer’s disease and cutaneous photosensitivity. Abnormal HFE protein resulting from the mutant HFE gene leads to formation of excess iron which has been postulated as likely mechanism for these diseases. Although there is evidence of iron overload in Africans, only few studies have explored possible genetic causes, and prevalence of rs1800562 is not known in West African population. Hence the need to determine the prevalence of rs1800562 in Nigeria using computational and wet laboratory approach.

Methods/Case Report

Details of rs1800562 were retrieved from Ensembl Genome Browser version 99. Severity of the consequences of this SNP on protein product was determined using bioinformatics tools including SIFT, Polyphen, Mutation Assessor, HOPE, I-mutant and MutPred2. Genotyping of rs1800562 was done In silico using restriction fragment length polymorphism (RFLP). Primer3plus was used for primer design, NCBI BLAST and SMS were used for primer validation. We used Webcutter 2.0 to determine suitable restriction enzymes. The genotyping was simulated using USCS virtual PCR and RestrictionMapper. Whole blood samples were obtained from 200 participants selected randomly from a pool of blood donors. DNA was extracted and flanking region of rs1800562 was amplified. The amplified product was digested by RSA1and fragments examined on agarose gel electrophoresis.

Results (if a Case Study enter NA)

The MAF was found to be 0.01 globally and 0.02 in Africa. In the two Nigerian population examined (Yoruba and Esan population), MAF was 0.00. Mutation Assessor and SIFT Polyphen consistently predicted the mutation to be of severe consequences. Analysis on HOPE, I-mutant and Mutpred2 revealed loss of protein stability, change in net charges affecting the HFE protein localization and its interaction with other proteins. All the participants in the wet laboratory analysis were homozygous for the wild type allele of rs1800562 (MAF=0).

Conclusion

This study confirmed the In silico prediction of the absence of rs1800562 in Nigeria. Future studies should focus on other SNPs of the HFE gene as well as other gene involved in iron metabolism.
A rare case of hairy cell leukemia variant with ATM nonsense mutation and ATM deletion, resulting in complete loss of function
Authors: Wei Q , Harada S , Reddy V
Published: October 2021

Introduction/Objective

Hairy cell leukemia variant (HCL-v) is a rare B cell lymphoproliferative disorder. Although HCL- v shares some overlapping features with classic HCL, it lacks the characteristic immune-morphology and pathogenic BRAF V600E mutation seen in classic HCL and recognized as a distinct entity in the 2017 WHO classification. Approximately half of HCL-v harbors MAPK2 mutation and one third has TP53 mutation. Cytogenetic abnormalities include gain of chromosome 5, loss of chromosome 7q, and deletion of 17p13/TP53 and 11q/22 ATM gene

Methods/Case Report

We report a case of HCL-v with total loss of ATM gene function. A 71-year-old male with splenomegaly presented with an outside diagnosis of splenic marginal zone lymphoma and underwent splenectomy. Flow cytometry on spleen demonstrated mature clonal B lymphoid cells and was positive for CD11c and CD103. However, unlike classical HCL, it was CD25 negative, which supports the diagnosis of HCL-v. Sections of spleen revealed most of the red pulp replaced by hairy cell leukemic infiltrate with minimal preservation of the normal lymphoid stroma. Peripheral blood smear showed atypical lymphocytosis with prominent nucleoli but most lacking circumferential shaggy contours, seen in classic HCL. Cytogenetic analysis identified a deletion in chromosome 11(q13q23), which deletes the ATM gene. NGS analysis demonstrated a nonsense variant in ATM (p. E888*). No significant sequence variants were identified in BRAF, MAP2K1 or TP53. This is the first case report of HCL-v with a deletion as well as a non-sense mutation in the ATM gene. ATM gene belongs to the PI3/PI4-kinase family, and is an important cell cycle checkpoint kinase that phosphorylates downstream proteins, including TP53, BRCA1 etc. Although detection of the ATM has therapeutic application for prostate cancer patient for poly [ADP]-ribose polymerase inhibitor treatment, the significance of loss of ATM function in hairy cell leukemia variant is unknown.

Results (if a Case Study enter NA)

N/A

Conclusion

In conclusion, our case demonstrates that molecular test in hairy cell leukemia variant can be helpful for confirming the diagnosis as well as providing predictive information for potential target therapy. In our case, lack of MAP2K1 mutation precludes the patient for targeted therapy. In addition, the finding of novel mutation might help to understand the molecular pathogenesis in this rare entity.
Non-IgG4-Related Fibrosing Mediastinitis Diagnosed on Needle Biopsy and Treated with Steroids
Authors: Wilhelm A , Muthukumarana P V , Uddin H , Okereke I
Published: October 2021

Introduction/Objective

Fibrosing mediastinitis (FM) is a rare and aggressive fibroinflammatory process of the mediastinum. It is a difficult diagnosis, especially on small biopsies as the presentation and the fibrosing background mimics malignancy. Although the exact etiology is unknown, prior fungal infections, certain medications and autoimmune conditions are documented. Significant histopathologic overlap with IgG4-related disease (IgG4-RD) can complicate the diagnosis. An accurate diagnosis is important for treatment as corticosteroids for IgG4-RD is proven to be effective; however, no effective treatment is described for FM. Additionally, immunosuppression would prove harmful in other conditions with fibrosing morphology. We report a very unique case of non-IgG4-related FM that was diagnosed on a needle biopsy and showed marked improvement with corticosteroids.

Methods/Case Report

A 65-year-old man with no significant medical history presented with syncope and recent weight loss. Imaging revealed a pericardial effusion and a heterogeneous 8.1 cm mediastinal mass encasing the great vessels and aortic arch. Needle biopsy showed fibrosis with keloid-type collagen, mixed inflammatory cells, and few benign lymphoid follicles. No evidence of hematolymphoid malignancy, carcinoma, or infection was seen. Serum and tissue IgG-4 studies were negative for IgG4-RD. A diagnosis of FM was made, and a trial of corticosteroids was initiated. Interestingly, the patient showed improvement with a 2.3 cm decrease in lesion size and near-complete resolution of pericardial effusion after one month of treatment.

Results (if a Case Study enter NA)

NA

Conclusion

FM should be included in the differential diagnosis of infiltrative mediastinal masses which presents a diagnostic challenge on limited needle biopsies. Although effective treatment for non-IgG4 related FM is not clearly established, corticosteroids may prove beneficial, as in this patient. Mortality from FM is directly related to the compressive effect on adjacent structures. Early diagnosis and treatment are therefore crucial. Further studies on the pathogenesis are necessary, as well as better directed therapeutic options.
Anaplastic lymphoma kinase rearrangements in non-small cell lung cancer in Jordan
Authors: Sughayer M A , Maraqa B , Al-Ashhab M
Published: October 2021

Introduction/Objective

ALK rearrangement is an important oncogenic driver in a substantial portion of non-small cell lung cancer (NSCLC) patients ranging from 2-7%. Treatment options such as ALK tyrosine kinase inhibitors (TKI) improve progression-free survival and overall survival. Candidates for such treatment are selected based on the identification of the ALK rearrangement. While fluorescence in situ hybridization (FISH) was considered the gold standard method, the availability of a robust FDA-approved companion diagnostic immunohistochemistry (IHC) assay has led to a paradigm shift in ALK testing. The purpose of this study is to determine the prevalence of ALK rearrangement in Jordanian NSCLC patients along with their clinicopathological characteristics and to compare the results of IHC and FISH methods for detecting ALK rearrangements.

Methods/Case Report

A retrospective study was conducted on 449 Jordanian King Hussein Cancer Center patients with NSCLC whose biopsy samples were tested for ALK rearrangement using FISH and or IHC (clone D5F3) in the period between 2018 and 2020.

Results (if a Case Study enter NA)

During the study period, the rate of ALK positivity by either IHC or FISH method was 4 percent (18 ALK positive cases out of 449 cases of non-small cell lung cancer). Seven cases were positive for both IHC and FISH, and nine cases were positive for IHC with no confirmation by FISH method; one case was ALK positive by IHC and negative by FISH with a significant response to ALK TKI; one case was IHC negative but FISH positive, with no ALK TKI therapy. The calculated sensitivity of ALK D5F3 immunostain compared to FISH results in the current study is 87.5% while the specificity is 96%.

ALK positive patients were significantly younger than those with negative results (p-value=0.051), and women were three times more likely than men to have the rearrangement (p-value=0.013). Rearrangement was more likely to be found in nonsmokers/light or ex-smokers (p-value= 0.013). All patients had clinical stage IV or III disease at presentation with stage IV found in tow thirds of the patients.

Conclusion

ALK rearrangement is found in 4% of all NSCLC in Jordan. Patients are more likely to be younger, females and light or nonsmokers with an advanced stage disease at presentation. IHC is an acceptable alternative to FISH for ALK testing with reasonable sensitivity and specificity in addition to its advantages in terms of robustness, turnaround time and cost savings
TAFRO syndrome: A case report with biopsy findings from three sites
Authors: Jadhav N C , Freeman J
Published: October 2021

Introduction/Objective

TAFRO syndrome (thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly) is a rare systemic inflammatory disorder. First reported in 2010, the majority of cases are from Japan, where it is currently regarded as a subtype of HHV8-negative Multicentric Castleman Disease.

Methods/Case Report

We report a case of TAFRO occurring in a 17-year old from Puerto Rico. She originally presented with abdominal pain, vomiting, and fever, and was also found to have splenomegaly, elevated sedimentation rate and C-reactive protein. Over the course of the next several days she developed respiratory distress, requiring ventilatory support, and anuric renal failure. Renal biopsy demonstrated evidence of thrombotic microangiopathy, which was interpreted as atypical hemolytic syndrome, requiring dialysis and treated with eculizumab. Because other diagnostic considerations at the time also included hemophagocytic lymphohistiocytosis and catastrophic antiphospholipid syndrome, bone marrow examination was performed which demonstrated increased megakaryocytes, without fibrosis or hemophagocytosis. Over the next two months, she developed anasarca with ascites, pleural effusion, pericardial effusion, multiple enlarged axillary and cervical lymph nodes, and persistence of splenomegaly. Biopsy of a left cervical lymph node demonstrated features suggestive of Castlemans disease, plasma cell variant. The constellation of symptoms, laboratory investigations and biopsy diagnoses support the diagnosis of TAFRO syndrome. The patient received rituximab, high dose steroids, and hemodialysis with clinical improvement. At 16 months follow up, the patient is in remission.

Results (if a Case Study enter NA)

NA

Conclusion

From a pathology standpoint, the case provides biopsy findings of three distinct organ systems all with relatively unusual findings that together are characteristic of TAFRO syndrome. It highlights the risk of accepting “biopsy proven” diagnoses such as atypical hemolytic uremic syndrome that also require significant laboratory and clinical correlation for true confirmation, and points out the importance and opportunity for pathologists to see the forest along with the trees in order to recognize rare diseases such as TAFRO.
Weakened IgG4 Staining and Positive PLA2R staining Indicate Resolving Primary Membranous Glomerulopathy (MGN)
Authors: Al-Othman Y , Li W , Kanaan H , Zhang P
Published: October 2021

Introduction/Objective

IgG4 related disease, a systemic autoimmune inflammatory disorders, can be identified by high% of IgG4 positive plasma cells, thus IgG4 staining in paraffin embedded tissue is widely available in the most of pathology labs. IgG4 staining has been found useful to identify primary MGN (PLA2R and/or THSD7A positive) by others and us. This study was to scrutinize the findings of primary vs secondary MGN needed for IgG4 staining as a screening tool in our renal pathology practice over pasts 5 years

Methods/Case Report

IgG4 staining in paraffin embedded tissue was performed in 45 primary MGN and 43 secondary MGN after the clinical history was reviewed and a possibility of primary MGN cannot be excluded. In addition, both groups of cases were also stained for PLA2R. Detail correlation with clinical history was analyzed.

Results (if a Case Study enter NA)

Totally 82 % (37/45) of primary MGN was found diffuse positive for IgG4 staining at 2+ intensity in the glomeruli. Seven out of eight remaining primary MGN cases with either negative or weak IgG4 stained MGN were found to have diffuse resolving features by electron microscopy but there was still positive PLA2R staining in the glomeruli. All secondary MGN were stained negatively for both IgG4 and PLA2R and we found that etiologies of the secondary MGN included membranous lupus nephritis, infection, GVHD, or variants of cancers.

Conclusion

Our data indicate that IgG4 staining along (without IgG1-3 staining) is a reliable screening tool to confirm the majority of primary MGN vs secondary MGN in paraffin embedded tissue. As both PLA2R+ and THSD7A+ primary MGN are both IgG4 related, the IgG4 staining may potentially represent a wider range of scope in identifying primary MGN. In addition, negative/weak IgG4 staining in PLA2R-positive MGN most likely represents a primary MGN with resolving features.
Red Blood Cell Alloimmunization in Sickle Cell Disease: Advantage of Ethnic Heritage between Donors and Patients in Jeddah, Saudi Arabia
Authors: Anwar F , Almohammadi M , Garni A , Jamallail S , Alsamkari W , Alsafi A
Published: October 2021

Introduction/Objective

Red blood cell (RBC) transfusion is frequently required for patients with sickle cell disease (SCD). Development of alloantibodies in these patients complicates the blood bank process needed to identify these antibodies and to find compatible RBC units. The rate of alloimmunization has been reported as high as 47% in one study and 34.2% in another study from Eastern region of Saudi Arabia. The purpose of this study was to determine incidence and rate of RBC alloimmunization in the Saudi population in the Western region in SCD.

Methods/Case Report

A retrospective analysis of the immunohematological and transfusion history of a total of 161 SCD patients was reviewed, of which there 95 males and 66 females. All patients had erythrocytapheresis, ranging from one to 24 full red cell exchange sessions. A total of 490 red cell exchanges were performed and 4,914 units of blood were used. Extended compatibility to RhCcEe and K antigen was performed. Patient who developed alloimmunization to any of RhCcEe and K antigen were matched for Kidd, Duffy and MSN antigens for subsequent RBC requirements.

Results (if a Case Study enter NA)

The RBC alloimmunization incidence was 18% with a rate of 0.6 antibodies per 100 RBC transfusions. Alloimmunization in females was significantly higher than in the patients. Eighteen (11.2%) female patients demonstrated antibodies as compared to eleven (6.8%) male patients. Twelve patients (7.4%) had a history of at least one alloantibody and 17 (10.6%) had more than one. Antibodies found were directed against E (7.4%), K (5.6%), and D, C, c, S, M, Lea, Jk a, Chido/Rodgers, Fy a. Seven (4.3%) patients also had warm autoantibodies.

Conclusion

RBC alloimmunization incidence and rate in our study was lower to those reported in less heterogeneous population of donors and patients. Nonetheless, RBC alloimmunization still occurs in patients with SCD, often due to Rh variants or lack of consistency in the application of prophylactic antigen matching between institutions. Therefore, we believe that this rate can still be further reduced if all centers in the region establish transfusion programs to include at least RhCcEe and K phenotypic compatibility and communication mechanisms between major treating centers and transfusion centers in smaller cities to minimize the risks of exposing the patient to different RhCcEe and K phenotype and of developing RBC alloimmunization.
The Immunohematologic and Clinical Evaluation of Increased Risk for Red Blood Cell Alloimmunization among Multi-Transfused Pediatric Patients in a Tertiary Medical Institution in Central Luzon, Philippines
Authors: Ebreo C C , Castillo R M , Dizon M D , Domasian A D , Floresta G P , Gallestre M C , Hernaez A M , Kasilag K V , Sison R G , Nieto X A
Published: October 2021

Introduction/Objective

Multiple blood transfusion is a therapeutic treatment for patients with hematologic disorders. However, patients who undergo repeated blood transfusions are at higher risk of developing red blood cell alloimmunization. This may further delay the availability of compatible blood and pose a higher risk for hemolytic transfusion reactions.

Methods/Case Report

A total of 55 patients, with an age range of 2-19 years, were included in the study. All patients had hematologic disorders and underwent chronic blood transfusion. In this retrospective study, Student’s t-test and Fisher’s exact tests were used to compare the means and percentages, respectively, under 5% level of significance. R ver 4.0.3 was utilized in the analyses.

Results (if a Case Study enter NA)

Out of the 55 pediatric patients, 10 (18.2%; CI95%: 9.1 to 30.9%) were found to be alloimmunized. Two patients had multiple antibodies whereas the others had one specificity, having a total of 12 detected alloantibodies. The most prevalent alloantibody was anti-E (60%), followed by anti-Jka (20%), anti-Mia (20%), anti-S (10%), and anti-K (10%). All obtained alloantibodies showed no correlation (p>0.05) with the age group of the patients. Subsequently, it was not evident that average blood transfusion (p=0.949) and packed red blood cells transfused per year (p=0.782) significantly differ between alloimmunized and non-alloimmunized patients. Similarly, there was no sufficient evidence that the age group (p=0.723), sex (p=1.000), blood group (p=1.000) and hematologic disorder (p=0.949) were associated with their alloimmunization status.

Conclusion

The alloimmunization incidence rate of 18.2% in this study was higher compared to related literatures, and most of the detected alloantibodies were of IgG type which are commonly clinically significant. The study showed no significant correlation between the specific risk factors and the development of alloimmunization. For patient care, it is imperative to include precautions such as extended RBC phenotyping and provision of antigen matched RBCs to prevent unnecessary transfusion reactions caused by alloimmunization.
Crossmatch to Transfusion Ratios in the Operating Room with an Expanding Hospital Footprint
Authors: Virk M S
Published: October 2021

Introduction/Objective

The crossmatch to transfusion (CT) ratio is one of several metrics used to assess the efficiency of blood product utilization. A ratio of 2:1 has been suggested as a hospitalwide limit that indicates appropriate use of red blood cells (RBCs), and the Q Probe Study found that the average CT ratio for hospitals in the United States is 1.85:1. This ratio can vary widely from one patient location to another due to anticipatory or unknown needs within the operating room (OR) compared to laboratory or symptomatic based orders for patients on the floor. Following a recent hospital expansion at Stanford University Hospital that led to the creation of multiple adult OR locations and a move of our blood bank, we monitored the CT ratios of various OR locations and surgical subspecialties to assess the drivers of increased blood product utilization and interventions that could improve these metrics.

Methods/Case Report

This quality improvement project involved the collection of blood product order information through our LIS (SafeTrace) and clinical system (EPIC), creation of an on-demand report to provide constant updates to that data, qualitative interviews with OR staff, and process mapping for multiple subspecialties.

Results (if a Case Study enter NA)

Prior to our hospital expansion there was an appropriate CT ratio of 2.1:1 in our adult ORs. Post-expansion, this ratio has been between 3-5:1, depending on the OR location. This led us to focus our efforts on the OR locations that were furthest from the blood bank and existed prior to the hospital expansion. In discussions with our OR colleagues, we found that the major driver for this increased ratio was a longer travel path for blood products and delays in delivery which led to larger amounts of anticipatory orders. In addition, these concerns caused duplicative efforts in the OR with surgical teams and Anesthesiologists placing pre-operative orders for the same cases. To address this concern, remote blood product dispensing was implemented. To address duplicative ordering workflows, we partnered with Anesthesiology to determine the blood ordering workflow agreements with each surgical subspecialty to ensure appropriate division of roles and responsibilities. Evaluation of CT ratios after these interventions is ongoing, but early data suggests a significant reduction.

Conclusion

Post-intervention phase ongoing and will be updated with additional data & conclusions prior to conference.