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The Molecular Pathology Section, Cleveland Clinic (Cleveland, OH), has undergone enhancement of its testing portfolio and processes. An Excel 2013- and paper-based data-management system was replaced with a commercially available laboratory information-management system (LIMS) software application, a separate bioinformatics platform, customized test-interpretation applications, a dedicated sample-accessioning service, and a results-releasing software application. The customized LIMS solution manages complex workflows, large-scale data packets, and process automation. A customized approach was required because, in a survey of commercially available off-the-shelf software products, none met the diverse and complex needs of this molecular diagnostics service. The project utilized the expertise of clinical laboratorians, pathologists, genetics counselors, bioinformaticians, and systems analysts in partnering with software-engineering consultants to design and implement a solution. Concurrently, Agile software-building best practices were formulated, which may be emulated for scalable and cost-effective laboratory-authored software.

Background Our understanding of SARS-CoV-2 evolution is limited. Most estimates arise from analysis of global databases populated with unrelated sequences and is currently estimated at ∼27.7 substitutions/genome/year. SARS-CoV-2 polymerase contains a proofreading function encoded by NSP-14 limiting change. Additionally, virus evolution may be influenced by patient comorbidity. Intra-host mutational rate (MR) during infection remain poorly studied. Methods To minimize effect of vaccination and/or natural immunity on MR analysis, paired samples from adults originating from the initial pandemic wave (3/17/2020 through 5/27/2020) were retrieved and analyzed at Cleveland Clinic. Viral genome analysis was performed using next generation sequencing, and mutations between paired samples were quantified at allele frequencies (AF) ≥ 0.1, ≥ 0.5 and ≥ 0.75 and compared. MR was calculated employing F81 and JC69 evolution­­­­ models and compared between isolates with (Δ NSP-14) and without (wildtype, wt) non-synonymous mutations in NSP-14 and by comorbidity. Results A total of 40 patients (80 sample pairs) were identified. Median interval between paired tests was 15 days [range 5-32]. The estimated MR by F81 modeling was 317.2 (95%CI 312.0-322.3), 54.6 (95%CI 52.5-56.7) and 45.1 (95%CI 43.1-47.0) substitutions/genome/year at AF of ≥0.1, ≥0.5, ≥0.75 respectively. Rates in ΔNSP-14 (n=13) vs wt (n=27) groups were 557.7 (95%CI 537.0-578.2) vs 193.1 (95%CI 187.1-199.1) p-value 0.001, 50.8 (95%CI 44.3-57.3) vs 56.3 (95%CI 53.1-59.4) p-value 0.144, and 31.0 (95%CI 25.9-36.1) vs 51.3 (95%CI 48.3-54.3) p- value < 0.001 at AF ≥0.1, ≥0.5, ≥0.75 respectively. Patients with immune comorbidities (n=6) had significantly higher MR of 137.6 (95%CI 114.6-160.5) vs 40.5 (95%CI 38.4-42.7) p-value < 0.001, and 113.7 (95%CI 92.8-134.5) vs 33.4 (95%CI 31.5-35.4) p-value < 0.001 at AF ≥0.5 and ≥0.75 respectively. Similar results were obtained when using the JC69 model. Conclusion Intra-host SARS-CoV-2 mutation rates are higher than those reported through population analysis. Virus strains with altered NSP-14 have accelerated MR at low AF. Immunosuppressed patients have elevated MR at higher AF. Understanding intra-host virus evolution will aid in current and future pandemic modeling. Disclosures Frank Esper, M.D, Johnson and Johnson: Advisor/Consultant Daniel D. Rhoads, M.D. PhD, Luminex: Advisor/Consultant|Talis Biomedical: Advisor/Consultant|Thermo Fisher: Advisor/Consultant.

Background Four severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants predominated in the United States since 2021. Understanding disease severity related to different SARS-CoV-2 variants remains limited. Method Viral genome analysis was performed on SARS-CoV-2 clinical isolates circulating March 2021 through March 2022 in Cleveland, Ohio. Major variants were correlated with disease severity and patient outcomes. Results In total 2779 patients identified with either Alpha (n = 1153), Gamma (n = 122), Delta (n = 808), or Omicron variants (n = 696) were selected for analysis. No difference in frequency of hospitalization, intensive care unit (ICU) admission, and death were found among Alpha, Gamma, and Delta variants. However, patients with Omicron infection were significantly less likely to be admitted to the hospital, require oxygen, or admission to the ICU (χ2 = 12.8, P < .001; χ2 = 21.6, P < .002; χ2 = 9.6, P = .01, respectively). In patients whose vaccination status was known, a substantial number had breakthrough infections with Delta or Omicron variants (218/808 [26.9%] and 513/696 [73.7%], respectively). In breakthrough infections, hospitalization rate was similar regardless of variant by multivariate analysis. No difference in disease severity was identified between Omicron subvariants BA.1 and BA.2. Conclusions Disease severity associated with Alpha, Gamma, and Delta variants is comparable while Omicron infections are significantly less severe. Breakthrough disease is significantly more common in patients with Omicron infection.

Background Methods for identifying gene fusion events, such as fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and transcriptome analysis, are either single gene approaches or require bioinformatics expertise not generally available in clinical laboratories. We analytically validated a customized next-generation sequencing (NGS) panel targeting fusion events in 34 genes involving soft-tissue sarcomas. Methods Specimens included 87 formalin-fixed paraffin-embedded (FFPE) tissues with known gene fusion status. Isolated total nucleic acid was used to identify fusion events at the RNA level. The potential fusions were targeted by gene-specific primers, followed by primer extension and nested PCR to enrich for fusion candidates with subsequent bioinformatics analysis. Results The study generated results using the following quality metrics for fusion detection: (a) ≥100 ng total nucleic acid, (b) RNA average unique start sites per gene-specific primer control ≥10, (c) quantitative PCR assessing input RNA quality had a crossing point Conclusions The test validation study demonstrated analytical sensitivity of 98.7% and analytical specificity of 90.0%. The NGS-based panel generated highly concordant results compared to alternative testing methods.

Inflammatory pseudotumor-like follicular/fibroblastic dendritic cell sarcoma (IPT-like FFDCS) is a rare, indolent neoplasm that occurs in the spleen or liver and harbors Epstein-Barr virus (EBV) integrated into the host genome. The molecular genetic characteristics of IPT-like FFDCS have not been well studied and there are no established and actionable molecular features to guide treatment decisions or diagnosis beyond the recognition of viral genome integration. We subjected two cases of IPT-like FFDCS to a comprehensive next-generation sequencing analysis. Several variants of uncertain clinical significance were detected in both tumors. No variants of potential or strong clinical significance were detected within the targeted regions of the evaluated genes. Additionally, no fusion events were detected involving the genes in either tumor. The performed molecular analysis identified no genetic aberrations in IPT-like FFDCS and its genomic landscape remains, with the exception of a monoclonal EBV gene, largely undefined.

This study piloted a pan-solid-tumor next generation sequence (NGS)-based laboratory developed test as a diagnostic aid in melanocytic tumors. 31 cases (4 "epithelioid" nevi, 5 blue nevi variants, 7 Spitz tumors [3 benign and 4 malignant] and 15 melanomas) were evaluated. All tumors [median diameter 7 mm (range 4-15 mm); median thickness 2.25 mm (range 0.25-12 mm)] yielded satisfactory results. The number of small nucleotide variants/tumor was significantly different between melanoma (median 18/tumor, range 4-71) and all other lesions (median 8/tumor, range 3-17) (P 0.004) and malignant (median 16/tumor, range 4-71) vs benign lesions (median 7/tumor, range 3-14) (P = 0.01). BRAF, MET, NTRK1, and ROS fusions only occurred in benign Spitz tumors; EML4 fusion, BRAF, MAP2K1 and TERT mutations occurred in malignant Spitz tumors and/or melanoma. Amplifications and NRAS and NF1 mutations only occurred in melanoma. Most melanomas contained1 pathogenic alteration. Developed NGS-based criteria correctly classified all malignant lesions in this series. 10/12 cases showed concordance with FISH; consensus diagnosis agreed with NGS classification in FISH-non-concordant cases. This pilot study suggests that NGS may be an effective diagnostic adjunct comparable to FISH, but further studies with larger numbers of cases are needed.

Context.— The College of American Pathologists (CAP), a laboratory accreditation organization with deemed status under the Clinical Laboratories Improvement Amendments of 1988 administers accreditation checklists. Checklists are used by laboratories to ensure regulatory compliance. Peer-level laboratory professionals audit laboratory records during inspections to assess compliance. Objective.— To identify the most frequently cited deficiencies for molecular oncology laboratories undergoing CAP accreditation inspections and describe laboratory improvement opportunities. Design.— The CAP Molecular Oncology Committee (MOC), which is involved in maintaining the Molecular Pathology checklist, reviewed data and inspector comments associated with the most frequently observed citations related to molecular oncology testing from laboratories inspected by the CAP during a 2-year period (2018–2020). Results.— Of 422 molecular oncology laboratories that underwent accreditation inspections, 159 (37.7%) were not cited for any molecular oncology–related deficiencies. For the All Common (COM) and Molecular Pathology checklists, there were 364 and 305 deficiencies, corresponding to compliance rates of 98.8% and 99.6%, respectively. The most frequently cited deficiencies are described. The COM checklist deficiencies were associated most often with the analytic testing phase; the MOL checklist deficiencies were more evenly distributed across the preanalytic, analytic, and postanalytic phases of testing. Conclusions.— Molecular oncology laboratories demonstrated excellent compliance with practices that support high-quality results for patients and the health care providers who use those test results in patient management. This review includes a critical assessment of opportunities for laboratories to improve compliance and molecular oncology testing quality.

Following bariatric and metabolic surgery (BMS), patients may develop persistent cytopenia(s) despite adequate micronutrient levels. A comprehensive analysis of laboratory and hematopathologic findings in BMS patients with unexplained cytopenia(s) has not been previously described.We reviewed the clinical and laboratory data, bone marrow histology, and used ancillary testing to characterize patients with a history of BMS who had subsequent bone marrow biopsies due to unexplained cytopenia(s).All patients had anemia and 59% (23/39) had additional cytopenias. Myelodysplastic syndrome (MDS) and clonal cytopenia of unknown significance (CCUS) were diagnosed in 8% (3/39) and 10% (4/39), respectively. Remaining cases were classified as idiopathic cytopenia of unknown significance (ICUS) with anemia alone (ICUS-A) in 47% (15/32) or multiple cytopenias (ICUS-PAN) in 53% (17/32). Time since surgery, age, or amount of weight loss was not associated with a specific diagnosis. No patient was vitamin B12 or folate deficient. However, vitamin B6 and zinc were decreased in 47% (5/11) and 29% (9/29), respectively. Examination of bone marrow aspirates revealed slight erythroid dyspoiesis affecting10% of precursors in 60% (9/15) ICUS-A and 59% (10/17) ICUS-PAN.Bone marrow findings in patients with unexplained cytopenia(s) after BMS are not specific in the majority of cases, and caution is advised when interpreting dyserythropoiesis. Levels of micronutrients and vitamins other than iron, folate and vitamin B12 are frequently disturbed in this patient cohort and warrant correction and close clinical follow-up.

AimVarious approaches have been reported for distinguishing separate primary lung adenocarcinomas from intrapulmonary metastases in patients with two lung nodules. The aim of this study was to determine whether histological assessment is reliable and accurate in distinguishing separate primary lung adenocarcinomas from intrapulmonary metastases using routine molecular findings as an adjunct.MethodsWe studied resected tumour pairs from 32 patients with lung adenocarcinomas in different lobes. In 15 of 32 tumour pairs, next-generation sequencing (NGS) for common driver mutations was performed on both nodules. The remainder of tumour pairs underwent limited NGS, or EGFR genotyping. Tumour pairs with different drivers (or one driver/one wild-type) were classified as molecularly unrelated, while those with identical low-frequency drivers were classified as related. Three pathologists independently and blinded to the molecular results categorised tumour pairs as related or unrelated based on histological assessment.ResultsOf 32 pairs, 15 were classified as related by histological assessment, and 17 as unrelated. Of 15 classified as related by histology, 6 were classified as related by molecular analysis, 4 were unrelated and 5 were indeterminate. Of 17 classified as unrelated by histology, 14 were classified as unrelated by molecular analysis, none was related and 3 were indeterminate. Histological assessment of relatedness was inaccurate in 4/32 (12.5%) tumour pairs.ConclusionsA small but significant subset of two-nodule adenocarcinoma pairs is inaccurately judged as related by histological assessment, and can be proven to be unrelated by molecular analysis (driver gene mutations), leading to significant downstaging.

Context.— Bone and soft tissue tumors are heterogeneous, diagnostically challenging, and often defined by gene fusions. Objective.— To present our experience using a custom 34-gene targeted sequencing fusion panel. Design.— Total nucleic acid extracted from formalin-fixed, paraffin-embedded (FFPE) tumor specimens was subjected to open-ended, nested anchored multiplex polymerase chain reaction and enrichment of 34 gene targets, thus enabling detection of known and novel fusion partners. Results.— During a 12-month period, 147 patients were tested as part of routine clinical care. Tumor percentage ranged from 10% to 100% and turnaround time ranged from 3 to 15 (median, 7.9) days. The most common diagnostic groups were small round blue cell tumors, tumors of uncertain differentiation, fibroblastic/myofibroblastic tumors, and adipocytic tumors. In-frame fusion transcripts were identified in 64 of 142 cases sequenced (45%): in 62 cases, the detection of a disease-defining fusion confirmed the morphologic impression; in 2 cases, a germline TFG-GPR128 polymorphic fusion variant was detected. Several genes in the panel partnered with multiple fusion partners specific for different diagnoses, for example, EWSR1, NR4A3, FUS, NCOA2, and TFE3. Interesting examples are presented to highlight how fusion detection or lack thereof was instrumental in establishing accurate diagnoses. Novel fusion partners were detected for 2 cases of solid aneurysmal bone cysts (PTBP1-USP6, SLC38A2-USP6). Conclusions.— Multiplex detection of fusions in total nucleic acid purified from FFPE specimens facilitates diagnosis of bone and soft tissue tumors. This technology is particularly useful for morphologically challenging entities and in the absence of prior knowledge of fusion partners, and has the potential to discover novel fusion partners.

We report the largest series to date (N = 6) of EWSR1-SMAD3 rearranged fibroblastic tumor. Initially described in 2018, the tumor features a marked female predominance (F:M, 5:1, mean age 44-years, median age 45.5 years; range 27-57), with most cases (5/6, 83%) arising in acral locations (4 on foot/toe, 1 on hand). One case presented on the lower extremity. The lesions presented as nodules and were composed of short, variably cellular, intersecting fascicles of uniform spindled cells in a collagenous to myxoid stroma. In four cases, the tumor abutted the epidermis without a grenz zone. In one case, there was an abrupt transition to a central, acellular hyalinized area. Two other cases had admixed smaller collagenous areas, reminiscent of collagen rosettes. One had a concentric arrangement of tumor cells around blood vessels. Mitotic activity was low (

BACKGROUND Noninvasive prenatal testing (NIPT) uses cell-free DNA (cfDNA) as an analyte to detect copy-number alterations in the fetal genome. Because maternal and fetal cfDNA contributions are comingled, changes in the maternal genome can manifest as abnormal NIPT results. Circulating tumor DNA (ctDNA) present in cases of maternal neoplasia has the potential to distort the NIPT readout to a degree that prevents interpretation, resulting in a nonreportable test result for fetal aneuploidy. METHODS NIPT cases that showed a distortion from normal euploid genomic representation were communicated to the caregiving physician as nonreportable for fetal aneuploidy. Follow-up information was subsequently collected for these cases. More than 450000 pregnant patients who submitted samples for clinical laboratory testing >3 years are summarized. Additionally, in-depth analysis was performed for >79000 research-consented samples. RESULTS In total, 55 nonreportable NIPT cases with altered genomic profiles were cataloged. Of these, 43 had additional information available to enable follow-up. A maternal neoplasm was confirmed in 40 of these cases: 18 malignant, 20 benign uterine fibroids, and 2 with radiological confirmation but without pathological classification. CONCLUSIONS In a population of pregnant women who submitted a blood sample for cfDNA testing, an abnormal genomic profile not consistent with fetal abnormalities was detected in about 10 out of 100000 cases. A subset of these observations (18 of 43; 41.9%) was attributed to maternal malignant neoplasms. These observational results suggest the need for a controlled trial to evaluate the potential of using cfDNA as an early biomarker of cancer.

This commentary highlights the article by Hsiao et al, who explore the recent migration of Current Procedural Terminology codes for reimbursement of genomic tests.

Renal cell carcinoma with (angio) leiomyomatous stroma (RCCLMS) is included as a provisional entity in the 2016 World Health Organization (WHO) classification of renal epithelial neoplasia; however, debate remains whether it represents a distinct entity or a heterogenous group of renal cell carcinomas (RCCs) with overlapping morphology. Also, its relationship to similar tumors occurring in the setting of tuberous sclerosis complex (TSC) is not fully addressed. We analyzed the clinicopathologic, immunohistochemical, and molecular characteristics of 23 sporadic RCCs associated with smooth muscle stroma and classified them into 2 groups, independent of molecular results: (1) RCCLMS (n=18) and (2) clear cell renal cell carcinoma (CCRCC) (n=5). The classification of a case as "RCCLMS" was based on morphologic comparison with 5 "index" RCCs from 3 patients with TSC showing similar features and the presence of diffuse CK7 expression. To investigate mutational and copy number alterations, a 170-gene solid tumor panel was utilized to sequence 14 RCCLMSs and control of 5 CCRCCs. Also, 4 RCCLMSs, suspicious for chromosome 8 monosomy, were further evaluated by a broader 479 gene sequencing panel that included ELOC (also referred to as TCEB1). Clinical information and follow-up data were obtained from electronic medical records. The mean age of patients with RCCLMS was 52 years (range, 33 to 69) with male:female ratio of 1:2. Macroscopically, all tumors were solitary and predominantly (82%) tan/red, circumscribed, and solid. The average tumor size was 2.3 cm (range, 1.1 to 4.5). Microscopically, the distinctive feature included tumor nodules of elongated and frequently branching tubules lined by cells with voluminous clear to mildly eosinophilic cytoplasm (100%), separated by focal to prominent smooth muscle stroma. Additional frequently identified features included: biphasic pattern of collapsed acini surrounding tubules with voluminous cytoplasm (50%), focal papillary architecture (39%), peritumoral lymphoid aggregates (39%), and hemosiderin-laden macrophages (33%). All 11 (100%) RCCLMSs with available staging information were pT1; 78% were WHO/International Society of Urologic Pathology (ISUP) grade 2 and 22% grade 3. Immunophenotypically, RCCLMSs were characterized by diffuse CK7, CAM5.2 and CD10 reactivity (100%). All patients with available follow-up (n=10) were alive and without disease progression after a mean and median follow-up of 25.2 (range: 1 to 58) and 25 months, respectively. The molecular results showed recurrent mutations in all RCCLMS: TSC1 (4), TSC2 (4), MTOR (6), and/or ELOC (2). Five control CCRCCs demonstrated primary alterations in VHL gene, while all 14 RCCLMS cases tested had intact VHL gene. Of 2 RCCLMSs with confirmed monosomy 8, 1 showed a hotspot ELOC mutation without TSC/MTOR mutations, and 1 showed a previously undescribed 3-bp in-frame ELOC deletion, along with a truncating TSC1 mutation. In conclusion, RCCLMS, as defined herein, harbors recurrent mutations of TSC1/TSC2, MTOR, and/or ELOC, consistent with hyperactive MTOR complex. Our findings argue that these tumors represent the sporadic counterpart to morphologically identical tumors occurring in TSC patients. Finally, the data support that RCCLMS is a novel subtype of RCC with unique morphologic, immunohistochemical, and molecular characteristics that is distinct from CCRCC and clear cell-papillary RCC.

CIC rearranged sarcomas have significant overlap with Ewing sarcoma, are aggressive, and typically present in deep soft tissue. They most commonly have a t(4 ; 19)(q35 ; q13) with CIC-DUX4 fusion. Superficial presentation is rare. We report eight (6F, 2M ; median 45-years-old, range 14-65) superficial CIC-rearranged sarcomas, involving the extremities (n = 4), vulva (n = 2), and trunk (n = 2). The tumors were composed of nodules/sheets of round cells with necrosis and hemorrhage separated by dense hyaline bands. Tumor cells had vesicular chromatin, prominent nucleoli and frequent mitotic figures. One showed pagetoid spread. Targeted next-generation sequencing was positive for CIC- DUX4 fusion (6/6) ; fluorescence in situ hybridization (FISH) was positive for CIC rearrangement (2/3). Eight of eight had evidence of CIC-DUX4 fusion/rearrangement by molecular techniques. Immunohistochemistry was positive for CD99+ (8/8) and DUX4+ (4/4). FISH for EWSR1 rearrangement was negative (5/5). Of five patients with at least 6 months follow-up, three of five died of disease, all within 2 years of presentation. One is alive with disease at 48 months. One is disease free at 3 months. Superficial CIC-rearranged sarcomas should be considered in cases exhibiting features reminiscent of Ewing sarcoma, but with increased pleomorphism and/or geographic necrosis. In contrast to superficial Ewing sarcomas, superficial CIC-rearranged sarcomas are aggressive.

e21560 Background: Prognosis in melanoma with brain metastasis is poor with a median survival of four months and a one-year survival rate of 10–20%. There is an unmet need for surveillance methods that can supplement imaging at regular intervals. Serial analysis of circulating tumor DNA (ctDNA) may aid surveillance and prognostication. A PCR-based, “specimen in/result out” testing device was employed to detect BRAF variants in plasma-derived ctDNA to evaluate the utility of rapid biomarker detection in the management of melanoma with brain metastasis. Methods: Serial blood samples from patients diagnosed with BRAF mutation-positive metastatic melanoma were collected at regular intervals. We employed a real-time PCR-based automated mutation detection system (Idylla; Biocartis, Belgium) to interrogate the plasma samples. The ctDNA mutation detection trend was analyzed relative to disease progression. Results: 39 patients with BRAF mutation positive melanoma were enrolled. 29 patients were treated in the metastatic setting, 10 in the adjuvant setting. 18 of the 29 patients with metastatic disease (62%) had brain metastases. Circulating BRAF mutation was detected in 17 of the 29 (59%) patients with metastatic disease, and was not detected in any patients treated adjuvantly. In the group with metastatic disease, this circulating biomarker changed from undetectable to detectable in eight (28%) and detectable to undetectable in three (10%). No change in circulating mutation status occurred in 18 (62%). In the eight patients who had an initial negative test that later became positive, seven (87%) had brain metastases. In three patients, ctDNA mutation detection occurred before the diagnosis of brain metastases on imaging, with a median lead time of five weeks (range, 3-12 weeks). In one patient with de novo metastatic disease admitted to the ICU, tissue was unavailable for BRAF testing but plasma was found to be positive for ctDNA BRAF detection. BRAF/MEK targeted therapy resulted in a sustained objective response. Five of six (83%) patients that had persistent ctDNA positivity had brain metastases. Among patients with brain metastases, median overall survival (mOS) of patients demonstrating >50% test positivity was numerically longer than those with

Key Points Question Are SARS-CoV-2 variants, virus clades, or clade groups associated with disease severity and patient outcomes? Findings In this cross-sectional study of 302 SARS-CoV-2 isolates, 6 different Global Initiative on Sharing All Influenza Data clades circulated in the community followed by a rapid reduction in clade diversity. Several variants, including 23403A>G (D614G), were significantly associated with lower hospitalization rates and increased patient survival. Meaning These findings suggest that SARS-CoV-2 clade assignment is an important factor that may aid in estimating patient outcomes., This cross-sectional study examines the association of identified SARS-CoV-2 variants, virus clades, and clade groups with disease severity and patient outcomes., Importance Understanding of SARS-CoV-2 variants that alter disease outcomes are important for clinical risk stratification and may provide important clues to the complex virus-host relationship. Objective To examine the association of identified SARS-CoV-2 variants, virus clades, and clade groups with disease severity and patient outcomes. Design, Setting, and Participants In this cross-sectional study, viral genome analysis of clinical specimens obtained from patients at the Cleveland Clinic infected with SARS-CoV-2 during the initial wave of infection (March 11 to April 22, 2020) was performed. Identified variants were matched with clinical outcomes. Data analysis was performed from April to July 2020. Main Outcomes and Measures Hospitalization, intensive care unit (ICU) admission, mortality, and laboratory outcomes were matched with SARS-CoV-2 variants. Results Specimens sent for viral genome sequencing originated from 302 patients with SARS-CoV-2 infection (median [interquartile range] age, 52.6 [22.8 to 82.5] years), of whom 126 (41.7%) were male, 195 (64.6%) were White, 91 (30.1%) required hospitalization, 35 (11.6%) needed ICU admission, and 17 (5.6%) died. From these specimens, 2531 variants (484 of which were unique) were identified. Six different SARS-CoV-2 clades initially circulated followed by a rapid reduction in clade diversity. Several variants were associated with lower hospitalization rate, and those containing 23403A>G (D614G Spike) were associated with increased survival when the patient was hospitalized (64 of 74 patients [86.5%] vs 10 of 17 patients [58.8%]; χ21 = 6.907; P = .009). Hospitalization and ICU admission were similar regardless of clade. Infection with Clade V variants demonstrated higher creatinine levels (median [interquartile range], 2.6 [−0.4 to 5.5] mg/dL vs 1.0 [0.2 to 2.2] mg/dL; mean creatinine difference, 2.9 mg/dL [95% CI, 0.8 to 5.0 mg/dL]; Kruskal-Wallis P = .005) and higher overall mortality rates (3 of 14 patients [21.4%] vs 17 of 302 patients [5.6%]; χ21 = 5.640; P = .02) compared with other variants. Infection by strains lacking the 23403A>G variant showed higher mortality in multivariable analysis (odds ratio [OR], 22.4; 95% CI, 0.6 to 5.6; P = .01). Increased variants of open reading frame (ORF) 3a were associated with decreased hospitalization frequency (OR, 0.4; 95% CI, 0.2 to 0.96; P = .04), whereas increased variants of Spike (OR, 0.01; 95% CI, G (D614G) specific genotypes occurred. Replaced clades were associated with worse clinical outcomes, including mortality. These findings help explain persistent hospitalization yet decreasing mortality as the pandemic progresses. SARS-CoV-2 clade assignment is an important factor that may aid in estimating patient outcomes.

Objective A novel algorithm to identify fetal microdeletion events in maternal plasma has been developed and used in clinical laboratory‐based noninvasive prenatal testing. We used this approach to identify the subchromosomal events 5pdel, 22q11del, 15qdel, 1p36del, 4pdel, 11qdel, and 8qdel in routine testing. We describe the clinical outcomes of those samples identified with these subchromosomal events. Methods Blood samples from high‐risk pregnant women submitted for noninvasive prenatal testing were analyzed using low coverage whole genome massively parallel sequencing. Sequencing data were analyzed using a novel algorithm to detect trisomies and microdeletions. Results In testing 175 393 samples, 55 subchromosomal deletions were reported. The overall positive predictive value for each subchromosomal aberration ranged from 60% to 100% for cases with diagnostic and clinical follow‐up information. The total false positive rate was 0.0017% for confirmed false positives results; false negative rate and sensitivity were not conclusively determined. Conclusion Noninvasive testing can be expanded into the detection of subchromosomal copy number variations, while maintaining overall high test specificity. In the current setting, our results demonstrate high positive predictive values for testing of rare subchromosomal deletions. © 2015 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd., What's already known about this topic? Circulating cell free DNA is a powerful clinical tool that can detect whole chromosomal aneuploidies in a fetus as early as 10 weeks with high specificity and high sensitivity. Subchromosomal events in the fetus have been shown to be detected via ccfDNA via whole genome sequencing. What does this study add? This study shows that ccfDNA whole genome analysis for fetal subchromosomal aneuploidies including clinically significant microdeletions can be tested clinically with high specificity and high positive predictive value (PPV).

In conclusion, to maximize the benefit of the genomic era, the molecular laboratory director will continue to be essential in the generation, analysis, and interpretation of patient results, which now include genomic data obtained through NGS approaches. That includes integrating this information as part of the complete care of the patient and communicating and interacting with professionals across disciplines. In addition, the molecular laboratory director must continue to provide training and education to current and future colleagues, within and outside of molecular pathology and molecular genetics. Professionalism includes volunteerism in professional organizations and education and advocacy to policy makers, health administrators, payers, and the public. It also includes efforts to increase visibility of the profession to our colleagues from other medical disciplines and the public at large. Thus, the role of the molecular laboratory professional is multifaceted, but, above all, it is to ensure the access to and quality of molecular pathology testing, the responsible implementation of expanded test modalities such as genome sequencing, and the interpretation thereof to aid the clinician in the medical management of the patient and ultimately to benefit the society by providing precision patient care.

In late 2013, the Association for Molecular Pathology’s Professional Relations Committee formed a combined working group of members from the Economic Affairs, Professional Relations, and Clinical Practice Committees to develop a framework that could be used to demonstrate the clinical utility of molecular genetic tests for both inherited disorders and cancers. The charge to this diverse task force of laboratory experts from academic, commercial, and private practice settings, although concise, proved to be large in scope. The definitions for clinical utility and clinical validity are frequently misused or viewed interchangeably. The task force recommendations seek to clarify these terms specifically as applied to the various roles for molecular genetic test results in supporting patient care decisions. The task force’s work-product appears in this issue of The

Purpose The accuracy of predicting conversion from early-stage age-related macular degeneration (AMD) to the advanced stages of choroidal neovascularization (CNV) or geographic atrophy (GA) was evaluated to determine whether inclusion of clinically relevant genetic markers improved accuracy beyond prediction using phenotypic risk factors alone. Design Cohort study. Participants White, non-Hispanic subjects participating in the Age-Related Eye Disease Study (AREDS) sponsored by the National Eye Institute consented to provide a genetic specimen. Of 2415 DNA specimens available, 940 were from disease-free subjects and 1475 were from subjects with early or intermediate AMD. Methods DNA specimens from study subjects were genotyped for 14 single nucleotide polymorphisms (SNPs) in genes shown previously to associate with CNV: ARMS2 , CFH , C3 , C2 , FB , CFHR4 , CFHR5, and F13B . Clinical demographics and established disease associations, including age, sex, smoking status, body mass index (BMI), AREDS treatment category, and educational level, were evaluated. Four multivariate logistic models (phenotype; genotype; phenotype + genotype; and phenotype + genotype + demographic + environmental factors) were tested using 2 end points (CNV, GA). Models were fitted using Cox proportional hazards regression to use time-to-disease onset data. Main Outcome Measures Brier score (measure of accuracy) was used to identify the model with the lowest prediction error in the training set. The most accurate model was subjected to independent statistical validation, and final model performance was described using area under the receiver operator curve (AUC) or C-statistic. Results The CNV prediction models that combined genotype with phenotype with or without age and smoking revealed superior performance (C-statistic = 0.96) compared with the phenotype model based on the simplified severity scale and the presence of CNV in the nonstudy eye (C-statistic = 0.89; P ARMS2 genotype had less of an impact on the prediction of GA compared with CNV. Conclusions Inclusion of genotype assessment improves CNV prediction beyond that achievable with phenotype alone and may improve patient management. Separate assessments should be used to predict progression to CNV and GA because genetic markers and smoking status do not equally predict both end points. Financial Disclosure(s) Proprietary or commercial disclosure may be found after the references.

Molecular pathology is based on the principles, techniques, and tools of molecular biology as they are applied to diagnostic medicine in the clinical laboratory. These tools were developed in the research setting and perfected throughout the second half of the 20th century, long before the Human Genome Project was conceived. Molecular biology methods were used to elucidate the genetic and molecular basis of many diseases, and these discoveries ultimately led to the field of molecular diagnostics. Eventually the insights these tools provided for laboratory medicine were so valuable to the armamentarium of the pathologist that they were incorporated into pathology practice. Today, molecular diagnostics continues to grow rapidly as in vitro diagnostic companies develop new kits for the marketplace and as the insights into disease gained by the progress of the Human Genome Project develop into laboratory tests.

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications.

Objective To examine the performance of the SensiGene Fetal RHD Genotyping Laboratory Developed Test (RHD Genotyping LDT) using circulating cell-free fetal DNA (ccff DNA) extracted from maternal plasma. Methods ccff DNA was extracted from maternal blood from non-sensitized women with singleton pregnancies in two cohorts, one with a serotype reference (11–13 weeks' gestation) and one with the reference source (6–30 weeks' gestation). The presence of three RHD exon sequences (exons 4, 5, 7), the psi-pseudogene, three Y-chromosome sequences (SRY, DBY and TTTY2), and the X/Y-chromosome TGIF gene control were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry—the RHD Genotyping LDT. Results The cohort with a serotype RhD reference showed correct classification in 201 of 207 patients, a test accuracy of 97.1%, with a sensitivity and specificity for prediction of RhD serotype of 97.2 and 96.8%, respectively. The cohort with a genotype RHD reference showed correct classification in 198 of 199 patients, indicating a test accuracy of 99.5% with a sensitivity and specificity for prediction of RHD genotype of 100.0 and 98.3%, respectively. Conclusion Fetal RHD genotyping can accurately be determined using ccff DNA in the first and second trimesters of pregnancy. Copyright © 2011 John Wiley & Sons, Ltd.

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research.

Objective To examine the performance of a mass spectrometry-based detection platform using three Y-chromosome sequences for fetal sex determination from circulating cell-free fetal DNA (ccffDNA) in maternal blood in the first trimester of pregnancy. Methods We extracted ccffDNA for the determination of fetal sex from stored maternal plasma obtained at 11 to 13 weeks' gestation from singleton pregnancies with documented fetal gender. Mass spectrometry was used to examine 236 specimens for the presence of three Y-chromosome sequences (SRY, DBY and TTTY2). The sample was classified as male, female or inconclusive depending on the detection of three, one/none and two sequences, respectively. Results Three (1.3%) of the 236 cases were classified as invalid due to the absence of a well-defined spectral peak for TGIF and 22 (9.3%) were reported as inconclusive. In the 211 cases with a valid result, the fetal sex was correctly identified in 90 of 91 male babies and 119 of 120 female babies giving an accuracy of 99.1% and sensitivity and specificity for prediction of male fetuses of 98.9 and 99.2%, respectively. Conclusion Fetal sex determination can be accurately determined from maternal ccffDNA in the first trimester of pregnancy using mass spectrometry analysis. Copyright © 2010 John Wiley & Sons, Ltd.

Validation of the MycoAlign assay, a newly developed Mycobacterium spp. identification system based on internal transcribed spacer-1 sequencing, was performed using 50 acid-fast bacilli (AFB)-positive clinical laboratory specimens. Forty-three (86%) diagnostic-level results were obtained, including 38 Mycobacterium spp. and 5 other AFB-positive genera. Three isolates (6%) had suboptimal identity scores with high probability (81-87% identity score). Four (8%) mixed-pattern results were obtained. Forty-five (90%) observations were concordant with the species identification by standard methods, including all controls.

Purpose: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community. Methods: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps. Results: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step. Conclusions: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

In conclusion, to maximize the benefit of the genomic era, the molecular laboratory director will continue to be essential in the generation, analysis, and interpretation of patient results, which now include genomic data obtained through NGS approaches. That includes integrating this information as part of the complete care of the patient and communicating and interacting with professionals across disciplines. In addition, the molecular laboratory director must continue to provide training and education to current and future colleagues, within and outside of molecular pathology and molecular genetics. Professionalism includes volunteerism in professional organizations and education and advocacy to policy makers, health administrators, payers, and the public. It also includes efforts to increase visibility of the profession to our colleagues from other medical disciplines and the public at large. Thus, the role of the molecular laboratory professional is multifaceted, but, above all, it is to ensure the access to and quality of molecular pathology testing, the responsible implementation of expanded test modalities such as genome sequencing, and the interpretation thereof to aid the clinician in the medical management of the patient and ultimately to benefit the society by providing precision patient care.

A novel platform for the electronic detection of nucleic acids on microarrays is introduced and shown to perform well as a selective detection system for applications in molecular diagnostics. A gold electrode in a printed circuit board is coated with a self-assembled monolayer (SAM) containing DNA capture probes. Unlabeled nucleic acid targets are immobilized on the surface of the SAM through sequence-specific hybridization with the DNA capture probe. A separate signaling probe, containing ferrocene-modified nucleotides and complementary to the target in the region adjoining the capture probe binding site, is held in close proximity to the SAM in a sandwich complex. The SAM allows electron transfer between the immobilized ferrocenes and the gold, while insulating the electrode from soluble redox species, including unbound signaling probes. Here, we demonstrate sequence-specific detection of amplicons after simple dilution of the reaction product into hybridization buffer. In addition, single nucleotide polymorphism discrimination is shown. A genotyping chip for the C282Y single nucleotide polymorphism associated with hereditary hemochromatosis is used to confirm the genotype of six patients' DNA. In addition, a gene expression-monitoring chip is described that surveys five genes that are differentially regulated in the cellular apoptosis response. Finally, custom modification of individual electrodes through sequence-specific hybridization demonstrates the potential of this system for infectious disease diagnostics. The versatility of the electronic detection platform makes it suitable for multiple applications in diagnostics and pharmacogenetics.

Background: A bioelectronic detection platform has recently been developed that facilitates the detection and characterization of nucleic acids. The DNA chip platform is compatible with homogeneous assays because separate labeling and wash steps are not required. A one-step, bioelectronic detection assay was developed to genotype patient samples with respect to the H63D polymorphism of the Hfe gene, associated with hereditary hemochromatosis. Methods and Results: Electrode arrays were modified with DNA capture probes that were perfectly matched to the wild-type or mutant allele of H63D. Amplicons containing the polymorphic site were hybridized with the capture probes on the electrode arrays in the presence of electronically labeled reporter (signaling) probes. Voltammetric analysis of the electrode arrays was conducted first at ambient temperature and then at elevated temperature. The electronic signal was preferentially diminished at elevated temperature from electrodes that hybridized with mismatched target amplicons. Conclusion: An assay for bioelectronic genotyping of the H63D polymorphism was developed and used with six patient specimens to show the feasibility of this system as a model for point mutation detection.

The complete sequencing of the human genome in 2003 was highly publicized; however, many people misinterpreted that milestone as the “complete understanding of everything in the human genome.” In actuality, our professional community's great progress in molecular diagnostics has been tempered by the reality that we still have much to learn about the relationships between genes and disease. Recently, a set of commentaries in the New England Journal of Medicine reiterated just how elusive genotype/phenotype correlations, when applied to complex diseases, can be.1,2,3 Information culled from such genome-wide association studies is often repackaged using standard molecular diagnostic lab techniques, co-opted by direct-to-consumer (DTC) genomic testing companies, and sold to consumers. Is that information of value to the “patient” or is it merely recreational genomics? Amid the seemingly incongruent mix of mature and clinically useful tests for some diseases and paucity of knowledge for others, DTC genomic testing companies have come to market offering testing for genetic markers for health and risk assessment. Certainly we can agree that people should be proactive in making their own health care decisions and have a right to information about their individual genomes; yet, the question remains whether DTC genomic tests are beneficial in making those decisions. As with any retail commercial offering, there are different types of DTC genomic testing. One example is testing directed toward an individual with a family member affected by a specific disease, prompting worry of inheriting the same disease. An Internet search may uncover a laboratory that offers testing for that mutation without a doctor's order. Whether the result is positive or negative has all manner of interpretation ramifications. Without a physician and/or (better yet) a genetic counselor in the mix to aid in interpretation, how useful to the lay “patient” is the test result? There's another type of DTC genomic testing that is equivalent to genetic fortune telling. Several companies now offer DNA analysis with a variety of claims ranging from determining ancestry to choosing cosmetics based on DNA sequence (the former is possible, but not the latter). These examples are fairly harmless, and if people choose to spend their money for these purposes, that's their prerogative. Indeed, some have termed this “recreational genomics,” and if one accepts that moniker, then caveat emptor. But what about companies that make broad claims regarding genetics and health? That's more serious than whether one is descendant from royalty or has a skin DNA pattern that demands a certain moisturizer.

Background: Testing for immunoglobulin and T-cell-receptor gene rearrangement is a useful adjunct to morphologic evaluation of bone marrow involvement by non-Hodgkin's lymphoma. At this institution, each sample from a patient undergoing bilateral bone marrow sampling for lymphoma staging is assayed separately to avoid possible false negatives from dilution, if, for example, only one sample is positive. Methods and Results: Gene rearrangement results were reviewed for 140 patients, to assess if pooling the two aspirate samples from bilateral biopsies could be implemented. Discordant results between right and left iliac crest samples were found in 4 of 140 (2.9%) patients. Morphologic assessment was performed for 138 of the 140 patients. In 64 cases there were no abnormal findings. Four of these 64 (6.3%) had monoclonality demonstrated by genotyping or immunophenotyping. Of 134 patients for which both tests were performed, 17 (12.7%) showed discordance: 6 were negative by immunophenotyping, positive by genotyping (3 cases with malignant morphology, 1 with suspicious morphology, and 2 without evidence of malignancy); 11 positive by phenotyping but not genotyping (6 cases with malignant morphology, 3 with suspicious morphology, and 2 without evidence of malignancy). Conclusions: These findings support the role for these ancillary studies; in some cases both may be needed to demonstrate monoclonality. Based on this low occurrence of discordant findings between right and left samples, we feel that bilateral specimens can be pooled without unacceptable risk of false negatives due to possible dilution.

Background: Chemiluminescence detection systems are rapidly gaining popularity as safer alternatives to isotopic methods in molecular diagnostics with equal sensitivity and specificity. In addition, they offer versatility of detection because of the availability of different haptens for labeling the probes, the antihapten antibodies conjugated with either alkaline phosphatase (AP) or horseradish peroxidase (HRP), and their respective chemiluminescent substrates. A novel dual chemiluminescent substrate (AP and HRP based) and probe systems to distinguish genotypes of cystic fibrosis ΔF508 mutation are described. Methods and Results: Two methodologies have been formulated to identify positively the genotypes of the cystic fibrosis ΔF508 mutation. In method 1, a pair of oligonucleotides designed to anneal to the fanking regions of ΔF508 mutation are differentially labeled with the hapten biotin or fluorescein and ligated using the template DNA of wild-type (N/N), heterozygous (N/ΔF508), and homozygous (ΔF508/ΔF508) genotypes. The ligated product containing both labels is detected by first binding with avidin-HRP and anti-fluorescein-AP followed by reaction with the dual substrate. As expected, the ligation products are detected only in N/ΔF508 and ΔF508/ΔF508 genotypes but not in N/N, where the ligation is precluded by the presence of three intervening nucleotides. In method 2, the three genotypes are hybridized on a membrane simultaneously with uniquely labeled (biotin or digoxigenin) oligonucleotides each designed to bind either the normal or the mutant allele. On treatment with HRP- and AP-conjugated antibodies followed by reaction with the dual substrate, only the band from N/ΔF508 genotype emitted a strong signal because of the binding of both oligonucleotides. Conclusions: The ligation and hybridization methods in conjunction with the dual substrate can detect and differentiate the genotypes with the ΔF508 mutation. These formats may be valuable for distinguishing normal individuals from carriers in population screening and fetuses that are heterozygous, from those that are homozygous for cystic fibrosis ΔF508 in prenatal and neonatal diagnosis.

Daniel Farkas, PhD, and David Bernard, MD, PhD, discuss strengths, weaknesses, opportunities, and threats associated with molecular diagnostics.

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications.

In continuation of an educational endeavor initiated in 1992 with the presentation of William Beaumont Hospital’s first DNA symposium, physicians, laboratories, and technologists gathered at the hospital on March 25–26, 1994, to discuss current trends and new directions in DNA technology as applied to the clinical setting. The results are reported in this special issue of Laboratory Medicine .

We performed a correlative study to assess HER-2/neu oncogene amplification and protein overexpression. Gene amplification was determined by simultaneous hybridization of human breast tumor DNA with probes for the oncogene and a single copy reference gene, lmmunohistochemistry with a murine anti–HER-2/neu antibody was done on different portions of the same tumors. It has been demonstrated that the extent of gene amplification plays a role in disease outcome in breast cancer patients. The results of our investigation allowed this controversial, but sometimes useful, prognostic indicator to be used by a hospital’s medical staff.

Laboratory markers help medical oncologists predict chemotherapy resistance. The multidrug resistance gene (MDR1) and its P-glycoprotein product (MDR1) hold the promise of being such a marker. The only studies that have correlated the expression of MDR with clinical response to chemotherapy have used immunohistochemical reactions on tissue sections with monoclonal antibodies to MDR1. We have conducted such a study with a polyclonal antibody. The presence of MDR1 in our study did not define a population of chemotherapy- resistant patients. Correlative laboratory and clinical studies need to be performed to define the role of MDR in human breast cancer.

To examine the performance of the SensiGene Fetal RHD Genotyping Laboratory Developed Test (RHD Genotyping LDT) using circulating cell-free fetal DNA (ccff DNA) extracted from maternal plasma.ccff DNA was extracted from maternal blood from non-sensitized women with singleton pregnancies in two cohorts, one with a serotype reference (11-13 weeks' gestation) and one with the reference source (6-30 weeks' gestation). The presence of three RHD exon sequences (exons 4, 5, 7), the psi-pseudogene, three Y-chromosome sequences (SRY, DBY and TTTY2), and the X/Y-chromosome TGIF gene control were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-the RHD Genotyping LDT.The cohort with a serotype RhD reference showed correct classification in 201 of 207 patients, a test accuracy of 97.1%, with a sensitivity and specificity for prediction of RhD serotype of 97.2 and 96.8%, respectively. The cohort with a genotype RHD reference showed correct classification in 198 of 199 patients, indicating a test accuracy of 99.5% with a sensitivity and specificity for prediction of RHD genotype of 100.0 and 98.3%, respectively.Fetal RHD genotyping can accurately be determined using ccff DNA in the first and second trimesters of pregnancy.

We designed a laboratory developed test (LDT) by using an open platform for mutation/polymorphism detection. Using a 108-member (mutation plus variant) cystic fibrosis carrier screening panel as a model, we completed the last phase of LDT validation by using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Panel customization was accomplished via specific amplification primer and extension probe design. Amplified genomic DNA was subjected to allele specific, single base extension endpoint analysis by mass spectrometry for inspection of the cystic fibrosis transmembrane regulator gene (NM_000492.3). The panel of mutations and variants was tested against 386 blinded samples supplied by "authority" laboratories highly experienced in cystic fibrosis transmembrane regulator genotyping; >98% concordance was observed. All discrepant and discordant results were resolved satisfactorily. Taken together, these results describe the concluding portion of the LDT validation process and the use of mass spectrometry to detect a large number of complex reactions within a single run as well as its suitability as a platform appropriate for interrogation of scores to hundreds of targets.