The role of CD8+ T-cell clones in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multidimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterized patients with ITP and compared them with age-matched controls using immunophenotyping, next-generation sequencing of T-cell receptor (TCR) genes, single-cell RNA sequencing, and functional T-cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon gamma, tumor necrosis factor α, and granzyme B, defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the TCR showed expanded T-cell clones in patients with ITP. T-cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon gamma, and trigger platelet activation and apoptosis via the TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

The highs and lows of cyclic thrombocytopenia.

Cyclic thrombocytopenia (CTP) is characterized by periodic platelet oscillation with substantial amplitude. Most CTP cases have a thrombocytopenic background and are often misdiagnosed as immune thrombocytopenia with erratically effective treatment choices. CTP also occurs during hydroxyurea treatment in patients with myeloproliferative diseases. While the aetiology of CTP remains uncertain, here we evaluate historical, theoretical and clinical findings to provide a framework for understanding CTP pathophysiology. CTP retains the intrinsic oscillatory factors defined by the homeostatic regulation of platelet count, presenting as reciprocal platelet/thrombopoietin oscillations and stable oscillation periodicity. Moreover, CTP patients possess pathogenic factors destabilizing the platelet homeostatic system thereby creating opportunities for external perturbations to initiate and sustain the exaggerated platelet oscillations. Beyond humoral and cell-mediated autoimmunity, we propose recently uncovered germline and somatic genetic variants, such as those of MPL, STAT3 or DNMT3A, as pathogenic factors in thrombocytopenia-related CTP. Likewise, the JAK2 V617F or BCR::ABL1 translocation that drives underlying myeloproliferative diseases may also play a pathogenic role in hydroxyurea-induced CTP, where hydroxyurea treatment can serve as both a trigger and a pathogenic factor of platelet oscillation. Elucidating the pathogenic landscape of CTP provides an opportunity for targeted therapeutic approaches in the future.

Impact of C-reactive Protein on Anticoagulation Monitoring in Extracorporeal Membrane Oxygenation.

OBJECTIVE: To evaluate the impact of inflammation on anticoagulation monitoring for patients supported with extracorporeal membrane oxygenation (ECMO). DESIGN: Prospective single-center cohort study. SETTING: University-affiliated tertiary care academic medical center. PARTICIPANTS: Adult venovenous and venoarterial ECMO patients anticoagulated with heparin/ MEASUREMENTS AND MAIN RESULTS: C-Reactive protein (CRP) was used as a surrogate for overall inflammation. The relationship between CRP and the partial thromboplastin time (PTT, seconds) was evaluated using a CRP-insensitive PTT assay (PTT-CRP) in addition to measurement using a routine PTT assay. Data from 30 patients anticoagulated with heparin over 371 ECMO days was included. CRP levels (mg/dL) were significantly elevated (median, 17.2; interquartile range [IQR], 9.2-26.1) and 93% of patients had a CRP of ≥5. The median PTT (median 58.9; IQR, 46.9-73.3) was prolonged by 11.3 seconds compared with simultaneously measured PTT-CRP (median, 47.6; IQR, 40.1-55.5; p < 0.001). The difference between PTT and PTT-CRP generally increased with CRP elevation from 2.7 for a CRP of <5.0 to 13.0 for a CRP between 5 and 10, 17.7 for a CRP between 10 and 15, and 15.1 for a CRP of >15 (p < 0.001). In a subgroup of patients, heparin was transitioned to argatroban, and a similar effect was observed (median PTT, 62.1 seconds [IQR, 53.0-78.5 seconds] vs median PTT-CRP, 47.6 seconds [IQR, 41.3-57.7 seconds]; p < 0.001). CONCLUSIONS: Elevations in CRP are common during ECMO and can falsely prolong PTT measured by commonly used assays. The discrepancy due to CRP-interference is important clinically given narrow PTT targets and may contribute to hematological complications.

The role of genetics in refractory immune thrombocytopenia.

Patients with refractory immune thrombocytopenia (rITP) have increased morbidity and mortality. Currently, there is limited understanding of the cause of refractoriness and no markers to help direct novel treatment options. Understanding the reason(s) for refractoriness is crucial to determining novel treatment options. The pathogenesis underlying rITP has generally been thought to be an underlying genetic predisposition with an environmental trigger. Familial ITP remains rare, and there are few twin studies, suggesting that a simple genetic cause is unlikely. However, genetic mutations provide the background for several autoimmune diseases. In this review, we explore the evidence of either an inherited genetic cause of rITP or an acquired mutation, in particular one resulting in clonal expansion of cytotoxic T cells.

Neutral-Coating Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Top-Down Identification of Hemoglobin Variants.

BACKGROUND: Identification of hemoglobin (Hb) variants is of significant value in the clinical diagnosis of hemoglobinopathy. However, conventional methods for identification of Hb variants in clinical laboratories can be inadequate due to the lack of structural characterization. We describe the use of neutral-coating capillary electrophoresis coupled with high-resolution mass spectrometry (CE-HR-MS) to achieve high-performance top-down identification of Hb variants. METHODS: An Orbitrap Q-Exactive Plus mass spectrometer was coupled with an ECE-001 capillary electrophoresis (CE) unit through an EMASS-II ion source. A PS1 neutral-coating capillary was used for CE. Samples of red blood cells were lysed in water and diluted in 10 mM ammonium formate buffer for analysis. Deconvolution of raw mass spectrometry data was carried out to merge multiple charge states and isotopic peaks of an analyte to obtain its monoisotopic mass. RESULTS: The neutral-coating CE could baseline separate individual Hb subunits dissociated from intact Hb forms, and the HR-MS could achieve both intact-protein analysis and top-down analysis of analytes. A number of patient samples that contain Hb subunit variants were analyzed, and the variants were successfully identified using the CE-HR-MS method. CONCLUSIONS: The CE-HR-MS method has been demonstrated as a useful tool for top-down identification of Hb variants. With the ability to characterize the primary structures of Hb subunits, the CE-HR-MS method has significant advantages to complement or partially replace the conventional methods for the identification of Hb variants.

Multiplex Epstein-Barr virus BALF2 genotyping detects high-risk variants in plasma for population screening of nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) exhibits unusual geographic restriction despite ubiquitous lifelong infection. Screening programs can detect most NPC cases at an early stage, but existing EBV diagnostics are limited by false positives and low positive predictive value (PPV), leading to excess screening endoscopies, MRIs, and repeated testing. Recent EBV genome-wide association studies (GWAS) suggest that EBV BALF2 variants account for more than 80% of attributable NPC risk. We therefore hypothesized that high-risk BALF2 variants could be readily detected in plasma for once-lifetime screening triage. METHODS: We designed and validated a multiplex genotyping assay to detect EBV BALF2 polymorphisms in human plasma. Targeted next-generation sequencing was used to validate this assay, conduct association studies with clinical phenotype, and longitudinally genotype plasma to assess within-host haplotype stability. We examined the association between NPC and BALF2 haplotypes in a large non-endemic population and three prior EBV GWAS. Finally, we estimated NPC mortality reduction, resource utilization, and cost-effectiveness of BALF2 variant-informed screening using a previously-validated cohort model. RESULTS: Following analytical validation, the BALF2 genotyping assay had 99.3% concordance with sequencing in a cohort of 24 NPC cases and 155 non-NPC controls. BALF2 haplotype was highly associated with NPC in this non-endemic population (I613V: odds ratio [OR] 7.9; V317M: OR 178.8). No other candidate BALF2 polymorphisms were significantly associated with NPC or hematologic disorders. Longitudinal genotyping revealed 97.8% within-host haplotype concordance, indicative of lifelong latent infection. In a meta-analysis of 755 NPC cases and 981 non-NPC controls, BALF2 I613V and V317M were significantly associated with NPC in both endemic and non-endemic populations. Modeled variant-informed screening strategies achieved a 46% relative increase in PPV with 7% decrease in effective screening sensitivity, thereby averting nearly half of screening endoscopies/MRIs among endemic populations in east/southeast Asia. CONCLUSIONS: EBV BALF2 haplotypes are temporally stable within hosts and can be readily detected in plasma via an inexpensive multiplex genotyping assay that offers near-perfect sequencing concordance. In endemic and non-endemic populations, I613V and V317M were highly associated with NPC and could be leveraged to develop variant-informed screening programs that mitigate false positives with small reductions in screening sensitivity.

Evaluation of a Rapid and Accessible Reverse Transcription-Quantitative PCR Approach for SARS-CoV-2 Variant of Concern Identification.

The ability to distinguish between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) is of ongoing interest due to differences in transmissibility, responses to vaccination, clinical prognosis, and therapy. Although detailed genetic characterization requires whole-genome sequencing (WGS), targeted nucleic acid amplification tests can serve a complementary role in clinical settings, as they are more rapid and accessible than sequencing in most laboratories. We designed and analytically validated a two-reaction multiplex reverse transcription-quantitative PCR (RT-qPCR) assay targeting spike protein mutations L452R, E484K, and N501Y in reaction 1 and del69-70, K417N, and T478K in reaction 2. This assay had 95 to 100% agreement with WGS for 502 upper respiratory tract swab samples collected between 26 April 2021 and 1 August 2021, consisting of 43 Alpha, 2 Beta, 20 Gamma, 378 Delta, and 59 non-VOC infections. Validation in a separate group of 230 WGS-confirmed Omicron variant samples collected in December 2021 and January 2022 demonstrated 100% agreement. This RT-qPCR-based approach can be implemented in clinical laboratories already performing SARS-CoV-2 nucleic acid amplification tests to assist in local epidemiological surveillance and clinical decision-making.

A Multiplex SNaPshot Assay is a Rapid and Cost-Effective Method for Detecting POLE Exonuclease Domain Mutations in Endometrial Carcinoma.

Determining the replicative DNA polymerase epsilon ( POLE) mutation status in endometrial carcinomas (ECs) has important clinical implications given that the majority of "ultramutated" tumors harboring pathogenic exonuclease domain mutations in POLE ( POLE mut) have a favorable prognosis, even among high-grade histotypes. Currently, there are no specific morphologic or immunophenotypic features that allow accurate detection of POLE mut tumors without molecular testing. Consequently, identifying POLE mut tumors has been challenging without employing costly and/or time-consuming DNA sequencing approaches. Here we developed a novel SNaPshot assay to facilitate routine and efficient POLE mutation testing in EC. The SNaPshot assay interrogates 15 nucleotide sites within exons 9, 11, 13, and 14 encoding the POLE exonuclease domain. The variant sites were selected based on recurrence, evidence of functional impact, association with high tumor mutation burden and/or detection in EC clinical outcome studies. Based on the pathogenic somatic variants reported in the literature, the assay is predicted to have a clinical sensitivity of 90% to 95% for ECs. Validation studies showed 100% specificity and sensitivity for the variants covered, with expected genotypic results for both the positive (n=11) and negative (n=20) patient controls on multiple repeat tests and dilution series. Analytic sensitivity was conservatively approximated at a 10% variant allele fraction (VAF), with documented detection as low as 5% VAF. As expected, the SNaPshot assay demonstrated greater sensitivity than Sanger sequencing for VAFs below 20%, an important characteristic for somatic mutation detection. Here we have developed and validated the first SNaPshot assay to detect hotspot POLE mutations. While next-generation sequencing and Sanger sequencing-based approaches have also been used to detect POLE mutations, a SNaPshot approach provides useful balance of analytical sensitivity, cost-effectiveness, and efficiency in a high-volume case load setting.

Next-generation sequencing confirms T-cell clonality in a subset of pediatric pityriasis lichenoides.

BACKGROUND: Pityriasis lichenoides (PL) is a papulosquamous disease that affects both adults and children. Previous studies have shown a subset of this entity to have clonal T-cell populations via PCR-based assays. In this study, we sought to implement next-generation sequencing (NGS) as a more sensitive and specific test to examine for T-cell clonality within the pediatric population. METHODS: We identified 18 biopsy specimens from 12 pediatric patients with clinical and histopathologic findings compatible with PL. Patient demographics, clinical features, management, and histopathologic findings were reviewed. All specimens were analyzed for clonality with NGS of T-cell receptor beta (TRB) and gamma (TRG) genes. RESULTS: Of the 12 patients, 9 (75%) had complete resolution of lesions at the time of data collection (mean follow-up 31 months). The remaining three patients significantly improved with methotrexate (with or without acitretin). Interestingly, 7 of 12 patients (58%) and 9 of 17 biopsy specimens (53%) showed evidence of T-cell clonality. Two patients showed matching TRB clones from different anatomic sites. CONCLUSIONS: T-cell clonality is a common finding in PL, probably representing a "reactive clonality" rather than a true lymphoproliferative disorder. Clonality alone cannot be used as a means to distinguish PL from lymphomatoid papulosis or cutaneous lymphoma.

Ultra-sensitive Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antigen Detection for the Diagnosis of Coronavirus Disease 2019 (COVID-19) in Upper Respiratory Samples.

An ultra-sensitive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antigen assay (S-PLEX, MesoScale Diagnostics) was evaluated in 250 retrospective and 200 prospective upper respiratory specimens. In samples with cycle threshold <35, there was 95%-98% positive and 93%-96% negative percent agreement with reverse transcription-polymerase chain reaction. S-PLEX may provide a high-throughput alternative to nucleic acid-based testing for coronavirus disease 2019 (COVID-19) diagnosis.

High Frequency of SARS-CoV-2 RNAemia and Association With Severe Disease.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in blood, also known as RNAemia, has been reported, but its prognostic implications are poorly understood. This study aimed to determine the frequency of SARS-CoV-2 RNA in plasma and its association with coronavirus disease 2019 (COVID-19) clinical severity. METHODS: An analytical cross-sectional study was performed in a single-center tertiary care institution and included consecutive inpatients and outpatients with confirmed COVID-19. The prevalence of SARS CoV-2 RNAemia and the strength of its association with clinical severity variables were examined and included intensive care unit (ICU) admission, invasive mechanical ventilation, and 30-day all-cause mortality. RESULTS: Paired nasopharyngeal and plasma samples were included from 85 patients. The median age was 55 years, and individuals with RNAemia were older than those with undetectable SARS-CoV-2 RNA in plasma (63 vs 50 years; P = .04). Comorbidities were frequent including obesity (37.6%), hypertension (30.6%), and diabetes mellitus (22.4%). RNAemia was detected in 28/85 (32.9%) of patients, including 22/28 (78.6%) who required hospitalization. In models adjusted for age, RNAemia was detected more frequently in individuals who developed severe disease including ICU admission (32.1 vs 14.0%; P = .04) and invasive mechanical ventilation (21.4% vs 3.5%; P = .02). All 4 deaths occurred in individuals with detectable RNAemia. An additional 121 plasma samples from 28 individuals with RNAemia were assessed longitudinally, and RNA was detected for a maximum duration of 10 days. CONCLUSIONS: This study demonstrated a high proportion of SARS-CoV-2 RNAemia, and an association between RNAemia and clinical severity suggesting the potential utility of plasma viral testing as a prognostic indicator for COVID-19.

Occurrence and Timing of Subsequent Severe Acute Respiratory Syndrome Coronavirus 2 Reverse-transcription Polymerase Chain Reaction Positivity Among Initially Negative Patients.

Using data for 20 912 patients from 2 large academic health systems, we analyzed the frequency of severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction test discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and was similar across institutions.

Strand-Specific Reverse Transcription PCR for Detection of Replicating SARS-CoV-2.

We developed an assay that detects minus-strand RNA as a surrogate for actively replicating severe acute respiratory syndrome coronavirus 2. We detected minus-strand RNA in 41 persons with coronavirus disease up to 30 days after symptom onset. This assay might inform clinical decision-making about patient infectiousness.

Large-Scale Testing of Asymptomatic Healthcare Personnel for Severe Acute Respiratory Syndrome Coronavirus 2.

Large-scale, 1-time testing of >12,000 asymptomatic healthcare personnel in California, USA, during April-June 2020 showed that prevalence of severe acute respiratory syndrome coronavirus 2 was low (<1%). Testing might identify asymptomatic and presymptomatic persons, including some with high viral burden, enabling prompt implementation of measures to limit nosocomial spread.

Performance of Nucleic Acid Amplification Tests for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Prospectively Pooled Specimens.

Pooled nucleic acid amplification tests for severe acute respiratory syndrome coronavirus 2 could increase availability of testing at decreased cost. However, the effect of dilution on analytical sensitivity through sample pooling has not been well characterized. We tested 1,648 prospectively pooled specimens by using 3 nucleic acid amplification tests for severe acute respiratory syndrome coronavirus 2: a laboratory-developed real-time reverse transcription PCR targeting the envelope gene, and 2 commercially available Panther System assays targeting open reading frame 1ab. Positive percent agreement (PPA) of pooled versus individual testing ranged from 71.7% to 82.6% for pools of 8 and from 82.9% to 100.0% for pools of 4. We developed and validated an independent stochastic simulation model to estimate effects of dilution on PPA and efficiency of a 2-stage pooled real-time reverse transcription PCR testing algorithm. PPA was dependent on the proportion of tests with positive results, cycle threshold distribution, and assay limit of detection.

Multiplex SARS-CoV-2 Genotyping Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic Surveillance.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for a pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a nucleic acid amplification test (NAAT) to genotype mutations in the viral spike protein could facilitate high-throughput variant surveillance. We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase PCR (RT-qPCR) to detect three nonsynonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2-positive specimens from our San Francisco Bay Area population. Between 1 December 2020 and 1 March 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K plus N501Y mutations. The assay had perfect (100%) concordance with whole-genome sequencing of a validation subset of 229 specimens and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed the rapid emergence of the L452R variant in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.

SARS-CoV-2 Nucleocapsid Plasma Antigen for Diagnosis and Monitoring of COVID-19.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antigen in blood has been described, but the diagnostic and prognostic role of antigenemia is not well understood. This study aimed to determine the frequency, duration, and concentration of nucleocapsid antigen in plasma and its association with coronavirus disease 2019 (COVID-19) severity. METHODS: We utilized an ultrasensitive electrochemiluminescence immunoassay targeting SARS-CoV-2 nucleocapsid antigen to evaluate 777 plasma samples from 104 individuals with COVID-19. We compared plasma antigen to respiratory nucleic acid amplification testing (NAAT) in 74 individuals with COVID-19 from samples collected ±1 day of diagnostic respiratory NAAT and in 52 SARS-CoV-2-negative individuals. We used Kruskal-Wallis tests, multivariable logistic regression, and mixed-effects modeling to evaluate whether plasma antigen concentration was associated with disease severity. RESULTS: Plasma antigen had 91.9% (95% CI 83.2%-97.0%) clinical sensitivity and 94.2% (84.1%-98.8%) clinical specificity. Antigen-negative plasma samples belonged to patients with later respiratory cycle thresholds (Ct) when compared with antigen-positive plasma samples. Median plasma antigen concentration (log10 fg/mL) was 5.4 (interquartile range 3.9-6.0) in outpatients, 6.0 (5.4-6.5) in inpatients, and 6.6 (6.1-7.2) in intensive care unit (ICU) patients. In models adjusted for age, sex, diabetes, and hypertension, plasma antigen concentration at diagnosis was associated with ICU admission [odds ratio 2.8 (95% CI 1.2-6.2), P=.01] but not with non-ICU hospitalization. Rate of antigen decrease was not associated with disease severity. CONCLUSIONS: SARS-CoV-2 plasma nucleocapsid antigen exhibited comparable diagnostic performance to upper respiratory NAAT, especially among those with late respiratory Ct. In addition to currently available tools, antigenemia may facilitate patient triage to optimize intensive care utilization.

Case-Control Study of Individuals with Discrepant Nucleocapsid and Spike Protein SARS-CoV-2 IgG Results.

BACKGROUND: Laboratory-based methods for SARS-CoV-2 antibody detection vary widely in performance. However, there are limited prospectively-collected data on assay performance, and minimal clinical information to guide interpretation of discrepant results. METHODS: Over a 2-week period, 1080 consecutive plasma samples submitted for clinical SARS-CoV-2 IgG testing were tested in parallel for anti-nucleocapsid IgG (anti-N, Abbott) and anti-spike IgG (anti-S1, EUROIMMUN). Chart review was conducted for samples testing positive or borderline on either assay, and for an age/sex-matched cohort of samples negative by both assays. CDC surveillance case definitions were used to determine clinical sensitivity/specificity and conduct receiver operating characteristics curve analysis. RESULTS: There were 52 samples positive by both methods, 2 positive for anti-N only, 34 positive for anti-S1 only, and 27 borderline for anti-S1. Of the 34 individuals positive for anti-S1 alone, 8 (24%) had confirmed COVID-19. No anti-S1 borderline cases were positive for anti-N or had confirmed/probable COVID-19. The anti-N assay was less sensitive (84.2% [95% CI 72.1-92.5%] vs 94.7% [95% CI 85.4-98.9%]) but more specific (99.2% [95% CI 95.5-100%] vs 86.9% [95% CI 79.6-92.3%]) than anti-S1. Abbott anti-N sensitivity could be improved to 96.5% with minimal effect on specificity if the index threshold was lowered from 1.4 to 0.6. CONCLUSION: Real-world concordance between different serologic assays may be lower than previously described in retrospective studies. These findings have implications for the interpretation of SARS-CoV-2 IgG results, especially with the advent of spike antigen-targeted vaccination, as a subset of patients with true infection are anti-N negative and anti-S1 positive.

Identification of a pathogenic TUBB1 variant in a Chinese family with congenital macrothrombocytopenia through whole genome sequencing.

Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. We herein report a large Chinese family presented with phenotypic variability involving thrombocytopenia and/or giant platelets. Whole genome sequencing (WGS) of the proband and one of his affected brothers identified a potentially pathogenic c.952 C > T heterozygous variant in the TUBB1 gene. This p.R318W ß1-tubulin variant was also identified in three additional siblings and five members of the next generation. These findings were consistent with an autosomal dominant inheritance with incomplete penetrance. Moreover, impaired platelet agglutination in response to ristocetin was detected in the patient's brother. Half of the family members harboring the p.R318W mutation displayed significantly decreased external release of p-selectin by stimulated platelets. The p.R318W ß1-tubulin mutation was identified for the first time in a Chinese family with congenital macrothrombocytopenia using WGS as an unbiased sequencing approach. Affected individuals within the family demonstrated impaired platelet aggregation and/or release functions.

Retrospective Screening for SARS-CoV-2 RNA in California, USA, Late 2019.

To investigate the possibility of earlier cases of severe acute respiratory syndrome coronavirus 2 infection than previously recognized, we retrospectively tested pooled samples from 1,700 persons with respiratory signs/symptoms seen at Stanford Health Care, Palo Alto, California, USA, during the last 2 months of 2019. We found no evidence of earlier infection.