More Than Results: The Clinical and Research Relationship in the Evolving Detection and Surveillance of SARS-CoV-2.

INTRODUCTION: During the coronavirus disease 2019 (COVID-19) pandemic, real-time reverse transcription polymerase chain reaction (RT-PCR) became an essential tool for laboratories to provide high-sensitivity qualitative diagnostic testing for patients and real-time data to public health officials. Here we explore the predictive value of quantitative data from RT-PCR cycle threshold (Ct) values in epidemiological measures, symptom presentation, and variant transition. METHODS: To examine the association with hospitalizations and deaths, data from 74,479 patients referred to the Avera Institute for Human Genetics (AIHG) for COVID-19 testing in 2020 were matched by calendar week to epidemiological data reported by the South Dakota Department of Health. We explored the association between symptom data, patient age, and Ct values for 101 patients. We also explored changes in Ct values during variant transition detected by genomic surveillance sequencing of the AIHG testing population during 2021. RESULTS: Measures from AIHG diagnostic testing strongly explain variance in the South Dakota state positivity percentage (R2 = 0.758), a two-week delay in hospitalizations (R2 = 0.856), and a four-week delay in deaths (R2 = 0.854). Based on factor analysis of patient symptoms, three groups could be distinguished which had different presentations of age, Ct value, and time from collection. Additionally, conflicting Ct value results among SARSCoV- 2 variants during variant transition may reflect the community transmission dynamics. CONCLUSIONS: Measures of Ct value in RT-PCR diagnostic assays combined with routine screening have valuable applications in monitoring the dynamics of SARS-CoV-2 within communities.

A Laboratory-Friendly CTC Identification: Comparable Double-Immunocytochemistry with Triple-Immunofluorescence.

The source of circulating tumor cells (CTC) in the peripheral blood of patients with solid tumors are from primary cancer, metastatic sites, and a disseminated tumor cell pool. As 90% of cancer-related deaths are caused by metastatic progression and/or resistance-associated treatment failure, the above fact justifies the undeniable predictive and prognostic value of identifying CTC in the bloodstream at stages of the disease progression and resistance to treatment. Yet enumeration of CTC remains far from a standard routine procedure either for post-surgery follow-ups or ongoing adjuvant therapy. The most compelling explanation for this paradox is the absence of a convenient, laboratory-friendly, and cost-effective method to determine CTC. We presented a specific and sensitive laboratory-friendly parallel double-detection format method for the simultaneous isolation and identification of CTC from peripheral blood of 91 consented and enrolled patients with various malignant solid tumors of the lung, endometrium, ovary, esophagus, prostate, and liver. Using a pressure-guided method, we used the size-based isolation to capture CTC on a commercially available microfilter. CTC identification was carried out by two expression marker-based independent staining methods, double-immunocytochemistry parallel to standard triple-immunofluorescence. The choice of markers included specific markers for epithelial cells, EpCAM and CK8,18,19, and exclusion markers for WBC, CD45. We tested the method's specificity based on the validation of the staining method, which included positive and negative spiked samples, blood from the healthy age-matched donor, healthy age-matched leucopaks, and blood from metastatic patients. Our user-friendly cost-effective CTC detection technique may facilitate the regular use of CTC detection even in community-based cancer centers for prognosis, before and after surgery.

Identification and Morphological Characterization of Features of Circulating Cancer-Associated Macrophage-like Cells (CAMLs) in Endometrial Cancers.

The blood of patients with solid tumors contains circulating tumor-associated cells, including epithelial cells originating from the tumor mass, such as circulating tumor cells (CTCs), or phagocytic myeloid cells (differentiated monocytes), such as circulating cancer-associated macrophage-like cells (CAMLs). We report for the first time the identification and in-depth morphologic characterization of CAMLs in patients with endometrial cancers. We isolated CAMLs by size-based filtration on lithographically fabricated membranes followed by immunofluorescence, using a CD45+/CK 8,18,19+/EpCAM+/CD31+/macrophage-like nuclear morphology, from > 70 patients. Irrespective of the histological and pathological parameters, 98% of patients were positive for CAMLs. Two size-based subtypes of CAMLs, <20 µm (tiny) and >20 µm (giant) CAMLs, of distinctive polymorphic morphologies with mononuclear or fused polynuclear structures in several morphological states were observed, including apoptotic CAMLs, CAML−WBC doublets, conjoined CAMLs, CAML−WBC clusters, and CTC−CAML−WBC clusters. In contrast, CAMLs were absent in patients with non-neoplastic/benign tumors, healthy donors, and leucopaks. Enumerating CTCs simultaneously from the same patient, we observed that CTC-positive patients are positive for CAMLs, while 55% out of all CAML-positive patients were found positive for CTCs. Our study demonstrated for the first time the distinctive morphological characteristics of endometrial CAMLs in the context of the presence of CTCs in patients.