Comparison of enzyme-linked fluorescent assay and electrochemiluminescence immune assay in procalcitonin measurement

Background Procalcitonin (PCT) measurement is required for intensive care patients with systemic inflammation symptoms, early diagnosis of possible infections, and evaluation of sepsis severity and prognosis. Objectives We aimed to determine the analytical performance of PCT measurement in a Roche Modular E170 (ECLIA) analyzer and compare the performance with VIDAS (BRAHMS/ELFA) analyzer findings. Material and methods Within-day and between-day precision value, linearity was determined, and two methods were compared with regression and Bland–Altman analysis. Results Both ECLIA and ELFA assays indicated excellent precision, where within-day precision varied between 1.18% and 3.97% CV, and between-day precision varied between 1.77% and 3.93% CV. The ECLIA method was linear up to 62.15 ng/mL. The arithmetic mean was 6.02 ng/mL with the ECLIA method and 8.02 ng/mL with the ELFA method. The correlation coefficient was r=0.996 and p=0.001. The correlation was linear between the two methods. Regression equation was found y=0.78x − 0.23. The Bland–Altman figure was revealed the difference between the methods was specifically in lower concentrations (<0.15 ng/mL). Conclusions Both methods show good precision and correlation. It was determined that the difference between methods was significant, especially at <0.15 ng/mL concentration.

Reinvestigation of Carbohydrate Specificity of EBCA-1 Monoclonal Antibody Used for the Detection of Candida Mannan

Monoclonal antibody EBCA-1 is used in the sandwich immune assay for the detection of circulating Candida mannan in blood sera samples for the diagnosis of invasive candidiasis. To reinvestigate carbohydrate specificity of EBCA-1, a panel of biotinylated oligosaccharides structurally related to distinct fragments of Candida mannan were loaded onto a streptavidin-coated plate to form a glycoarray. Its use demonstrated that EBCA-1 recognizes the trisaccharide β-Man-(1→2)-α-Man-(1→2)-α-Man and not homo-α-(1→2)-linked pentamannoside, as was reported previously.

Developing a Novel Multiplexed Immune Assay Platform to Screen Kinase Modulators of T Cell Activation

T cell activation plays a central role in inflammation, autoimmune diseases and cancer. Cancer immunotherapies, such as immune checkpoint inhibitor, bi-specific antibody, chimeric antigen receptor T (CAR T) cell, and adoptive tumor-infiltrating lymphocyte (TIL) therapies require the characterization and monitoring of T cell activation. Here we describe a novel, multiplex immune assay platform based on high-throughput flow cytometry technology and advanced computational algorithms for data analysis. The assay simultaneously measures T cell dynamics including phenotype, time-dependent expression of activation markers, secreted effector cytokines, and proliferation. The assay screened a kinase chemogenomic library and identified 25 kinase inhibitors with distinct inhibition profiles on early (CD69) and late (CD25) activation markers and the cytokines IFNγ and TNFα. We identified 5 kinase inhibitors with dissimilar effects on CD69 and CD25 expression, and a cluster of total 4 MEK1//2 inhibitors with similar activation profiles. The screening revealed 3 kinase inhibitors for PKC, IKK2, and MEK1/2 respectively, all with a phenotypic signature similar to ruxolitinib, a Jak1/2 inhibitor used to treat myelofibrosis disease. These results suggest this multiplexed assay platform, combined with a chemogenomic library screening, may be used as primary screen for phenotypic or target-based drug discovery, target identification, and potential drug repositioning.

Clinical utility of ANA-ELISA vs ANA-immunofluorescence in connective tissue diseases

We investigated the performance of ANA-ELISA for CTDs screening and diagnosis and comparing it to the conventional ANA-IIF. ANA-ELISA is a solid-phase immune assay includes 17 ANA-targeted recombinant antigens; dsDNA, Sm-D, Rib-P, PCNA, U1-RNP (70, A, C), SS-A/Ro (52 and 60), SS-B/La, Centromere B, Scl-70, Fibrillarin, RNA Polymerase III, Jo-1, Mi-2, and PM-Scl. During the period between March till December 2016 all requests for ANA from primary, secondary, and tertiary care centers were processed with both techniques; ANA-IIF and ANA-ELISA. The electronic medical record of these patients was reviewed looking for CTD diagnosis documented by the Senior rheumatologist. SPSS 22 is used for analysis. Between March and December 2016, a total of 12,439 ANA tests were requested. 1457 patients were assessed by the rheumatologist and included in the analysis. At a cut-off ratio ≥ 1.0 for ANA-ELISA and a dilutional titre ≥ 1:80 for ANA-IIF, the sensitivity of ANA-IIF and ANA-ELISA for all CTDs were 63.3% vs 74.8% respectively. For the SLE it was 64.3% vs 76.9%, Sjogren’s Syndrome was 50% vs 76.9% respectively. The overall specificity of ANA-ELISA was 89.05%, which was slightly better than ANA-IIF 86.72%. The clinical performance of ANA-ELISA for CTDs screening showed better sensitivity and specificity as compared to the conventional ANA-IIF in our cohort.

PENGARUH BUAH OKRA (Abelmoschus Esculantus)TERHADAP INSULIN C-PEPTIDA TIKUS PUTIH WISTAR (Rattus norvegicus) DIABETES YANG DIINDUKSI DENGAN STREPTOZOTOCIN

Kejadian diabetes mellitus diakibatkan oleh beberapa faktor risiko baik itu yang bisa dikendalikan maupun yang tidak bisa dikendalikan. Usaha pengendalian bisa dilakukan baik secara farmakologi atau nonfarmakologi. Tatalaksana pengobatan diabetes mellitus tipe 2 dimaksudkan untuk mengembalikan fungsi sel beta pankreas dalam mengsekresikan insulin serta meningkatkan sensitivitas reseptor insulin pada sel. Sedangkan untuk tatalaksana nonfarmakologi umumnya dilakukan dengan mengkonsumsi bahan yang dapat menekan faktor pencetus kejadian tersebut. Okra (Abelmoschus esculentus) merupakan tanaman yang secara empiris telah terbukti dapat digunakan untuk mengendalikan penyakit diabetes mellitus, namun pembuktian secara ilmiah dari efektivitas tanaman ini masih sangat kurang. Oleh sebab itu, penelitian ini bertujuan untuk mendapatkan informasi terkait pengaruh ekstrak buah okra terhadap kadar insulin c-peptida dan kadar gula darah pada tikus diabetes mellitus yang diinduksi dengan streptozotosin intraperitoneal dalam rentang waktu 25 hari. Kadar c-peptida diukur pada hari ke 25 dengan metode Electro Chemiluminesencent Immune Assay (ECLIA). Berdasarkan hasil penelitian yang diperoleh menunujukkan tidak adanya perbedaan yang bermakna pada kadar insulin c-peptida pada semua kelompok uji (0,01 ng/ml). Hasil yang mengesankan diperoleh bahwa okra kering memiliki efek lebih kuat dibandingkan metformin (p = 0,002), okra cair (p = 0,754), dan blangko (p = 0.01) dalam menurunkan kadar gula darah. Oleh sebab itu, dapat disimpulkan bahwa ekstrak okra baik kering (serbuk) atau cair dapat menurunkan kadar gula darah pada tikus DM yang diinduksi dengan streptozotosin.

Floor of Mouth Neurofibroma: A Rare Presentation

Solitary sporadic neurofibromas are uncommon benign tumours of the neural sheaths of the peripheral nervous system. Solitary sporadic neurofibromas of the oral cavity and floor of mouth are extremely rare. We present the case of a 36-year-old male who presented with a 3-month history of a soft non-tender floor of mouth mass. MRI demonstrated a cystic septated lesion filling the left sublingual space. The lesion was excised via an external approach. Histology and immune assay unexpectedly demonstrated a neurofibroma.

Heparin Induced Thrombocytopenia in Patients with COVID-19

Background The Coronavirus disease-2019 (COVID-19) is a global pandemic caused by novel coronavirus SARS-CoV-2. Acute respiratory and renal failure as well as systemic coagulopathy are critical aspects of the morbidity and mortality in patients with COVID-19. Heparin Induced Thrombocytopenia (HIT) occurs when IgG antibodies form against platelet factor 4-Heparin complex, resulting in platelet activation and removal, leading to a prothrombotic state. HIT is suspected when there is a platelet count decrease of more than 50% after exposure to heparin, along with hypercoagulability. Clinical Scoring systems like 4T Score (Thrombocytopenia, Timing, Thrombosis, no other cause of Thrombocytopenia) have been developed to assess the pretest probability of HIT. The use of unfractionated heparin, post-orthopedic and post-cardiac surgery state, female gender, and old age are recognized as risk factors for HIT. There is a nine-fold increased risk of developing HIT in patients requiring hemodialysis. ICU patients and patients with VTE without thrombocytopenia are considered to have low pre-test probability for HIT. Studies have shown that only 6% who are investigated serologically for HIT actually have the diagnosis. We conducted this study to assess the incidence and risk factors for HIT in COVID-19 positive patients and its impact on mortality. Methods A retrospective analysis was performed on all patients who were COVID-19 positive and hospitalized between March 1, 2020 and June 26, 2020 at our institution. Patients with intermediate or high suspicion for HIT, based on 4T score of 4 or higher, underwent IgG-specific platelet factor 4(PF4)-dependent enzyme immune assay (EIA). Washed platelet assays such as serotonin release assay (SRA) and heparin-induced platelet aggregation (HIPA) were used as confirmatory tests in cases with intermediate or low optical density (OD) with EIA. The incidence of HIT, its impact on mortality, and positivity of IgG-specific PF4-EIA in COVID-19 patients were studied, and statistical analysis was done with X2 testing. Subgroup analysis was performed based on demographic factors and risk factors for HIT, including exposure to heparin or low molecular weight heparin (LMWH), history of or cancer, recent orthopedic or cardiac surgery, exposure to renal replacement therapy (RRT), and severity of disease (D-dimer &gt;6 ULN, Acute Kidney Injury, ICU admission, and mechanical ventilation requirement). These factors were analyzed by Fisher's exact test to determine their impact on mortality. The hospital course for HIT antibody-positive patients was further analyzed to study the impact of COVID-19 related therapy, such as Remdesivir, Tocilizumab, Hydroxychloroquine, Steroids, and anticoagulation after diagnosis of HIT. Results WEight out of 1265 hospitalized COVID-19 positive patients tested positive for IgG-specific platelet factor 4(PF4)-dependent enzyme immune assay (EIA+). Incidence of EIA+ in COVID-19 patients was 0.6%, which is significantly higher than in the general population 0.2% (p&lt;.0001, 95% CI 0.25-1.20%). The incidence of thromboembolic events in EIA+ patients was 87.5%, significantly higher than the rate of 10.90% in all COVID-19 patients (p&lt;.0001, CI 41.96- 86.98%). The mortality rate in EIA+ patients was 50%, significantly greater than the mortality rate of 12% in all hospitalized COVID-19 patients (p=.0011, CI 9.46-66.53). Serological confirmation of HIT diagnosis was 37.5% (2 had OD&gt;1, 1 was SRA positive) which is significantly higher than confirmation of HIT in nonCOVID-19 patients 6% (p&lt;.0001, 95% CI 29.57-85.32%). All 3 confirmed HIT patients (100%) had severe disease (3/3 required ICU admission, 2/3 required mechanical ventilation, the one not requiring mechanical ventilation required RRT). Conclusion Our study indicates incidence of HIT is higher in the COVID-19 population. The incidence of positive EIA for patients with intermediate to high 4T scores is also higher in COVID-19 positive patients. This can be attributed to the cytokine storm and severe sepsis seen in critically ill COVID-19 patients. Our study also suggests that development of HIT can contribute to increased risk for thromboembolic events as well as mortality of COVID-19 patients, however, our study is limited due to small sample size. Therefore, prospective studies are needed to analyze the impact of HIT on morbidity, mortality and long-term outcomes in COVID-19 patients. Table Disclosures No relevant conflicts of interest to declare.

Rapid and Sensitive Multiplex Assay for the Detection of B. anthracis Spores from Environmental Samples

Prompt and accurate detection of Bacillus anthracis spores is crucial in the event of intentional spore dissemination in order to reduce the number of expected casualties. Specific identification of these spores from environmental samples is both challenging and time-consuming. This is due to the high homology with other Bacillus species as well as the complex composition of environmental samples, which further impedes assay sensitivity. Previously, we showed that a short incubation of B.anthracis spores in a defined growth medium results in rapid germination, bacterial growth, and secretion of toxins, including protective antigen. In this work, we tested whether coupling the incubation process to a newly developed immune-assay will enable the detection of secreted toxins as markers for the presence of spores in environmental samples. The new immune assay is a flow cytometry-based multiplex that simultaneously detects a protective antigen, lethal factor, and edema factor. Our combined assay detects 1 × 103–1 × 104/mL spores after a 2 h incubation followed by the ~80 min immune-multiplex detection. Extending the incubation step to 5 h increased assay sensitivity to 1 × 102/mL spore. The protocol was validated in various environmental samples using attenuated or fully virulent B. anthracis spores. There was no substantial influence of contaminants derived from real environmental samples on the performance of the assay compared to clean samples, which allow the unequivocal detection of 3 × 103/mL and 3 × 102/mL spores following 2 and 5 hour’s incubation, respectively. Overall, we propose this method as a rapid, sensitive, and specific procedure for the identification of B. anthracis spores in environmental samples.

Case 5

Erythema migrans is the most common skin manifestation of Lyme disease caused by Borrelia spp. There are some differences in the clinical presentation and aetiology between cases in Europe and the United States as the vectors and Borrelia spp. vary. The diagnosis is usually confirmed by serology, that is, enzyme immune-assay and Western blot, although there are other approaches such as PCR. Apart from erythema migrans, other manifestations include meningitis, heart block, and arthritis. There are a number of options for treatment, including tetracyclines and penicillins, and duration depends on the presence of complications. There is a limited if any role for prophylactic antibiotics to prevent acquisition after exposure to a tick. Much controversy surrounds chronic Lyme disease which may not be a real clinical entity and may mimic other conditions such as connective tissue diseases. There is no evidence that ongoing symptoms are due to persistent infection.