Clot Waveform Analysis Demonstrates Low Blood Coagulation Ability in Patients with Idiopathic Thrombocytopenic Purpura

Background: Although platelets, which contain large amounts of phospholipids, play an important role in blood coagulation, there is still no routine assay to examine the effects of platelets in blood coagulation. Methods: Hemostatic abnormalities in patients with thrombocytopenia, including those with idiopathic thrombocytopenic purpura (ITP), were examined using clot wave analysis (CWA)–small-amount tissue-factor-induced FIX activation (sTF/FIXa) and thrombin time (TT). Results: Although there were no marked differences in the three parameters of activated partial thromboplastin time (APTT) between normal healthy volunteers and typical patients with ITP, the peak heights of the CWA-sTF/FIXa were markedly low in patients with ITP. The three peak times of the CWA-sTF/FIXa in patients with a platelet count of ≤8.0 × 1010/L were significantly longer than those in patients with a platelet count > 8.0 × 1010/L and the peak heights of the CWA-sTF/FIXa in patients with a platelet count of ≤8.0 × 1010/L were significantly lower than those in patients with >8.0 × 1010/L. The peak heights of the CWA-APTT in patients with ITP were significantly lower than in patients with other types of thrombocytopenia. The three peak heights of the CWA-sTF/FIXa in ITP patients were significantly lower than those in patients with other types of thrombocytopenia. The CWA-TT showed lower peak heights and longer peak times in patients with ITP in comparison to patients with other types of thrombocytopenia. Conclusions: The CWA-sTF/FIXa and CWA-TT results showed that blood coagulation is enhanced by platelets and that the blood coagulation ability in ITP patients was low in comparison to healthy volunteers and patients with other types of thrombocytopenia.

The Reevaluation of Thrombin Time Using a Clot Waveform Analysis

Object: Although thrombin burst has attracted attention as a physiological coagulation mechanism, clinical evidence from a routine assay for it is scarce. This mechanism was therefore evaluated by a clot waveform analysis (CWA) to assess the thrombin time (TT). Material and Methods: The TT with a low concentration of thrombin was evaluated using a CWA. We evaluated the CWA-TT of plasma deficient in various clotting factors, calibration plasma, platelet-poor plasma (PPP), and platelet-rich plasma (PRP) obtained from healthy volunteers, patients with thrombocytopenia, and patients with malignant disease. Results: Although the TT-CWA of calibration plasma was able to be evaluated with 0.01 IU/mL of thrombin, that of FVIII-deficient plasma could not be evaluated. The peak time of CWA-TT was significantly longer, and the peak height significantly lower, in various deficient plasma, especially in FVIII-deficient plasma compared to calibration plasma. The second peak of the first derivative (1st DP-2) was detected in PPP from healthy volunteers, and was shorter and higher in PRP than in PPP. The 1st DP-2 was not detected in PPP from patients with thrombocytopenia, and the 1st DP-2 in PRP was significantly lower in patients with thrombocytopenia and significantly higher in patients with malignant disease than in healthy volunteers. Conclusion: The CWA-TT became abnormal in plasma deficient in various clotting factors, and was significantly affected by platelets, suggesting that the CWA-TT may be a useful test for hemostatic abnormalities.

UV Spectrophotometric Analysis and Validation of Dapsone in Semisolid Dosage Form

Objectives: A new, economical, sensitive, simple, rapid UV spectrophotometric method has been developed for the estimation of Dapsone in pure form and Semisolid Dosage form. Method: This UV method was developed using methanol: RO water (75:25) as a solvent. In the present method the wavelength selected for analysis was 260nm. UV-Visible double beam spectrophotometer (Systronic 2201) was used to carry out spectral analysis. The ICH guidelines were used to validate the method. Results: The method was validated for linearity, range, accuracy, precision, robustness, LOD and LOQ. Linearity was found in the range of 3-18µg/ml. Accuracy was performed by using recovery study. The amount of drug recovered was found to be in the range of 100.1-100.5 %. The %RSD value was found to be less than 2. Conclusion: The proposed UV spectroscopic method was found to be accurate, precise, stable, linear, specific, and simple for quantitative estimation of Dapsone in bulk and Semisolid dosage form. Hence the present UV spectroscopic method is suitable for routine assay of Dapsone in bulk and Semisolid dosage form.

Osteopontin as a Regulator of Colorectal Cancer Progression and Its Clinical Applications

A high expression of the phosphoprotein osteopontin (OPN) has been associated with cancer progression in several tumor types, including breast cancer, hepatocarcinoma, ovarian cancer, and colorectal cancer (CRC). Interestingly, OPN is overexpressed in CRC and is associated with a poor prognosis linked to invasion and metastasis. Here, we review the regulation and functions of OPN with an emphasis on CRC. We examine how epigenetic and genetic regulators interact with the key signaling pathways involved in this disease. Then, we describe the role of OPN in cancer progression, including proliferation, survival, migration, invasion, and angiogenesis. Furthermore, we outline the interest of using OPN as a clinical biomarker, and discuss if and how osteopontin can be implemented as a routine assay in clinical laboratories for monitoring CRC patients. Finally, we discuss the use of OPN an attractive, but challenging, therapeutic target.

P03.07 Fast automated microfluidic-based multiplexed immunofluorescence for tumor microenvironment analysis

BackgroundImmuno-oncology and targeted molecular therapies have acquired a central role in the treatment of multiple cancers. Consequently, high-throughput biomarker analysis and tumor immune profiling have seen an increased demand. Multiplexed immuno-assays are a powerful tool to address these needs, but still time- and resource-consuming. Our goal is to develop a fast and automated high-plex fluorescent immunostaining procedure, using a microfluidic-based device, that can be easily implemented as routine assay.Materials and MethodsProtocol optimization has been performed on FFPE sections of human tonsil. Slides were manually deparaffinized before being entirely processed (antigen-retrieval, staining, elution and counterstaining) by Lunaphore’s autostainer, LabSatTM. The OPAL® tyramide signal amplification (TSA) system was used as detection method. Signal analysis was done on Mantra® workstation. The 6-plex panel was composed of FoxP3, PD-L1, PD-1, CD68, CD8 and pan-CK, plus DAPI counterstaining. Protocols were subsequently transferred on NSCLC representative specimens and finally assessed on a TMA cohort.ResultsOur platform allowed to reduce drastically the incubation times due to active transport of reagents across the tissue. Thereby, the automated 6-plex assay could be performed in less than 4h30min, within the timeframe of a single IHC standard assay. Protocol optimization resulted in high signal-to-background ratio for each marker and removal of previous step antibodies over 99%. LabSatTM also guaranteed remarkable signal uniformity, even over large tissue sections with less than 10% signal gradient over 1 cm. On NSCLC samples, the detected pattern and expression level for all six biomarkers were comparable to the standard chromogenic stainings performed with standard automated tissue stainer.ConclusionsLabSatTM autostainer enables multistaining runs in a timely manner, opening the perspective of rapid simultaneous detection of multiple markers in their morphological context on a routine-based approach. This versatile analysis tool can offer a better and more quantitative understanding of tumor heterogeneity and microenvironmental interactions, allowing advances in targeted therapy for lung cancer as well as broader spectrum of malignancies.Disclosure InformationA. Kehren: A. Employment (full or part-time); Significant; Lunaphore Technologies SA. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Akoya Biosciences. M.G. Procopio: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; KTI grant (Schweiz). B. Pelz: A. Employment (full or part-time); Significant; Lunaphore Technologies SA. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Akoya Biosciences. Z. Siddiqui: A. Employment (full or part-time); Modest; Lunaphore Technologies SA. K. Roman: A. Employment (full or part-time); Significant; Akoya Biosciences. S. Adnane: A. Employment (full or part-time); Modest; Lunaphore Technologies SA. S. Brajkovic: A. Employment (full or part-time); Significant; Lunaphore Technologies SA. C. Hoyt: A. Employment (full or part-time); Significant; Akoya Biosciences. D.G. Dupouy: A. Employment (full or part-time); Significant; Lunaphore Technologies SA. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Lunaphore Technologies SA. A. Soltermann: A. Employment (full or part-time); Significant; Institut für Klinische Pathologie Universitätsspital Zürich.

An improved method for preparation of mouse retinal cryosections

Immunohistochemistry using mouse retinal cryosections is a routine assay used in vision research. However, retinal tissues are fragile, and it is difficult to obtain an ideal retinal cryosection. Here, we developed a modified method for preparing retinal cryosection. Super Glue was applied on the surface of the sclera before the cornea and the lens are removed from either the unfixed or PFA-fixed mouse eyeballs. The new methods largely prevented retinal detachment in mouse retinal cryosections. Immunostaining of retinal cryosections derived from PFA-fixed mouse eyes using rod and cone markers yielded high-quality immunofluorescent images. Immunolabeling of retinal cryosections obtained from unfixed mouse eyes using a cilium-specific marker had improved orientations of photoreceptor connecting cilia. This new method substantially improves the morphology and immunostaining results of fixed and unfixed mouse eyes.

Anti-annexin A5, Antiphosphatidylinositol Antibodies and Haematological Parameters in Preeclampsia Patients: A Case-Control Study

Background: Prevalence and the risk of common antiphospholipid antibodies (aPLs) such as lupus anticoagulant (LA), anticardiolipin (aCL) and anti-β2-glycoprotein I (β2-GPI) antibodies in preeclampsia is a matter of debate. Recently, interests have expanded in evaluating the risks of presenting non-classic aPLs in preeclampsia patients. Objective: The objective of this case-control study was to evaluate some haematological parameters besides assessing the presence of anti-annexin A5 and antiphosphatidylinositol (aPI) antibodies in preeclampsia patients compared to nonpreeclampsia subjects. Methods: From two hospitals in Malaysia, a total of 84 subjects were recruited in this case-control study including 42 preeclampsia and 42 age-matched non-preeclampsia subjects. Some haematological parameters [i.e., haemoglobin, total white blood cell, platelet, prothrombin time and activated partial thromboplastin time (APTT)] were assessed besides screening for anti-annexin A5 and aPI antibodies. Results: Among the haematological parameters, APTT was significantly high in mild preeclampsia when compared to severe preeclampsia subjects (p=0.007). IgG anti-annexin A5 antibody was detected in a single preeclampsia subject only (2.4%) and none in non-preeclampsia subjects. Conclusion: Because of the low prevalence, non-classic aPLs should not be considered as a risk factor in developing preeclampsia and not justifiable to consider as a routine assay in pregnant women.

Selective synthetic growth medium “Acinetobacter phenylalanine agar” for isolation and identification of Acinetobacter calcoaceticus — Acinetobacter baumannii complex species

The aim of the study was to carry out clinical and microbiological testing of selective growth medium Acinetobacter phenylalanine agar to isolate and identify bacterial species belonging to the Acinetobacter calcoaceticus — Acinetobacter baumannii complex (ACB complex). For this, 400 samples of clinical material (wound discharge, blood, urine, bronchoalveolar lavage) were examined in 2018 in the Clinical and Bacteriological Laboratory of the Military Medical Academy by using routine assays (seeding on blood agar growth medium, pure culture isolation and identification by using biochemical assays as well as VITEK 2 microbial identification system, bioMerieux) and plating together with growth medium Acinetobacter phenylalanine agar selective to Acinetobacter spp. owing to L-phenylalanine as a sole nitrogen and carbon source additional selected with trimethoprim. ACB complex Acinetobacters were identified 18–24 hours later after incubation at 37°C by emergence of typical colonies on selective medium. Control tests for cytochrome oxidase as well as oxidative/fermentation (OF)-glucose test by using peroxide-hydrogen microvolume method (for 1 h) allowed to rapidly distinguish ACB complex Acinetobacters from other bacteria as well as from Acinetobacter spp. unable to glucose oxidation. Next, species identity for all isolated Acinetobacter strains was established by using matrix-activated laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS). It was found that by using novel vs. routine assay Acinetobacter spps. were isolated in 26 (18 samples — in monoculture, 8 — in association with Pseudomonas aeruginosa or Klebsiella pneumoniae subsp. pneumoniae with tiny associate colonies) vs. 20 (7 — in monoculture, 13 — in association with Pseudomonas aeruginosa, Klebsiella, Escherichia, Citrobacter, Providencia, Staphylococcus, Enterococcus, C. albicans). Using MALDI-TOF MS method revealed that 25 out of 26 Acinetobacter strains isolated on selective growth medium belonged to the ACB complex (A. baumannii — 23, A. pittii — 2), whereas one strain (A. baylyi) did not belong to ACB complex. Hence, the diagnostic specificity of the Acinetobacter phenylalanine agar synthetic growth medium for isolation and identification of the A. calcoaceticus — A. baumannii complex species comprised 96.2%, with diagnostic sensitivity exceeding that one for routine assay by 25%. Use of selective growth medium accelerates research by allowing to isolate and identify ACB complex Acinetobacter spp. 18–24 h later after plating clinical material. Selectivity of growth medium was potentially stable, as its major trophic selection factor did not depend on acquired bacterial antibiotic resistance, which is also suitable as a synthetic growth medium for standardized studies.

Toward comparability of anti-drug antibody assays: is the amount of anti-drug antibody–reagent complexes at cut-point (CP-ARC) the missing piece?

Immunogenicity testing is a mandatory and critical activity during the development of therapeutic proteins. Multiple regulatory guidelines provide clear recommendations on appropriate immunogenicity testing strategies and required bioanalytical assay performances. Unfortunately, it is still generally accepted that a comparison of the immunogenicity of different compounds is not possible due to apparent performance differences of the used bioanalytical methods. In this perspective, we propose the ‘cut-point anti-drug antibody–reagents complex’ (CP-ARC) concept for technical comparability of the bioanalytical methods. The feasibility and implementation in routine assay development is discussed as well as the potential improvement of reporting of bioanalytical immunogenicity data to allow comparison across drugs. Scientific sound comparability of the bioanalytical methods is the first step toward comparability of clinical immunogenicity.