Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.

Abstract OR-2: The Formation of Dps-DNA Complexes under Different Conditions According to Cryo-EM and SAXS

Background: The effect of Dps-DNA co-crystals formation, which occurs in stressed Escherichia coli cells exposed to extreme conditions, is well described in the literature. However, the exact mechanisms of co-crystals formation are yet to be postulated remaining largely unknown. Here we summarize the results obtained by our group over the last few years using cryo-Electron Microscopy (cryo-EM) and Small Angle X-ray Scattering (SAXS). Methods: Samples for cryo-EM were plunge frozen in liquid ethane with Vitrobot Mark IV and studied with Titan Krios (ThermoFisher Scientific, US) cryo-EM, equipped with Falcon 2 direct electron detector, Image corrector (CEOS, Germany), and Volta phase plate. Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET) studies were conducted with 300 kV accelerating voltage in low dose mode using EPU and Tomography software (ThermoFisher Scientific, US). Cryo-EM data processing was conducted using Warp, CryoSPARC, IMOD, EMAN, and Relion software packages. SAXS measurements were performed at the EMBL on the P12 BioSAXS beam line at the PETRAIII storage ring (DESY, Hamburg). Results: In this work, Dps-DNA complex formation is thoroughly studied using complementary cryo-EM (including SPA, cryo-ET, and subtomogram averaging) and SAXS methods. The formation of individual complexes of Dps with small linear DNA fragments and the Dps-Dps interaction was visualized using cryo-EM. It was found that Dps-DNA complex remains stable under various conditions and while the addition of different ions leads to the disruption of co-crystals, the process is completely or partially reversible. Conclusion: Recent studies conducted by our group showed that Dps-DNA co-crystals adopt triclinic or cubic crystal lattice (FEBS Lett., 2019; Biomolecules, 2020). Here we present the results on the studies of Dps interaction with small linear DNA fragments, demonstrate the effects of MgCl2, FeSO4, and EDTA on the Dps-DNA complex and individual Dps protein structure, discuss the influence of the temperature and time on the co-crystals.

Proliferation of SARS-CoV-2 B.1.1.7 Variant in Pakistan-A Short Surveillance Account

The emergence of a more transmissible variant of SARS-CoV-2 (B1. 1.7) in the United Kingdom (UK) during late 2020 has raised major public health concerns. Several mutations have been reported in the genome of the B.1.1.7 variant including the N501Y and 69-70deletion in the Spike region that has implications on virus transmissibility and diagnostics. Although the B.1.1.7 variant has been reported by several countries, only three cases have been reported in Pakistan through whole-genome sequencing. Therefore, the objective of the study was to investigate the circulation of B.1.1.7 variant of concern (VOC) in Pakistani population. We used a two-step strategy for the detection of B.1.1.7 with initial screening through TaqPathTM COVID-19 CE-IVD RT-PCR kit (ThermoFisher Scientific, Waltham, US) followed by partial spike (S) gene sequencing of a subset of samples having the spike gene target failure (SGTF). From January 01, 2021, to February 21, 2021, a total of 2,650 samples were tested for SARS-CoV-2 and 70.4% (n = 1,867) showed amplification of all the 3 genes (ORF, N, and S). Notably, 29.6% (n=783) samples have been SGTF that represented numbers from all the four provinces and suggest a rather low frequency during the first 3 weeks of January (n = 10, n = 13, and n = 1, respectively). However, the numbers have started to increase in the last week of January, 2021. During February, 726 (93%) cases of SGTF were reported with a peak (n = 345) found during the 3rd week. Based on the partial sequencing of SGTF samples 93.5% (n = 29/31) showed the characteristic N501Y, A570D, P681H, and T716I mutations found in the B.1.1.7 variant. In conclusion, our findings showed an upsurge of B.1.1.7 cases in Pakistan during February, 2021 affecting 15 districts and warranting large scale genomic surveillance, strengthening of laboratory network and implementation of appropriate control measures in the country.

150 Changes in pyruvate metabolism alters the epigenetic and molecular maturation of bovine oocytes

During invitro maturation (IVM), bovine oocytes undergo important metabolic, epigenetic, and transcriptional changes for the acquisition of developmental competence. Particularly, metabolic changes that alter the availability of cytoplasmic acetyl-CoA, the main substrate for histones acetylation, may alter the epigenetic profile of the oocyte, with consequences for correct molecular maturation. To test this hypothesis, cumulus–oocyte complexes (COCs) were IVM in three experimental groups: Control [IVM medium (TCM-199-Bicarbonate, 10% fetal bovine serum, 1µg mL−1 oestradiol, 10µg mL−1 FSH, and 10µg mL−1 human chorionic gonadotrophin)], DCA (IVM medium supplemented with 1.5mM sodium dichloroacetate, a pyruvate to acetyl-CoA conversion stimulator) and IA (IVM medium supplemented with 5µM sodium iodoacetate, a glycolysis inhibitor). Cumulus cells (CC) and oocytes (Oo) were analysed separately at 24h (mitochondrial activity, MA; MitoTracker Red CMXRos, ThermoFisher Scientific] and at 0, 4, 8, 16, and 24h of IVM [lysine 9 histone 3 acetylation (H3K9ac immunofluorescence) and new transcript synthesis (only CC; Click-iT® RNA, ThermoFisher Scientific). The images were acquired using a fluorescence microscope and analysed by Image J software. The results from at least 3 replicates were compared by Student’s t-test (treatment vs. control) or by ANOVA followed by Tukey’s test (comparison within the same group in different time points) considering P<0.05. As expected, DCA treatment led to an increase in MA in CC and oocytes (CC control vs. DCA, P=0.003; Oo control vs. DCA, P=0.003). In CC, during the first 4h, H3K9ac increased significantly in the treated group and decreased in the control group. At 8, 16, and 24h, both groups presented similar tendencies, although H3K9ac levels remained higher in DCA compared with control at all time points (P<0.001). The synthesis of new transcripts in CC was stimulated by DCA compared with control at 8h (P=0.02) and particularly at 16h (P=0.002), when acetylation levels were at the lowest point. Interestingly, in oocytes, the initial trend was reversed. An increase was observed in the H3K9ac levels of the control group (P=0.014), whereas no difference was observed for DCA in the first 4h. Moreover, although acetylation levels followed a downward tendency with time in both groups, oocytes treated with DCA showed lower H3K9ac levels at 4 and 8h and a higher level at 24h (P=0.04) compared with control. Regarding IA, lower MA were verified in CC whereas oocytes had the opposite profile (CC control vs. IA: P=0.0035; Oc control vs. IA: P<0.001). In CC, this decrease in MA was not accompanied by a decrease in H3K9ac. In contrast, H3K9ac increased compared with the control group at 8 and 16h (control 8h vs. IA 8 h: P=0.019 and control 16h vs. IA 16 h: P=0.019). These changes were accompanied by an increase in the synthesis of new transcripts in the IA group over the time of IVM. Based on these data, we can conclude that changes in pyruvate metabolism caused by manipulation of the IVM system lead to epigenetic and molecular changes in both CC and oocytes.

P05.01 Comparative analysis of RNA versus DNA as input material for IGH repertoire sequencing panels for immuno-oncology applications and rare clone detection

BackgroundRecent progress in tumor immunotherapies have shown the importance of next generation sequencing (NGS) T cell repertoire profiling to characterize T cell immune response to treatment. Understanding the role of the B cell repertoire upon stimulation of the immune system by checkpoint blockade is paramount for immunotherapeutic approaches in treatment of B cell malignancies, as well as understanding B cell function within traditional I/O strategies. The ability to detect low frequency B cell clones enables numerous hematology/oncology research applications, including identification of potential biomarkers and minimal residual disease (MRD) research. Historically, efforts to track the frequency of malignant B cells by IGH chain sequencing have utilized DNA input given potential challenges in accurately quantifying template copy number from RNA data owing to B cell subtype specific variation in the expression of the B cell receptor. Hypothetically, however, RNA input based monitoring could be advantageous both owing to reduced input requirements and superior ability to detect B call malignancies of plasmablast and plasma cell origin, where the BCR is robustly expressed. Here we compared the ability of RNA and DNA based IGH chain sequencing to detect Burkitt’s Lymphoma cell lines and Chronic Lymphocytic Leukemia samples at a frequency of 10-6 from peripheral blood.Materials and MethodsHere we present performance for rare clone detection utilizing the Ion OncomineTM BCR IGH-SR assay and the Ion OncomineTM BCR IGH-LR assay. These assays use multiplex primers targeting all known IGH germline variable genes in the framework 1 (FR1) or framework 3 (FR3) regions of the B cell receptor using either DNA or RNA as input. To evaluate detection sensitivity of the IGH-SR assay we utilized DNA or RNA from Burkitt’s lymphoma cell lines as well as clinical chronic lymphocytic leukemia (CLL) samples controllably added to a background of peripheral blood leukocytes (PBL) by mass ratio to create specimens with a known target B cell frequency. Automated downsampling analysis was used to confirm libraries were sequenced to saturation. Library preparation and analysis was performed in replicate to quantify sensitivity of detection.ResultsFor each cell line, we prepared and sequenced (1) 30 libraries derived from amplification of 2ug gDNA spiked with 2pg cell line gDNA and (2) 10 libraries derived from amplification of 100ng RNA spiked with 0.1pg cell line total RNA. The Burkitt’s lymphoma cell line and CLL samples were detected in 10/30 and 8/30 libraries respectively, consistent with the performance of orthologous DNA-based sequencing approaches. For RNA libraries, the Burkitt’s lymphoma and CLL samples were detected in each library (10/10 and 10/10, respectively).ConclusionsHere we demonstrate the ability to detect B cell clones down to 10-6 from gDNA and RNA inputs utilizing the Ion OncomineTM BCR IGH-SR assay. Feasibility for rare clone detection is shown in gDNA or RNA enabling B cell minimal residual disease research, and high sensitivity characterization the B cell role in response to checkpoint blockade within the tumor microenvironment. Importantly, we find that RNA based IGH sequencing may significantly reduce input requirements for rare clone detection, potentially enabling routine detection of clones at 10-6 frequency from a single library.Disclosure InformationG.M. Lowman: A. Employment (full or part-time); Significant; ThermoFisher Scientific. L. Pickle: A. Employment (full or part-time); Significant; ThermoFisher Scientific. M. Toro: A. Employment (full or part-time); Significant; ThermoFisher Scientific. J. Chang: A. Employment (full or part-time); Significant; ThermoFisher Scientific. D. Topacio-Hall: A. Employment (full or part-time); Significant; ThermoFisher Scientific. T. Looney: A. Employment (full or part-time); Significant; ThermoFisher Scientific.

Personalized approach to drug therapy for multimorbid patients with left ventricular non-compaction cardiomyopathy

Некомпактный миокард левого желудочка (НМЛЖ) характеризуется развитием ряда осложнений, что в сочетании с сопутствующими заболеваниями обычно приводит к полипрагмазии. Фармакогенетическое тестирование (ФГТ), направленное на выявление вариантов генов, ассоциированных с метаболизмом лекарственных препаратов (ЛП), может позволить подобрать наиболее эффективную и безопасную лекарственную терапию в каждом конкретном случае. В исследование было включено 16 больных с НМЛЖ, его осложнениями и другими заболеваниями. Всем пациентам проводилась оценка 60 однонуклеотидных полиморфизмов (SNP) с помощью полимеразной цепной реакции в реальном времени в амплификаторе QuantStudio 12KFlexReal-TimePCRSystem (ThermoFisherScientific, США). По результатам ФГТ выявлено 62,5% пациентов с НМЛЖ, генотипы которых ассоциированы с изменением метаболизма ЛП. У 12,5% пациентов в анамнезе прием ЛП, требующих коррекции дозы или замены на другой ЛП с учетом результатов ФГТ. A left ventricular non-compaction cardiomyopathy (LVNC) is characterized by the development of a number of complications, which in combination with concomitant diseases usually leads to polypharmacy. Pharmacogenetic testing (PGT), aimed at identifying variants of genes associated with the metabolism of drugs, allows to choose the most effective and safe drug therapy in each case. The study includes 16 patients with LVNC, its complications and other diseases. All patients were assessed for 60 single nucleotide polymorphisms (SNPs) using real-time polymerase chain reaction in a QuantStudio 12KFlexReal-TimePCRSystem thermocycler (ThermoFisher Scientific, USA). According to the results of PGT 62.5% of the patients with LVNC and genotypes associated with a change in the metabolism of drugs were revealed. 12.5% of the patients had a history of taking drugs that required dose adjustment or replacement with another drug, taking into account the results of PGT.

Minimal Residual Disease Detection at RNA and Leukemic Stem Cell (LSC) Level. Comparison of Qpcr, d-PCR and CD26 Stem Cell Measurements in Chronic Myeloid Leukemia (CML) Patients in Deep Molecular Response (DMR)

Background. A Deep Molecular Response (DMR), defined as BCR-ABL1 p210 transcript at levels <= 0.01% by QPCR, is the prerequisite for a successful interruption of treatment in patients with CML. However, approximately 50% of patients in Treatment Free Remission (TFR) studies had to resume therapy after BCR-ABL1 p210 transcript levels rose above the 0.01% DMR threshold. To improve transcript detection sensitivity, transcript levels were analyzed using D-PCR. D-PCR increased BCR-ABL1 transcript detection sensitivity by 10-100 fold, however its ability to better select successful TFR patients remains unclear. Beyond the role of the immune system, relapse may be due to the presence of residual leukemic stem cells (LSCs) that are transcriptionally low or silent, insensitivity to tyrosine kinase inhibitors (TKIs) or other CML-causing transcripts not easily detectable by BCR-ABL1 PCR techniques Another approach is to use flow cytometry to detect and quantify bone marrow Ph+ LSCs CD34+/CD38− that co-express CD26 (dipeptidylpeptidase-IV) although its meaning in TFR is unclear. Aim. To compare and examine values of the three different methods of detecting minimal residual disease (MRD) in CML at RNA and LSC levels in patients in TFR or DMR. Patients and Methods. The twenty-seven patients in this study received treatment with either Imatinib (12), Dasatinib (6), Nilotinib (7), Bosutinib (1) or Interferon (1). Twelve patients were in TFR, while the rest were in DMR. TFR patients had stopped therapy for less than 1 year (3), <3 years (2), 6 years (6) and 17 years (1). Blood samples were collected and tested 3 times. BCR-ABL1 transcript quantification was performed using the automated Xpert Ultra BCR-ABL1 MonitorTM Cepheid method and calibrated for the ABL reference gene and BCR-ABL1 target gene. Samples were analyzed according to the manufacurer with results expressed in BCR-ABL1/ABL %IS, or undetectable transcript (U), with PCR sensitivity of 5.0 (>250,000 ABL copy numbers). D-PCR analysis used TaqMan-MGB probes targeting the BCR-ABL1 transcript. A custom assay was designed and produced with a FAM-label, basing on the sequence of routinely-used probes. BCR-ABL1 quantifications were performed analyzing 50ng of cDNA on a QuantStudio 3D Digital PCR System (ThermoFisher Scientific). BCR-ABL1 transcript values using dPCR were expressed as number of copies/ul. The secondary analysis were carried out by AnalysisSuite Cloud Software (ThermoFisher Scientific). To detect peripheral blood circulating CD34+/CD38-/CD26+ LSCs, cells were incubated with anti-CD45 (BD Biosciences), anti-CD34 (581), anti-CD38 (HIT2) and anti-CD26 (M-A261) (BD Pharmigen). Acquisition and analysis were performed by FACSCanto II flow cytometer (BD Biosciences, NR Nannini). CD45+ cells acquired for each sample ranged from 500,000 to 1,000,000. Isotype controls were included in each staining. Median absolute number of CD26+ cells/μL were calculated as follows: (# WBCs/μL) × (% CD34+/CD38−/CD26+ stained CD45+ cells). Results. TFR status, the type of TKI treatment, QPCR results, dPCR data and LSCs quantification are summarized in Table 1. Both d-PCR and LSCs showed higher sensitivity than QPCR, exhibiting positive results when transcript levels using QPCR were undetectable (16). None of the patients tested negative with d-PCR, however 14/16 were under the threshold of 0.468 copies/uL, corresponding to a stable DMR. LSC levels were negative in 5 patients, 3 of which also tested negative with QPCR. In all patients QPCR ranged from 0-0.0068, d-PCR from 0.073 to 0.943, and LSCs from 0 to 0.156. Results were divided in quartiles, depending on molecular response for QPCR, the 0.468 threshold for dPCR and the distribution for the LSCs. The 2 lowest quartiles defined the lowest detectable DMR. A strong correlation of these data in TFR patients was noted (10/12 concordant) while 8/15 DMR patients were discordant. Conclusions To our knowledge this is the first attempt to analyze and compare DMR in a CML population using standard (QPCR) and highly sensitive (dPCR and LSCs) methods. TFR patients, some lasting up to 17 years, were in the lowest detectable DMR categories. Very little is known about the biology of CML implications of circulating LSCs. Larger studies and dynamic scoring will help define their informative and predictive value. Disclosures Abruzzese: Pfizer: Consultancy; Ariad: Consultancy; Novartis: Research Funding; BMS: Consultancy.

Molecular tools for phytoplankton monitoring samples

HABs can have severe impacts in fisheries or human health by the consumption of contaminated bivalves. Monitoring assessment (quantitative and qualitative identification) of these organisms, is routinely accomplished by microscopic identification and counting of these organisms. Nonetheless, molecular biology techniques are gaining relevance, once these approaches can easily identify phytoplankton organisms at species level and even cell number quantifications. This work tests 12 methods/kits for genomic DNA extraction and seven DNA polymerases to determine which is the best method for routinely use in a common molecular laboratory, for phytoplankton monitoring samples analyses. From our work, Direct PCR master mix for tissue samples, proved to be the most adequate by its velocity of processivity, practicability, reproducibility, sensitiveness and robustness. However, brands such as Omega Biotek, GRISP, Qiagen and MP Biomedicals also showed good results for conventional DNA extraction as well as all the Taq brands tested (GRISP, GE Healthcare Life Sciences, ThermoFisher Scientific and Promega). Lugol’s solution, with our tested kits did not show negative interference in DNA amplification. The same can be said about mechanical digestion, with no significant differences among kits with or without this homogenization step.

An alternative inhibition method for determining cross-reactive allergens

Background:Inhibition assays are an useful tool to identify the allergen of primary sensitization of cross-reactive allergens. Classical ELISA-based inhibition assays are limited by both the availability of commercial standardized allergen extracts and the experience and knowledge needed for making home-made extracts. Moreover the direct comparison of the inhibition ELISAs outcomes between different laboratories is difficult because of different sources of used allergen extracts and a number of methodological variations. Therefore, we propose a novel ImmunoCap (Phadia, Thermofisher Scientific) based immunoinhibition method with the use of commercially available Caps as the allergen source.Methods:The novel ImmunoCap based immunoinhibition method was developed and tested with sera from patients with a well-known cross-reactive sensitization for fig (Results:The amount of allergens (fig and ficus extracts) needed to reach a similar degree of inhibition was comparable for both inhibition methods.Conclusions:The ImmunoCap based inhibition assay, in addition to classical inhibition methods, is a valuable tool as the ImmunoCap analyzer and commercial allergens (Caps) are more widely available which makes the outcomes of inhibition tests comparable between different laboratories. Furthermore, in the ImmunoCap inhibition method the same protein source is used for both the inhibition of sIgE and sIgE measurement, which might be even more relevant when multiple cross-reactive allergens are tested.

Απομόνωση σε καλλιέργειες και ανοσοϊστοχημική μελέτη των νεοπλασματικών κυττάρων σε ασθενείς με ηπατοκυτταρικό καρκίνο

Η καλλιέργεια ηπατοκυττάρων υπήρξε πάντα δυσχερές εργαστηριακό εγχείρημα, παρά τις επίμονες προσπάθειες πολλών ερευνητών διεθνώς. Στο Πειραματικό Εργαστήριο της Α΄Προπαιδευτικής Χειρουργικής Κλινικής, τουΠανεπιστημίου Αθηνών (Δ/ντής ο καθηγητής κ. Γεώργιος Κ. Ζωγράφος), στο ΙΠΠΟΚΡΑΤΕΙΟ Γ.Ν.Α Νοσοκομείο Αθηνών, οι υπεύθυνοι Βιολόγοι κ.κ. Αγάπη Κατάκη και Αναστασία Δεβετζή, ανέπτυξαν μία απολύτως αξιόπιστη και αποτελεσματική μέθοδο, την οποία και ακολουθήσαμε στην παρούσα εργασία. ΑΣΘΕΝΕΙΣ - ΥΛΙΚΟ ΚΑΙ ΜΕΘΟΔΟΙΟ σκοπός ήταν να μελετήσουμε τα ιδιαίτερα χαρακτηριστικά των νεοπλασματικών ηπατοκυττάρων σε ασθενείς με πρωτοπαθή ηπατοκυτταρικό καρκίνο(ΗΚΚ), με προοπτική τα αποτελέσματα να αποτελέσουν ένα πιθανό οδηγό στην φαρμακευτική αντιμετώπιση της νόσου. Ως γνωστόν, η συγκεκριμένη κακοήθης πάθηση του ήπατος, είναι ανθεκτική στην χημειο-ακτινοθεραπεία και η ηπατεκτομή αποτελεί σήμερα την πρώτη γραμμή θεραπείας. Για τον σκοπό αυτόν, εκτός της επιτυχούς απομόνωσης, σε μονοκαλλιέργειες φυσιολογικών και νεοπλασματικών ηπατοκυττάρων, από υγιές ηπατικό παρέγχυμα (16 μάρτυρες που υπεβλήθησαν σε λαπαροσκοπική χολοκυστεκτομή, κατόπιν έγγραφης συναινεσης) και νεοπλασματικό ιστό από 20 ασθενείς μετα ηπατεκτομή για ΗΚΚ, διενεργήθη και ανοσο-ιστοχημική μελέτη με ειδικά αντισώματα για την ολοκλήρωση της αποκάλυψης κάποιων ιδιαιτέρων χαρακτηριστικών των καρκινικών κυττάτων. Τα αντισώματα που χρησιμοποιήσαμε ήταν τα Hep-Par 1, CD90, Oct ¾, CXCR4/CD184. Ένζυμα απομονώσεως για τις μονοκαλλιέργειες χρησιμοποιήθηκαν κολλαγενάση τύπου IV και θρυψίνη 0.25% σε EDTA. Ως θρεπτικά μέσα χρησιμοποι-ήθηκαν DNEM-HAMS F12, RPMI 1640 και το Hank’s Balanced Salt Solution(HBSS).Από τον νεοπλασματικό ιστό και το υγιές παρέγχυμα τα κυτταρα διαχωρίστικαν, μέχρι την πλήρη διάσταση της μάζης, αδρανοποιήθηκαν κατάλληλακαι φιλτραρίστηκαν σε ειδικό ηθμό 70 μικρομέτρων, σύμφωνα με την μεθοδολογία του εργαστηρίου. Μετά από διαδοχικές φυγοκεντρήσεις, εκπλύσειςκαι εναιωρήσεις, τα ηπατοκύτταρα απομονώθηκαν με τριπλή φυγοκέντρηση113 στις 700 σ.α.λ. γιά 12΄. Η κυτταρική συγκομιδή και η βιωσιμότητα των απομονωθέντων ηπατοκυττάρων εκτιμήθηκε με Tryptan blue και 7-ΑΑD.Tόσο τα καρκινικά, όσο και τα υγιή ηπατοκύτταρα εναιωρήθηκαν εκ νέου και προστέθηκαν ανθρώπινη ινσουλίνη, μείγμα Pen/Strept, γενταμυκίνη και L-γλουταμίνη. Η απόδοση των ηπατοκυττάρων εκτιμήθηκε με καταμέτρηση κατά Neubauer. 2Χ10(5) ηπατοκύτταρα τοποθετήθηκαν σε 24 επικαλυμμένους δοκιμαστικούς σωλήνες και αφέθηκαν να προσκολληθούν για 48 ώρες. Ακολούθως αναρροφήθηκαν προσεκτικά και τοποθετήθηκαν σε διάλυμα καλλιεργείας ηπατοκυττάρων για άλλες 16 ώρες για σταθεροποίηση. Η τελική μέτρηση έγινε με κυτταρομετρία ροής ηπατοκυττάρων.΄Ολα τα ηπατοκύτταρα καρκινικά και μή, όπως και τα φυσιολογικά ηπατοκύτταρα υποβλήθηκαν σε επεξεργασία συλλογής, έκπλυσης και φυγοκέντρη-σης, ώστε το ηπατικό ίζημα να δεχθεί τριπλή ανοσο-ιστοχημική χρώση με αντι-ανθρώπινη αλβουμίνη FITC και 7- αμινοακτινομυκίνη. Ακολούθησε επώαση, μονιμοποίηση και διάτρηση των κυτταρικών μεμβρανών. Η επώαση έγινε σε θερμοκρασία δωματίου και στο σκοτάδι, για 24, 48 και 72 ώρες, σύμφωνα με το πρωτόκολλο της μελέτης και την μεθολογία του εργαστηρίου.Τα ηπατοκύτταρα αναγνωρίστηκαν με Hep-Par-1 από την κυτταρομετρία ροής και έγινε χρώση αιματοξυλίνης για την βαφή των πυρήνων. Τελικά παρατηρήθηκαν στο μικροσκόπιο. Με ανοσο-ιστοχημική τεχνική, αναζητήθηκε η έκφραση του CD90 σε τομέςπαραφίνης, δειγμάτων ηπατοκυτταρικού καρκινώματος. Χρησιμοποιήθηκε τοκεκαθαρμένο αντίσωμα CD90 (Thy-1) της affymetrix eBioscience, με αρ. Καταλόγου 14-0909, κλώνος eBio5E10 (5E10), σε συγκέντρωση 0,5 mg/ml καιμε Host/Isotype: mouse IgG1, Kappa, με αναμενόμενη μεμβρανική και κυτταροπλασμική χρώση. Επίσης με την ίδια τεχνική αναζητήθηκε η έκφραση του υποδοχέα Oct3/4 και CXCR4/CD 184.Οι συνεχείς μεταβλητές των ευρημάτων αναφέρονται σαν μέσος όρος μετυπικές αποκλίσεις. Οι συζευγμένες συγκρίσεις μεταξύ ποιοτικών μεταβλητών, έγιναν με την χρήση προσημασμένης δοκιμασίας Wilcoxon. ΑΠΟΤΕΛΕΣΜΑΤΑ H κυτταρική απόδοση υγρού ιστού μετά την κατεργασία με κολλαγενάση ήταν κατά μεγάλη προσέγγιση 7Χ10(6) για τα καρκινικά ηπατοκύτταρα και 5Χ10 (5) για τα υγιή. Αμέσως μετά την απομόνωση η μέση βιωσιμότητα ήταν 90% και 92% αντιστοίχως για τα καρκινικά και υγιή ηπατοκύτταρα. Τα κύτταρα από ΗΚΚ και τα υγιή ηπατοκύτταρα εξέφρασαν την αλβουμίνη σε επίπεδα 94% και 92% αντίστοιχα, όπως τεκμηριώθηκε με την χρήση κυτταρομετρίας ροής.Τα ανοσο-ιστοχημικά αποτελέσματα έδειξαν ότι τα CD90(+) κύτταρα ηπατοκυτταρικού καρκινώματος ανέδειξαν ογκογόνο (tumorigenic) ικανότητα, που αντίστοιχα δεν ανέδειξαν τα CD90(-) κύτταραΤο υγρό μονοκλωνικό αντίσωμα ποντικού OCT ¾ της Novocastra, με κωδικό ΝCL-L-OCT ¾, κλώνος Ν1ΝΚ, που χρησιμοποιήσαμε, στα αποτελέσματά μας είχαμε 10 περιπτώσεις με πολλά κύτταρα εντόνως θετικά, ενώ σε άλλες 5 περιπτώσεις παρατηρήσαμε ασθενώς θετικά κύτταρα και άλλη 1 με σπανιώτατα μεμονωμένα μετρίως θετικά.Το πολυκλωνικό αντίσωμα CD184/CXCR4 της ThermoFisher SCIENTIFIC,με αρ. Καταλόγου PA3-305, Host/Isotype:Rabbit που χρησιμοποιήσαμε, σε 9 περιπτώσεις μας εκφράσθηκε κυτταροπλασμικά σε πολλά από τα νεοπλασματικά κύτταρα και σε άλλες 6 εκφράσθηκε σε μεμονωμένα νεοπλασματικά κύτταρα. Σε όλες τις θετικές περιπτώσεις παρατηρήθηκε και θετική έκφρασή τουστους διαύλους του Ηering, προφανώς σε δυσδιάκριτα προγονικά/βλαστικάκύτταρα (stem/progenitor cells).Η αποκάλυψη των παραπάνω υποδοχέων στά νεοπλασματικά ηπατοκύτταρα, από ασθενείς με ΗΚΚ, (in vitro), ανιχνεύθησαν κοινοί με υποδοχείς στααρχέγονα καρκινικά ηπατοκύτταρα. Η παρατήρηση αυτή πιθανώς να αποτελέσει σημαντικό στοιχείο στην εξέλιξη της χημειοθεραπείας, με πιό στοχευμένη και εξειδικευμένη δράση, στις περιπτώσεις του ανθεκτικού μέχρι σήμερα ΗΚΚ.