Veren ja litium-hepariinin suhteen vaikutus kalium-, natrium-, kreatiniini- ja CRP-tuloksiin kliinisen kemian plasma-analyyseissä

Tässä opinnäytetyössä tutkittiin veren ja antikoagulantin suhteen vaikutusta analyysituloksiin litium-hepariiniputkissa, koska verinäyteputkia ei aina saada täytettyä optimaalisesti. Opinnäytetyössä verrattiin myös vakuumin vaikutusta analyysituloksiin sekä kahden eri putkivalmistajan tulostasoja. Tavoite oli parantaa potilasturvallisuutta lisäämällä laboratoriotulosten luotettavuuden arviointia. Tutkimusmenetelmä oli kvantitatiivinen. Näytteet otettiin 20 vapaaehtoiselta tutkimushenkilöltä oppilaitoksen tiloissa. Näytteet käsiteltiin ISLABin Kuopion aluelaboratorion Puijon laboratoriossa ja analysoitiin Cobas c 501 -analysaattorilla. Tulokset käsiteltiin Excel-taulukko-ohjelmalla tekemällä tarvittavat laskutoimitukset. Näytteenotto-ohjeen mukaisesti optimaalisesti täytettyyn näyteputkeen verrattiin ½ täytettyjä, ¼ täytettyjä sekä ¼ täytettyjä ja ilmattuja näyteputkia. Verinäyteputkeen jäävän vakuumin vaikutus selvitettiin vertaamalla ¼ täytettyjä näyteputkia keskenään. Putkivalmistajavertailu tehtiin samoin kriteerein täytettyjen putkien kesken. Näytetilavuus vaikutti kaliumpitoisuuteen ollessaan alle puolet optimaalisesta; muihin analyytteihin sillä ei ollut vaikutusta. Vakuumilla ei ollut vaikutusta. Putkivalmistajien tulostasoilla ei ollut eroa. Johtopäätöksenä kaliumtuloksia ei voi pitää luotettavina näytetilavuuden ollessa alle ½ optimaalisesta. Tutkimuksen luotettavuutta lisäisi suurempi otoskoko. Jatkotutkimuksena tulisi selvittää näytetilavuuden vaikutus CRP-pitoisuuteen suuremmalla otoskoolla siten, että näytteiden CRP-pitoisuudet kattavat koko analysaattorin mittausalueen. Putkivalmistajavertailu tulisi tehdä myös Terumon näyteputkilla. Analyyttien säilyvyyttä vajaaksi jääneissä näyteputkissa tulisi tutkia.This quantitative study was undertaken to investigate the effects of incomplete filling of lithium heparin tubes during blood sample collection on the results of certain clinical analyses. If the removal of the vacuum from a blood sample collection tube affects the results of the analyses was also investigated. Further, the results obtained from the samples taken into the blood sample tubes purchased from two separate manufacturers were compared. On the basis of the results achieved, the necessity to modify the guidelines for blood sampling procedures was evaluated. The blood samples were collected from 20 volunteers. The samples were pretreated and analyzed at the laboratory of clinical chemistry (ISLAB, Kuopio, Finland). The analytical results were processed by using Excel-spreadsheet. The results obtained from the samples intentionally taken by underfilling (50 % and 25 % of the recommended sample volume) the collection tubes, and both by underfilling (25 % of the recommended sample volume) were compared to those from optimally filled tubes. The affect of vacuum was determined by comparing the results obtained from both underfilled (25 % of the recommended volume) tubes. Differences between two separate manufacturers were examined from the tubes filled with same criteria. The results of the analyses show that, among the analyzed compounds, the underfilling of the blood sample collection tubes during sampling only has an impact on the concentrations of potassium-ions. Neither discarding the vacuum from the test tubes nor the manufacturer of the collection tubes have an impact on the concentrations of the compounds analyzed. The reliability of the results should maybe be verified with a larger group of trial patients. Further research is needed to clarify the possible alterations of pathologically elevated CRP-concentrations under the conditions described in this study. In addition, the stability of the analytes in the cases of delayed analyses would be necessary to clarify

Cytokines impact natural killer cell phenotype and functionality against glioblastoma in vitro

ObjectiveNatural killer (NK) cells are a part of the innate immune system and first-line defense against cancer. Since they possess natural mechanisms to recognize and kill tumor cells, NK cells are considered as a potential option for an off-the-shelf allogeneic cell-based immunotherapy. Here, our objective was to identify the optimal cytokine-based, feeder-free, activation and expansion protocol for cytotoxic NK cells against glioblastoma in vitro.MethodsNK cells were enriched from human peripheral blood and expanded for 16 days with different activation and cytokine combinations. The expansion conditions were evaluated based on NK cell viability, functionality, expansion rate and purity. The cytotoxicity and degranulation of the expanded NK cells were measured in vitro from co‑cultures with the glioma cell lines U‑87 MG, U‑87 MG EGFR vIII, LN-229, U-118 and DK-MG. The best expansion protocols were selected from ultimately 39 different conditions: three magnetic cell‑selection steps (Depletion of CD3+ cells, enrichment of CD56+ cells, and depletion of CD3+ cells followed by enrichment of CD56+ cells); four activation protocols (continuous, pre-activation, re-activation, and boost); and four cytokine combinations (IL-2/15, IL‑21/15, IL‑27/18/15 and IL-12/18/15).ResultsThe expansion rates varied between 2-50-fold, depending on the donor and the expansion conditions. The best expansion rate and purity were gained with sequential selection (Depletion of CD3+ cells and enrichment of CD56+ cells) from the starting material and pre-activation with IL‑12/18/15 cytokines, which are known to produce cytokine-induced memory-like NK cells. The cytotoxicity of these memory-like NK cells was enhanced with re-activation, diminishing the donor variation. The most cytotoxic NK cells were produced when cells were boosted at the end of the expansion with IL-12/18/15 or IL-21/15.ConclusionAccording to our findings the ex vivo proliferation capacity and functionality of NK cells is affected by multiple factors, such as the donor, composition of starting material, cytokine combination and the activation protocol. The cytokines modified the NK cells' phenotype and functionality, which was evident in their reactivity against the glioma cell lines. To our knowledge, this is the first comprehensive comparative study performed to this extent, and these findings could be used for upscaling clinical NK cell manufacturing

Contribution of astrocytes to familial risk and clinical manifestation of schizophrenia

Previous studies have implicated several brain cell types in schizophrenia (SCZ), but the genetic impact of astrocytes is unknown. Considering their high complexity in humans, astrocytes are likely key determinants of neurodevelopmental diseases, such as SCZ. Human induced pluripotent stem cell (hiPSC)-derived astrocytes differentiated from five monozygotic twin pairs discordant for SCZ and five healthy subjects were studied for alterations related to high genetic risk and clinical manifestation of SCZ in astrocyte transcriptomics, neuron-astrocyte co-cultures, and in humanized mice. We found gene expression and signaling pathway alterations related to synaptic dysfunction, inflammation, and extracellular matrix components in SCZ astrocytes, and demyelination in SCZ astrocyte transplanted mice. While Ingenuity Pathway Analysis identified SCZ disease and synaptic transmission pathway changes in SCZ astrocytes, the most consistent findings were related to collagen and cell adhesion associated pathways. Neuronal responses to glutamate and GABA differed between astrocytes from control persons, affected twins, and their unaffected co-twins and were normalized by clozapine treatment. SCZ astrocyte cell transplantation to the mouse forebrain caused gene expression changes in synaptic dysfunction and inflammation pathways of mouse brain cells and resulted in behavioral changes in cognitive and olfactory functions. Differentially expressed transcriptomes and signaling pathways related to synaptic functions, inflammation, and especially collagen and glycoprotein 6 pathways indicate abnormal extracellular matrix composition in the brain as one of the key characteristics in the etiology of SCZ.Peer reviewe

DataSheet_1_Cytokines impact natural killer cell phenotype and functionality against glioblastoma in vitro.docx

ObjectiveNatural killer (NK) cells are a part of the innate immune system and first-line defense against cancer. Since they possess natural mechanisms to recognize and kill tumor cells, NK cells are considered as a potential option for an off-the-shelf allogeneic cell-based immunotherapy. Here, our objective was to identify the optimal cytokine-based, feeder-free, activation and expansion protocol for cytotoxic NK cells against glioblastoma in vitro.MethodsNK cells were enriched from human peripheral blood and expanded for 16 days with different activation and cytokine combinations. The expansion conditions were evaluated based on NK cell viability, functionality, expansion rate and purity. The cytotoxicity and degranulation of the expanded NK cells were measured in vitro from co‑cultures with the glioma cell lines U‑87 MG, U‑87 MG EGFR vIII, LN-229, U-118 and DK-MG. The best expansion protocols were selected from ultimately 39 different conditions: three magnetic cell‑selection steps (Depletion of CD3+ cells, enrichment of CD56+ cells, and depletion of CD3+ cells followed by enrichment of CD56+ cells); four activation protocols (continuous, pre-activation, re-activation, and boost); and four cytokine combinations (IL-2/15, IL‑21/15, IL‑27/18/15 and IL-12/18/15).ResultsThe expansion rates varied between 2-50-fold, depending on the donor and the expansion conditions. The best expansion rate and purity were gained with sequential selection (Depletion of CD3+ cells and enrichment of CD56+ cells) from the starting material and pre-activation with IL‑12/18/15 cytokines, which are known to produce cytokine-induced memory-like NK cells. The cytotoxicity of these memory-like NK cells was enhanced with re-activation, diminishing the donor variation. The most cytotoxic NK cells were produced when cells were boosted at the end of the expansion with IL-12/18/15 or IL-21/15.ConclusionAccording to our findings the ex vivo proliferation capacity and functionality of NK cells is affected by multiple factors, such as the donor, composition of starting material, cytokine combination and the activation protocol. The cytokines modified the NK cells' phenotype and functionality, which was evident in their reactivity against the glioma cell lines. To our knowledge, this is the first comprehensive comparative study performed to this extent, and these findings could be used for upscaling clinical NK cell manufacturing.</p