Abstract
The effect of different suspending and washing procedures for recovery of sensitized sheep erythrocytes (EA) after freezing at −196°C was investigated. Best results were obtained using gelatin-veronal-buffered saline-sucrose containing 0.15 mM-Ca and 1 mM-Mg (GVBSM++-sucrose) as the suspending and first washing buffer. The cryoprotective agents tested were polyvinylpyrrolidone (PVP), neutralized PVP, purified PVP and a gum product, Avelex 1030. All PVP preparations tested gave good results as cryoprotectants in terms of cell recovery after thawing whereas Avelex 1030 was less satisfactory. The EA cells frozen in the presence of untreated PVP showed, however, increased susceptibility to the hemolytic action of complement, whereas cells frozen with purified or neutralized PVP gave titers similar to that obtained with fresh cells. Good results were also obtained with Avelex 1030. Complement titrations with frozen EA cells were more reproducible than titrations with fresh cells.
Keywords: sensitized sheep erythrocytes (EA), freezing at −196°C, storing at −90°C, complement titrations
Sammendrag
Betydningen av suspensjonsmiddel og vaskeprosedyre for gjenvinning av sensibiliserte røde saueblodlegemer (EA) etter nedfrysing til −196°C ble undersøkt. Best resultat ble oppnådd med gelatin-veronalbuffer-sukrose med 0.15 mM Ca og 1 mM Mg (GVBSM++-sukrose) som suspensjonsmiddel for cellene før frysing og som vaskebuffer etter tining. Som kryoprotektive midler ble brukt polyvinylpyrrolidone (PVP), renset PVP, nøytralisert PVP og et gummiprodukt, Avelex 1030. Cellegjenvinningen var uavhengig av hvilken PVP som ble brukt, mens Avelex 1030 ga dårligere resultat enn PVP. Den hemolytiske virkning av komplement økte ved bruk av EA celler som hadde vært frosset i nærvær av ubehandlet PVP, mens celler som hadde vært frosset med renset eller nøytralisert PVP eller med Avelex 1030 ga samme titre i komplementtitreringer som ufrosne celler. Komplementtitreringer med frosne EA celler ga mer konstante resultater enn når ufrosne celler preparert separat ble brukt som reagens.
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