Jacqueline O'Brien

Conditions for measuring DNA synthesis in PHA stimulated human lymphocytes in 20 μl hanging drops with various cell concentrations and periods of culture

Abstract We have studied conditions for measuring the uptake of [3H]thymidine ([3H]Tdr) by human lymphocytes in inverted microcultures, varying cell concentrations and periods in culture. Analysis of variance of the log values for [3H]Tdr uptake may be used to separate effects of variables and their interactions. A pulse time of 2 h, a total thymidine concentration of about 1 μg/ml and a specific activity of [3H]Tdr of 2 Ci/mmole were optimal. Variables such as cell concentration, period of culture, type of serum and dose of PHA were shown to interact, suggesting that these variables should be examined together especially when comparing different samples of lymphocytes. Conditions for doing this simply in small volumes are now available for culturing, thymidine pulsing, harvesting and analysis of data.

A simple technique for harvesting lymphocytes cultured in terasaki plates

A simple method has been developed for harvesting lymphocytes from Terasaki microplates based on a culture technique where the plates are inverted and the cells grown on the hanging meniscus of the medium in each well. Sixty individual filter discs were prepared in wells in a polycarbonate harvesting plate. A dry Terasaki plate was used to cut these filters with the harvester acting as a die. The inverted culture plate was then lowered onto the harvester to allow the cells on the meniscus from each well to be absorbed on the appropriate filter disc. The culture plate was then discarded and the cells on the filters washed in the harvester. The wash solutions were removed by suction through holes below each filter. The time taken to harvest the cultures from 60 wells of a plate was 3--5 min.

Injury to human granulocytes at low temperatures

Abstract Granulocytes differ from other blood cells in that they are more sensitive to injury on freezing and thawing. Previous studies suggest that the difficulty in preserving them is related to their sensitivity to osmotic stress. A miniaturized system both for freezing granulocytes and testing their function in the same Terasaki plates has been developed. This allowed study of several factors simultaneously including concentration of protective additive, different cooling conditions, and dilution conditions on rewarming. We observed two types of injury to granulocytes frozen to higher subzero temperatures and thawed directly. The first type was initially severe but decreased with time in the frozen state under some conditions and appears not to have been reported in other cell systems. The second type of injury consists of conventional loss of function with longer holding times after freezing. Cells surviving these two classes of injury could be protected against the further stress of rapid cooling into liquid nitrogen, but this protection required a longer time during cooling in the frozen state than with other cell types. We have studied the interactions between several variables, e.g., time in DMSO before freezing and dilution rate after thawing in an attempt to characterize the unusual injurious mechanism at high subzero temperatures that, we believe, is the real cause of the difficulty of preserving these cells.

Selection of Leukaemic Cell Populations by Freezing and Thawing

THE co-existence of normal and abnormal cell populations in the peripheral blood of leukaemic patients may explain the variations in the reactivity to mitogens (discussed by Ling1). One example is the low or delayed response of cells from chronic lymphocytic leukaemic (CLL) patients to phytohaemagglutinin (PHA) in comparison with normal cells. The variations presumably result from a combination of the response of the underlying normal population to mitogens and the different responses (inhibition, no effect or stimulation) of the abnormal cells. Analysis of abnormal cell populations and their interactions, together with the study of serum factors, is hindered by the heterogeneity of leukaemic cell suspensions. We have now investigated the differential survival of leukaemic and normal cells from leukaemic blood following freezing and thawing using controlled cooling conditions. Previously we established the cooling rate at which normal human peripheral lymphocytes are recovered optimally using dimethylsulphoxide (DMSO) as a protective additive2,3. Lymphocytes activated by mitogen or specific antigen before freezing required different cooling rates for optimal recovery. We show here that cooling rates both faster and slower than those required for the recovery of normal lymphocytes permit the differential survival of abnormal cells from leukaemic blood and that this technique may clarify the study of serum factors.

The Effect of Veiled Cells on Lymphocyte Function

The response of lymphocytes to stimulation with Con A has been studied in the presence of veiled cells collected from the afferent lymph. In these enriched cultures the response occurred earlier with smaller numbers of lymphocytes and at lower concentrations of Con A. Veiled cells also caused clumping of lymphocytes in unstimulated cultures. In stimulated cultures small cells with veil-like projections appeared after 48 hr, but were not seen in unstimulated cultures.

HL‐A Groups in Pernicious Anaemia

Summary. There is a significant association with HL‐A3 type and pernicious anaemia. In addition there is a negative association with HL‐A2. There is no association between HL‐A type and the presence or absence of immunity to intrinsic factor.