In vitro quantitation of Theileria parva sporozoites for use - TropMed ...

Chapter 8: SNA and refinement of titration protocol 113
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Table 15: Antibody detection of post inoculation sera as detected by the three assays.
ANTIGEN SELISA P67 PIM
Live sporozoites + + +
Dead sporozoites - - -
Schizonts + - +
8.4. Discussion
Storage of PBMC at 4°C seems to preserve cell vitality better than at 37°C. The comparison of cell
samples kept at 4°C and 37°C showed that the refrigerated sample had a higher population of live
cells. Hunt and her colleagues (2005) using rodent and human cells have shown that "pausing" of
mammalian cells by storing at 4°C maintains functionality up to nine days. Our results are in
agreement and indications are that, in the short term, refrigeration preserves cell vitality better than
an overnight incubation at 37°C (5% CO 2 ). The lower staining of the 37°C sample could be due to
CFSE leakage from a proportion of cells which may indicate cell deterioration because the stain
does not leak, in appreciable amounts, from healthy cells (Vander Top et al., 2006). Alternatively,
the diluted stain in the 37°C sample could be due to a higher number of daughter cells. A shift
towards higher granularity was evident in the cells kept at 37°C. Cells undergoing apoptosis
become more granular because nuclear chromatin is condensed, the organelles are compacted and
the cell shrinks (Ormerod, 1994). CFSE staining was preferred to labelling with monoclonal
antibodies that discriminate PBMC subpopulations because the focus was on the effect of storage
conditions on the whole system and not necessarily on a particular cell phenotype.
The exclusion of Con-A activation of cells shortened the procedure by 48 hours in addition to
making slides easier to read. The density of cells when activated had made it difficult to spot the
infected ones.
Replacing microtubes with microtitration plates makes the test more user friendly as it cuts out use
of thermoshakers dilutions in separate sets of tubes and laborious transfer from these to plates for
final. Shaking the plates for 1 minute outside the incubator appears to result in a higher proportion
of positive wells for sucrose protected stabilates relative to glycerol stabilates. This could be due to
conditions such as better pH maintenance because the plates are placed at 37°C in a CO 2 incubator
for primary incubation. However, with the 1 min shaking, it was observed for the glycerol stabilates
that some wells with higher sporozoite dose gave false negatives. The cause for these false
negatives is not known but it was speculated that some factor in the glycerol stabilate negatively