I have been reading the BD Retic Count protocol and it seems it does not take into account the white cells. Is the assumption that their presence in relation to rbc is very minimal that in the end it doe not matter? What if the patient has malaria infection too?
The FSC and SSC gates used in flow cytometry mean that WBC don't overlap with reticulocytes on size. Since BD's ReticCount is eye-wateringly expensive, we have used "home-made" Retic Count (it's just Thiazole Orange in PBS/azide) and used it to estimate malaria parasitaemia in cultures. Reticulocytes and parasites completely overlap in size and fluorescence, so anti-CD71 can be used to differentiate. If you want more detailed protocols let me know, and some of them are on our website. There are other dyes we have used which are better than thiazole orange.
A note of caution - don't use DAPI to count live cells - it massively underestimates cell numbers in live cells.
See paper below for details of the thiazole orange staining method.
Arnot DE, Cavanagh DR, Remarque EJ, Creasey AM, Sowa MP, Morgan WD, Holder AA,
Longacre S, Thomas AW. Comparative testing of six antigen-based malaria vaccine
candidates directed toward merozoite-stage Plasmodium falciparum. Clin Vaccine
Immunol. 2008 Sep;15(9):1345-55. doi: 10.1128/CVI.00172-08. Epub 2008 Jun 11.
PubMed PMID: 18550731; PubMed Central PMCID: PMC2546674.
http://www.malariaresearch.eu
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