Improved light microscopy counting method for accurately counting Plasmodium parasitemia and reticulocytemia

Abstract

Even with the advances in molecular or automated methods for detection of red blood cells of interest (such as reticulocytes or parasitized cells), light microscopy continues to be the gold standard especially in laboratories with limited resources. The conventional method for determination of parasitemia and reticulocytemia uses a Miller reticle, a grid with squares of different sizes. However, this method is prone to errors if not used correctly and counts become inaccurate and highly time-consuming at low frequencies of target cells. In this report, we outline the correct guidelines to follow when using a reticle for counting, and present a new counting protocol that is a modified version of the conventional method for increased accuracy in the counting of low parasitemias and reticulocytemias.

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays

“…Cytospins were made after 44 h of maturation, methanol fixed, and then stained using Hemacolor. Parasitemia and staging were assessed via light microscopy and whole-field counting (32).…”

CRISPR/Cas9 knockouts reveal genetic interaction between strain-transcendent erythrocyte determinants of Plasmodium falciparum invasion

“…Cytospins were prepared immediately upon mixing the schizonts and target cells as well as 18-24 h post invasion. Slides were stained with May-Grünwald Giemsa as described, and parasitemia was evaluated by reticle counting (92,93). For invasion inhibition assays, the MEM6/6 clone of the α-BSG antibody (preservative free) was used (Invitrogen) along with a matched isotype control antibody (preservative free) (Invitrogen).…”

Monitoring Plasmodium vivax resistance to antimalarials: Persisting challenges and future directions

“…Ex vivo assays of P. vivax drug susceptibility are carried out over 42–46 h with patient-derived parasite samples cultured in the presence of increasing drug concentrations, but the low parasitemias that are characteristic of clinical isolates (often <0.1% and are rarely >0.5%) severely limit their use. Moreover, test results are affected by factors such as previous patient use of antimalarials, the synchronicity and blood stage composition of clinical infections, time delays in processing clinical samples, variations in methods used for sample cryopreservation and thawing and differences in assay protocols, including the choice of culture media and methods for quantifying parasite growth ( Russell et al, 2008 ; Russell et al, 2012 ; Lim et al, 2016b ; Rangel et al, 2018 ; Thomson-Luque et al, 2019 ).…”