Calculating Hematocrit When Passaging Plasmodium falciparum Cultures
When culturing Plasmodium falciparum in the lab, maintaining the right hematocrit and parasitemia is essential. This guide explains how to calculate hematocrit in routine culture setups and how to adjust both hematocrit and parasitemia when passaging cells to reduce parasite density.
What Is Hematocrit in Malaria Culture?
Hematocrit refers to the percentage of red blood cells (RBCs) in the total culture volume. In Plasmodium cultures, typical hematocrits range from 2% to 5%, depending on the purpose (routine culture, drug assay, synchronization).
Formula:
Hematocrit (%) = (Volume of packed RBCs / Total culture volume) × 100
For example, to prepare a 10 mL culture at 5% hematocrit:
Total RBC volume needed = 0.05 × 10 = 0.5 mL
Add 9.5 mL of complete culture medium (RPMI + supplements)
Hematocrit vs. Parasitemia
Hematocrit = total RBCs (infected + uninfected) as % of total volume
Parasitemia = % of RBCs that are parasitized
When passaging cultures to reduce parasitemia, both values must be considered.
How to Passage Cultures to Reduce Parasitemia
Let’s say you have a culture at 10% parasitemia and want to reduce it to 1%, while maintaining a 5% hematocrit in a 10 mL flask.
Step 1: Calculate Volume of Infected RBCs to Transfer
Volume of iRBCs to transfer = (Desired parasitemia / Current parasitemia) × Final volume
= (1% / 10%) × 10 mL = 1 mL
So, 1 mL of culture from the 10% flask will give 1% parasitemia in the new flask.
After spinning, assume you obtain 0.1 mL of iRBC pellet from this 1 mL.
Step 2: Calculate Total RBC Volume for Desired Hematocrit
Hematocrit volume = (Desired hematocrit %) × Final volume
= 5% × 10 mL = 0.5 mL RBCs total
You already have 0.1 mL of iRBCs, so you need:
0.5 - 0.1 = 0.4 mL of fresh uninfected RBCs
Step 3: Add Culture Medium to Reach Final Volume
You now have:
0.1 mL infected RBCs
0.4 mL uninfected RBCs
Total RBCs = 0.5 mL
To reach 10 mL:
10 mL - 0.5 mL = 9.5 mL of complete culture medium
Final Setup Summary
0.1 mL iRBCs from high parasitemia culture
0.4 mL uninfected RBCs
9.5 mL culture medium
Final parasitemia = 1%
Final hematocrit = 5%
Tips
Spin down culture at ~500 × g for 5 min to pellet RBCs
Use fresh human RBCs within 1-2 weeks (stored at 4°C)
Parasitemia too high (>5–10%)? Passage before culture crashes
Keep hematocrit consistent across assays for reproducibility
Acknowledgement
Many thanks to Felix Zoiku from the Noguchi Memorial Institute for Medical Research (NMIMR) for the detailed explanation of the staining and culture process for Plasmodium species.
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