Calculating Hematocrit When Passaging Plasmodium falciparum Cultures
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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
Preparing Red Blood Cells for Culture
Before the red blood cells (RBCs) can be used in culturing, the cells must be rigorously washed and separated from unwanted components like white blood cells and plasma.
Initial Collection: Blood is collected using anticoagulants to prevent clotting. Normal human erythrocytes used for dilution are collected in acid, citrate, and dextrose (ACD) or CPDA-1 (citrate, phosphate, dextrose, and adenine) and stored at 4°C. While various blood types can be used, Type O+ erythrocytes are often preferred because they are compatible with all serum sources. While erythrocytes stored in CPDA-1 remain suitable for culture for up to 35 days, fresher cells (less than 10 days old) are generally recommended for optimal parasite growth and better results in synchronization techniques like gelatin flotation
Centrifugation and "Buffy Coat" Removal: The blood is washed in 10 volumes of RPMI 1640 by centrifugation (specifically at 1000g for 10 minutes for the human cells), to separate the layers. The researchers then carefully remove the supernatant (plasma) and the buffy coat, which contains the white blood cells.
Washing the Cells: The remaining red blood cells are resuspended in an equal volume of RP medium (RPMI 1640 powder supplemented with Hepes buffer and NaHCO3). The cells are centrifuged again, the supernatant is discarded, and this washing process is repeated to ensure the cells are clean and the medium is fully integrated.
Hematocrit (or haematocrit) is defined as the percentage of the total volume of the culture medium that is composed of red blood cells (RBCs)
Storage Dilution: For storing cells at a 50% hematocrit, the packed RBCs are diluted with an equal volume of incomplete culture medium
Storage: Normal human erythrocytes can be stored at 4°C for up to two weeks before they are processed and used in the culture system. Once the red blood cells have been washed and prepared, they should be used within 5 days.
When pelleting parasites, understanding xg vs RPM is crucial. Learn more in our centrifuge speed conversion guide: https://adwoabiotech.blogspot.com/2026/01/xg-to-rpm-centrifuge-conversion-guide.html
Wondering when this xg to RPM knowledge comes in handy? You'll need it when culturing parasites or mammalian cells.
Parasite Culture Maintenance: Culturing malaria parasites requires gentle, precise centrifugation. Too fast damages the parasites; too slow leaves debris in your culture.
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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- Park, Y. H., Shi, Y. P., Liang, B., Medriano, C. A. D., Jeon, Y. H., Torres, E., Uppal, K., Slutsker, L., & Jones, D. P. (2015). High-resolution metabolomics to discover potential parasite-specific biomarkers in a Plasmodium falciparum erythrocytic stage culture system. Malaria Journal, 14, Article 122. https://doi.org/10.1186/s12936-015-0651-1
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