.png)
As a translational scientist, you’ve probably asked yourself some version of this question:
“How much blood do I actually need to collect to get reliable data?”
Under-collect, and you risk missing critical signals. Over-collect, and you may frustrate clinicians, slow down enrollment, and create unnecessary patient burden.
So how do you calculate the blood volume required to achieve reliable measurements?
That’s where the coefficient of variation (CV) comes in.
Lower CV = higher reliability. A bad CV is 20%+. A good number is less than 5%.
But CV isn’t just random, it depends on:
- How common the cell type is (its frequency in blood)
- How many cells you collect (based on volume and White Blood Cell (WBC) count)
- Assay precision and event loss
Calculating Required Blood Volume from a Target CV
Let’s walk through a concrete example.
Imagine you’re measuring B-cells, which make up about 4.03% of total leukocytes in peripheral blood. You want your measurements to have a CV of 5%, tight enough to be used as a critical endpoint.
Here’s the conceptual model behind the volume calculation.
Step 1: Determine how many cells are needed for a given CV
- Cells = (100 / CV%)² (Hedley and Keeney, 2013).
For a 5% CV: Cells = (100 / 5)² = 400 cells
Step 2: Convert cell count to total leukocytes
- WBCs = Cells / Frequency of population
WBCs = 400 / 0.0403 ≈ 9,930
Step 3: Determine how much blood is needed based on WBC count per mL
- Assuming WBC concentration = 5 million cells/mL
- Volume = 9,930 / 5,000,000 = ~2 µL
What If You Can’t Choose the Blood Volume?
Sometimes, the sample volume isn’t flexible. You’re capped at, say, 12 mL per patient.
Now the question becomes:
“Given the blood volume and estimated WBC count, what CV can I expect for each cell population?”
This is the reverse problem, you already know the input (volume), and you’re trying to estimate the output (CVs).
Here’s an example (Hedley and Keeney, 2013).
Step 1: Estimate Total Leukocytes Collected
Start by estimating how many white blood cells are available:
- 12 mL × 5,000,000 WBCs/mL = 60 million WBCs
This gives you the theoretical upper bound before any processing or acquisition loss.
Step 2: Account for Instrument Acquisition Limits
In practice, cytometry platforms do not acquire all available cells. Each run is limited by throughput, time, and instrument efficiency.
For example:
- Mass cytometry
- Typical acquisition target: ~1,000,000 cells loaded
- Estimated event loss: ~70%
- Final acquired events: ~300,000
The acquired events, not total WBCs in blood, are what ultimately determine measurement reliability.
Step 3: Estimate Events per Cell Population
Now distribute those acquired events based on population frequency.
For example, using mass cytometry:
- CD8+ T cells (~16.4% of leukocytes) = 49,000 CD8+ T cell events
Step 4: Convert Event Counts to Expected CV
Once you know the number of events for a population, you can estimate reliability using the same statistical relationship, applied in reverse:
CV = 100 / √(number of events)
For CD8+ T cells:
- CV ≈ 0.45%
This level of precision is well within the “optimal” range for highly quantitative endpoints.
Plan for the Data You Want
Because blood samples sit at the center of trial design and ultimately support regulatory submissions, precision and sample quality cannot be treated as afterthoughts. The amount of blood collected directly determines how reliably immune cell populations can be quantified.
Teams can plan around fixed blood draws and use those constraints to estimate the precision they can expect for each population. Alternatively, they can start with a target CV and calculate the blood volume required to reliably quantify the data of interest. Tools like our blood volume calculator make both approaches explicit, helping teams align blood volume, assay precision, and study endpoints from the outset.
Citation:
Hedley, B.D. and Keeney, M. (2013), Technical issues: flow cytometry and rare event analysis. Int. Jnl. Lab. Hem., 35: 344-350. https://doi.org/10.1111/ijlh.12068
