Are Absolute Cell Counts Essential In Drug Discovery?
By Ed Miseta, Chief Editor, Clinical Leader
When you hear the phrase absolute cell counts, what comes to mind for most people is how it is used clinically to explain how many cells of a specific cell type are accurately present in a defined volume of whole blood (e.g. a patient may have a measured CD4 count of 500 cells/μL). But what does this mean? First, an appreciation of what the different whole blood cell types are is required. In whole blood, there are three major cell types, red blood cells, white blood cells and platelets. In the white blood cell category, there are five main cell populations: neutrophils, eosinophils, basophils, monocytes and lymphocytes.
“Historically, clinicians have used the ability to separate out those cell types physiologically, or on specialized cell counting equipment, into those five main populations, and from that have been able to assess the overall health of an individual as it specifically relates to blood counts,” says Ren Capocasale, founder and CEO of FlowMetric. “When you hear the term lymphopenia, or neutropenia, those are individuals that have abnormally low numbers of either lymphocytes or neutrophils.”
According to Capocasale, the advent of AIDS diagnosis has made absolute cell counts more significant. HIV is monitored via the overall T-cell population ratio, and how it gradually goes up or down based on disease progression or treatment. CD4 T cells are monitored and the overall number of CD4 cells is important to the overall immune health of the individual. “The ratio between CD4 and CD8 counts was traditionally used, but we eventually realized that at a certain point, there’s kind of a point of no return,” he says. “Once your absolute CD4 T cell counts drop below 200 for a microliter of blood, that’s when an HIV patient is in real trouble and susceptible to opportunistic infection.”
Clinical Uses In Drug Development
While absolute cell counts have been a part of diagnosis for a long time, what is new are absolute count relevance to drug discovery and development. Absolute cell counts now have the ability to combine cell concentration determination from a specific flow cytometry tool, with population data, so as to get an absolute cell count across the board.
“We can now tell how many CD4 cells there are, how many M1 or M2 macrophages there are, or how many eosinophils, basophils, or neutrophils there are,” notes Capocasale. “More importantly, the ability exists to determine the effects of a drug on the specific population.”
To perform absolute cell counting in drug development scenarios requires a bit more work. Many CROs will tend to not report absolute cell counts because of the additional steps required in preparation of a sample. And this additional effort entails additional expense. There is precision involved that is essential, so traditionally CROs have felt that the ROI of the additional cost was not justified.
Capocasale notes his company performs cell counting across the board for all assays when requested by the client, noting he believes it is absolutely essential in making go/no go decisions for the experimental therapeutics he tests. “It’s not enough to understand the percentage of a certain population within the context of whole blood,” he states. “You need to understand the absolute number of that population relative to everything else.”
As an example, he notes that you may evaluate a drug by flow cytometry that is known to deplete B cells. If you solely study percentages, it may appear that there are other effects as well, such as the increase in T and NK cells which is occurring because B cells are being depleted. However, if you enumerate cell numbers, then you can, in fact, understand real changes in those lymphocytes based on drug effect.
Few Flow Cytometry Experts
Capocasale notes the FDA is currently an under-funded, under-manned, and an over-worked agency. One of the pressing issues for them, when analysing and performing due-diligence on drug INDs, is in critically understanding the flow cytometry data.
“Very few people have a great command of this analytical technique,” he says. “If you look at how many people call themselves flow cytometrists versus molecular biologists, it’s a significantly smaller population. This is a speciality that many folks simply do not have.”
This lack of expertise can also create issues. When the NME (New Molecular Entity) data gets to the FDA, one of the comments often made refers to the potential subjectivity of the data itself. Capocasale notes part of that weakness is due to the fact that many CROs report flow data as simply a change in percentage of a population.
“If the population went down by 50%, but the overall cell numbers only went from 10 cells per microliter to 5 cells per microliter, you’re hard pressed to prove that data is significant when the total cell number is 4,000 to 11,000 cells per microliter,” he says.
“Percentages are misleading. If you’re going from 100 down to 50, that’s a 50% drop, but 100 cells out of 10,000 cells isn’t a comparably significant population.”
Don’t Underestimate the Importance
Capocasale cautions clinicians not to underestimate the importance of absolute cell counts. Minimal residual disease, or MRD, refers to the circulating tumor cells from tumors that have metastasized in the body and is specifically how many circulating tumor cells there are at any given time following therapy. It is essential to the prognosis of certain cancer types to know whether there are greater than or less than five circulating tumour cells per microliter of blood. Absolute counting is the only way to understand that difference in prognosis.
“For us, absolute cell counts are routinely incorporated into the way we undertake and execute our projects,” he states. “It’s an added value to clients that enables them to have actionable data reviewed by the FDA. The FDA is increasingly savvy to the pitfalls of simply recording percent positives, so in more and more scenarios, absolute cell counts are being requested.”
“More and more I think you will see the FDA and others working on developing flow assays for clinical trials recognizing the need for reporting absolute cell counts,” adds Capocasale. “If the FDA is going to request it across the board, then eventually labs are going to be forced into doing it. Maintaining extremely tight correlation and variability counts is critical. As a result, I believe its importance in certain scenarios will continue to grow.”