A new point of care CD4 test
Polly Clayden, HIV i-Base
One of the most exciting sessions at the conference Point of care CD4 testing in resource-poor settings – was hidden away in a mini room at 7 am on Wednesday morning. 
This satellite session presented the work to date on the development of a CD4 point of care test, appropriate for use in resource-limited settings. This programme is funded and led by the CD4 Initiative, Imperial College London, managed by Dr Hans-Georg Batz and Dr Steven Reid, and Zyomyx Inc. in San Francisco is developing the assay.
This initiative, established in 2005, set out with the ambitious target to develop a point of care test in four years (from January 2007) to a set of predetermined specifications. Three prototypes were assessed against flow cytometry and the Zyomyx test compared favourably so the initiative is proceeding with its development.
We describe the CD4 Initiative in the TAG Pipeline Report 2010. 
Peter Wagner from Zyomyx presented a project review from the company. Besides the specifications necessary for the health system and operator of a point of care test simple to use and maintain, low cost, not dependent on electricity etc he outlined the technical challenges and solutions in terms of point-of-care cell counting.
He explained that cells are not soluble markers. They are large, sticky, prone to lysis and gravitate to the bottom of any flow cell. This makes them difficult to process, mix and capture within integrated devices and machines. Additionally, in order to count CD4 cells and meet the specifications for simplicity, it is necessary to remove monocytes with no reagents or extra steps for the operator.
The assay needs to allow full capture of CD4 cells without loss due to lysis, entrapment or non-specific binding in order to make a complete cell count. Dr Wagner noted that in general, reacting cells with reagents or beads in solution works better technically than approaches that require cells to find a cartridge surface for sorting. He also said, gravity is your enemy(e.g. for proper sample mixing) unless you can use it to your advantage for the test principle which this device does.
The assay utilises a proprietary cell stacking approach to CD4 counting. A finger prick blood sample is taken. This is introduced into a one-piece, injection-molded, closed system cartridge, at the blood inlet port, which closes with a cap. The cartridge is placed in the mixer/spinner device. This is hand powered with no batteries required or does not need to be kept cool. It performs two functions, mix and spin.
In the cartridge, CD4 beads bind to CD4 cells to selectively transfer them to a volumetric measurement compartment forming a compact stack. Monocytes are
removed through binding of magnetic CD14 beads for magnetic separation within the flow path. The height of the stack gives the CD4 count readout by eye as a dark line similar to a mercury thermometer.
The assay is fully quantitative, which surpassed the original specifications for a cut-off test. Therefore it can be used for treatment decisions and monitoring. No external reagents are required and it can give a result in less than 10 minutes.
Dr Wagner reported similar performance to flow cytometry in collaboration with Steven Deeks UCSF group with 100% sensitivity, 94.34% specificity, 92.31% positive predictive value and 100% negative predictive value.
The group will begin field-testing the device in Malawi in early 2010.
A CD4 test that is not reliant on electricity, that is easy to use, fast, disposable, that requires no cold chain or maintenance and is point of care, offers enormous possibilities for decentralisation of treatment delivery.
For a health worker, the skills required are performing a finger stick, twisting a lid, using a mechanism not unlike a lettuce spinner and reading off a gauge like a thermometer. Less than two hours of training is required. So use in the community seems completely feasible.
The fast turnaround would mean no return visit by patients for results, which has the potential to reduce loss to follow up that happens frequently at this stage. Although DART demonstrated that ART can be delivered safely without routine monitoring, a test is still required to start treatment. That this test is quantitative means it is useful beyond the decision of when to start treatment, and DART investigators also suggested that there is a role for CD4 testing from the second year on ART to guide the switch to a second line regimen. This will become more important as programmes mature.
The CD4 Initiative came about through a generous grant from the Gates Foundation. Additional funds are needed to finalise the work and bring the
project to commercial production. It is absolutely critical that this funding is found.
- Point of care CD4 testing in resource-poor settings. 18th IAS, July 2010, Vienna, Austria. Satellite session WESA06.
- Clayden P. HIV diagnostics pipeline. TAG 2010 Pipeline Report, page 25-28. https://i-base.info/htb/13421