By Wayne Ryan, PhD, and Dominic Warrino, PhD
As every laboratory professional knows, reliable results begin with reliable samples, and nowhere is this more evident than in today’s flow cytometry lab. Flow cytometry is used to determine the types and quantities of antigens on the surface of white blood cells (WBC) through a process known as immunophenotyping. Antigens present on the cell surface signal activation of the immune response system, and the technique of identifying these antigens through their cluster of differentiation (CD) pattern has become an essential part of diagnosing and monitoring a wide range of conditions, including transplant acceptance, cancers, and infectious diseases. The CD4+ lymphocyte count, in particular, has emerged as the gold standard for monitoring HIV/AIDS antiretroviral treatment, while absolute counts, percentages, and ratios for a growing number of CD markers are being incorporated into testing panels over a wide field of high value diagnostic applications.
The key challenge to the clinical lab arises from the fact that these surface epitopes can begin to deteriorate very rapidly, leading to sample integrity issues. Often within 48 hours, whole blood samples collected into conventional K3EDTA tubes display loss of stability, resulting in variable antibody binding and leading to inaccurate measurement of CD markers. The race against the clock to analyze patient specimens has been costly, forcing weekend/holiday staffing calls, expensive sample transport choices, and patient redraws. Sample deterioration can force the choice between assuring sample quality and realizing the efficiencies of batching for flow cytometric tests that are performed less frequently. In underserved geographic areas, the timeliness of sample shipment to a flow cytometry lab has been the limiting factor in providing patient access to the most appropriate medical testing. This has had severe consequences in the HIV/AIDS arena.
Finding a Solution
To address the WBC preservation problem, Streck Laboratories of Omaha, Neb, applied its cell stabilization technology, developed over many years, to the production of a new, FDA-cleared and CE marked blood-collection tube, Cyto-Chex® BCT. These standard-size vacuum-assisted tubes are specifically designed for flow cytometry applications, effectively preserving surface epitopes for up to 7 days. The 13 x 75 mm tube is configured for a 5 mL draw, and includes 60 µL of concentrated reagent with a cell preservative and anticoagulant. While stabilizing whole blood samples at room temperature to accommodate batching or shipping needs, the small volume of reagent allows direct analysis from the collection tubes without the need for dilution corrections. The Cyto-Chex BCT is designed for the collection and storage of blood specimens for typing of immunophenotypic markers of WBC by flow cytometry. In particular, the ability to recover the lymphocyte subset cell markers of the HIV panel has been demonstrated to extend to at least 7 days at 18° to 30°C, and 3 days at 37°C following collection. This may be important when transporting samples where the temperature is difficult to control.
Figure 1. Typical patient data.
Study Results Show No Compromise in Quality
Streck Laboratories selected the HIV marker panel defined by the Centers for Disease Control (CDC) for studies that included 20 HIV-positive and 25 noninfected donors. All samples were maintained at room temperature (18°–22°C) throughout the 7 days of the studies. Samples drawn into K3EDTA tubes were used as controls for both the infected and noninfected groups. Analysis was performed on the BD FACSCalibur flow cytometer. The absolute cell counts of CD3+, CD4+, and CD8+ lymphocyte subsets in whole blood drawn into Cyto-Chex BCT were compared at 7 days postdraw to the values obtained 6 hours after collection in a K3EDTA tube. The results for typical patients are shown in Figure 1. For each marker and in both normal and HIV donor blood, the cell counts at 7 days in the Cyto-Chex BCT are nearly identical to those at 6 hours in EDTA. When a statistical comparison of 6-hour EDTA versus 7-day BCT data for all 25 normal and 20 HIV-infected patients is carried out, R2 values (R2=1 for complete equivalence) shown in Table 1 convincingly demonstrate that, for the markers tested, the Cyto-Chex BCT can preserve peripheral whole blood immune epitopes for up to 7 days.
Of the CD3, CD4, CD8, CD19, CD45, and CD16+56 immune epitopes tested, all showed uncompromised results up to 7 days when drawn into the Cyto-Chex BCT. In normal and infected donor types, 7-day Cyto-Chex BCT results are indistinguishable from 6-hour K3EDTA results based on correlation coefficients.
Although the study reported here focused on the recovery of HIV markers CD3, CD4, and CD8, it is important to recognize that preservation characteristics of Cyto-Chex BCT may be applicable to other cell markers as well.
Real Relief in the Real World
Could the Cyto-Chex BCT be the answer to the laboratorian’s weekend prayers? With 7-day stability, samples may be collected on any day of the week even if further storage and shipping will be required—with no worry about loss of sample reliability. Moreover, the Cyto-Chex BCT can practically and economically address some of the critical concerns about delivering high-quality care to those in rural populations in the United States or in underserved areas of the developing world. If advanced flow cytometry is not performed either frequently or locally, the Cyto-Chex BCT can save samples through the batching or shipping delays that would otherwise degrade them.
Along with enabling the delivery of leading-edge care for a range of disease states in the US, Cyto-Chex BCT can be a tremendous tool in the global fight against HIV/AIDS.
In HIV infection, the CD4 marker is the critical laboratory measurement used for clinical prognosis, therapeutic monitoring, and entry into clinical trials. To provide the best standard of care, this testing must be made available to those living with HIV/AIDS across the United States. Worldwide, according to UNAIDS estimates for 2003, there are 40 million people infected with HIV/AIDS, with approximately two thirds living in sub-Saharan Africa. Highly active antiretroviral therapy (HAART) has considerably reduced mortality and morbidity in HIV-infected persons in developed countries, and although the use of HAART remains limited in Africa, there are global efforts to make these drugs available to several million HIV-infected Africans. To effectively deliver the care that HAART promises, CD4 testing must be available as well. The lack of stability of phenotypic markers that would present a major obstacle to CD4 testing in remote and underserved areas worldwide is a problem that can be addressed with the Cyto-Chex BCT.
Stability in a Changing Future
The relationship of enhanced laboratory testing, targeted drug therapy, and treatment monitoring continues to mature. For the clinical laboratory, this increases both the potential contribution toward preserving life and wellness, and the demands placed on budget-strapped, short-staffed, or remote laboratories striving to play their part in meeting the best standards of medical care. It is in this environment of higher expectations and tight resources that Streck Laboratories developed Cyto-Chex BCT, a simple but profound technological advance in blood collection that enables improvement in both sample integrity and laboratory workflow.
Wayne Ryan, PhD, is chairman and CEO, and Dominic Warrino, PhD, is research scientist at Streck Labs.