A New No-Lyse, No-Wash Flow-Cytometric Method for the Determination of CD4 T Cells in Blood Samples

Abstract

Background: Commonly used flow-cytometric methods for immunophenotyping are based on erythrocyte lysing reagents. It is known from the literature that these reagents result in a significant loss of leucocytes caused by membrane destruction. Although the dual-platform method should compensate this phenomenon, the subset-specific individual differences in sensitivity to lysing reagents lead to incorrect values. In order to overcome this problem we introduce a no-lyse, no-wash procedure in combination with absolute true volumetric counting (TVC) for the enumeration of CD4 T cells. Material and Methods: Whole-blood samples of 50 blood donors and 20 samples of patients with acquired immunodeficiency syndrome (AIDS) were treated with both a lyse, no-wash and a nolyse, no-wash procedure. Then, CD4 T cells were counted with a TVC flow cytometer (CyFlow Counter). 30 blood samples of blood donors were treated with a lyse and wash protocol and measured by the CyFlow Counter and FACS-Calibur system (reference method). A new gating strategy was used for the data analysis for both, the lyse and no-lyse method and compared to the traditional gating procedure. Results: The TVC method showed a good reproducibility for both, the lyse (CV 1.9%) and the no-lyse procedure (CV 1.5%). CD4 counts measured by the no-lyse procedure are on average 10% higher than by using the lysing protocol. The comparison of the CyFlow Counter and FACS-Calibur results showed a correlation of r = 0.961. The simplified gating strategy shows a good correlation to the traditional gating procedure (r = 0.998). Conclusion: Erythrocyte lysing procedures cause substantial cell loss with individual values for every single subclass and patient. Therefore, the use of a no-lyse procedure is recommended. The new gating strategy is fully comparable to the traditional one and simplifies enumeration of CD4 T cells. Using the no-lyse procedure in combination with the new gating strategy, it is possible to reduce the costs per sample from EUR 30.– to below EUR 2.–.

Evaluation of a New Single-Parameter Volumetric Flow Cytometer (CyFlow green ) for Enumeration of Absolute CD4 + T Lymphocytes in Human Immunodeficiency Virus Type 1-Infected Thai Patients

“…A quality control measure recommended by CDC, but not included in this CD4 strategy, is the use of isotype controls. However, cursors can be set without the use of isotype controls, since discrimination from non-CD4 ϩ T lymphocytes and of dim CD4 monocytes and dendritic cells can be achieved readily due to the bright expression of CD4 on CD4 ϩ T lymphocytes (14,17,20). In this study, the absolute CD4 ϩ and CD8 ϩ T-lymphocyte counts were Ϫ21.8 and Ϫ7.2 cells/l less from the CyFlow green system than the two predicate systems, respectively (Fig.…”

“…The identification of CD4 ϩ and CD8 ϩ T lymphocytes on the basis of one-parameter CD4 expression is not novel. Several studies (9,14,16,24) used the concept of primary CD4 gating by reliably identifying CD4 ϩ or CD8 ϩ T lymphocytes by the high level of CD4 or CD8 expression. In one study (16), a series of more than 600 blood samples from both healthy and HIV-infected patients analyzed with the CytoronAbsolute system by primary CD4 gating with a single CD4 monoclonal antibody yielded absolute CD4 counts that were almost identical to those generated by a standard three-tube protocol with nine monoclonal antibodies (r 2 ϭ 0.999, with a minimal bias of ϩ4 CD4 ϩ cells/l).…”

“…However, there are two main drawbacks of such a method: (i) many studies have highlighted problems associated with the DPderived CD4 ϩ T-lymphocyte count, especially in relation to the generation of an absolute lymphocyte count by the hematology analyzer (1,3,11,16,23); and (ii) the cost of DP FCM CD4 testing in Thailand remains relatively high ($12 to $20). Several single-platform (SP) flow cytometric technologies, including FCM volumetric counting and microfluorometry (8,9,13,14,19), as well as, most commonly, the bead-based flow cytometric method for measurement of the absolute CD4 ϩ T-lymphocyte counts, have been developed and successfully evaluated (21,25). Although this bead-based flow cytometric method eliminates the need for multiple technologies, it is still limited by the high cost of the currently available FCMs and fluorescent beads.…”

Comparison of a New, Affordable Flow Cytometric Method and the Manual Magnetic Bead Technique for CD4 T-Lymphocyte Counting in a Northern Nigerian Setting

“…As many as 200 samples can be analyzed per day compared to 10 to 15 samples with the Dynabead techniques (6). The cost per test for Cyflow is between $3.00 and $5.00, whereas that of Dynabead ranges from $12.00 to $22.00 and other flow cytometry techniques are as high as $30.00 to $100.00 per test (1,6). Therefore, Cyflow is three-to fourfold more cost-effective.…”

Comparison of two alternative methods for CD4+ T‐cell determination (Coulter manual CD4 count and CyFlow) against standard dual platform flow cytometry in Uganda

“…Even though some of them show promising results, data evaluating their use and reliability in remote rural areas with low standards of health care are still scarce (3,5,(7)(8)(9)(10)(11)(12)(13). The ideal method for CD4 þ cell counting in such areas has not yet been identified.…”