Lab Med Online.  2022 Jan;12(1):11-19. 10.47429/lmo.2022.12.1.11.

Comparison of Automated and Manual Gating of Lymphocyte Subsets in Hematopoietic Stem Cell Transplantation Recipients

• Lee H
• Kim J
• Lee J
• Jung J
• Oh EJ

• KMID: 2538578
• DOI: http://doi.org/10.47429/lmo.2022.12.1.11

Abstract

Background
Lymphocyte subset analysis is essential to evaluate the engraftment status in hematopoietic stem cell transplantation (HSCT). Automated gating tools are widely used for flow cytometry analysis. Unlike healthy individuals, different cell populations and aberrant expressions may occur in HSCT samples. In the present study, we evaluated the applicability of automated gating in HSCT recipients by comparing it to expert-based manual gating.
Methods
Lymphocyte subset was performed using Beckman Coulter Navios (Beckman Coulter, USA) flow cytometry. Data files from 22 patients with hematologic malignancies were analyzed in parallel by manual gating and automated gating using Navios Tetra software. Quality control results and reproducibility were evaluated using IMMUNO-TROL controls.
Results
Spearman rank correlation coefficients between the two gating methods were >0.970 in all cell populations except CD8+ T cells. CD8+ T cell counts via automated gating were higher than those of manual gating in all cases due to the T cell populations with reduced CD8 expression. Automated gating program failed to identify CD4+CD8+ double-positive T cell population. Moreover, it excluded certain lymphocytes with low forward scatter (FSC) and high side scatter (SSC). Furthermore, two HSCT recipients revealed a high percentage of CD56−CD16+ NK cells, we found the need to add CD16 reagent to the Navios system. All coefficients of variation were <10% except for CD56+ NK cells via automated gating.
Conclusions
Manual gating confirmation via flow cytometry histogram is necessary to identify the aberrant phenotypes and unexpected cell populations in HSCT recipients.

Keyword

Lymphocyte subset; Flow cytometry; Automated gating; Hematopoietic stem cell transplantation

Figure

• 

  Fig. 1 The difference in CD8+ T cells between automated and manual gating. (A) Correlation of CD8+ T cells between automated and manual gating. CD8+ T cells analyzed via automated gating were higher than those evaluated by manual gating (Y (automated gating)=9.504+0.925 X (manual gating)), r=0.862, P<0.001). The automated gating program (B) included CD8 dim population in CD8+ T cell, but we excluded them in manual gating (C) (data from Case 4).

• 

  Fig. 2 CD8 T cell errors in automated gating. Automated gating (A and B) failed to identify CD8+ T cells. In manual gating (C and D), we could identify the exact number of CD8+ T cells after compensation. (A) and (C) represent Case 16; (B) and (D) represent Case 18.

• 

  Fig. 3 Gating of total lymphocytes via automated gating by excluding some lymphocytes. Certain proportions of total lymphocytes were excluded in gating (A) and (B) (data from Case 16). Abbreviations: GADJ, gate adjust; LADJ, lymphocyte adjust; FSC, forward scatter; SSC, side scatter.

• 

  Fig. 4 CD3+ T cell difference in a case with tube-to-tube variability. In automated gating, CD3+ T cell fraction from tube 1 (A) was overestimated compared to that in tube 2 (B), as certain CD3− cell fraction was included in the analysis of tube 1 (data from Case 6).

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