CD45 is Essential for Lymphocyte Gating in a T-lymphocyte Subset Assay of Bronchoalveolar Lavage Fluid by Flow Cytometry

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CD45 is Essential for Lymphocyte Gating in a T-lymphocyte Subset Assay of Bronchoalveolar Lavage Fluid by Flow Cytometry

Hyoeun Shim, Young Hyun Choi, Chan-Jeoung Park, Ji-Sun Lee, Sang Hee Han, Keumrock Hwang, Seongsoo Jang, and Hyun-Sook Chi

Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea

유세포검사에 의한 기관지폐세척액의 림프구아형검사에 있어서 CD45 사용의 필요성

심효은․최영현․박찬정․이지선․한상희․황금록․장성수․지현숙 울산의대 서울아산병원 진단검사의학과

접수일: 11 / 6 / 16

최종재심접수일: 11 / 12 / 20 게재승인일: 12 / 1 / 2

본 논문은 대한진단검사의학회 신임위원 회 질향상 연구사업 과제(2010년)로 지 원받아 이루어진 것임.

교신저자：박찬정

우)138-736 서울시송파구아산병원길86, 울산의대 서울아산병원 진단검사의학과 전화 : 02)3010-4508

FAX : 02)478-0884

E-mail : cjpark@amc.seoul.kr

Background: Proper gating is important in flow cytometric assays of lymphocyte subsets. Forward light scatter (FSC)/side light scatter (SSC) gating requires application of a lymphocyte purity correction when lymphocyte purity is less than 95%. We compared 3 different gating methods to establish an accurate gating method appropriate for a T-lymphocyte subset assay of bronchoalveolar lavage (BAL) fluid.

Methods: Leukocyte numbers and subtypes in 31 BAL fluid samples were assessed manually and by using an automatic hematology analyzer. T-lymphocyte subsets (T cells, T helper/inducer cells [Th], and T suppressor/cytotoxic cells [Tc]) were assessed by flow cytometry. We compared 3 methods of lymphocyte gating:

CD45/SSC gating (reference method), FSC/SSC gating, and FSC/SSC gating with application of a lymphocyte purity correction. Lymphocyte purity was determined by CD45/CD14 staining of BAL fluid.

Results: We observed a significant correlation between lymphocyte percentage and lymphocyte purity (r = 0.453, P = 0.011). T-cell results obtained using the reference method were not correlated with the results of the other 2 gating methods (r = 0.189 each, P = 0.308 for FSC/SSC gating and P = 0.310 for FSC/SSC gating with purity correction). Mean differences between the reference method and FSC/SSC gating (T cells: 14.4%, P = 0.002; Th cells: 7.7%, P = 0.006; Tc cells: 7.1%, P = 0.001) were greater than those between the reference method and FSC/SSC gating with purity correction (T cells: 12.1%, P = 0.004; Th cells: 1.7%, P = 0.608; Tc cells: 0.2%, P = 0.957).

Conclusions: Lymphocyte purity correction after FSC/SSC gating improved the accuracy of Th- and Tc-cell measurements, but not T-cell measurements. CD45 is essential for lymphocyte gating in T-lymphocyte subset assays of BAL fluid.

Key Words: Bronchoalveolar lavage fluid, Flow cytometry, Lymphocyte gating, T-lymphocytes subsets

J Lab Med Qual Assur 2012 ; 34：1-8

INTRODUCTION

Assays of lymphocyte subsets in bronchoalveolar lavage fluid (BAL) are important in the differential diagnosis of pulmonary disorders, including sarcoidosis [1,2], idiopathic pulmonary fibrosis [3], and allergic alveolitis [4]. Compared to other methods used to evaluate BAL lymphocyte subsets, flow cytometry offers the advantages of being objective, fast, and accurate [5]. Although the gating of specific cells is very important, there are no clear guidelines on optimal gating strategies for BAL lymphocyte subset analyses.

According to Clinical and Laboratory Standards Institute (CLSI) guidelines, lymphocyte gating using bright CD45 expression and low sideward light scatter (SSC) can increase the yield and purity of lymphocytes, obviating the need to correct lymphocyte subset values for contamination by other white blood cells [6]. However, CD45/SSC gating has not completely replaced the forward light scatter (FSC)/SSC gating method in lymphocyte subset analyses [7-9]. Notably, during activation or degradation of the immune system, or when the number of lymphocytes is reduced (for example during viral infection, atypical infection, and use of immunosuppressants), accurate lymphocyte subset analysis is impossible by FSC/SSC gating alone.

The flow cytometry checklist of the College of American Pathologists (CAP) has suggested that appropriate gating techniques be used to select cell populations for analysis and has recommended using a combination of light scatter and/or fluorescence measurements for lymphocyte gating.

Particularly in samples containing low numbers of lymphocytes and/or high numbers of monocytes and granulocytes, lymphocyte gates may be validated using CD45-FITC and CD14-PE monoclonal antibodies (FLO.30460). This checklist also suggests that the results of lymphocyte subset analysis be corrected for gate purity as appropriate (FLO.30470) [10]. Here, we have compared these 3 gating methods-CD45/SSC gating, FSC/SSC gating, and FSC/SSC gating followed by purity cor- rection-in lymphocyte subset analysis of BAL fluid.

MATERIALS AND METHODS

1. Patients

BAL fluid samples were obtained from 31 patients, including 16 patients with diffuse interstitial lung disease, 4 with lung tumors, 2 with chronic obstructive pulmonary disorder, and 9 with other diseases, including pneumonia, sarcoidosis, diffuse alveolar hemorrhage, and Steven-Johnson syndrome.

2. Bronchoalveolar lavage

Flexible bronchoscopy was performed, and BAL fluid was collected into 150 mL of sterile physiological saline solution in 3 consecutive 50 mL aliquots. After filtering through sterile gauze to remove mucus, BAL fluid was transported to the laboratory.

3. White blood cell counts and differential analysis

White blood cell counts (WBC) and differential analysis of cellular subsets were performed using an autoanalyzer (Cell-Dyn Sapphire; Abbott Labora- tories, Abbott Park, IL, USA) and manually using a hemocytometer and microscope with Wright–

Giemsa-stained cytospin slides. The results of these 2 methods were compared.

4. Flow cytometric analysis

For flow cytometry, the specimens were centrifuged at 1,800 rpm for 5 min, after which the supernatant was decanted and the cell pellets were resuspended in phosphate buffered saline. Flow cytometric analyses of lymphocyte subsets (T cells, T helper/inducer cells [Th], and T suppre- ssor/cytotoxic cells [Tc]) and lymphocyte gate purity were performed using a FACS Canto instrument and FACS Diva software (Becton- Dickinson, San Jose, CA, USA). A CD4-FITC/

CD8-PE/CD3-PerCP/CD45-APC panel was used for

4-color multiparameter flow cytometry, and a

CD45-FITC/CD14-PE panel was used for

determining lymphocyte gate purity (Fig. 1). All

monoclonal antibodies were purchased from

Becton-Dickinson. BAL fluid (100 µL) was incubated

with fluorochrome-labeled monoclonal antibodies

(10 µL) for 20 min in the dark at room temperature,

(A) (B)

(C)

Fig. 1. Three different methods of lymphocyte gating. (A) CD45/side light scatter (SSC) gating, (B) forward light scatter (FSC)/SSC gating, (C) FSC/SSC lymphocyte gating followed by purity correction (lymphocyte purity:

CD45(+)/CD14(-) proportion of FSC/SSC‐gated lymphocytes).

followed by the addition of 450 µL 1 × FACS Lysing Solution (Becton-Dickinson) and incubation for 10 min in the dark at room temperature. For each assay, 20,000 events per tube were acquired immediately and then analyzed.

Lymphocyte purity was defined as the percentage of CD45(+)bright/CD14(-) mononuclear cells after FSC/SSC lymphocyte gating. CD45(+)bright/

CD14(-) backgating was performed such that the CD45(+)bright/CD14(-) portion was further gated based on the FSC/SSC portion. Purity-corrected lymphocyte values were calculated by dividing the T-lymphocyte values (%) of the second FSC/SSC gating by lymphocyte purity (%). Lymphocyte subset percentages obtained using the 3 gating methods (CD45/SSC gating, FSC/SSC gating, and

FSC/SSC gating with purity correction) were compared. Differences in the percentages of T lymphocyte subsets between the different gating methods were calculated by subtracting T lymphocyte subset percentages of FSC/SSC gating or FSC/SSC gating followed purity correction from those of CD45/SSC gating.

5. Statistical analysis

All statistical analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA).

Pearson’s correlation was used to analyze correlations between continuous parameters.

Differences between results and their significances

were calculated by paired t tests. P-values less than

0.05 were considered statistically significant.

Fig. 2. Relationship between lymphocyte percentage and purity. Lymphocyte purity was correlated with lymphocyte percentage as determined by autoanalyzer.

Table 1. Correlations and differences in T-lymphocyte subset results obtained using 3 different gating methods Correlation of results using CD45/SSC gating Differences in results using CD45/SSC gating Mean ± SD, median (%)

FSC/SSC gating FSC/SSC gating followed by purity correction FSC/SSC gating FSC/SSC gating followed by purity correction

T cells 0.189

( P = 0.308) 0.189

( P = 0.310) 14.4 ± 22.9, 4.6

( P = 0.002) 12.1 ± 23.2, 2.1 ( P = 0.004) T helper/inducer cells 0.646

( P = 0.000) 0.485

( P = 0.006) 7.7 ± 14.3, 2.4

( P = 0.006) -1.7 ± 18.7, -4.1 ( P = 0.608) T suppressor/

cytotoxic cells 0.776

( P = 0.000) 0.618

( P = 0.000) 7.1 ± 10.5, 3.3

( P = 0.001) 0.2 ± 15.1, 1.0 ( P = 0.957) Abbreviations: SSC, side light scatter; FSC, forward light scatter.

RESULTS

1. Correlations between autoanalyzer and manual results

We found that correlations between autoanalyzer and manual methods were 0.871 (P = 0.000) for WBC counts and 0.524 (P = 0.002) for lymphocyte percentages. WBC counts ranged from 13 to 6,650/µL and 20 to 6,390/µL for autoanalyzer and manual counts, respectively, and the corresponding lymphocyte percentage ranges were 0.4% to 99.4%

and 1% to 89%, respectively.

2. Lymphocyte purity after FSC/SSC and CD45/SSC gating

The average purity of lymphocytes, as determined by CD14 and CD45 labeling after FSC/SSC gating,

was 76.5% ± 26.3% (median, 86.8%; range, 2.4%

-98.9%). The average granulocyte contamination was 20.5% ± 24.1%, and the average monocyte contamination was 2.6% ± 4.4%. In contrast, the average purity of lymphocytes after CD45/SSC gating was 98.8% ± 2.0% (median, 99.5%; range, 90.3%-100.0%).

3. Relationship between lymphocyte percentage and purity

We observed significant positive correlations between lymphocyte purity and lymphocyte percentage in both autoanalyzer (r =0.453; P= 0.011) (Fig. 2) and manual counts (r = 0.402; P = 0.025). The correlation between the number of lymphocytes and their purity was r= 0.322 (P = 0.078) using the autoanalyzer and r = 0.365 (P = 0.044) with the manual method.

4. Correlations and differences in T-lymphocyte subset results among the 3 gating methods (Table 1)

T cell percentages obtained using CD45/SSC gating were not significantly correlated with those obtained using FSC/SSC gating or FSC/SSC gating followed by lymphocyte purity correction. However, percentages of Th and Tc cells obtained using CD45/SSC gating were significantly correlated with those obtained using FSC/SSC gating and FSC/SSC gating with purity correction (Fig. 3).

T cell percentages obtained using CD45/SSC

gating differed significantly from those obtained by

FSC/SSC gating and FSC/SSC gating follo-

wed by lymphocyte purity correction. Th and Tc cell

(B)

(C) (A)

Fig. 3. Correlations between CD45/side light scatter (SSC) gating and forward light scatter (FSC)/SSC gating followed by lymphocyte purity correction for T cells (A), T helper/inducer cells (B), and T suppressor/cytotoxic cells (C).

(A)

(B)

(C)

Fig. 4. Correlations between lymphocyte percentages

and differences between CD45/side light scatter

(SSC) gating and forward light scatter (FSC)/SSC

gating with lymphocyte purity correction for T cells (A),

T helper/inducer cells (B), and T suppressor/cytotoxic

cells (C).

percentages obtained using CD45/SSC gating differed significantly from those obtained using FSC/SSC gating, but not from those obtained using FSC/SSC gating with purity correction.

5. Correlations between lymphocyte percentages and differences in T-lymphocyte subset values obtained using the different gating methods (Fig. 4)

Differences in Th cell values obtained using 2 different gating methods (CD45/SSC gating and FSC/SSC gating followed by lymphocyte purity correction) decreased as the lymphocyte percentage increased (r =-0.409, P =0.022). However, differences in T cells and Tc cells between the 2 different gating methods did not correlate with the corresponding lymphocyte percentages (T cells: r = -0.250, P=0.176; Tc cells: r =-0.285, P= 0.120).

Differences between values obtained by CD45/SSC gating and FSC/SSC gating also showed a decreasing trend as the percentage of lymphocytes increased, but none of the correlations between T cells (r = -0.273, P = 0.137), Th cells (r = -0.349, P = 0.054), and Tc cells (r = -0.105, P = 0.573) showed statistical significance.

DISCUSSION

Accurate analysis of lymphocyte subsets in BAL is important for the differential diagnosis and assessment of pulmonary disorders. Among the many factors influencing subset analysis results, the difference in gating methods is one of the most important factors. FSC/SSC gating uses clusters based on the FSC and SSC characteristics of leukocytes to distinguish between granulocyte, lymphocyte, and monocyte populations [11].

CD45-CD14 backgating was introduced to optimize the recovery of lymphocytes within the lymphocyte gate and to identify cells other than lymphocytes within the light-scatter gate [12]. CD45-SSC gating identifies lymphocytes by their CD45 and SSC characteristics (i.e., CD45

and SSC

), and more than 95% of cells identified by these characteristics should be lymphocytes [13].

CD45/SSC gating plus CD3 discrimination is considered the “gold standard” for the accurate determination of absolute CD4 and CD8 T cell

counts [14].

In the current study, differences between the results of CD45/SSC gating and those of the other 2 gating methods were statistically significant for T cells. For Th and Tc cells, however, the differences between the results of CD45/SSC and FSC/SSC gating were significant, but the differences between the results of CD45/SSC gating and FSC/SSC gating followed by lymphocyte purity correction were not.

Correcting for lymphocyte purity with CD45/CD14 narrowed the difference between CD45/SSC gating and FSC/SSC gating. These differences were calculated by subtracting the results of FSC/SSC gating or FSC/SSC gating followed by lymphocyte purity correction from those of CD45/SSC gating.

Most means and medians were positive, indicating that CD45/SSC gating resulted in a greater proportion of subsets than did FSC/SSC gating.

Setting an overly restricted FSC/SSC gate on lymphocytes to avoid contamination by monocytes has been reported to lead to an underestimation of lymphocytes [15]. The FSC/SSC gating strategy is somewhat subjective, further necessitating the use of anti-CD45 monoclonal antibodies in gating.

The correlations between CD45/SSC gating and FSC/SSC gating were lower for T cells (r =0.189) than for Th (r =0.646) and Tc (r =0.776) cells, and the same trend was observed for the correlation between FSC/SSC gating followed by lymphocyte purity correction. T cells measured with CD3 alone had larger cellular components; these cells were filtered and selected because they are then further measured with CD4 or CD8, resulting in a higher correlation among gating methods.

Correlations between the percentages of

lymphocytes and differences in T cells and Tc cells

determined by CD45/SSC gating and FSC/SSC

gating followed by lymphocyte purity correction were

not significant. However, the differences in Th cell

results obtained using these 2 gating methods

decreased as the proportion of lymphocytes

increased. When the proportion of lymphocytes was

high, lymphocyte purity, determined by CD45/CD14

on FSC/SSC gating, was also high. However, a

certain percentage of lymphocytes that consistently

showed high lymphocyte gate purity could not be

identified. When the proportion of lymphocytes was

low, FSC/SSC-gated cells contained many other cell types, including granulocytes and monocytes, hindering the accurate measurement of lymphocyte subsets. These findings indicate the necessity for CD45/SSC gating of BAL fluid, especially for samples containing low percentages of lymphocytes.

CD45-directed gating has been reported to reliably distinguish lymphocytes from other BAL leukocytes, even when specimens contain relatively low percentages of lymphocytes [16].

In summary, we found that FSC/SSC gating alone produced T-lymphocyte subset values that differed from those obtained by CD45/SSC gating.

Correcting for lymphocyte purity using CD45/CD14 after FSC/SSC gating decreased the discrepancy between CD45/SSC gating and FSC/SSC gating, but significant differences in the proportions of T cells still remained. In addition, T cell results obtained using CD45/SSC gating did not correlate with those obtained using FSC/SSC gating or FSC/SSC gating followed by lymphocyte purity correction. Thus, the use of CD45 is necessary for accurate analysis of T-lymphocyte subsets in BAL fluid.

요 약

배경: 정확한 게이팅법은 림프구아형을 유세포분석함에 있어 매우 중요하다. Forward light scatter (FSC)/side light scatter (SSC) 게이팅을 이용하여 측정할 경우, 림프 구의 순도가 95% 미만일 때 측정값을 순도로 보정해야 한 다. 본 연구는 폐포세척액의 T-림프구 및 T-림프구아형 측정 시 세 가지 다른 게이팅법을 비교하여 가장 정확한 게이팅법 을 찾고자 하였다.

방법: 자동혈구측정기와 수기법으로 31개의 폐포세척액 내의 백혈구수를 측정하고 감별 계산하였고 유세포분석기로 T림프구 및 T림프구 아형(도움T세포, 억제T세포)을 측정하 였다. 림프구 게이팅 방법을 비교하기 위하여 CD45/SSC 게이팅(표준방법), FSC/SSC 게이팅, 림프구 순도로 보정한 FSC/SSC 게이팅, 세가지 방법을 실시하여 결과를 분석하였 다. 림프구의 순도는 CD45/CD14를 이용하여 측정하였다.

결과: 전체 백혈구에서 림프구가 차지하는 비율과 림프구 의 순도는 의미 있는 상관성을 보였다(r =0.453, P = 0.011). 표준방법을 이용해 측정한 T세포의 결과와 두 가지 다른 게이팅 방법으로 측정한 결과들은 서로 상관성이 낮았 다 (FSC/SSC 게이팅과는 r =0.189, P=0.308, 순도 보 정한 FSC/SSC 게이팅과는 r =0.189, P =0.310). 표준 방법과 FSC/SSC 게이팅 사이의 차이는(T세포- 평균 14.4%, P =0.002; 도움T세포- 평균 7.7%, P = 0.006;

억제T세포- 평균 7.1%, P = 0.001), 표준방법과 순도를 보정한 FSC/SSC 게이팅 사이의 차이보다 높았다(T세포- 평균 12.1%, P= 0.004; 도움T세포- 평균 1.7%, P = 0.608; 억제T세포- 평균 0.2%, P =0.957).

결론: 림프구 순도로 보정한 FSC/SSC 게이팅에 의한 측 정값은 도움 T세포와 억제 T세포 측정 결과를 더 정확하게 했지만 T세포 측정의 정확도는 개선하지 못했다. 따라서, 유 세포분석을 이용하여 폐포세척액의 T세포 및 T세포 아형을 측정 시 CD45를 이용한 림프구 게이팅은 필수적이다.

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