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Keywords:

  • paroxysmal nocturnal hemoglobinuria;
  • monocyte analysis;
  • flow cytometry;
  • CD64;
  • standardization

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Background:

Assays of antigen expression on myeloid cells have an underlying premise that the assay integrates high purity gating of the leukocyte subpopulation in question. While CD45/side scatter (SSC) gating provides sufficient gating purity for qualitative assays of antigen expression; it is unsuitable for quantitative assays of antigen changes, especially monocytes. We have validated a monochromatic gating approach combining CD45 and CD64 labeled with the same fluorochrome that allows for high purity monocyte gating.

Methods:

Twenty-five blood samples were stained using three different antibody combinations (CD45 FITC + CD163 PE; CD45 FITC + CD64 PE; CD45 FITC + CD64 FITC). Data analysis focused on the percentage of “monocytes” defined by the various antibody and SSC gating combinations.

Results:

Percent monocyte recovered by monochromatic CD64 gating was not statistically different from two-color CD45 + CD64 or CD45 + CD163 gating. All three methods of immunologic monocyte identification yielded a 12.93%–15.15% reduction in the “monocyte” percentage compared to CD45/SSC gating.

Conclusions:

A monochromatic combination of CD45 and CD64 antibodies with scatter signals allows higher purity monocyte gating by flow cytometry (FC) compared to CD45/SSC gating. This approach allows for the development of a high resolution four-color assay, such as for detection of paroxysmal nocturnal hemoglobinuria, whereby a single four-color tube will allow simultaneous high purity monocyte (CD64+) and neutrophil (CD15+) analysis of both phosphatidylinositol (PI) linked protein expression and FLAER binding. © 2013 International Clinical Cytometry Society


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Evaluation of cellular antigen expression is increasingly becoming an important diagnostic tool. Accurate assays of antigen expression on myeloid cells require a high purity gating of leukocyte subpopulations. While CD45/side scatter (SSC) gating is appropriate for qualitative assays of antigen expression, it is unsuitable for quantitative assays of antigen changes, especially monocytes, due to overlapping with other leukocyte populations such as NK cells, activated T or B cells, dendritic cells, mast cells, and basophils. When the NK cells overlap the monocyte population, neither a FSC/SSC nor a CD45/SSC gate will be able to sufficiently separate the two cell populations (1).

We investigated several gating methods that yield a greater purity gating of the monocytes by incorporating anti-CD45 and a monoclonal antibody (CD163 or CD64) with a common fluorochrome shared by the reagents that bind to antigens expressed constitutively on monocytes. These antibodies increase the fluorescence intensity of monocytes relative to other leukocyte populations, allowing for a purer gating of monocytes through the exclusion of non-monocytic cells. Furthermore, we discovered that a monochromatic labeling approach was highly comparable to a dual-fluorochrome approach.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

High Purity Monocyte Gating

Specimens

Blood samples from 25 adults were collected in 3.0 ethylene-diaminetetra-acetic acid (EDTA) Vacutainers (BD Medical Systems, Franklin Lakes, NJ) and stored at room temperature until processing. Blood donors include 7 females and 15 males with three additional donors of unknown gender with specimens collected in accordance with institutional guidelines. The ages of blood donors ranged from 21 to 60 years.

Reagents

The following monoclonal antibodies to human antigens were used: fluorescein isothiocyanate (FITC) conjugated CD45 (clone ML2) and purified CD45 (clone ML2) from IQ Products (Groningen, Netherlands); phycoerythrin (PE) conjugated CD163 (clone Mac2-158); PE conjugated CD64 (clone 22); FITC conjugated CD64 (clone 22) (Trillium Diagnostics, Brewer, ME). All monoclonal antibodies were titered to determine the optimal staining concentration, requiring a final staining volume of 10 μl per sample (see Fig. 1).

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Figure 1. Determining the optimal CD45 concentration: separation between leukocytes and red blood cells. All samples were stained with the optimal concentration of CD64, while the labeled CD45 was titered. The optimal CD45 concentration defined as the amount of antibody that maximizes the separation between the monocytes and the other leukocytes, while maintaining their separation from the red blood cells. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]

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Sample preparation and flow cytometry

All samples were stained with varying amounts of unconjugated anti-CD45 and anti-CD45 FITC; this allows for antigen saturation, but lowers the effective fluorochrome:protein ratio. In addition to the anti-CD45 reagents, the 100 μl blood samples were stained with anti-CD163 PE, anti-CD64 PE, or anti-CD64 FITC to create three different reagent combinations for each blood donor (combinations shown in Table 1). The samples were vortexed after the addition of the reagents and incubated in the dark for 15 min. Thereafter, Trillium Lyse, an ammonium chloride based erythrocyte-lysing buffer was added (Trillium Diagnostics). The samples were gently vortexed, followed by another 15-min incubation period at room temperature. Flow cytometry of the samples was completed within an hour of sample preparation. Samples were protected from light up until flow cytometry acquisition. All the samples were analyzed on a FACScan flow cytometer with CellQuest software (BD Biosciences, San Jose, CA), collecting at least 25,000 cellular events. Prior to acquisition, cytometer linearity and sensitivity was optimized by running CaliBRITE beads with FACSComp software (BD Biosciences, San Jose, CA). Listmode analysis of the files was performed using with WinList (Verity Software House, Topsham, ME). All results were expressed as percent monocytes of total leukocytes.

Table 1. Summary of the Percent Monocyte Decrease for the Four Different Gating Methods
Gating methodAverage % monocyte decrease
CD45/SSC
CD163PE +CD45 FITC12.93
CD64PE + CD45 FITC15.15
Monochromatic CD45 + CD6414.74
Gating strategy

On a two-parameter correlated histogram (FSC vs. SSC), a gate was set around the leukocyte population to exclude cell debris and cell aggregates. For sample Tube 1, consisting of CD45FITC + CD163PE, leukocytes were replotted on FL2 v. SSC and FL2 vs. FL1 histograms (Figs. 2B and 2C). The percent monocyte was determined as an average of the results from these two histograms. For the CD45FITC + CD64PE sample, leukocytes were also replotted on FL2 vs. SSC and FL2 vs. FL1 (Figs. 2E and 2F) with the results averaged from the two histograms. For sample Tube 3, gated cells were replotted on a histogram of FL1 vs. SSC to determine the monocyte region for the CD45 + CD64FITC monochromatic gating method (Fig. 2D). CD45/SSC gating was determined using the results of sample Tube 1 plotted on a FL1 vs. SSC histogram (Fig. 2A).

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Figure 2. Gating techniques for determining monocyte percent of total leukocytes. Single donor showing identification of different analysis methods based upon CD45/SSC gating (A), CD163 gating (B and C, results averaged), CD 64+ and CD45+ monochromatic gating (D), and CD64 two color gating (E and F, results averaged). [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]

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Co-expression of CD64 and CD163

To evaluate the co-expression of CD64 and CD163 on monocytes, we dual-stained 50 samples with anti-CD64 and anti-CD163. Staining was performed using the Leuko64 kit (Trillium Diagnostics). The kit contains Reagent A, a cocktail comprised of antibodies with affinities to CD64 (FITC conjugated) and CD163 (PE conjugated), and Reagent B, an ammonium chloride lysing buffer. Fifty microliters of fresh peripheral blood were pipetted into the bottom of a test tube. Fifty microliters of Reagent A was added to the blood aliquot. Samples were gently vortexed upon the addition of the antibody cocktail and incubated in the dark at room temperature for 15 min. Thereafter, erythrocytes were lysed with Reagent B. The addition of lysed buffer was followed by another 15-min incubation period in the dark. All the samples were analyzed on a BD FACScan flow cytometer with CellQuest software, collecting at least 50,000 cellular events.

Listmode analysis of the files was performed using with WinList (Verity Software House, Topsham, ME). Monocytes and lymphocytes were identified by SSC and their expression of CD64 and CD163. Lymphocytes, the negative population, were used to define the gate for monocytes. CD163+ monocytes were replotted on a CD64 FITC single-parameter histogram to determine the percentage of CD163-gated monocytes that are also CD64+. CD64+ monocytes were replotted on a CD163 PE single-parameter histogram to determine the percentage of CD64-gated monocytes that are also CD163+.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Percent Monocyte Decrease

The CD45/SSC gating method yielded a percent monocyte range (lower quartile to upper quartile) of 6.79–7.89. The CD64PE + 45FITC, CD163PE + 45FITC, and CD64FITC + 45 FITC methods resulted in lower percent monocyte ranges, respectively, 5.74–6.78, 5.87–6.88, and 5.74–6.78 (Fig. 3). In comparing immunological gating of monocytes with CD45/SSC, the average percent decrease in monocyte ranged from 12.93% to 15.15% (Table 1).

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Figure 3. Box and whisker plots showing the range of monocyte percent for the four different methods. Immunologic gating of monocytes consistently yielded lower results relative to CD45/SCC gating. Monocyte percentages in blood samples vary according to whether specific monocyte identification (CD163 or CD64) is employed or only CD45 SSC gating is employed. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]

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Inter-Method Correlation

We performed an inter-method correlation analysis, evaluating the percent monocyte detected by each gating method. CD64 and CD45 combination gating, while highly correlated to CD45/SSC gating, yields significantly less monocytes (Table 2). There is no positive bias evident between the CD163 and CD45, dual-chromatic and monochromatic CD64 and CD45 gating approaches (Fig. 4). Thus immunologic gating with monocyte markers in any combination reduced the cells in the defined monocyte gate by approximately 15%, which was the expected finding when other cell types such as NK cells, apoptotic PMNs, dendritic cells are not included in the monocyte region.

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Figure 4. Passing Bablock regression comparing monocyte gating methods: CD64 two color gating (average of 1B and 1C) to CD64 FITC monochromatic gating (as in Fig. 1C) to derive monocyte percent. A Bland–Altman plot demonstrates no significant bias between the two approaches. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]

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Table 2. Summary of Method Statistics
Gating methodsCD45/side scatterCD64 PE & CD45 FITCCD163 PE & CD45 FITCMonochromic CD64 & CD45
  1. The range of results for each comparison is depicted by the values in boldface. R2: relation coefficients; the level of significant difference between the results of the two comparative methods. P < 0.05 is considered a significant difference.

CD45/side scatter6.79–7.89P < 0.0001P < 0.0001P < 0.0001
CD64 PE & CD45 FITCR2 = 0.8925.74–6.78P = 0.3789P = 0.6003
CD163 PE & CD45 FITCR2 = 0.719R2 = 0.8585.87–6.88P = 0.4942
Monochromic CD64 & CD45R2 = 0.896R2 = 0.928R2 = 0.8055.74–6.78

Co-expression of CD64 and CD163

The expression of CD64 and CD163 is highly correlated. The percentage of CD163-gated monocytes that are also CD64+ ranged from 97.01 to 100.0 (median: 99.97). The percentage of CD64-gated monocytes that are also CD163+ ranged from 96.57–100.0 (median: 99.82) (see Fig. 5). The findings support the concept that nearly all CD163+ monocytes co-express moderate levels of CD64, but that only minor subset (<3%) of CD64+ monocytes have no or very low expression of CD163.

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Figure 5. Co-expression of CD163 and CD64 in 50 blood samples. Samples were dual stained with CD64 FITC and CD163 PE and show most samples to have simultaneous expression for both antigens. There is a small subset of CD64+ monocytes that in some cases had very low or absent CD163 expression in some cases (9 of 50 cases had <99% CD163 expression), whereas the corollary was not found. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

In this study, we demonstrated a simple and reliable method for gating monocyte populations. While there is currently little interest in the immunophenotyping of monocytes other than for research work, there are some applications where high purity identification of monocytes in blood or bone marrow samples may be of interest by labs having relatively limited color detection on the instrument. Monocytes are a heterogeneous population, with various subsets (2–4). High purity gating of monocytes requires an antigen that is constitutively expressed on all monocyte subsets. We previously have reported that immunologic gating using CD64 and CD163 is superior to CD14 (5, 6); this is likely due to the fact that CD14 is not expressed on all blood monocytes and CD14 can be expressed on other myeloid cells, including activated neutrophils (7).

CD45/SSC gating is useful for as distinguishing leukocytes from erythrocytes and for making qualitative representations of lymphocyte, monocyte, and neutrophil populations. The monocyte region in a CD45/SSC bivariate plot contains cells from other leukocyte lineages (1). This study demonstrates that monochromatic CD64 and CD163 immunologic gating of monocytes is superior to CD45/SSC gating by eliminating the 5–15% of cells in the CD45/SCC “monocyte” gate that are of non-monocytic lineages, such as NK cells, activated T or B cells, dendritic cells, mast cells, and basophils. We determined that monocytic expression of CD64 and CD163 expression is highly correlated, indicating that these antigens are markers of a homogenous population, as previously reported (5, 6). While CD163 displays improved specificity over CD14, it has a low expression with only 2,000–15,000 sites per monocyte, particular in cases with monocyte activation where CD163 is shed from monocytes. The fact that the majority of circulating monocytes express CD163 has been debated due to the unfortunate commercial availability of CD163 clones with heterogeneous quality regarding the binding to the CD163 epitope (8). Our studies used the Mac2-158 clone, which is superior to the RM3/1 and GHI/61 clones for detection of CD163 (8), thus explaining discordant results from those studies mistakenly reporting CD163 to be expressed on a minority of blood monocytes.

Human monocytes have also been classified based on their CD64 (FcγRI) expression (4). As with CD163, CD64 expression is localized to monocytes, making it an accurate and precise monocyte marker. Furthermore, there are more binding sites for CD64, leading to relatively constant expression, which becomes further elevated with monocyte activation. A possible limitation of using CD64 expression to gate monocytes is that PMN antigenic changes lead to the up-regulation of CD64 during pathohysiological acute inflammatory response or innate immune response under the influence of inflammatory related cytokines such as interleukin 12, interferon gamma, and granulocyte colony stimulating factor (9). However, in these authors' experience, when the PMN CD64 expression increases, a parallel upregulation is seen with monocyte CD64 expression. Additionally, in our experience is it a rare case, usually those with monocytic leukemia, where monocytes cannot easily be identified in a CD64/SSC bivariate plot.

We increased the purity of monocyte gating by using CD64, an antigen that is constitutively expressed on monocytes, to increase monocyte fluorescence intensity relative to other leukocyte populations. In order to further increase the purity of the monocyte population, we diminished the fluorescence intensity of the other leukocyte populations (lymphocytes, neutrophils, and eosinophils) using unlabeled anti-CD45, which effectively decreases the F/P ratio of the CD64 reagent. CD45 expression is the highest in mature lymphocytes and monocytes, with CD45 protein comprising 5–10% leukocyte surface proteins (10, 11). The fluorescence intensity of leukocytes can be reduced by altering the CD45 reagents. The monocyte population, however, maintains their fluorescence intensity because of their constitutive CD64 expression. Our study determined the reagent combinations that maximize the separation between monocytes and other leukocyte populations while maintaining their separation from red blood cells by CD45 expression.

The use of the markers CD45 and CD64 in a paroxysmal nocturnal hemoglobinuria (PNH) assay is not a novel combination, as they are monocyte markers recommended by the ICCS in their guidelines for PNH testing, in which they suggest combining light scatter and CD45 expression with lineage markers, such as CD64 or CD33, to further improve the accuracy of gating (12). Building upon the ICCS recommendations for PNH testing, Sutherland et al. elucidated the clones and antibody dilutions that comprise a “perfect cocktail” for high sensitivity PNH testing, incorporating CD45 PECy7 and CD64PECy5 into their WBC cocktail (13). The utility of CD64 monocyte gating in PNH was also recently reported to be superior to CD33 also on the basis of its high purity of monocyte in cells having moderate expression (14).

Our study serves to validate a monochromatic gating approach for monocytes, minimizing the number of flourochromes needed for analysis. We demonstrate that CD64 in combination with CD45, both labeled with the same flourochrome, serves to increase the fluorescence intensity of monocytes sufficient to derive similar monocyte frequency measurements as other methods requiring a dual labeled approach. There was no statistically significant difference between the CD163 PE + CD45 FITC, CD64 PE + CD45 FITC, and CD64 FITC + CD45 FITC gating methods, demonstrating one color is equally effective as two color gating. One practical use of this finding is that a leukocyte screening tube for PNH that can be designed to examine both a PI linked protein, such as CD157, and FLAER™, while gating on monocytes using one color (CD45+, CD64+) and neutrophils in another color with CD15 (13). Thus, PNH leukocytes can be studied in a single tube assay using instruments restricted to only four-color detection and avoiding the use of more complex fluorochrome combinations.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED
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    Zarez PV, Davis BH. Comparative study of monocyte enumeration by flow cytometry: Improved detection by combining monocyte-related antibodies with anti-CD163. Lab Hematol 2004; 10: 2431.
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    Schiff DE, Rae J, Martin TR, Davis BH, Curnutte JT. Increased phagocyte FcγRI expression and improved Fcγ-receptor-mediated phagocytosis after in vivo recombinant human interferon-γ treatment of normal human subjects. Blood 1997; 90: 31873194.
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    Caldwell C, Patterson W. Relationship between T200 antigen expression and stages of B cell differentiation in resurgent hyperplasia of bone marrow. Blood 1987; 70: 11651172.
  • 11
    Caldwell C, Patterson W, Toalson B, Yesus Y. Surface and cytoplasmic expression of CD45 antigen isoforms in normal and malignant myeloid cell differentiation. Am J Clin Pathol 1991; 95: 180187.
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    Borowitz MJ, Craig FE, DiGiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry Part B Clin Cytom 2010; 78B: 211230.
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    Sutherland R, Keeney M, Illingworth A. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry Part B Clin Cytom 2012; 82B: 195208.
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    Dalal B, Khare N. Flow cytometric testing for paroxysmal nocturnal hemoglobinuria: CD64 is better for gating monocytes than CD33. Cytometry Part B Clin Cytom 2013; 84B: 3336.