High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections
- Authors:
- Published online on: January 24, 2018 https://doi.org/10.3892/br.2018.1047
- Pages: 224-234
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Copyright: © Chan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Multicolor flow cytometry provides detailed phenotypic information on the cellular composition of a sample. It allows for the identification of cellular subsets based on specific markers both on the surface of and inside cells (1). With the development of novel classes of fluorochromes and advancements in cytometer design, the use of more than 8 colors for flow cytometry should become common practice in clinical labs and increase the sensitivity of current assays (2).
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare stem cell disorder caused by acquired mutation in the X-linked phosphatidylinositol glycan complementation class A gene, with an annual incidence rate of 1.3 new cases per million of the population per year (3–8). The acquired mutation generates a defect in the expression of glycol-phosphatidyl inositol (GPI)-linked structures such as the complement regulatory proteins, CD55 and CD59 (9–12). A lack of these GPI-linked complement regulatory proteins on red blood cells (RBCs) may cause intravascular complement-mediated hemolysis, resulting in the release of free hemoglobin and leading to the symptoms of PNH, which may include the development of anemia, hemoglobinuria, thrombosis, dysphagia, abdominal pain, pulmonary hypertension, renal impairment and erectile dysfunction (4,13–16). Previous PNH assays have focused on loss of CD55 and CD59 on CD235a red cells or granulocytes (17,18). However, pathological hemolysis of PNH red cells may generate inaccurate results, as many patients who are screened for PNH have received erythrocyte transfusions, which may contribute to underestimating the size of the PNH clone population. As a result, PNH erythrocytes may be undetectable even when a substantial PNH population is present (19). Furthermore, CD55 expression cannot distinguish intermediate complement sensitivity (type II) and more abnormal PNH-type III RBC clones (20), and the sensitivity of CD55 alone is not high enough to detect PNH population sizes of less than 1% (21). Despite there being therapeutic support for patients with PNH, including transfusions and anticoagulation management (22), the survival rate remains low, with 35% of cases being fatal within 5 years of diagnosis (23–25). Small PNH clones may grow over time and benefit from eculizumab treatment (25). Thus, high-sensitivity PNH assays are required to detect PNH populations of less than 1% in order to follow up closely and to provide early treatments for patients.
To improve the resolution of PNH detection, the present study formulated an 8-color combination for the detection of PNH white blood cells (WBCs), which included lineage-gating antibodies (CD64 for monocytes and CD15 for granulocytes) together with fluorescein-labeled proaerolysin (FLAER) and three GPI-linked markers, namely CD157 [both granulocyte and monocyte-specific (26)], CD24 [granulocyte-specific (27)] and CD14 [monocyte-specific (28)]. This combination matches the International Clinical Cytometry Society (ICCS) guidelines for the diagnosis and monitoring of PNH and related disorders by flow cytometry (20).
To the best of our knowledge, this present report is the first to identify a high-sensitivity 8-color antibody cocktail for PNH flow detection with the use of a dump channel. It may be a useful tool for PNH detection and has the potential to improve the accuracy, work flow, cost and diagnostic process while saving the labor and time of technicians and clinical pathologists.
Materials and methods
Blood samples and antibodies
Whole blood (WB) samples (100 µl) from PNH patients were provided by the College of American Pathologists (CAP; Northfield, IL, USA) and UK NEQAS (London, UK) between May 2017 and August 2017. WB samples (100 µl) from 10 healthy donors (aged 58.4±11.1 years) were also collected. The samples were stored in BD Vacutainer™ blood collection tubes containing EDTA as an anti-coagulant (BD Biosciences, Franklin Lakes, NJ, USA) at 4°C and tested within a month. For certain experiments, PNH proficiency test samples were obtained from CAP and UK NEQAS. Alexa Fluor (A)488-conjugated FLAER was purchased from Cedarlane (Burlington, ON, Canada). Phycoerythrin (PE)-conjugated anti-CD157 (SY/11B5), Allophycocyanin (APC)-conjugated anti-CD24 (ALB9) and APC-A750-conjugated anti-CD64 (clone 22) were purchased from Beckman Coulter, Inc. (Brea, CA, USA). Peridinin chlorophyll protein complex (PerCP)-conjugated anti-CD14 (MfP9), PE-cyanine (Cy)7-conjugated anti-CD5 (L17F12), PE-Cy7-conjugated anti-CD19 (SJ25C1), V450-conjugated anti-CD15 (MMA), V500-C-conjugated anti-CD45 (2D1), PerCP-Cy5.5-conjugated anti-CD3 (SK7), APC-conjugated anti-CD4 (SK3), APC-H7-conjugated anti-CD8 (SK1), V450-conjugated anti-CD7 (M-T701), PE-conjugated anti-CD55 (IA10) and APC-conjugated anti-CD14 (MfP9) were purchased from BD Biosciences. All the antibodies were titrated to optimize the cocktail performance. The present study was reviewed by the Research Determination Office of Kaiser Permanente (Oakland, CA, USA) and determined not to be human research due to the use of secondary de-identified samples; therefore, the requirements of informed patient consent and ethical approval were waived.
Cell staining with 8-color antibody cocktail and acquisition
The 8-color antibody cocktail for PNH detection was prepared according to the configuration listed in Table I. A total of 1×106 cells in the EDTA-whole blood samples were transferred to 12×75 mm Falcon tubes and stained with the 8-color cocktail antibody cocktail for 15 min at room temperature in dark. The whole blood was then lysed with 2 ml BD FACS™ Lysing Solution (BD Biosciences) for 10 min at room temperature. After washing the cells twice with phosphate-buffered saline (PBS; Beckman Coulter, Inc.) and resuspending in 0.5 ml 0.5% paraformaldehyde as fixation buffer (Polysciences, Inc., Warrington, PA, USA), data were acquired on a BD FACSCanto™ II flow cytometer with standard configuration (BD Biosciences). A total of 100,000 events in the monocyte and granulocyte cell gates were acquired. BD FACSDiva™ v.6.1.3 software under standard 8-color configuration (BD Biosciences) was used for acquisition and analysis. For compensation, a BD™ Comp Beads kit was used to create cocktail-specific compensation matrices on whole blood from the healthy donors utilizing batch-matched single color reagents following the manufacturer's instructions. For a 4-color control experiment, PE-CD55 (10 µl), APC-CD14 (5 µl), FLAER-A488 (10 µl) and V500-C-CD45 (5 µl) were used to stain four normal whole blood samples (100 µl each). For certain experiments, whole blood was stained with PerCP-Cy5.5-CD3, APC-CD4, V450-CD7 and APC-H7-CD8.
 | Table I.Configuration of 8-color flow assay cocktail for paroxysmal nocturnal hemoglobinuria detection. |
Sensitivity test
The percentage of PNH clones [FLAER−/CD157− on CD64+ granulocytes (Grans)] was determined using the 8-color cocktail listed in Table I. Three PNH samples were spiked in 100 µl normal whole blood to obtain a final concentration of PNH clones of 0.5–2%. They were then serial diluted at 1:1 (v:v) using normal whole blood as diluent up to 0.005–0.01% expected PNH percentage (Table II). Unspiked normal whole blood was used as a negative control. Following preparation of the serial dilutions, the samples were stained with the 8-color cocktail following the staining procedure described above. A total of 100,000 events in the monocyte and granulocyte gates were acquired for each sample. CD15+ Grans FLAER−/CD157− PNH clones were identified on CD157/FLAER dot plots.
| Table II.Target number of events for paroxysmal nocturnal hemoglobinuria populations with high sensitivity. |
Data acquisition
Data acquisition was performed with FCS Express 5 (De Novo Software, Glendale, CA, USA) and BD FACSDiva™ v.6.1.3 using gating strategy on dot plots, after which appropriate subsets were identified.
Statistical analysis
To assess the correlation of CD15+ Grans FLAER−/CD157− PNH clones between expected and actual percentages, a linear regression analysis was performed. All statistical calculations were performed with Excel 2016 (Microsoft Corporation, Redmond, WA, USA) and Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). R2>0.95 and P-value <0.0001 were considered to indicate significant correlation.
Results
Normal blood exhibits positive staining for CD55 using the 4-color PNH panel
In the present study, a whole blood sample from a healthy donor was stained with a 4-color cocktail containing FLAER-A488, PE-CD55, APC-CD14 and V500-C-CD45. From this, a minor CD55− granulocyte population was observed (Fig. 1). However, this population was FLAER+, suggesting that it was not representative of PNH clones, which have been determined to be FLAER− (26). Three other normal samples exhibited the same FLAER−/CD55+ population of granulocytes (data not shown).
Artifacts on granulocyte and monocyte staining with fluorochrome-conjugated antibodies
To increase the sensitivity of multicolor flow cytometry, the present study aimed to remove non-specific binding and artifacts. A normal blood sample was stained with a multicolor cocktail targeting T cell markers (CD3, CD4, CD8 and CD7). A number of artifacts were identified in the granulocytes and monocytes regions on CD3/SSC, CD4/SSC, CD8/SSC and CD7/SSC plots (Fig. 2, indicated in red). Other lymphocyte markers including CD25 exhibited the same artifacts (data not shown). Some of those artifacts were doublets and could be eliminated by doublet discrimination (data not shown). However, some artifacts remained following gating out of all doublets as observed on the CD19+CD5/SSC plot in Fig. 3 (green arrow).
Granulocyte and monocyte PNH clones were identified with the 8-color flow assay
To determine whether the 8-color PNH panel was able to identify PNH clones, blood samples spiked with PNH clones were stained with an 8-color PNH cocktail (Table I). The forward scatter width and height (FSC-W and -H) were used to gate the singlets population. Monocytes and granulocytes were gated together and non-specific binding events were removed by gating the negative population on the ‘dump’ channel (CD5 and CD19; Fig. 3). A number of artifacts were identified on the right side of the granulocyte and monocyte region on the CD5+CD19/side scatter (SSC) plot, and those events were eliminated by gating the negative populations of monocytes and granulocytes, resulting in reduced background (Fig. 3). The monocyte antigen CD64 and granulocyte antigen CD15 further separated monocytes and granulocytes, respectively. The PNH clones exhibited negativity for FLAER and GPI-linked antigens (CD157, CD24 and CD14; Fig. 3). The CD5+CD19+ population was present together with CD15+ Grans, CD64+ Mono and affected FLAER−/CD157− quadrant as indicated in Fig. 4 (top panel; green population). There were FLAER−/CD157− differences that ranged from 0.014 to 1.28% when excluding or including the CD5+CD19+ populations (Fig. 4, lower panel). By contrast, normal whole blood stained with the same 8-color PNH cocktail did not exhibit any events on the lower left quadrant of FLAER/CD157, FLAER/CD24 and FLAER/CD14 plots (Fig. 5, top panel). The samples of 10 normal donors were stained with this 8-color PNH cocktail and exhibited an average of 0.002773±0.001743% PNH clones (FLAER−/CD157−) in the CD15+ Gran population and 0.000764±0.002415% in the CD64+ monocyte population, respectively (Fig. 5, lower panel). These results suggest that the 8-color assay has high specificity.
Sensitivity of the WBC 8-color PNH flow assay
The percentage of PNH clones in three PNH samples were determined using the 8-color panel. The PNH samples were then spiked into normal whole blood to attain a final PNH concentration of 0.5–2%, and serial dilutions were performed. The first tube of sample 1 exhibited 2.2% PNH clones (FLAER−/CD157−), and was subsequently diluted in 50% increments until it reached 0.01% (Fig. 6). The normal whole blood control did not exhibit a PNH population on the left lower quadrant of the FLAER/CD157 plot (Fig. 6). The actual number of FLAER−/CD157− events in sample 1 was 10/69,216 CD15+ Grans at the lower limit of detection (LOD) of 0.014% (Fig. 6 and Table III). The number of FLAER−/CD157− events in samples 2 and 3 at the corresponding LODs were 10/73,469 CD15+ Grans and 8/77,229 CD15+ Grans, respectively (Table III); samples 1–3 exhibited an LOD for FLAER−/CD157−CD15+ Grans of 0.01% (Table III) and 0.05% for CD64+ Mono (data not shown) on the 8-color flow assay. The expected PNH clone percentage was significantly correlated with the actual value, R2 value of 0.9839 and p value less than 0.0001 on a linear curve (Fig. 7), suggesting that the 8-color PNH flow assay had high sensitivity, detecting a PNH clone population as low as 0.01% in CD15+ Grans.
Accuracy of the WBC 8-color PNH flow assay
The percentages of PNH clones in the CAP and UK-NEQAS samples were determined using the 8-color panel. The percentage of FLAER−/CD157− Grans in the CAP sample was 8.79%, which matched the CAP FLAER/CD157 granulocytes specification (4.70–10.00%; Fig. 8). The percentage of FLAER−/CD157− Mono in the CAP sample was 7.89%, which matched the CAP FLAER−/CD157− monocytes specification (7.39–10.13%; Fig. 8). Furthermore, the percentage of two UK-NEQAS samples identified by the 8-color assay matched the UK-NEQAS specifications (Fig. 8), suggesting that the 8-color flow assay had high accuracy for PNH clone identification.
Discussion
Flow cytometry has become a standard tool for the analysis of cellular phenotype and function within the immune system, among other applications. As more fluorochromes have been developed with advances in cytometer design and lasers, the identification of more rare cell populations has become achievable, and the sensitivity of existing assays has increased (1,2). For PNH, as a rare disease, a high sensitivity assay with short acquisition time is required.
In the present study, it was demonstrated that the 4-color combination (FLAER, CD55, CD14 and CD45) was not accurate in identifying PNH populations in whole blood samples. Earlier methods of PNH detection have relied on the loss of CD55 and CD59 on RBCs and granulocytes (17,18,20). However, the present study identified that CD55 negative populations were not representative of PNH clones, suggesting that a novel assay is required to aid more accurate clinical diagnosis of PNH. Additionally, CD55 is not an adequate marker for PNH as it cannot distinguish PNH type II (partially deficient GPI-anchored proteins) from type III (lacking expression of GPI-anchored proteins) RBCs (20).
The improved methods in flow cytometry may enable the measurement of increased markers at a single cell level in less time (2,29,30). In the present study, an 8-color configuration was designed for PNH identification. With increased photomultiplier tubes in the assay, it allowed the inclusion of other antigens that could increase sensitivity of the flow assay. However, artifacts were observed on monocyte and granulocyte surface staining even when using lymphocytes markers such as CD3 and CD7. These artifacts were composed of doublets and non-specific staining signals, which were present on the positive side of each channel. To generate a high sensitivity flow assay for PNH detection, the aim was to eliminate all potential non-specific binding events, as only 10 events in a 100,000 total population (granulocytes and monocytes) was required to achieve 0.01% sensitivity. A minority of false positive events would markedly decrease the sensitivity. The CD5+CD19+ populations were combined with CD15+ Grans, CD64+ monocytes and were even identified on the lower left quadrant of the CD157/FLAER plot used for PNH clone identification. The percentage differences in FLAER−/CD157− clones by excluding and including CD5+CD19+ populations were 0.014–1.28%, which are significant numbers when considering a 0.01% PNH clone population was being targeted. However, it was demonstrated using doublet discrimination (FSC-W/FSC-H) and a ‘dump’ channel (CD5 and CD19) to gate the negative population that the background staining was lowered markedly to detect 0.002773% (CD15+ Grans) and 0.000764% (CD64+ Mono) PNH populations (FLAER−/CD157−) in whole blood from 10 normal donors. Sutherland et al (31) described a 4-color combination of FLAER, CD14, CD64 and CD45 for high-resolution detection of PNH monocytes with a background rate of 0.0033%. Our current 8-color combination further improved the background rate for monocytes and granulocytes to 0.00076% and 0.00277%, respectively, with only 100,000 events of monocyte and granulocyte acquisition. Furthermore, the gating strategy using granulocyte antigen (CD15) and monocyte antigen (CD64) served a key role in separating the monocyte and granulocyte populations prior to assessment of the GPI-linked antigens. As a result, the 8-color panel improved sensitivity and specificity with a shorter acquisition time in PNH identification.
FLAER-A488 is a fluorescent aerolysin that directly binds GPI-linked anchors and has been suggested as an optimum reagent for PNH assays (20). In the current study, three additional GPI-linked antigens were included to improve specificity. CD157 is a GPI-linked protein expressed on both granulocytes and monocytes (32); CD14 is a GPI-protein expressed on monocytes (27); and CD24 is a GPI-protein expressed on granulocytes (28). The combination of FLAER, CD157, CD24 and CD14 provided a comprehensive panel for targeting of all major GPI-linked antigens on PNH samples.
Furthermore, PNH samples provided by CAP and UK NEQAS were also tested by the 8-color PNH flow assay. The percentage of PNH clones identified by the 8-color assay matched the specifications provided by CAP and UK NEQAS, suggesting that the assay is able to identify PNH populations with sufficient accuracy.
In conclusion, flow cytometry is an important tool for leukemia/lymphoma diagnosis (33–36). Abnormal cells may exhibit aberrant expression of certain markers and downregulate other markers, in comparison to normal samples (37). It is important that a pre-mixed reagent generates an accurate expression profile of the sample using a combination of multicolor cocktails, to enable pathologists to correctly identify different types of leukemia/lymphoma and also PNH in patient samples (26,38–42). However, the current strategies employing CD55 and CD59-based assays in certain laboratories do not have adequate sensitivity to detect PNH population sizes of less than 1%, and thus the 30–40% PNH samples that typically exhibit a lower than 1% PNH clone population go undetected (21,43). A high sensitivity flow assay is required to identify those populations, to allow early and suitable treatments to be provided for patients. The present results demonstrate that the 8-color PNH antibody cocktail is sensitive in distinguishing small PNH populations from normal samples with low background and short acquisition time. It may provide an effective tool for pathologists in the diagnosis of PNH, including in those cases with small percentages of PNH clones.
Acknowledgements
The authors are thankful to CAP and UK NEQAS for providing PNH samples for the study. All authors declare no conflicts of interest regarding the study described in this manuscript.
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