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Keywords:

  • high sensitivity 5-color flow;
  • paroxysmal nocturnal hemoglobinuria;
  • CD157

Abstract

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

Background

Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH.

Methods

CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15, and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages.

Results

Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R2 >0.99) with predicate values over a range (0.06%–99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples.

Conclusions

While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs. © 2013 International Clinical Cytometry Society

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare, acquired stem cell disorder caused by a somatic mutation in the X-linked PIG-A gene ([1-4]). Depending on whether this mutation is a mis-sense or nonsense variant (or both), there is a partial or absolute defect in the biosynthesis and expression of glyco-phosphatidyl inositol- (GPI) linked structures including the complement-defense structures CD55 and CD59 ([5-8]). Absence of CD59 in particular ([9, 10]) and CD55 on red cells is largely responsible for intravascular hemolysis associated with clinical PNH (reviewed in [11]).

Since the early 1990s, flow cytometry to detect GPI-deficient cells has been the method of choice to rapidly diagnose PNH ([12-15]). Early methods relied upon the loss of CD55 and CD59 on red blood cells and granulocytes ([12, 13]), but such approaches were neither sufficiently accurate nor sensitive to detect small PNH clones (<1%) in aplastic anemia (AA) and myelodysplastic syndrome (MDS) cases. While a variety of more sensitive approaches have recently been developed for PNH white blood cell (WBC) detection based on a fluorescent derivative of Pro-aerolysin, FLAER ([16-20]), FLAER-based assays are not yet universally deployed and recent data from external quality assurance (EQA) programs have highlighted the variable capabilities of laboratories to accurately detect WBC PNH clones in stabilized whole blood samples ([21, 22]).

To address these issues, the International Clinical Cytometry Society (ICCS) published guidelines for the diagnosis and monitoring of PNH and related disorders by flow cytometry ([23]). In order to improve PNH WBC detection, the ICCS Guidelines recommended the use of one lineage-gating antibody together with two GPI-linked reagents, one of which should preferably be FLAER. In developing a more detailed ‘Practical Guideline’, we detailed a 4-color combination for high-resolution detection of PNH granulocytes using FLAER, CD24, CD15 and CD45 that is highly sensitive and that can be deployed on a variety of clinical cytometers ([24]). Similarly, a 4-color combination of FLAER, CD14, CD64 and CD45 was developed for high-resolution detection of PNH monocytes ([24]). The granulocyte assay can reliably detect PNH phenotypes at the 0.02% level or higher, while the monocyte assay can reliably detect PNH phenotypes at ≥ 0.04% ([24]). Background rates for the granulocyte and monocyte assays were assessed in 10 normal samples and found to be <0.0013% and <0.0033% respectively ([24]).

The expression of variety of other GPI-linked structures on various blood cell lineages has been documented in peripheral blood ([25, 26]) and bone marrow ([27]). Of these, CD66b was extensively deployed in the detection of PNH granulocytes ([21, 28]). However, until recently, CD66b was available only in FITC-conjugated form, ruling out its use in FLAER-Alexa488-based assays. The recent availability of a PE-conjugated form allowed us to test its utility in comparison with the predicate FLAER/CD24-based granulocyte assay.

CD157 is another GPI-linked structure, expressed on both granulocytes and monocytes, identified previously as a potential reagent for the detection of GPI-deficient cells ([25-27]). The recent availability of PE-conjugated CD157 allows direct comparison with CD24PE and with CD14PE in the 4-C granulocyte and 4-C monocyte protocols respectively.

In this study, we compare the predicate granulocyte assay with one in which CD66bPE was substituted for CD24PE and show herein that it is inferior to the predicate method. We have also compared the predicate granulocyte and monocyte assays with CD157-based equivalents on PNH and normal samples, and show that the CD157 assays perform in an equivalent manner with similar sensitivities on PNH samples and similar low background rates on normal samples.

For those with cytometers equipped with 5 or more photomultiplier tubes (PMTs), it is possible to use CD157 in combination with FLAER, CD15, CD64, and CD45 to detect both granulocytes and monocytes with high sensitivity in a single 5-color tube.

Finally, we have compared the utility of the predicate 4-color granulocyte and monocyte assays with the 4- and 5-color CD157-based assays on stabilized whole blood from PNH patients and show that the CD157 assays perform equally as well as the 4-C predicate assays on such samples.

MATERIALS AND METHODS

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

Antibodies and Conjugates

FLAER was obtained from Cedarlane, Burlington, Ontario, Canada. CD14PE (clone RMO2), CD24PE (clone ALB9), CD15PECy5 (clone 80H5), CD45 PECy7 (clone J33) and CD64PECy5 (clone 22) were obtained from Beckman Coulter, Miami, FL. CD157PE (clone SY11B5), CD24PE (clone SN3), CD15eFluor450 (clone MMA) and CD14PE (clone 61D3) were generously provided by A. Salazar, eBioscience, San Diego, CA. CD66b FITC and PE conjugates (clone G10F5) were generously provided by A. Chen, Biolegend, San Diego, CA. CD45APC (clone 2D1) was obtained from BD Biosciences San Jose, CA. All individual antibodies and FLAER were verified for appropriate reactivity with target cells and titrated to optimize specific staining performance prior to being cocktailed for use in the high-sensitivity granulocyte and monocyte assays as detailed elsewhere ([24]).

WBC Staining Procedure for Predicate Granulocyte and Monocyte Cocktails

100 μL of fresh (less than 24-h-old) EDTA anti-coagulated peripheral blood was carefully pipetted into the bottom of a test tube. An appropriate volume of “granulocyte” (FLAERAlexa488, CD24PE, CD15PECy5, and CD45PECy7) or “monocyte” (FLAERAlexa488, CD14PE, CD64PECy5, and CD45PECy7) cocktail was added directly to the blood and mixed gently. For experiments performed on the FACSCalibur (BD Biosciences San Jose, CA), CD45APC was used in place of CD45PECy7. After a 20-min incubation period in the dark, the RBCs were lysed and the samples fixed with Immunoprep or Versalyse (Beckman Coulter), washed once with PBS, resuspended in 1 mL of PBS and acquired on the cytometer.

CD66b-Based Granulocyte Assay

CD66bPE was cocktailed with FLAER, CD15PECy5, and CD45APC for the granulocyte assay. Sample staining and processing was performed as above.

CD157-Based Cocktails for Granulocyte and Monocyte Assays

CD157PE was cocktailed with FLAER, CD15PECy5 and CD45PECy7 (granulocyte assay) or with FLAER, CD64PECy5, and CD45PECy7 (monocyte assay). CD45APC was substituted for CD45PECy7 for samples analyzed on the FACSCalibur. Samples were stained and processed exactly as described above for the predicate 4-color granulocyte and monocyte assays.

CD157-Based Cocktails for 5-Color Granulocyte/Monocyte Assay

A 5-color cocktail comprised of FLAER, CD157PE, CD64ECD, CD15PECy5, and CD45PECy7 was prepared and sample staining and processing was performed as above.

Verification and Validation of CD157-Based High-Sensitivity Granulocyte and Monocyte Assays

Frequencies of cells with PNH phenotype in normal samples

The frequency of cells with PNH phenotype among granulocytes and monocytes in 10 normal samples was determined as described previously using the predicate FLAER-CD24-CD15-CD45 granulocyte and FLAER-CD14-CD64-CD45 monocyte assays ([24]). The normal samples were similarly screened using the 4-color and 5-color CD157-based granulocyte and monocyte assays.

For the 4-color granulocyte assays, data was acquired for 100,000 CD15-gated granulocytes and the number of FLAER-negative/CD157-negative events was determined. For the monocyte assays, data was acquired until at least 20,000 CD64-gated monocytes were acquired or for a maximum of 10 minutes and the number of FLAER-negative/CD157-negative events was determined.

For the 5-color granulocyte/monocyte assays, data was collected until at least 20,000 CD64-gated monocytes were collected, or for a maximum of 10 min.

CD157-based WBC assay sensitivity

A fresh PNH sample was diluted serially 1:10, 1:100, 1:1,000, and 1:10,000 (vol:vol) with a normal blood sample as described previously ([24]), and stained with CD157-based cocktails to establish the sensitivity of the CD157-based assays. At least 100 FLAER-negative/CD157-negative PNH granulocytes or FLAER-negative/CD157-negative PNH monocytes were collected except at greatest dilutions, at which data was collected for a maximum of 10 min per tube.

Evaluation of High-Sensitivity White and Red Blood Cell Assays on Stabilized Whole PNH Blood

A sample of PNH blood was stabilized within 24 h of sample draw using a previously described process ([29]). The stabilized sample was obtained from Dr. J. Azcona-Olivera, R&D Systems Inc., Minneapolis, MN and was tested for PNH WBC content (granulocytes and monocytes) using the predicate 4-color granulocyte and monocyte assays as well as the CD157-based granulocyte/monocyte assays.

We also tested our high-sensitivity red blood cell assay on the stabilized material using a combination of CD235aFITC and CD59PE ([24]). Two combinations were assessed: Combination A, CD235a (clone KC16, Beckman Coulter) and CD59 (clone MEM43, Invitrogen); Combination B, CD235a and CD59 (clones 10F7MN and OV9A2 respectively, eBioscience).

Flow Cytometry

Initial assessments, titrations and validation of individual antibody conjugates as well as initial validation of instrument platform-specific cocktails were performed on both the FC500 (Beckman Coulter) and FACSCalibur cytometers. Samples stained with 4- and 5-color assays were analyzed on an FC500 cytometer (Beckman Coulter) equipped with a single 488 nm laser and 5 PMTs. In some cases, samples stained with the 5-color cocktail were analyzed on a Navios cytometer (Beckman Coulter) equipped with 3 lasers and 10 PMTs. A 5-color assay comprising FLAER, CD157PE, CD45PerCP (clone 2D1, BD Biosciences), CD64APC (clone 10.1, BD Biosciences) and CD15eFluor450 (clone MMA, eBiosciences) was used to stain cells for analysis on a FACSCanto II cytometer (BD Biosciences) equipped with 3 lasers and 8 PMTs.

RESULTS

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

Comparison of CD66bPE and CD157PE with Predicate Granulocyte and Monocyte Assays

To address the utility of CD66b we first titrated the PE-conjugated form alongside the FITC-conjugated version. The PE-conjugated form was no brighter than the FITC form, nor did it improve the delineation of granulocytes from monocytes over the range of antibody concentrations tested (data not shown).

Similarly, we titrated the CD157PE reagent to optimize the specific staining of granulocytes and monocytes in comparison with the internal unstained lymphocytes. CD157 very effectively separated stained granulocyte and monocyte populations from the unstained lymphocytes at all concentrations (data not shown).

We then compared the ability of optimally titrated CD66bPE or CD157PE to substitute for CD24PE in the predicate 4-color granulocyte assay on the FACSCalibur instrument (FLAER, CD24PE, CD15PECy5, CD45APC). As shown in Figure 1, a PNH sample was stained with the 3 cocktails. Granulocytes were identified by a combination of light scatter, CD45 staining and bright CD15 gating as described previously ([24]). Gated granulocytes were analyzed for PNH phenotypes by either the predicate cocktail (FLAER and CD24PE), or by FLAER and CD66bPE, or by FLAER and CD157PE. Compared to the predicate CD24-based assay, CD66b was much less capable of separating the PNH from normal cells. In contrast, the CD157 very clearly showed excellent separation of the PNH and normal granulocytes.

image

Figure 1. Comparison of CD24, CD66b, and CD157 in 4-color PNH granulocyte assay. A fresh sample from a long-term PNH patient was stained with the predicate 4-color granulocyte assay (FLAER, CD24PE, CD15PECy5, and CD45APC), or in duplicate assays in which CD24PE was replaced by either CD66bPE or CD157PE as described in the Materials and Methods. Light scatter voltages were established so that all nucleated cells were visible above the forward scatter threshold (top left) and debris excluded with a combination of light scatter and CD45 gating (top middle). CD45+ events were displayed on CD15 versus SS plot (top right) and granulocytes (bright CD15, high SS) were gated. Gated granulocytes were displayed on a FLAER versus CD24 plot (bottom left) or FLAER versus CD66b (bottom middle) or FLAER versus CD157 (bottom right). PNH granulocytes were enumerated in the lower left quadrant of each plot. Normal granulocytes were enumerated in the upper right quadrant. Data acquisition was performed on a FACSCalibur cytometer and analysis performed with Flowjo software (v 9.4.1).

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In a second set of experiments, CD157 exhibited very good separation of PNH from normal monocytes, comparable with that obtained with CD14 in the predicate monocyte assay (FLAER, CD14PE, CD64PECy5, CD45APC) (data not shown).

WBC Clone Sizes with CD157-Based Assays

Given the very good separation between PNH and normal granulocytes and between PNH and normal monocytes with the CD157-based 4-color cocktails, we instituted a more extensive study comparing the predicate CD24 (granulocytes) and CD14 (monocytes) assays with those incorporating CD157 (Fig. 2). Granulocytes (right column) were gated as described in Figure 1. PNH granulocyte clone sizes were 90.1% and 89.1% using the CD24- and CD157-based assays respectively.

image

Figure 2. Comparison of 4-color CD157-based assays with predicate 4-color CD24-based granulocyte and CD14-based monocyte assays. A fresh sample from a long-term PNH patient was stained with the predicate monocyte (top left) and granulocyte assays (top right) [24], or the CD157-based monocyte (bottom left) and granulocyte (lower right) assays. Data was acquired on FC500 and analyzed with Flowjo software. Very similar numbers of PNH monocytes (bottom left) and granulocytes (bottom right) were detected with the CD157-based assays as were detected with their respective predicate equivalents (monocytes top left, granulocytes top right).

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Monocytes (left column) were gated as described ([24]) using a combination of light scatter, CD45 staining and CD64 staining. PNH monocyte clone sizes were 88.2% and 87.8% for the CD14- and CD157-based assays respectively.

Comparison of WBC Clone Sizes with Predicate and 4-Color CD157-Based Assays

20 PNH samples were assessed with 4-color CD157-based granulocyte and monocyte assays and compared with predicate 4-color CD24- and CD14-based granulocyte and monocyte assays. As shown in Figure 3A and 3B, a good correlation was observed between the two methods over a wide range of PNH clone sizes (range, 0.17%–99.8% for PNH granulocytes; 0.35%–99.8% for PNH monocytes). A strong correlation (R2 = 0.999) and little bias was found between the CD157-based assays and the predicate assays for both granulocytes (Fig. 3A, y = 0.9987x–0.2012) and monocytes (Fig. 3B, y = 1.0039x–0.0446).

image

Figure 3. Correlation of PNH granulocyte and monocyte clone sizes detected with predicate and CD157-based assays. A: Clones detected with predicate CD24-based and 4-color CD157-based granulocyte assays. B: Clones detected with predicate CD14-based and 4-color CD157-based monocyte assays. C: Granulocyte clones detected with predicate CD24-based and 5-color CD157-based assays. D: Monocyte clones detected with predicate CD14-based and 5-color CD157-based assays.

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Assessment of Background PNH Phenotypes in Normal Samples Using 4-Color CD157-Based Assays

We next assessed the rate of background PNH phenotypes with the 4-color CD157-based granulocyte and monocyte assays (Fig. 4). In 10 normal samples tested in this study, background rates for both granulocyte and monocyte assays were 0–10 events per million (data not shown). These data are very similar to those already published for the predicate CD24 based granulocyte and CD14-based monocyte assays ([24]). Of note, as shown in Figure 4, a second population of cells was identified within the CD15-positive “granulocyte” population (upper and lower right plots). When CD15-gated “granulocytes” were stained with the predicate CD24-based assay (upper right) this extra population was only partially resolved with slightly increased CD24 expression that overlapped the main neutrophil cluster. When the CD15-positive cells were stained with the CD157 cocktail (lower right), this second population resolved with lower CD157 expression. This extra population is most likely eosinophils, as they expressed less CD15, greater CD45 and generally higher side scatter than bona fide neutrophils.

image

Figure 4. Comparison of background PNH phenotypes in normal samples using predicate and 4-color CD157-based assays. A normal PB sample was stained with predicate monocyte (top left) or granulocyte (top right) assays or CD157-based 4-color monocyte (bottom left) or granulocyte (bottom right) assays. All data acquired on FC500 and analyzed with Flowjo.

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WBC Clone Sizes with Single Tube 5-Color CD157-Based Assay

The utility of CD157 to serve as the universal GPI-specific reagent for both granulocytes and monocytes raised the possibility of developing a single tube 5-color cocktail to simultaneously detect PNH cells in both populations with high sensitivity. A combination of FLAER, CD157PE, CD64ECD, CD15PECy5 and CD45PECy7 was evaluated for this purpose. An example of the 5-color assay performed on an FC500 cytometer is shown in Figure 5. A Boolean gating strategy similar to the individual 4-color assays was used to identify CD15+ granulocytes and CD64+ monocytes. Gated granulocytes and monocytes were examined with FLAER versus CD157 plots to identify PNH granulocyte and monocyte populations based on their FLAER-negative, CD157-negative phenotypes. Lymphocytes, used as internal negative controls for the assay, are also displayed to demonstrate optimal PMT set-up, optimal compensation set-up, and verify expected reagent performance.

image

Figure 5. 5-color single tube CD157-based assay for PNH granulocyte and monocytes. A fresh sample from a long-term PNH patient was stained with FLAER, CD157PE, CD64ECD, CD15PECy5, and CD45PECy7 and data acquired on FC500. Debris removed with light scatter (top row, left) and CD45 gating (top row, middle). Granulocytes (middle row, left) and monocytes (middle row, middle) were identified and gated based on CD15 and CD64 expression respectively. Gated granulocytes (bottom row, left) and monocytes (bottom row, middle) were displayed on FLAER versus CD157 plots and PNH granulocytes and monocytes (FLAER-negative, CD157-negative) identified. Gated lymphocytes were identified and sequentially gated based on CD45 and SS (top row middle) and lack of CD64 staining (not shown) and displayed on FLAER versus CD157 (top row right), or FLAER versus CD64 (middle row, right) and FLAER versus CD15 (bottom row, right).

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Comparison of WBC Clone Sizes with Predicate and 5-Color CD157-Based Assays

Nineteen PNH samples were assessed with the predicate 4-color granulocyte and monocyte assays and the single tube CD157-based 5-color assay. As shown in Figures 3C and 3D, very similar clone sizes were observed with the predicate 4-C assays and the 5-C assay over a range of PNH clone sizes (range, 0.06%–99.8% for PNH granulocytes and 0.07%–99.49% for PNH monocytes). A similar strong correlation (R2 = 0.999) was found between the 5-C CD157-based assay and the predicate 4-C assays for both granulocytes, Figure 3C (y = 0.9943x–0.0331), and monocytes, Figure 3D (y = 0.9899x + 0.5843).

Rate of Background PNH Phenotypes in Normal Samples Using 5-Color CD157-Based Assay

We assessed the background levels of PNH granulocytes and monocytes in normal samples with the single tube 5-color CD157-based assay. In the example from data acquired on a Navios cytometer shown in Figure 6, only 1 event with a PNH granulocyte phenotype was detected among almost 150,000 CD15-gated granulocytes while zero PNH monocyte phenotypes were detected in almost 20,000 CD64-gated monocytes. Among 10 normal samples in which a mean of 160,000 granulocytes were collected (range, 68,000–254,000) only a mean of 1 PNH granulocyte was detected (range 0 – 2). A mean of 18,000 monocytes were collected (range, 12,250–28,500) and only a single PNH monocyte was detected over the same ten samples. For granulocytes, these data are very similar to those published previously with the predicate 4-color assay. For monocytes, the background rates with the 5-color assay are even lower than observed with the predicate 4-color assay ([24]).

image

Figure 6. Background rate of PNH granulocytes and monocytes with 5-Color CD157-based assay. Normal PB sample stained with FLAER, CD157PE, CD64ECD, CD15PECy5, and CD45PECy7 and data acquired on Navios cytometer. Analysis performed as described in legend to Figure 5.

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Assay Sensitivities of 4- and 5-Color CD157-Based Assays

A fresh PNH sample containing ∼89% PNH granulocytes and 89% PNH monocytes was serially diluted from 1:10 to 1:10,000 with a normal peripheral blood sample as described previously ([24]), then stained using the 4-C and 5-C CD157-based granulocyte and monocyte assays. As shown in Table 1, granulocyte assay sensitivities for the 4- and 5-C assays were very similar at around 0.01%, as were the 4- and 5-C monocyte assays at around 0.03%. These findings are very similar to the predicate 4-C CD24- and CD14-based granulocyte and monocyte assays respectively ([24]).

Table 1. Assay Sensitivities for 4- and 5-Color CD157-Based WBC Assays
Sample dilutionGranulocyte assayMonocyte assay
4-Color CD157 Assay Sensitivities
1:103.76%3.65%
1:1000.29%0.276%
1:10000.034%0.039%
1:100000.003%ND
5-Color CD157 assay sensitivity
1:103.4%3.99%
1:1000.26%0.32%
1:10000.03%0.03%
1:100000.003%ND

“Type II” PNH Populations in Granulocyte and Monocyte Lineages

The presence of cells with partial FLAER/CD24 deficiency (type II cells) has been reported in the granulocyte and to a lesser extent in the monocyte lineages ([30]). The ability to delineate type II phenotypes has been improved with the use of FLAER-based assays that better separate PNH clones from normal cells compared with traditional flow assays that employed antibodies to individual GPI-linked structures ([30]). Thus, when using the predicate 4-color CD24- and CD14-based granulocyte and monocyte assays on samples containing type II phenotypes, most of the separation was determined by differential FLAER staining rather than by the CD24 and CD14 on the type II granulocytes and monocytes respectively. The substitution of CD24 and CD14 with CD157 in the 4-color and 5-color assays resulted in excellent separation between type III, type II and normal cells for both FLAER and CD157 expression in patient samples containing type II populations. An example from such a sample acquired on an FC500 is shown in Figure 7. Although the clinical significance of the type II white blood cells is uncertain at this time, it is important to recognize these type II populations when present and include them in the total PNH clone.

image

Figure 7. Comparison of predicate WBC assays with 5-color CD157-based assay on PNH sample containing Type II WBCs. Top row shows predicate granulocyte (right) and monocyte (left) assays on long-term PNH patient with PNH Type II WBCs. Data acquired on FC500 and analyzed as described in legend to Figure 2. Bottom row shows the analysis of granulocytes (right) and monocytes from the same sample stained with CD157-based 5-color assay. Data acquired on Navios cytometer and analyzed as described in legend to Figure 6.

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Comparison of 4- and 5-C WBC Assays on Stabilized Whole PNH Blood

A stabilized PNH whole blood sample manufactured to contain ∼5% PNH granulocytes was tested with the various assay formats.

As shown in Figure 8 top row, all three assays detected very similar levels of PNH granulocytes in the stabilized sample. Assessment of monocytes also resulted in the generation of very similar data (data not shown). Analysis of two other stabilized samples manufactured to contain 3% and 0.5% PNH granulocytes respectively showed that all 4-C and the 5-C assays generated very similar data for PNH clone sizes for both granulocyte and monocyte lineages (data not shown). Some minor adjustments to the acquisition protocols relative to fresh samples were required before and after data acquisition. Specifically, the forward scatter gain was increased to account for the lower scatter of the stabilized cells. Additionally, minor adjustments to the compensation matrices were also required due to increased autofluorescence of the fixed cell populations, but these compensation adjustments were made post-data acquisition.

image

Figure 8. Comparisons of PNH granulocyte clone size with predicate, 4-color CD157- and 5-color CD157-based assays on stabilized PB. Top row: A sample from a PNH patient was stabilized and admixed with stabilized normal blood as described in the Materials and Methods to generate a PNH granulocyte content of about 5%. The sample was stained with the predicate granulocyte (FLAER, CD24PE, CD15PECy5, and CD45PECy7), 4-color CD157 granulocyte (FLAER, CD157PE, CD15PECy5, and CD45PECy7) and 5-color granulocyte (FLAER, CD157PE, CD64ECD, CD15PECy5, and CD45PECy7) assays. CD15-gated granulocytes from each assay were displayed on FLAER versus CD24 plot (top left), FLAER versus CD157 plot (4-color assay, top middle) and FLAER versus CD157 (5-color assay, top right). Bottom row: A sample of the stabilized blood was stained with CD235FITC (clone 10F7MN) and CD59PE (clone OV9A2) as described previously for high-sensitivity PNH RBC detection ([24]). Red blood cells were identified by a combination of light scatter (left) and CD235 expression (middle) and assessed for CD59 expression (right). All data acquired on FC500 and analyzed in Flowjo.

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Finally, the stabilized PNH blood samples were also assessed with the previously described high-sensitivity RBC assay ([24]). As shown in Figure 8 (stabilized sample containing 5% PNH granulocytes), these stabilized samples were suitable for the clear assessment and enumeration of PNH RBC clones.

SUMMARY

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

The development and validation of sensitive, standardized, flow cytometric methodologies to detect PNH was until recently, hampered by the rarity of this disease and technical difficulties in the accurate identification of PNH cells. This was addressed in 2010 by the publication of the ICCS Guidelines for the diagnosis and monitoring of PNH by flow cytometry ([23]). The subsequent publication of the ‘Practical Guidelines’ describing specific reagent cocktails and detailed analytic strategies has further promoted the use of simple yet robust assays to detect PNH red and white blood cells that can be deployed on a range of flow cytometers with 4 or more PMTs ([24]). These assays could reliably detect PNH granulocyte and monocyte clones at levels of 0.02% and 0.04%, respectively.

In this study we have addressed the utility of CD66bPE and CD157PE as potential alternative GPI-specific structures to CD24PE in the granulocyte assay and CD14PE in the monocyte assay. Unfortunately, we found the separation between PNH and normal granulocytes with CD66b to be sub-optimal compared with the predicate CD24PE reagents (clone ALB9, Beckman Coulter and clone SN3, eBioscience). Therefore we do not endorse the use of CD66b as an improved reagent over the currently recommended CD24.

CD157, a member of the CD38 supergene family and an ectoenzyme, functions as an integrin receptor and signaling molecule ([31]). It is known to be expressed at relatively high antigen density on neutrophils and monocytes ([25-27]), although these studies were performed with a FITC conjugate of a different clone (RF3, Beckman Coulter). We show here that CD157-based assays perform at least as well as our CD24- and CD14-based predicates with PNH samples containing a wide range of clone sizes. Furthermore, background rates of PNH phenotypes in normal samples showed the same very low levels as the predicate assays. Overall, the CD157-based assays performed equally as well as the existing 4-color predicate assays previously published ([24]) that form the basis of recommendations on PNH testing in the Clinical and Laboratory Standards Institute H52-A2 document ([32]). All 4-color assays described in this study also performed with similar efficiency on the FACSCalibur, platform either in combination with FLAER, CD15PECy5 (or CD64PECy5 for the monocyte assay) and CD45APC, or with FLAER, CD45PerCP, and CD15APC (or CD64APC for the monocyte assay) ([33]).

The ability of CD157 to simultaneously serve as a GPI-specific marker for both neutrophil and monocyte lineages raised the possibility of a single tube 5-color PNH WBC assay. We found that the 5-color CD157-based assay detects both PNH neutrophils and PNH monocytes with high sensitivity and with the same low background rates on normal samples as noted with the predicate 4-color assays ([24]). This is advantageous for laboratories with flow cytometers equipped with 5 or more PMTs, which perform frequent testing for PNH and related disorders. Indeed, the 5-color assay described herein, based on FLAER, CD157PE, CD64ECD, CD15PECy5, and CD45PECy7 generated equivalent data to the predicate and CD157-based 4-color assays on samples containing a wide range of PNH clone sizes on our FC500 instruments equipped with a single laser and 5 PMTs. This same combination was also assessed on a Navios cytometer equipped with 10 PMTs, deriving virtually identical data on the same samples.

In other experiments, the 5-color cocktail of FLAER, CD157PE, CD45PerCP, CD64APC, and CD15eFluor450 was capable of performing the same high-sensitivity analysis on a FACSCanto II instrument (data not shown).

One aspect in which the CD157-based assays differed from the CD24- and CD14-based predicates was in the ability of CD157-based assays to better delineate type II and type III granulocytes and type II and type III monocytes. While the clinical significance of the presence of type II white blood cells is unknown, it is important to include these cells in the overall assessment of PNH clone sizes for both granulocyte and monocyte lineages.

In our previous publication ([24]), we recommended that laboratories use a tiered or sequential approach to the analysis of samples submitted for PNH testing. This approach involved testing all samples with the high sensitivity 2-color RBC and 4-color granulocyte assays, followed by a reflex monocyte assay in cases where PNH phenotypes were identified with RBC and/or granulocyte assays. In this setting, we recommended pre-cocktailed 2-color RBC and pre-cocktailed 4-color granulocyte reagents, while the reflex monocyte reagents were only utilized on an ‘as needed’ basis. We continue to believe that this is an efficient and cost-effective approach for laboratories that do not perform frequent PNH testing, as recently underscored by the study of Marionov et al. who demonstrated good intra- and inter-laboratory correlation, precision and reproducibility between laboratories in a multi-center study, independent of PNH clone sizes ([34]).

However, for laboratories in major centers or reference laboratories that perform routine testing for PNH and related diseases, the single tube 5-color approach we describe herein has some significant advantages. Only a single 5-color WBC reagent cocktail is needed, thereby reducing sample preparation time, opportunities for pipetting errors, technical time, time needed for data acquisition/analysis, and significantly improving overall laboratory efficiency.

While not a critically important aspect of this study, stabilized whole blood samples are increasingly used by EQA schemes such as UK NEQAS ([21, 33]), QMP-LS ([22]), and others. As shown above, all 4-color assays used in this study were able to detect and enumerate PNH granulocyte and monocyte phenotypes in a series of stabilized whole blood samples manufactured to contain a range (0.5%, 3% and 5%) of PNH granulocyte clones (22, MK and DRS unpublished data). Similarly, the CD157-based 5-color assay was able to accurately enumerate PNH granulocytes and monocytes in these stabilized samples. We previously assessed the stabilized samples with the high-sensitivity RBC assay and found this material to be suitable for the assessment of PNH RBCs ([22]). Thus, all WBC and RBC assays described herein and previously ([24]) can be used with stabilized PNH whole blood preparations that are increasingly used in EQA programs.

ACKNOWLEDGMENTS

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

The authors gratefully acknowledge Ms. Angela Salazar (eBioscience) for providing several of the antibody conjugates used in this study. They also acknowledge Mr. Andrew Chang (Biolegend) for providing the CD66b conjugates. They thank Mr. Juan Azcona-Olivera (R&D Systems) for the stabilized PNH blood samples used in this study. They are especially grateful to Mr. John Quinn (Tree Star Inc.) for help with FlowJo analysis. DRS, AI, and MK have consulted for Alexion Pharmaceuticals and have taken part in Advisory Board meetings and received speaker fees.

LITERATURE CITED

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