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Keywords:

  • paroxysmal nocturnal hemoglobinuria;
  • standardization;
  • quality control;
  • quality assessment;
  • EQA

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

Background

Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol-deficient structures on red blood cells and white blood cells in paroxysmal nocturnal hemoglobinuria (PNH) were recently published.

Methods

UK NEQAS LI issued three stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific Standard Operating Procedures (SOPs) and pretitered antibody cocktails to 19 international laboratories experienced in PNH testing. Samples were tested using both standardized protocol/reagents and in-house protocols. Additionally, samples were issued to all participants in the full PNH External Quality Assessment (EQA) programs.

Results

Expert laboratory results showed no difference in PNH clone detection rates when using standardized and their “in-house” methods, though lower variation around the median was found for the standardized approach compared to in-house methods. Neutrophil analysis of the sample containing an 8% PNH population, for example, showed an interquartile range of 0.48% with the standardized approach compared with 1.29% for in-house methods. Results from the full EQA group showed the greatest variation with an interquartile range of 1.7% and this was demonstrated to be significantly different (P < 0.001) to the standardized cohort.

Conclusions

The results not only demonstrate that stabilized whole PNH blood samples are suitable for use with currently recommended high-sensitivity reagent cocktails/protocols but also highlight the importance of using carefully selected conjugates alongside the standardized protocols. While much more variation was seen among the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories. © 2014 International Clinical Cytometry Society

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening acquired hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidylinositol glycan complementation Class A (PIG-A) gene [1-4]. In normal individuals, this gene encodes an enzyme involved in the first stage of glycophosphatidylinositol (GPI) biosynthesis. In PNH, as a result of PIG-A mutation(s), there is a partial or absolute inability to make GPI-anchored proteins, including complement-defense structures such as CD55 and CD59 on red blood cells (RBCs) and white blood cells (WBCs) [5-8]. Absence of CD59 in particular [9] and CD55 on RBCs is largely responsible for intravascular hemolysis associated with clinical PNH (reviewed in 10).

Clinical features of PNH include complement mediated intravascular hemolysis, a thrombotic tendency, and bone marrow failure with associated cytopenias. These clinical features negatively affect the quality of life and the life expectancy of PNH patients, with thrombosis being the major cause of death [11, 12].

There is a well-documented relationship between aplastic anemia (AA) and PNH [11, 12], with some studies reporting up to 70% of AA patients having PNH clones (reviewed in 13). Additionally, small PNH clones have been reported in patients with early stage myelodysplastic syndrome (MDS), particularly the refractory cytopenias with unilineage dysplasia variant [12, 14]. However, a recent statistical reanalysis of data from a large study (EXPLORE) of patients with AA, MDS, and other bone marrow failure syndromes found only a low incidence of PNH clones in MDS [15].

Since the early 1990s, flow cytometry to detect cells lacking GPI-anchored surface molecules has become the method of choice for the diagnosis and monitoring of PNH and related disorders [16-19]. However, many laboratories retained the use of CD55- and CD59-based assays to detect GPI-deficient RBCs and WBCs rather than making use of the increasingly wide range of monoclonal antibodies now available to detect GPI-anchored proteins on WBCs in particular [20]. While a variety of more sensitive approaches have subsequently been developed for PNH, WBC detection based on the fluorescent derivative of the bacterial lysin Proaerolysin (FLAER) [21-26] (FLAER-based assays) are still not widely deployed. Recent data from the UK NEQAS PNH proficiency testing programs have highlighted wide variations in reagent selection, testing protocols for neutrophils, and variable capabilities of laboratories to accurately detect WBC PNH clones in stabilized whole blood samples [27].

To address these issues, the International Clinical Cytometry Society (ICCS) published flow cytometric guidelines for the diagnosis and monitoring of PNH and related disorders by flow cytometry [28]. For PNH WBC detection, the ICCS Guidelines recommended the use of one antibody for lineage gating together with two GPI-linked reagents, one of which should be FLAER. This reagent has proven to be well suited to detect GPI-deficient cells in peripheral blood samples as it has an excellent signal to noise ratio [21-26]. However, SOPs utilizing specific assay cocktails were not identified in the ICCS Guidelines. In developing more detailed “Practical Guidelines,” a 4-color combination using FLAER, CD24, CD15, and CD45 was proposed for high-sensitivity detection of PNH neutrophils that could be deployed on a variety of clinical cytometers [29]. Similarly, a 4-color combination using FLAER, CD14, CD64, and CD45 was proposed for high-sensitivity detection of PNH monocytes and a 2-color combination using CD235aFITC and CD59PE was developed for high-sensitivity detection of PNH RBCs [29]. While specific clones and conjugates were identified along with detailed SOPs for these assays, the degree of implementation and adoption of these guidelines is not yet known.

With the recent availability of stabilized whole blood PNH preparations from UK NEQAS LI [27] and others that are suitable for testing the standardized reagent combinations [29, 30], we have attempted to determine the effect of using standardized conjugates and protocols for the detection of GPI-deficient PNH phenotypes among 19 experienced laboratories.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

As of June 2013, the UK NEQAS LI PNH program had 156 international participants. For the purpose of the study, the decision was made to examine the effects of using a standardized protocol among experienced laboratories only. A questionnaire was issued to all participants inquiring how many PNH tests were performed per annum. The laboratories with the highest throughput of new PNH cases per annum were short listed. This list was refined further by selecting those centers demonstrating satisfactory performance within the PNH EQA program over the previous 12 months. Finally, as the intention was to assess platform-specific protocols and reagents, the selected laboratories were placed into one of two groups depending on the flow cytometer platform being used.

After obtaining informed consent, blood samples were collected from patients with known PNH at St James' University Hospital, Leeds. These were immediately transported a distance of 35 miles to UK NEQAS LI in Sheffield. The samples were stabilized using a procedure previously described [31]. Stabilized PNH material was admixed into stabilized whole blood to create samples with approximately 0.1% and 8% GPI-deficient PNH leucocyte populations. For a normal sample (0% GPI-deficient cells), stabilized whole blood from a normal donor was used. Aliquots (1 ml) of the samples were prepared and tested to ensure they were suitable for use based upon flow cytometric staining characteristics.

As mentioned previously, CD55 and CD59 have historically been used for detecting GPI-deficient neutrophils in PNH [16, 17]. For this study, a number of other clones/conjugates were evaluated (generously donated by Beckman Coulter, High Wycombe, England; BD Biosciences, Oxford, England; and eBioscience, San Diego, CA).

After extensive validation, verification, and titration to optimize individual reagent performance the following conjugates were selected to analyze GPI-deficient neutrophils on Beckman Coulter cytometers: FLAER (Cedarlane Laboratories Burlington, Ontario, Canada), CD24PE (clone SN3, eBioscience), CD15PECy5 (clone 80H5, Beckman Coulter), and CD45PECy5 (clone J33, Beckman Coulter). To detect GPI-deficient monocytes on the Beckman Coulter platform FLAER, CD14PE (clone 61D3, eBioscience), CD64PECy5 (clone 22 Beckman Coulter), and CD45PECy5 were selected. To detect GPI-deficient neutrophils on the BD Biosciences cytometers, FLAER, CD24PE, CD15APC (clone HI98, BD Biosciences) and CD45PerCP (clone 2D1, BD Biosciences) were selected. To detect GPI-deficient monocytes on the BD Biosciences cytometers, FLAER, CD14PE, CD64APC (clone 10.1, BD Biosciences) and CD45PerCP, and were selected.

Once optimal performance of the selected individual antibodies had been verified, a series of 4-color cocktails of the reagents were made in an instrument platform-specific manner (Table 1). Subsequently, the four cocktails were evaluated on both normal and several fresh PNH samples to verify their performance on their respective instruments. SOPs and examples of the staining patterns for each of the cocktails generated on their respective instruments with fresh samples are shown in Appendix A (Supporting Information). Although exact cocktail volumes were prescribed as shown in Appendix A (Supporting Information), it is important to note that individual laboratories that wish to generate their own reagent cocktails should determine optimal reagent volumes only after appropriate verification and titration of their own individual conjugates. Gating strategies used were exactly as those previously detailed in the Practical Guidelines [29].

Table 1. Standardized Panels for Each Flow Cytometer Type with Details of Conjugate and Clone of Each Reagent Shown in Parentheses
Standardized Reagent Panels
Beckman Coulter CytometersBecton Dickinson Cytometers
NeutrophilsMonocytesNeutrophilsMonocytes
FLAER (Alexa 488)FLAER (Alexa 488)FLAER (Alexa 488)FLAER (Alexa 488)
CD24 (PE/SN3)CD14 (PE/61D3)CD24 (PE/SN3)CD14 (PE/61D3)
CD15 (PECy5/80H5)CD64 (PECy5/22)CD15 (APC/HI98)CD64 (APC/10.1)
CD45 (PECy7/J33)CD45 (PECy7/J33)CD45 (PerCP/2D1)CD45 (PerCP/2D1)

The 19 international centers selected for the study were issued with 1 ml aliquots of each of the three stabilized whole blood samples prepared as described above. Pretitrated aliquots of antibody cocktails for 4-color neutrophil and 4-color monocyte assays together with SOPs for both assays optimized for each instrument platform (for either Beckman Coulter or BD Biosciences instruments) were also provided. Each center was requested to test the samples using the provided standardized reagents and SOPs and with their current in-house methodologies. Results were then submitted to UK NEQAS in terms of both “PNH clone size” and classification of “clone present/clone absent.” The testing labs were blinded to the actual values of each sample.

To further compare how standardization of the 19 centers improved their performance, we also issued the three samples to all laboratories in the UK NEQAS LI PNH programs requesting them to assay the samples with their routine procedures. These results were then compared to the 19 laboratory cohort.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

Three stabilized samples were supplied to participants: Sample A was a normal sample, whereas Samples B and C were manufactured to contain approximately 0.1% and 8% GPI-deficient neutrophil populations, respectively. An example of the staining pattern obtained with the standardized neutrophil and monocyte cocktails on an FC500 platform is shown for Sample C in Figure 1. In the analysis shown, a PNH neutrophil clone of 7.95% was detected. In the same sample, a monocyte clone of 8.96% was detected.

image

Figure 1. Example of stabilized sample (Sample C) with large (8%) PNH clone stained with the standardized neutrophil and monocyte cocktails. For both assays, debris was removed by a combination of light scatter and CD45PECy7 versus side scatter gating (top row). For neutrophil assay, neutrophils were gated based on bright CD15PECy5 expression and high side scatter (middle, left), and assessed for FLAER and CD24PE staining (middle). PNH neutrophils were gated based upon lack of staining with FLAER and CD24PE. Lymphocytes (internal control) were gated based on a Boolean gated combination of CD45PECy7, low side scatter and lack of CD15PECy5 staining and assessed for FLAER and CD24PE staining (middle right). For monocyte assay, monocytes were gated based on bright CD64PECy5 staining and intermediate side scatter (bottom, left) and assessed for FLAER and CD14PE staining (middle). PNH monocytes were gated based on lack of staining with FLAER and CD14PE. Lymphocytes (internal control) were gated based on bright CD45PECy7 staining, low side scatter and lack of CD64PECy5 staining and assessed for FLAER and CD14PE staining (bottom, right). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The qualitative results returned for leucocyte analysis, identifying the presence or absence of “PNH clones,” showed that the standardized protocol and standardized reagents were in consensus with results returned for in-house protocols and local reagents, with all correctly reporting “clone present/clone absent” using both methods. Furthermore, no center detected the presence of a PNH clone in the normal Sample A and all participants of the study correctly reported “clone present” for the Samples B and C containing 0.1% and 8% PNH clones, respectively. The median neutrophils and monocytes PNH clone size in both the samples was similar for the standardized approach (8.05% and 8.57%, respectively) and the in-house protocols (8.10% and 8.80%, respectively). Importantly, however, there was a difference between the standardized and in-house techniques in the range of results returned for PNH clone size. While this was shown to be “not significant” by applying an f-test, it is evident that both B and C samples (0.1% and 8% PNH clone, respectively) showed lower variation around the median for the standardized approach when compared to in-house methods (Table 2). This was true for both the neutrophil and monocyte populations (Figs. 2 and 3, respectively). The reagents and protocols were designed to be platform specific; the data for neutrophil and monocyte analysis on both platforms are shown in Tables 3 and 4, respectively.

image

Figure 2. Box and whisker plot illustrating range of results obtained for in-house and standardized testing of Sample B (0.1% PNH). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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image

Figure 3. Box and whisker plot illustrating range of results obtained for in-house and standardized testing of Sample C (8% PNH).

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Table 2. Comparison of Range of Results Obtained for In-House and Standardized Testing
 Sample A (normal)
 NeutrophilsMonocytes
 In-houseStudyIn-houseStudy
Sample A (Normal)
Median (%)0.000.000.000.00
Range (%)0.010.010.040.00
Interquartile range (%)0.000.000.010.00
Sample B (0.1% PNH)
Median (%)0.100.090.110.13
Range (%)0.330.150.260.20
Interquartile range (%)0.030.020.080.08
Sample C (8% PNH)
Median (%)8.108.058.808.57
Range (%)2.531.694.573.95
Interquartile range (%)1.290.481.530.91
Table 3. Comparison of Results for Neutrophil Analysis Obtained for the Study Group from Both Analytical Platforms
 Becton DickinsonBeckman Coulter
 In-houseStudyIn-houseStudy
Sample A (normal)—Neutrophil Population
Median (%)0.000.000.000.00
Range (%)0.010.000.000.01
Interquartile range (%)0.000.000.000.00
Sample B (0.1% PNH)—Neutrophil Population
Median (%)0.110.100.110.08
Range (%)0.100.040.330.15
Interquartile range (%)0.020.020.020.03
Sample C (8% PNH)—Neutrophil Population
Median (%)8.268.158.358.16
Range (%)2.531.401.091.69
Interquartile range (%)1.550.600.620.50
Table 4. Comparison of Results for Monocyte Analysis Obtained for the Study Group from Both Analytical Platforms
 Becton DickinsonBeckman Coulter
 In-houseStudyIn-houseStudy
Sample A (Normal) Monocyte Population
Median (%)0.000.000.000.01
Range (%)0.000.030.000.04
Interquartile range (%)0.000.000.000.04
Sample B (0.1% PNH) Monocyte Population
Median (%)0.100.100.170.13
Range (%)0.260.260.090.18
Interquartile range (%)0.030.060.030.09
Sample C (8% PNH) Monocyte Population
Median (%)8.708.558.309.25
Range (%)3.771.902.923.95
Interquartile range (%)2.030.491.441.46

The normal Sample A and PNH Sample C (8% clone) used in the study were also issued as part of an EQA trial to all 156 participants within the PNH program (June 2012). Sample B was issued to 40 participants in the high resolution PNH program (July 2012). Leucocyte data from the full EQA program contrasted to that of the 19 labs in the first phase of the study in that the latter correctly identified the presence/absence of PNH clones using both in-house and standardized protocols. However, in the full program, 18 centers incorrectly reported the presence of a neutrophil PNH clone in the normal Sample A. These results ranged from 1% clone detection to as high as 32%. For Samples B and C, containing a 0.1% and 8% PNH clone, respectively, four labs failed to detect PNH phenotypes in Sample B with one participant even failing to detect the PNH clone in Sample C. Results from the quantitative returns from the EQA participants are shown in Table 5 and it is noted for both Samples B and C, that these show a greater variation around the median than the 19 international laboratories in the study group for either the in-house or standardized methods (0.078%, 0.03%, and 0.2%, respectively, for Sample B; 1.7%, 1.29%, and 0.48%, respectively, for Sample C). Using the f-test for statistical analysis of variance between the data sets demonstrated that the EQA data returns were significantly different (p < 0.001) to the data from both the in-house and standardized methods. The data shown in Figures 4 and 5 are for neutrophils only as monocyte data are not, at present requested in the full EQA program.

image

Figure 4. Box and whisker plot incorporating P-values illustrating range of results obtained for in-house and standardized testing of Sample B (0.1% PNH) in comparison to the data from the EQA send out. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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image

Figure 5. Box and whisker plot incorporating P-values illustrating range of results obtained for in-house and standardized testing of Sample C (8% PNH) in comparison to the data from the EQA send out. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Table 5. Summary of Results Submitted for Neutrophil Analysis of Samples Issued to 156 Labs in the EQA Send Out
 Sample A (normal) (Neutrophils)Sample B (0.1% PNH) (Neutrophils)Sample C (8% PNH) (Neutrophils)
n15640156
Median (%)0.000.18.08
Range32.04.933.0
Interquartile range (%)0.000.0781.7

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

The ability to perform high-sensitivity flow cytometric analysis for PNH is important because in a recent large study, 40% of samples containing GPI-deficient WBCs did so at levels of 1% or less [32]. UK NEQAS LI has previously evaluated the efficacy of FLAER as a single color reagent to detect GPI-deficient PNH neutrophils in stabilized whole PNH and normal blood samples. The aim of the current study was to ascertain if highly standardized and instrument-specific FLAER-based, 4-color neutrophil and monocyte protocols could be deployed to accurately detect GPI-deficient cells in stabilized whole blood samples “spiked” to contain PNH cells in the 0.1% and 8% ranges. In also supplying a normal sample, we sought to assess whether participants would correctly identify it as such.

The 4-color protocols selected for this study utilized CD15 as a neutrophil-gating reagent and CD64 for gating monocytes due to the superior performance of these reagents over CD33 in high-sensitivity assays [29, 33, 34]. While it is known that CD15 does not fully delineate neutrophils from eosinophils and there may be low-level inclusion of eosinophils, the gating strategy used excludes the majority [29].

The results returned demonstrated that the overall medians between the in-house and standardized approaches for detection of GPI-deficient leucocyte (neutrophils and monocytes) were similar in a cohort of 19 laboratories chosen because of their expertise in flow analysis of PNH. Additionally, statistical analysis showed no significant difference between the two groups, justifying their selection as expert testing centers. While this outcome was unsurprising, the variation around the medians was lower with the standardized approach than with the in-house methods. Thus for the two populations studied (neutrophils and monocytes) the standardized approach demonstrated an improvement in precision across all laboratories. If such an improvement can be demonstrated among an expert group of laboratories then one would expect a significant improvement if a less experienced group adopted the standardized protocols. Additionally, reagents and protocols were designed to be specific to the flow cytometer being used; no significant differences between the platforms were noted.

The stabilized samples used within the study were also distributed to all 156 participants in the UK NEQAS LI EQA program. Using only in-house methods, this group showed much greater variation around the median. Within this group, however, 18 participating centers incorrectly reported the presence of a neutrophil PNH clone in the normal Sample A. These results ranged from 1% clone detection to as high as 32%. This kind of error, if reproduced in the analysis of clinical samples, could lead to a misdiagnosis of PNH. For Samples B and C (containing a 0.1% and 8% PNH clone, respectively), four labs failed to detect PNH phenotypes in Sample B with one participant even failing to detect the PNH clone in Sample C. This error, if reproduced on a clinical sample would result in a failure to detect PNH, even when large clones are present. Overall, the results from the full EQA group showed a significant difference with regards variability (p < 0.001) when compared to the 19 laboratories using either their “in-house” method or the standardized protocol underscoring the need for wider deployment of standardized reagent sets and modern SOPs. Such an approach might be expected to lead to better laboratory performance in the field of PNH detection.

The ability to detect GPI-deficient cells and monitor PNH clone sizes in patients forms an essential part of their clinical management. The monitoring of RBCs in particular can be useful to assess the efficacy of complement blockade therapy for the control of hemolysis. Patients with classic PNH have large clone sizes and typically present with florid intravascular hemolysis [10-12, 19]. The development of an effective treatment, Eculizumab, a humanized antibody directed against complement component C5 (Soliris™, Alexion Pharmaceuticals, Cheshire, CT) has dramatically altered the course of the disease and improved life expectancy in PNH patients [13, 35-37]. This has underscored the importance of accurate detection and monitoring of patients with PNH and related diseases [28]. PNH clone size also appears to influence thrombophilic propensity [13, 35, 36]. Soliris is one of the world's most expensive drugs and with the potential cost ramifications of any alterations in treatment, it is essential that the assays used to detect PNH phenotypes are both accurate and precise. In addition, given the rare incidence of the disease, a significant amount of international collaboration may be required to fully monitor disease course progression and therapy benefits. As such, it is in the best interest of patients and health service providers to use monitoring techniques that have the lowest amount of intra- and interlaboratory variation such as the standardized protocol and reagent selection process described in this study.

It has previously been shown that using stabilized EQA material together with standardized protocols and targeted training in flow cytometric rare event enumeration of CD34+ stem cells a significant reduction of intra- and interlaboratory variation can be achieved [38]. This study now highlights the importance of both reagent choice and the use of a standardized approach to undertake PNH analysis and has the potential to reduce such variability, even among experienced laboratories. It has also demonstrated, for the first time, that the range of results obtained from a standardized protocol are more precise with lower variation around the median than other nonstandardized approaches. For laboratories where poor performance in EQA is a problem, this multicenter validated protocol, if adopted, provides a basis for improvement. Therefore, it would be beneficial for all centers involved in the testing of PNH to revisit their current techniques and ensure that they are performing at a level of variation similar or better than when using a highly standardized approach described in this study.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

The authors would like to thank Alexion Pharmaceuticals, Beckman-Coulter, BD Biosciences, eBioscience, for providing the antibodies used in this study, and all the laboratories that participated in this study, taking the time to submit results for review, without whom, this study could not have been undertaken.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
  8. Supporting Information

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