How to cite this article: Tsagarakis NJ, Paterakis G. A simple flow cytometric assay for routine paroxysmal nocturnal hemoglobinuria testing based on immature reticulocytes and granulocytes. Cytometry Part B 2012; 82B: 259–263.
The aim of this study was to test an easy-to-perform flow cytometric (FCM) assay for the routine investigation for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), through the simultaneous detection of PNH clones on immature reticulocytes (i-RET) and granulocytes.
During the last 5 years, eight patients were diagnosed with PNH in our laboratory, among 90 patients prospectively studied for PNH. The determination of glycosylphosphatidylinositol (GPI) deficient cells on the erythroid lineage was made with a two-color FCM assay of CD71 and CD59, evaluating the PNH clone on i-RET. Three color combinations based on CD66b/CD16/CD45 and CD59/CD24/CD45 were used for the determination of GPI-deficient granulocytes.
In all the patients with PNH, the PNH clones determined with CD71(+)CD59(−) red blood cells (RBC) were nearly identical to the respective clones determined with CD16(dim/−)/CD66b(−) and CD59(−)/CD24(−) granulocytes, in contrast to the clones determined with CD59-deficient erythrocytes only, which were significantly lower.
Flow cytometry (FCM) plays a key role in the laboratory investigation of paroxysmal nocturnal hemoglobinuria (PNH), and rapid diagnosis of this condition is highly desirable. A definitive diagnosis of PNH can be established by demonstrating the absence of cell membrane glycosylphosphatidylinositol (GPI)-anchored proteins from granulocytes or red blood cells (RBC) (1). A consensus that describes FCM procedures for detecting PNH cells has been recently presented, which focuses on the analytical procedures important for analysis (2).
The affected reticulocytes have been proposed to be a reliable marker for the diagnosis of PNH and for the evaluation of erythropoiesis by PNH stem cells (3). The superiority of reticulocyte analyses over erythrocyte analyses, because of its transfusion and hemolysis independency has been suggested (4). Recently, CD71 intensity on immature reticulocytes (i-RET) was shown to be well correlated with their RNA content levels, indicating the usefulness of CD71 as an i-RET marker (5).
Focusing on the need for a simple and reproducible FCM method for the routine detection of robust PNH cell populations, we present a two-color CD71- and CD59-based evaluation of PNH-RBC clones, in parallel with study of GPI-deficient granulocytes with the use of previously established markers, such as CD16, CD24, CD66b, and CD59, with simple CD45-based three color combinations.
PATIENTS AND METHODS
Among 90 patients prospectively investigated for PNH, eight had significant PNH clones and were diagnosed as having PNH (two males, six females, with a median age of 41.5 years), seven had small GPI-deficient clones (<1%), in the context of aplastic anemia (3) or myelodysplastic syndrome (MDS) (4), whereas no GPI-deficient clone was detected in the remaining 75, based on simultaneous granulocyte and RBC analysis. All 90 patients were analyzed in the context of an extended investigation for unexplained anemia, cytopenia of unknown etiology, unexplained hemolysis or unexplained thrombosis. The validation for PNH diagnosis was conducted according to extended granulocyte testing (2), which was considered to be the reference test for the estimation of the true value of the PNH clone.
The PNH RBC two-color evaluation panel consisted of two tubes, one using CD59-FITC (clone P282E from Beckman Coulter) and CD71-PE (Monoclonal Mouse Anti-Human Transferrin Receptor Antigen, clone T56/14 from Invitrogen) and one using IgG1 isotype controls (FITC- and PE-conjugated, Dako, Denmark). The working solution was prepared by mixing 5 μl of peripheral blood diluted in 5 ml of phosphate buffered saline (PBS). Subsequently, 50 μl of the above diluted solution was stained with 10 μl labeled antibodies (besides CD59-FITC 20 μl, based on titration, not shown), without lysis, and after a one-wash step was finally analyzed after resuspension in 0.5 ml of PBS. It is important to underline that increased care is required not to leave blood traces in the interior on the walls of the tubes during pipetting, so as to avoid false CD59(−) results.
For detection of the reference percentage of the PNH clone on granulocytes, we used two tubes of three color combinations, using a common base of CD45-PE-Cy5 conjugate (clone J.33 from Beckman Coulter). The FITC and PE conjugates used were: CD59-FITC (clone P282E, Beckman Coulter) combined with CD24-PE (clone ALB9, Beckman Coulter) and CD66b-FITC (clone 80H3, Beckman Coulter) combined with CD16-PE (clone 3G8, Beckman Coulter). After a 15-min incubation of peripheral blood (100 μl) with 10 μl labeled antibodies at room temperature, the cells were lysed (BD FACS Lysing Solution, BD Biosciences, CA) for 10 min and centrifuged (1800 rpm, for 5 min). The supernatant fluid was discarded and the cells were resuspended in 0.5 ml of PBS for analysis.
For each sample, cells of intermediate to high forward scatter (FS) were initially gated in FS/side scatter (SS) plots, not only to exclude debris, dead cells, and platelets (low FS/SS) but also to include reticulocytes (high FS). We first analyzed the control tube of PNH RBC two-color evaluation panel, to avoid nonspecific fluorescence (false positive events) in FS/CON-PE-plots that may lead to misdiagnosis (false CD59 negative events). Thus, a different cutoff line was positioned for each pair of control and CD71 application, to obtain true positive events in the subsequent determination of FS/CD71-PE plots. We accepted the absence of positivity or positivity of less than 0.1% in the control tube. Whenever detected, CD71 positivity was sufficiently bright to be easily discriminated as a positive cluster. CD71(+) cells were further evaluated for CD59 negativity (Fig. 1). Considering that the CD71-positive population percentage was sufficiently low, usually in the range of 0.2–0.5%, the CV of the assay depended on the absolute number of CD71positive/CD59 negative acquired events which were used to determine the PNH clone percentage. To get an acceptable CV of 10% approximately 100 CD71+/CD59-events were needed. Aiming at a sensitivity of 5%, the number of the analyzed CD71(+) events should reach 2,000, corresponding to a total of 1 million acquired red cell events.
In the evaluation panel of PNH granulocytes, the PNH cells were identified as either CD16(dim/−)/CD66b(−) (Fig. 2) or CD24(−)/CD59(dim/−) (not shown), whereas the normal granulocytes were discriminated by their high CD16 and CD66b expression (Fig. 2) and their positivity for CD24 and CD59 (not shown). Eosinophils were discriminated as CD16(−)/CD66b(+) (Fig. 2). The cutoff value used to characterize a PNH-granulocyte clone was 1% for any marker examined (aiming at a sensitivity of 1%).
All laboratory data [i.e., RBC, hemoglobin (Hb), hematocrit (Hct), and reticulocytes] were obtained using a Cell-Dyn 4000 Hematology Analyzer (Abbott Laboratories, Abbott Park, IL).
The Bland–Altman assessment for agreement was used to compare the results (percentages of estimated clones) obtained by i-RET (CD71+/CD59− RBC) versus granulocytes (CD16dim/−), and whole RBC (simply CD59−) versus granulocytes (CD16dim/−). A range of agreement was defined as mean bias ±2 SD. The Wilcoxon signed-rank test was used to compare the values obtained by CD71(+)/CD59(−) RBC and CD59(−) RBC as a whole, as obtained by the PNH-RBC evaluation panel. A value of P < 0.05 was considered statistically significant.
The measurements on the eight patients with PNH are shown in Table 1. In all eight, significant PNH-clones (>30%) were detected in the neutrophilic lineage. The neutrophilic PNH clones obtained by CD16(dim/−), CD66b(−), CD59(dim/−), and CD24(−) expression revealed the following median (%) values: 93.8(30.9–99), 74.7(31.8–98.9), 58.5(24–72.6), and 94.6(40.3–99), respectively. The median Hb of the eight patients with PNH at the time of diagnosis was 10.1 (8.1–11.8). The median percentage (%) of the measured reticulocytes of these patients was 3.8 (range 2.5–11.3). The median (%) values of CD71(+) cells, CD59(−) cells as a whole and CD71(+)/CD59(−) cells were 0.7 (range 0.2–2.1), 53.4 (range 8.7–97.3), and 85.2 (range 34.1–98.3), respectively (Table 1). Significantly higher proportions of PNH clones were obtained with the evaluation of CD59-deficient/CD71(+) RBC, compared to CD59-deficient RBC as a whole (P < 0.05). It was also important that the mean bias ±2 SD among the determined clones (%) on i-RET versus granulocytes, was much smaller than that among the determined clones (%) on total RBC versus granulocytes (3.81 ± 15.06 and 28.09 ± 43.78, respectively). This indicates a stronger agreement among the estimated PNH clones on i-RET and granulocytes than among the estimated PNH-clones on total RBC and granulocytes.
Table 1. The Obtained Measurements of the Eight PNH Patients
Obtained by Cell-Dyn 4000 (Abbott) hematology analyzer.
RBC CD71(+) (i-RET) were obtained by FCM and reticulocytes (Retics) were obtained by hematology analyzer (ratio).
Among the remaining patients investigated for PNH clone, seven patients revealed small granulocyte clones (<1%) and CD71(+)/CD59(−) RBC clones (<5% of CD71-positive cells, as mentioned above) and 75 patients revealed no CD71(+)/CD59(–) events (mostly because of the absence of CD71 positivity), and <0.2% GPI-deficient granulocytes. The prospective evaluation of the assay in an increased number of patients will permit us to orientate more accurate and appropriate cutoff values, to reassess the sensitivity of the assay and evaluate possible GPI-deficient cells in patients with aplastic anemia or hypoplastic MDS.
Although erythroid and granulocyte bone marrow precursors have been proposed to have an equivalent proliferative advantage in PNH (6), granulocytes clones are frequently detected when RBC clones are not (7). The recently published consensus guidelines propose glycophorin A (CD235a) and CD59 for routine PNH analysis on RBC and CD45/SS or CD15/SS with fluorescinated inactive aerolysin variant (FLAER), CD24, CD66b, and CD16, for PNH clone analysis on granulocytes (2).
The basic advantage of our method is the PNH clone assessment on reticulocytes, in accordance with previous publications (3–5, 8–11). The total reticulocyte count has been suggested as a useful marker for PNH clone size evaluation (9). The population of affected RBC was found low in patients with PNH who received transfusions or suffered from hemolytic precipitation, whereas the population of affected reticulocytes was unchanged (3). PNH-affected RBC have shortened life-span in the circulation, and the percentage of PNH-affected CD59(−) population has been found higher in reticulocytes than in total RBC (11). This was confirmed in our study, where the estimated CD59 negativity was always higher in i-RET than in the total RBC population. Comparison between CD59 negativity on total RBC and i-RET could reliably indicate the extent of hemolysis. The guidelines propose study on total RBC, which may have a bias effect of hemolysis and transfusion on the PNH clone size. However, one limitation of our method is the apparent uncertainty in the determination of Type-II RBC (partly GPI-deficient), as it can provide clear distinction only between Type-III cells (completely deficient) and Type-I cells (normal). The vast majority of reticulocytes in patients with PNH are considered to be completely GPI-deficient (Type-III cells) (6), which supports the robustness of our method focused on estimation of the completely deficient RBC clones to determine the presence of a PNH clone.
The second advantage of our method is the selection of CD71 to detect i-RET. Erythroid cells in the marrow express CD71, transferrin receptor, and reticulocytes released from the marrow lose their expression during maturation (5). CD71 intensity on i-RET is correlated with their RNA content, indicating the usefulness of CD71 as an i-RET marker (5). The preparation and application of CD71 is easier than that of RNA dye thiazole orange (TO) usually used to detect reticulocytes. TO is a known contaminant of the tubing of the analyzer (DNA/RNA fluorochrome), which needs meticulous cleaning before any subsequent count. Regarding the proposed use of glycophorin A by the guidelines, apart from the fact that it is used for total RBC analysis, the need for careful evaluation of the selected clone and conjugate, to determine the optimum concentration for limiting aggregates and providing an acceptable signal on positives (2), renders its use difficult, especially for laboratories of limited experience.
The third advantage is the better agreement of PNH clones evaluated on i-RET (CD71+ RBC) with PNH clones assessed on granulocytes, compared to clones assessed on total RBC. Data from evaluations on i-RET and granulocytes can be routinely cross-checked, due to the anticipated concordance of PNH clone measurements. Monocyte testing has been omitted to obtain a more cost-saving assay, compared to guidelines. CD45 was not used for the exclusion of white blood cells (WBCs) in RBC study, as their percentage within the RBC gate in whole blood was considered minimal, and CD71 was not expected to be expressed in such a high percentage of WBCs in peripheral blood as to mislead diagnosis.
Our study confirmed that CD16 and CD24 are among the most reliable markers for the determination of PNH clones on granulocytes, and these clones were found significantly related to the clones on i-RET. Although FLAER was not included in our diagnostic array, we do not question that it is a useful innovation for PNH analysis (7). However, FLAER is a specialty reagent, and the proposed protocol is specifically intended for laboratories with relatively limited resources, that need to be able to diagnose PNH clones. For this reason, we propose CD66b/CD16/CD45 as the minimum and most cost-effective combination for PNH clone determination on granulocytes. Granulocytes with polymorphic variants of CD16 will be distinguishable from PNH clones as CD16(−)/CD66b(+) populations, whereas PNH-clones will be CD66b(−).
In conclusion, for routine PNH screening, we recommend the simultaneous investigation for a PNH-clone on i-RET and granulocytes. The data from the two investigations should be routinely cross-checked, because of the anticipated concordance of PNH clone measurements. Considering that the proposed method can be easily performed in laboratories of limited experience, with commonly used antibodies, we propose its routine usage and prospective evaluation. The simplicity and limited cost of the method could presumably justify its use in PNH screening for every suspicious case, contributing to the detection of previously unrecognized PNH.