Analysis of platelets by flow cytometry in patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Journal Pre-proof Analysis of platelets by flow cytometry in patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

David J. Araten, Daniel Boxer, Leah Zamechek, Erik Sherman, Michael Nardi PII:

S1079-9796(19)30270-0

DOI:

https://doi.org/10.1016/j.bcmd.2019.102372

Reference:

YBCMD 102372

To appear in:

Blood Cells, Molecules and Diseases

Received date:

19 July 2019

Accepted date:

22 September 2019

Please cite this article as: D.J. Araten, D. Boxer, L. Zamechek, et al., Analysis of platelets by flow cytometry in patients with Paroxysmal Nocturnal Hemoglobinuria (PNH), Blood Cells, Molecules and Diseases(2019), https://doi.org/10.1016/j.bcmd.2019.102372

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© 2019 Published by Elsevier.

Journal Pre-proof Title: Analysis of platelets by flow cytometry in patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Authors: David J. Aratena,b, Daniel Boxera, Leah Zamecheka, Erik Shermana, Michael Nardia a

Division of Hematology, Department of Medicine, NYU School of Medicine, and the Laura and Isaac Perlmutter Cancer Center, NYU Langone Health, ACC Building, 240 East 38th Street, 19th floor, New York, NY 10016 b

New York VA Medical Center 423 East 23rd Street New York, NY 10016

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Corresponding Author: David J. Araten, MD, ACC Building, 240 East 38th Street, 19th floor, New York, NY 10016. 212-731-5186. Fax 212-731-5540. [email protected]

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Abstract:

The marked pro-thrombotic tendency in PNH is likely to be at least partly due to the population

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of platelets derived from the abnormal stem cell clone. However, identification of GPI (-)

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platelets by flow cytometry can be technically difficult. Here we describe a technique that involves the addition of aspirin immediately after the separation of platelet rich plasma and the

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use of gel filtration to isolate platelets away from plasma proteins and other blood cells. In a study of 92 analyses of samples from 50 patients, we have demonstrated that the percentage of

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PNH platelets correlates well with the percentage of PNH granulocytes. We also provide data on several cases where there was an extreme discrepancy between the proportion of PNH

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granulocytes and red cells; in these cases, the demonstration of abnormal platelets suggests that the patient is likely to be at risk of thrombosis. We believe this test will be potentially useful

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in the evaluation of samples from such patients and may serve as a tool to investigate the causes of hypercoagulability in PNH.

Keywords: PNH, Platelets, Flow cytometry, Hypercoagulability, Thrombosis, GPI Abbreviations: GPI: glycosylphosphatidylinositol, AA: aplastic anemia, PNH: paroxysmal noctural hemoglobinuria Acknowlegdments: This paper is dedicated to the memory of our late Division Chief, Dr. Simon Karpatkin, whose enthusiasm for platelet biology largely inspired this project. Funding: This work was supported by The PNH Research and Support Foundation Award from the Aplastic Anemia & MDS International Foundation.

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Journal Pre-proof Introduction: Paroxysmal Nocturnal Hemoglobinuria (PNH) is characterized by complement mediated hemolysis due to loss of the glycosylphosphatidylinositol (GPI)-linked complement inhibitors CD55 and CD59 on the surface of the red cell. This is due to an acquired somatic mutation in a stem cell that disrupts the PIG-A gene, which encodes an enzyme that is essential for the biosynthesis of the GPI anchor [1]. Aplastic anemia and PNH are both rare disorders, yet the

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two conditions commonly coincide in the same patient (referred to as AA/PNH). Therefore, it is

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thought that the opportunity for this stem cell clone to expand must be related to immune

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mediated marrow failure [2]. In many cases, however, the aplasia is mild or subclinical [3].

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There are several potential explanations for the marked hypercoagulable state seen in patients with PNH, but it seems likely that platelets derived from the mutant stem cell are playing a key

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and CD59 on their surface [4-8].

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role, since they may be susceptible to complement-mediated activation due to a lack of CD55

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Using reagents to identify the GPI anchor and GPI-linked proteins on red cells and granulocytes,

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flow cytometry can establish the diagnosis of PNH, but a similar analysis of platelets can be technically difficult for several reasons: (1) because platelets are the smallest circulating cell, they will have fewer antigens, and thus the fluorescence signal will be relatively lower on the normal platelet population; (2) because platelets express FcRIIa receptors, some monoclonal antibody reagents could bind non-specifically; (3) flow cytometry relies on the assumption that individual events represent single cells, and platelet agglutination may obscure the presence of a GPI (-) population; (4) since platelets may be activated by clotting factors, changes in temperature, and mechanical factors, it may be challenging to develop a flow cytometry

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Journal Pre-proof protocol that minimizes these variables, since centrifugation, vortexing, incubation on ice, and washing are commonly used in the preparation of cells for flow.

Historically, attempts to demonstrate a deficiency of GPI-linked proteins on platelets in patients with PNH have had mixed results. In some cases, the staining of the normal platelet population had produced only weak fluorescence, and in some cases it had not been possible to

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demonstrate distinct populations of platelets. Nevertheless, there are some reports of successful

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identification of PNH platelets in patients with this disorder, in which the percentage of abnormal

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populations among platelets and granulocytes are generally correlated (table 1). Of note,

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various techniques have been used, and there is currently no standard procedure for the

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analysis of PNH platelets, in contrast to the case for red cells and neutrophils [9] .

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Here we describe in detail our technique for the analysis of GPI (-) platelets and we have

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Material and Methods:

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applied this to a cohort of 50 patients.

After obtaining informed consent on an IRB-approved protocol, peripheral blood samples from 50 patients with PNH or AA/PNH were analyzed as well as samples from unaffected controls. To separate platelet rich plasma (PRP), whole blood samples were collected in purple top (EDTA) tubes and centrifuged at 200g for 7 minutes at room temperature with the brake turned off. After this step, there was no further centrifugation, manual agitation, or use of a vortex. The PRP was aspirated, and acetylsalicylic acid (Sigma) was added to it immediately from a freshly made 10 mM stock to a final concentration 0.5 mM.

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Journal Pre-proof For the gel columns, Sepharose 2B (GE-Healthcare, 170130-01) was washed in 10 times the volume of modified 1x Tyrode’s Buffer for 3 washes, allowing the sepharose to settle by gravity each time, in order to remove the alcohol preservative. The modified Tyrode’s buffer was made up from a 10x solution that had been balanced to pH 7.4 with NaOH and filtered through a 0.2 uM membrane prior to storage at room temperature, which was diluted for a final 1x concentration of 9.86 mMol HEPES, 150 mMol NaCl, 2.55 mMol KCl, 0.33 mMol NaH₂PO₄, 12

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mMol NaHCO₃, 2.53 mMol EDTA, and 5.55 mMol Glucose.

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For each column, the washed Sepharose was poured into a thin soft plastic cylinder with a

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7mm diameter and 210mm length, with a funnel top and nozzle at the bottom with a 1.5mm aperture, fitted with a polyethylene disk (obtained from Kimble Kontes) as previously described

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[10]. The washed sepharose suspended in 1x modified Tyrode's buffer was then poured into the

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column until it was about 1 cm from the top. After the sepharose settled, it was washed with buffer. As soon as the buffer had drained completely from the top of the column, the aspirin-

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treated PRP was slowly added directly to the top and allowed to absorb completely onto the

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sepharose. The funnel at the top of the column was then filled with the modified Tyrode’s buffer. The eluate from the bottom of the column was collected into 1.5cc Eppendorf tubes, which were observed for opacity, indicating the presence of collected platelets. The tubes with the collected filtered platelets were then set aside for the staining steps.

50 L of the buffer containing the gel-filtered platelets was stained concurrently with FLAERAlexa 488, (obtained from Pinewood Scientific Services, Victoria BC, Canada) at a dilution of 1:20, and with anti CD59-PE (Clone MEM-43, a mouse IgG2a antibody obtained from AbD

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Journal Pre-proof Serotec), used at a 1:10 dilution, for 30 minutes at room temperature, in the dark. In some experiments we used FITC-conjugated anti CD55 at a 1:10 dilution (Clone IA10, a mouse IgG2a antibody, obtained from BD Pharmingen, Franklin Lakes, NJ). Samples were gated based on forward scatter (FSC), side scatter (SSC), and FL1 and FL2 expression, all acquired on a loglog scale on a BD Facsan. Based on unstained platelets, gates were set so that negatives would appear in the first decade. Compensation controls were also made using red cells from whole

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blood stained with CD59-FITC (AbD Serotec, 1:10) and CD59-PE (1:10). To prevent doublets or

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clumps from confounding the analysis, samples were diluted 1 : 200 in Hanks + 0.1% Bovine

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Serum Albumin (BSA) or Tyrode’s buffer and passed through a filter top (35 m) cell strainer

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(obtained from Falcon) immediately before flow analysis, and we aimed to collect events at a rate of less than 1000 per second. In cases where distinct populations were not resolved, the

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platelets were diluted further to be sure that doublets were not confounding the analysis. The

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data was acquired using Cellquest and analyzed using FlowJo software.

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To analyze red cells, 4 L of whole blood was diluted into 50 L of cold 1X PBS. Samples were

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then stained with CD59-FITC (1:10) on ice for 30 minutes prior to analysis. For analysis of granulocytes, 50 L of whole blood was stained with FLAER Alexa 488 (1:20) at 37C for 30 minutes and with CD24-FITC (obtained from BioLegend) in separate tubes on ice for 30 minutes, followed by lysis with a Beckman-Coulter immunolyse kit.

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Journal Pre-proof Results: Clinical charateristics of the patients studied Of the 50 patients analyzed, 15 had had unprovoked thromboses. Two of these patients were known to have the factor V Leyden allele. One additional patient had a serious deep vein thrombosis occurring despite eculizumab in the setting of surgery for a hip fracture. Another had a minimally evident deep vein thrombosis of the lower extremity immediately after delivering a

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healthy neonate, despite eculizumab and aspirin. 19 patients were on anticoagulation (6 of

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these were patients on primary prophylaxis with anticoagulation because their symptoms,

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hemoglobin level, and transfusion history did not mandate the use of eculizumab). 27 patients

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were on eculizumab. Of 31 patients with more than 50% PNH granulocytes at the time of the platelet analysis, 20 had been on either eculizumab or anticoagulation to prevent thromboses

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and never had a thrombotic event. One developed an unprovoked episode of concurrent arterial

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and venous thromboses despite intermittently therapeutic warfarin, 8 had not been on anticoagulation or eculizumab prior to developing their first thrombotic event (either because

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they presented with thromboses or because their first thrombosis occurred in the years before

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primary prophylaxis became standard of care), and 2 patients were not on any prophylactic measures and did not develop a thrombosis. Four patients had been referred with a history of thrombotic events but were found to have <50% PNH granulocytes by the time of referral. Three of the 50 patients were eventually referred for allogeneic stem cell transplant due to the development of excess marrow blasts in association with a fall in the peripheral blood counts.

Validation of the flow cytometry method

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Journal Pre-proof Platelet populations were easily resolved on FSC/SSC, demonstrating lower scatter than red cells. Platelet populations from 15 unaffected donors bound to the anti-CD59 antibody sufficiently to shift FL2 fluorescence above the gates set by the unstained cells. In most experiments FLAER staining was less intense than for CD59, and the fluorescence levels of the two were correlated. In samples of platelets derived from controls not affected by PNH or aplastic anemia, after staining with these two reagents simultaneously, the mean percentage of

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events that fell within the GPI (-) gates was approximately 0.3% (range 0.015% to 0.83%,

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standard deviation 0.28%). Samples from patients with PNH and AA/PNH demonstrated GPI (-)

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platelet populations that could be easily resolved from the normal platelet populations (figure 1).

A total of 92 out of 99 analyses performed on samples from 50 patients gave interpretable

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results. For 7 samples, the results were considered to be uninterpretable for technical reasons.

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For 4 analyses, there was high background staining of the FLAER reagent on the GPI (-) population, which did not preclude interpretation using the anti-CD59 antibody. Considering

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together the GPI (+) platelet sub-populations that were present in most of these patients

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together with the platelets analyzed from the unaffected controls, there were 88 analyses where we could assess the adequacy of the staining of GPI (+) populations. For 77% of these analyses, both the CD59 antibody and the FLAER reagent clearly stained the GPI (+) platelets. For 19.5% of the analyses, the anti CD59 reagent stained the platelets adequately but the FLAER reagent did not. For 3.5% of the analyses, staining with FLAER was adequate but the fluorescence from the anti-CD59 reagent was inadequate.

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Journal Pre-proof For 6 samples (2 normal donors, 4 patients with PNH clones ranging from 16 to 99% of the granulocytes), we stained cells in a separate tube with anti CD55-FITC together with anti-CD59PE. The pattern was very similar to the results seen for FLAER/CD59-PE (figure 2), and the percentage of GPI (-) cells based on the two parallel analyses performed on the same day were highly correlated (r = 0.999).

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For 26 patients, between 1 and 5 repeat analyses were performed for a total of 68 analyses.

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The relative variation of the percentage of GPI(-) cells compared with the median of the set of

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analyses for any one patient was >±25% for 10% of the analyses for the platelets, in

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comparison with 13% of the analyses for the granulocytes and 36% of the analyses for the red cells (p = 0.001 for the comparison between the analysis of the red cells and the analysis of the

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platelets; p = 0.006 for the comparision between the analysis of the granulocytes and the

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analysis of the red cells by the X2 test). The proportion of GPI (-) granulocytes was more highly correlated with the proportion of GPI (-) platelets for each individual (Pearson's corection

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coefficient r = 0.93, 95% CI 0.89-0.96) than the correlation between the proportion of GPI (-)

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platelets and the proportion of GPI (-) red cells (r = 0.69, 95% CI 0.51-.81) or the correlation between the proportion of GPI (-) red cells and the proportion of GPI (-) granulocytes (r= 0.69, 95% CI 0.50-0.81, figure 3).

Relationship between flow cytometry studies and thrombotic history We were interested in whether the proportion of PNH blood cells would be predictive of thrombosis in these patients. The median proportion of PNH red cells, granulocytes and platelets was 21%, 84%, 70% respectively in the group without a history of thrombosis and

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Journal Pre-proof 16%, 84%, and 77% in the group with a history of thrombosis, suggesting that this was not the case. In contrast, the percentage of PNH granulocytes had been a strong predictor of thrombosis in our previous series of 92 patients [11] and in other published data sets [12]. We next considered whether the absolute count of abnormal platelets would be predictive of thrombosis, and we used the product of the platelet count and the percent of PNH platelets to estimate this parameter. This value was similar in the 2 groups: 85,000/l in those with a history

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of thrombosis and 89,000 /l in those without a history of thrombosis.

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Analysis of receiver operator curves

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Because most of the patients in the current study who had large PNH clones had been on either anticoagulation or anticomplement therapy, we considered that this had altered the natural

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history of the disease such that the proportion of PNH cells is now no longer predictive of

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thrombotic events. We therefore re-analyzed our previous data set of 92 patients from the days before patients had been treated with prophylactic anticoagulation or anticomplement therapy

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[11], to reexamine the optimal predictor of thrombosis. Data available from this set included the

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LDH, blood counts, and the proportion of PNH red cells and granulocytes. (Of these 92 patients, only 7 were still followed and available to give samples and to be analyzed among the set of 50 patients described above). We used this data to derive receiver operator curves based on the sensitivity and specificity of various parameters for predicting thrombosis, and calcuted a confidence interval for the area under the curves (AUC) as described[13]. The AUC was the highest, 0.81, for the relative percent of PNH granulocytes (95% CI 0.71 – 0.91), which was similar to the AUC using the relative percentage of PNH red cells (including PNH III and PNH II cells, AUC 0.79, 95% CI 0.68-0.89). In contrast, the percentage of PNH II cells, the lactate

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Journal Pre-proof dehydrogenase level, the absolute neutrophil count, and the platelet count all were associated with AUC's such that the lower 95% limits approached or were below 0.5 (0.5-0.76, 0.56-0.83, 0.46-0.73, 0.44-0.7 respectively) . We considered that based on the current data, the percent of PNH granulocytes would have been closely related to the percentage of PNH platelets in the previous data set and we used this to estimate an absolute PNH platelet count for the set of 92 previously studied patients. For the estimated absolute PNH platelet count, the AUC was 0.75

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(95% CI 0.63 – 0.87) and the AUC was 0.76 (95% CI 0.65 – 0.88) for the absolute count of

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circulating PNH granulocytes. Using the relative percent of PNH granulocytes to predict

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thrombosis, the commonly used cutoff value of 50% as an indication for prophylactic

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anticoagulation or anti-complement thereapy would be associated with a 90% sensitivity and a

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60% specificity for predicting thrombotic events.

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Analysis of platelets in patients with skewed proportions of PNH red cells and granulocytes It is commonly observed that the proportion of PNH granulocytes is greater than the proportion

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of PNH red cells, presumably due to hemolysis, and that was the case in the current study as

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well (figure 3C). However, for 12 patients, the skewing was defined as extreme based on an absolute difference between the proportion of PNH granulocytes and red cells that was >50% or a ratio between the percent PNH granulocytes and percent PNH red cells that was >50. The median value for the percent PNH platelets for this group was 81% (range 0.93% to 97%), and all except one of these patients had >60% PNH platelets.

Four notable patients of the group with extreme skewing had PNH red cells representing < 5% of the total; one was a 54 year old man who had a prior history of hemoglobinuria and had been

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Journal Pre-proof on primary prophylactic warfarin for over 10 years without any thromboses. It was considered that over time, the percentage of PNH red cells had declined. He was then found to have 92% PNH platelets, 2.5% PNH red cells, and 99.5% PNH granulocytes, and despite the resolution of hemolysis, he was continued on anticoagulation.

The second patient with extreme skewing was a 53 year old woman who had been treated

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successfully 6 years previously with immunosuppression for aplastic anemia. She had been on

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prophylactic low dose aspirin based on the presence of detectable PNH granulocytes without

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detectable abnormal red cells, with no thromboses detected by MRV imaging. She had never

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demonstrated a significant proportion of PNH red cells on any occasion and in contrast to the first patient, never had hemoglobinuria. 95% of the platelets from this patient were found to be

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GPI (-), as well as 0.08% of the red cell and 99.5% of the granulocytes. Based on these findings

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and recent evidence that such patients frequently have thrombotic complications without

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hemolysis [14, 15], it was recommened to her that she should be on full dose anticoagulation.

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A third patient with extreme skewing was a 32 year old woman who was a recent immigrant at the time of the analysis, who had a distant history of thrombosis and radiographic evidence of a previous portal vein thrombosis with cavernous transformation, but had never been given anticoagulation or anticomplement therapy. The platelet count was 38,000, and she had 1.5% PNH red cells, 93% PNH granulocytes, and a unimodal population of platelets, representing almost all of the gated events, which demonstrated intermediate levels of staining with FLAER, CD59, and CD55 compared with control GPI (+) and GPI (-) populations analyzed in the same

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Journal Pre-proof experiment. Based on these findings, given her history of thrombosis and her low platelet count, she was started on eculizumab without anticoagulation.

A fourth notable patient with extreme skewing had presented with hemolytic anemia and a positive HAM test 30 years prior to this analysis, and then developed autoimmune neutropenia associated with clonal large granular lymphocytes 15 years later, which had been the subject of

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a separate report [16]. At that time, there were 10% PNH red cells and 85% PNH granulocytes.

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On the current analysis, there were approximately 1% PNH red cells, 60% PNH granulocytes,

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and 0.93% GPI(-) platelets. She has never had a thrombotic event and has been on long-term

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prophylactic aspirin.

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Discussion:

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PNH is a highly prothrombotic condition, with an estimated incidence of 10% per year [17], occurring in approximately 50% of patients observed in large series. Risk factors include

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African-American ancestry [11] and the relative size of the PNH cell population. In non-

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randomized studies, anticoagulation with warfarin [18] and anticomplement therapy with eculizumab [17] are highly effective at preventing thrombosis. Occasionally, patients will present with thrombosis, which had been reported to be a poor prognostic indicator [19] but which may no longer be the case given the availability of combined treatment with anticomplement and anticoagulation therapy-- and tissue plasminogen activator for immediately life threatening or refractory thromboses [20].

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Journal Pre-proof While the presence of abnormal platelets is widely believed to be driving the thrombotic tendency, previous reports on flow cytometry to identify populations of GPI (-) platelets have utilized varying techniques, with varying results (summarized in table 1). Consequently, this is not currently part of the standard laboratory investigation for PNH. One previous report indicated that the percentages of abnormal PNH granulocytes and platelets are correlated [21], and so the proportion of PNH granulocytes is often used as a surrogate when considering thrombotic

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risk. Current recommendations for the initiation of prophylactic measures to prevent a primary

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thrombotic event (either anticoagulation or anticomplement therapy) are, therefore, based upon

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the percentage of abnormal granulocytes [22].

The results shown here demonstrate that the technique for the analysis of GPI (-) platelets can

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be robust and reliable. For example, for those patients in whom we had repeat analyses, the

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proportion of PNH platelets seemed to be at least as reproducible as the percentage of PNH granulocytes. Overall, only about 7 percent of the analyses performed represented technical

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failures. We attribute our ability to delineate separate GPI (+) and GPI (-) platelets to several

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factors: (i) treatment with aspirin; (ii) gel filtration; (iii) avoidance of cold, centrifugation and agitation. To our knowledge, this is the first analysis of platelets that also utilized the FLAER reagent, a fluorescent, non-toxic derivative of proaerlysin that integrates into the membrane of cells in a GPI-dependent manner [23]. FLAER, however, requires proteases for its activation, which are provided by the target cell population. In 19.5% of the analyses, there was poor staining with FLAER, perhaps due to inadequate level of the proteases in the platelet preparation.

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Journal Pre-proof Although in our initial studies the staining with a mouse anti-CD55 reagent (clone Bu14) suggested that this would not be a reliable method, we considered that this reagent is an IgG1 antibody, unlike the anti-CD59 reagent, which has an IgG2a heavy chain. We later repeated these experiments with a different mouse anti-CD55 reagent (clone IA10), which has an IgG2a heavy chain, and here the reagent provided reasonably intense staining of the normal platelet population and good delineation of bimodal populations in patients with PNH. Of note, mouse

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IgG2a antibodies may be less likely to bind to FCγIIA receptors on human platelets [24-26], and

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therefore these reagents may be preferable.

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In earlier studies, the percentage of GPI negative blood cells was a strong predictor of thrombosis in patients with PNH [11, 12]. It is now appreciated that anticomplement therapy

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and anticoagulation can prevent thrombotic events [17, 18]. Given that most of the patients in

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this study who had large PNH clones were on either anticoagulation or anticomplement therapy at the time of the flow analysis, this may explain why the size of the PNH clone was not

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predictive of thrombosis here. In a reanalysis of our earlier data set of patients who had not

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been prophylactically treated, the percentage of PNH granulocytes was the best predictor of thrombosis[11]. We do not know, however, how the assay for GPI (-) platelets would have performed in comparison, but one might predict that the absolute count of GPI (-) platelets would be the single best predictor.

The techniques described here were particulalry applicable to 4 patients with an extreme discrepency, such that there was a high percentage of GPI (-) granulocytes and barely

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Journal Pre-proof detectable GPI (-) red cells. In 3 of these patients, large populations of abnormal platelets were detected, confirming the clinical judgement that these patients remain at high risk of thrombosis.

Conclusions We have a developed a method that may be useful in 3 groups of patients: (1) those who have over 50% PNH granulocytes without detectable PNH red cells. While these patients are said to

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be at a very high risk of thrombosis[14, 15] it may be useful to demonstrate the presence of GPI

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(-) platelets, to justify the risks of prophylactic therapies for asymptomatic patients; (2) Patients

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who demonstrate a disappearance of the PNH red cell and granulocyte population [27] to the

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point where they might consider discontinuation of eculizumab or anticoagulation therapy. For these patients, the lack of a PNH patient population might confirm that it is safe to do so; (3)

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Patients whose proportion of PNH granulocytes is close to the recommended cut off for the

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initiation of prophylactic therapies—often cited as 50%. In this case, the demonstration of PNH platelets, or the lack of PNH platelets, might help with this decision. Further studies with this

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technique are warranted in such patients to further establish a role for platelet GPI analysis in

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the clinical care of patients with PNH.

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Journal Pre-proof Figure Captions Figure 1: flow cytometry analysis of platelets from an unaffected donor in comparison with platelets from a patient with a large PNH clone and a patient with AA/PNH with a smaller PNH clone, stained with anti-CD59-PE and FLAER-Alexa 488. Unstained platelets are shown in comparison.

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Figure 2: Platelets from a patient with PNH (top panels) and a patient with AA/PNH (bottom panels), stained in separate tubes with a combination of anti-CD59-PE with FLAER or antiCD59-PE with anti CD55-FITC. Distinct GPI (+) and GPI (-) populations are demonstrated with both methods, though the FL1 fluorescence is slightly higher using the CD59-PE/CD55-FITC combination.

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Figure 3: Comparisons of the relative frequency of GPI (-) red cells, granulocytes, and platelets determined by flow cytometry. Each point represents the results from an individual patient. For patients for whom there were repeat analyses, the median value is shown.

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Authors Nicolson-Weller [28, 29]

Year

reagents anti CD55 (DAF) polyclonal antiserum anti CD55 (DAF), secondary antibody CD59

cell preparation

Kinoshita [30] Fujioka &Yamada[31]

1985 1994

Hall & Rosse[32]

1996

1997

CD59, CD55 CD55, CD59, secondary antobodies CD55 CD59 CD58 secondary antibody CD55 CD59, PE secondary antibody

Maciejewski et al[33]

1996

Vu et al [34]

1996

Jin et al[21] Hernandez-Capo et al [35] Holada et al [36] Ruiz-Delgado et al[37]

2002 2009

CD59 CD55

2009

CD59, CD55

1985

# patient samples

washed platelets

3

washed platelets wash in PBS

2 17

wash in GVB-EDTA

54

l a

r P

n r u

9

13

f o

decreased expression in 3/3 patients bimodal distribution demonstrated low CD59 staining intensity low intensity on normal cells, populations in PNH patients not well distinguished

o r p

e 10

wash in PBS aspirin treatment, wash in PBS and EDTA stain without lysing or washing gel filtered

results

platelet flow less sensitive than other cell lineages GPI (-) platelet populations detected correlation between %CD59 and %CD55 deficient cells, correlation between % PNH plts and granulocytes

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weak expression distinct populations demonstrated

2

GPI(-) platelet populations seen in other hematologic disorders

Table 1 : summary of the prior literature on the identification of GPI (-) platelet populations in patients with PNH.

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Journal Pre-proof References

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