Received: 11 January 2022 Accepted: 29 April 2022

DOI: 10.1111/ijlh.13877

ORIGINAL ARTICLE

Validation of a single tube 3-colour immature red blood cell

screening assay for the detection and enumeration of small,
medium and large paroxysmal nocturnal haemoglobinuria
clones by flow cytometry

Iuri Marinov1 | Stephen J. Richards2 | Adam Pešek1 | Andrea J. Illingworth3 |

D. Robert Sutherland4

1
Clinical Department, Institute of
Haematology and Blood Transfusion, Prague,
Czech Republic
2
Division of Haematology and Immunology,
Leeds Institute of Medical Research at St
James's, University of Leeds, Leeds, UK
3
Dahl-Chase Diagnostic Services, Bangor,
Maine, USA
4
Department of Laboratory Medicine, Toronto
General Hospital, Toronto, Ontario, Canada
Correspondence
Iuri Marinov, Institute of Haematology and
Blood Transfusion, U nemocnice 1, 128
20, Prague 2, Czech Republic.
Email: iuri.marinov@uhkt.cz
Abstract
Introduction: The reliable diagnosis of paroxysmal nocturnal haemoglobinuria (PNH)
by flow cytometry is based on mandatory analysis of the erythroid, neutrophilic and
monocytic lineages. In this study, we have evaluated the performance characteristics
of a recently published immature red blood cell (iRBC) assay as a potential screening
test for PNH by flow cytometry.
Methods: Intra- and inter-assay imprecision were determined in five replicates of
small, medium and large PNH iRBC clones. Analytical and functional sensitivity was
assessed by performing spiking tests for five replicates. Thirty healthy donors and
441 PNH patients were tested for evaluation of clinical specificity, sensitivity, posi-
tive and negative predictive values.
Results: Coefficients of variation (CV) for intra-/inter-assay imprecision analyses
were 1.31/1.50, 3.19/2.61 and 3.99/1.58 for the big, medium and small clone sizes,
respectively. Absolute values (100%) were found for both clinical specificity and sen-
sitivity as well as for both positive and negative predictive values. The CV from 5 rep-
licate results for 10 clustered events was 15.7%. The coefficient of determination
(r2), Pearson's correlation coefficient (r) and Bland–Altman mean bias were 0.9436/
0.9234/1.7 for PNH iRBC compared to PNH neutrophils and 0.9553/0.9387/2.1 for
PNH iRBCs compared to PNH monocytes.
Conclusion: Our results confirm very good performance characteristics, high analyti-
cal and functional sensitivity, absolute clinical specificity and sensitivity as well as
favourable correlation between PNH iRBCs and both PNH neutrophils and mono-
cytes, suggesting that this cost-effective 3-colour iRBC assay can be used as a reli-
able screening test for evaluation of small, medium and large PNH clones by flow
cytometry.

KEYWORDS
flow cytometry, immature red blood cells, paroxysmal nocturnal haemoglobinuria, screening
assay

868 © 2022 John Wiley & Sons Ltd. wileyonlinelibrary.com/journal/ijlh Int J Lab Hematol. 2022;44:868–874.

MARINOV ET AL.
1 | I N T RO DU CT I O N
Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired, life-
threatening, haematopoietic stem cell disorder resulting from the
somatic mutation of the X-linked phosphatidylinositol glycan anchor
biosynthesis class A gene (PIGA) gene, which encodes an enzyme
involved in the first stage of glycosylphosphatidylinositol (GPI)
biosynthesis.1–3 The PIGA gene mutation(s) results in a partial or com-
plete inability to biosynthesize GPI-anchored proteins including
complement-regulatory structures such as CD55 and CD59
(in particular) on red blood cells (RBCs) and white blood cells (WBCs)
leading to complement-mediated, intravascular haemolysis of GPI-
deficient RBCs.4–6 Since the 1990s, when CD55/CD59-based flow
cytometry became the method of choice for identifying GPI-deficient
cells, a number of highly sensitive assays were developed to detect
PNH phenotypes in WBCs and RBCs.7–13
The ability of the PNH RBC assay to delineate type II PNH RBCs
(partial loss of CD59) with longer survival time and type III PNH RBCs
(complete loss of CD59) with shorter survival time provides clinically
more relevant information with regard to the severity of haemolysis
and risk of thrombotic complications. However, various factors such
as recent or regular red blood cell transfusions, RBC haemolysis, RBC
life-span, large type II PNH populations overlapping with normal RBCs
can hamper the accurate delineation of mature PNH RBC (mRBC)
subsets and generate misleading information concerning the bona fide
PNH clone sizes in the mRBC lineage.12 Although more representative
of disease extent with respect to the real PNH clone size, the PNH
WBC assay could also be challenged due to low numbers of acquired
monocytes resulting in discrepancies between PNH neutrophil and
PNH monocyte clone sizes. Therefore, the mandatory analysis of neu-
trophilic, monocytic and erythroid compartments using validated
reagents and appropriate panel designs as described in the recently
published ICCS/ESCCA guidelines for the diagnosis and monitoring of
PNH and related disorder is a fundamental aspect of robust assay
design for the accurate detection and quantification of PNH
clones.12–14 A variety of flow cytometry assays have been developed
for iRBCs (both nucleated RBCs and/or reticulocytes) based on
nucleic acid stain15,16 or more recently, antibodies targeting the trans-
17,18
ferrin receptor CD71.
Sutherland et al. recently demonstrated that addition of
CD71-APC to the ICCS/ESCCA guidelines PNH mRBC protocol
(CD235a-FITC/CD59-PE) significantly improved the ability to analyse
PNH clone sizes in the RBC lineage.19 Importantly, the assay retained
the ability to analyse mRBCs with high sensitivity in the same stained
sample, an important attribute when trying to detect very small
populations of PNH RBCs in cases with aplastic anaemia (AA) and
hypoplastic bone marrow failure syndrome (BMFS).
In this study, we report our results concerning the further valida-
tion of this cross-platform assay with respect to its potential role as a
simple, robust and cost effective single-tube, single-lineage PNH
screening assay for detection and enumeration of small, medium and
large PNH clones by flow cytometry. This assay would be particularly
suitable for laboratories with limited or no experience in performing
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869
more complex, multi-parameter 5-, 6- and 7-colour analysis of PNH
WBCs in PNH and related disorders.
2 | P A T I E N T S A ND M E T H O D S
2.1 | Patients
Peripheral blood (PB) from 30 healthy donors and 441 PNH patients
following informed consent was used for the study. The results were
acquired in the Leeds laboratory (S. Richards) from 2007 to 2018 and
in Prague laboratory (I. Marinov) from 2018 to 2020.
2.2 | Flow cytometry
Acquisition and analysis were performed on a BD FACSCanto™
cytometer equipped with three lasers and BD FACSDiva™ 6.1.3. soft-
ware (BD Biosciences, San Jose). Photomultiplier tube (PMT) voltage
optimization was performed using unstained and single stained cells,
standardization and spectral overlap compensation were done auto-
matically using the CS&T module.
2.3 | PNH neutrophil and monocyte analysis
The PNH neutrophils (Ne) and monocytes (Mo) were analysed using
the previously published 5-colour, FLAER/CD157-based approach.9,20
2.4 | PNH iRBC analysis
The monoclonal antibodies (MoAbs) used in the iRBC study are listed in
Table 1. Ethylenediamine tetra-acetic acid (EDTA) anti-coagulated whole
PB was diluted 1:100 with fresh clean PBS, and 100 μl was carefully pip-
etted using reverse-pipetting techniques directly into the bottom of the
staining tube taking care to avoid aerosols and blood trails on the inside
of the tube. The appropriate volume of diluted CD235a-FITC/CD59-PE/
CD71-APC was then pipetted directly into the bottom of the tube and
admixed with the diluted sample by gentle up-and-down pipetting. After
careful removal of the tip, the sample was gently ‘swirled ‘on a vortex
set at very low speed to avoid aerosol generation. After 20 min, the sam-
ple was washed twice with clean PBS, resuspended in 1 ml of PBS and
then ‘racked’ (the tube is dragged back and forth a few times across a
hard plastic or metal test tube rack) to disrupt any RBC aggregates gen-
erated during the staining process. iRBCs were analysed using sequential
gating on forward scatter (FSC) log/side scatter (SSC) log, FSC
log/CD235a, FSC log/FSC log (for doublet discrimination) and CD71/
CD235a bi-parametric plots (Figure 1). A mean of 3736 for intra-assay
imprecision, 4727 for inter-assay imprecision, 3366 for spiking test and
6351 for comparative analysis CD71/CD235a iRBCs were then acquired
and PNH iRBC clone determined by partial or complete CD59 deficiency
and expressed as percentage from total iRBCs.
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870 MARINOV ET AL.

T A B L E 1 Gating and GPI-informative

MoAb Purpose Clone-Fluorochrome Source
reagents and fluorochromes used in the
CD59 GPI-informative MEM-43-PE Exbio study
CD71 Gating OKT9-APC (older studies) eBioscience
MEM-75-APC (recent studies) Exbio
CD235a Gating KC16-FITC Beckman Coulter

F I G U R E 1 Representative bi-parametric plots for sequential gating of RBCs on FSClog/SSClog (A), FSClog/CD235a (B), CD71/CD235a
(D) and analysis of CD235a+/CD71- PNH mRBCs (E) and CD235a+/CD71+ PNH iRBCs (F)

TABLE 2 Results for intra-/inter-assay precision analysis for different target clone sizes

PNH clone Clone size N Min. Max. Mean SD CV (%)

iRBCs >50% 5/5 78.81/77.39 81.50/81.03 80.46/79.32 1.05/1.19 1.31/1.50
20%–50% 5/5 24.28/24.84 26.64/26.57 25.71/25.60 0.82/0.37 3.19/2.61
<20% 5/5 12.81/13.78 14.32/14.02 13.40/14.02 0.54/0.22 3.99/1.58

2.5 | Intra- and inter-assay imprecision analysis
Five replicates of small (<20%), medium (20%–50%) and large (>50%)
PNH iRBC clones were analysed in a single analytical run for intra-
assay imprecision and in separate analytical runs for inter-assay
imprecision.
2.6 | Analytical and functional sensitivity—
spiking test
Five replicates of a neat sample (PNH blood), control sample (normal
blood) and five serial dilutions (1:10, 1:100, 1:1000, 1:10000,
1:100000) were analysed to evaluate the limit of blank (LOB) defined
 1751553x, 2022, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ijlh.13877 by Estsp Politecnico Do Porto, Wiley Online Library on [21/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
MARINOV ET AL. 871

as the highest apparent signal detected in samples containing no

0.73
5.49
8.71
15.74
37.16
CV%
measurand and the limit of detection (LOD) defined as the lowest

NA
NA
level of target events, that can be reliably distinguished from LOB

PNH PNH

79.01 0.58
29.32 1.61
3.32 0.29
0.34 0.05
0.05 0.02
with acceptable CV < 30%.

mean SD

0
0
0
0
%
2.7 | Clinical specificity, clinical sensitivity and
positive and negative predictive values

iRBCs %

3.30
0.40
0.03
79.00
27.53
PNH

0
0
Clinical specificity, sensitivity and positive and negative predictive values
were evaluated according to the formulas: number of true negatives/

iRBCs
total without disease  100 (%), number of true positives/total with

12
335
102
12 324 9736

1
0
0
PNH
disease  100 (%), number of true positives/total test positive  100

1217
3091
2998
3071
2602
2785
(%) and number of true negatives/total test negatives  100 (%),

iRBCs
Run 5
respectively.

iRBCs %

3.34
0.33
0.04
79.10
27.88
PNH
2.8 | Statistical analysis

0
0
The results from intra-, inter-assay imprecision, analytical, functional

iRBCs

98
10
382
12 390 9800

1
0
0
PNH
sensitivity were expressed as CV, the results from clinical specificity,
clinical sensitivity and positive and negative predictive value were

1370
2937
3052
2717
2834
3052
iRBCs
Run 4
expressed as relative values (%). For comparison of results for PNH
iRBCS and PNH WBCs, we used the coefficient of determination (r2)
from linear regression analysis and the Pearson's correlation coeffi-

iRBCs %
cient (r) significant at level 0.01, the agreement of results was evalu-

2.92
0.25
0.07
79.61
28.98
PNH

0
0
ated by Bland–Altman analysis of the relationship between the
differences and the mean of differences. Mean bias equal to zero
iRBCs

shows absolute agreement between methods.

6
2
0
0
85
422
12 440 9904
PNH

1456
2912
2386
3023
2812
2408
iRBCs
Run 3

3 | RESULTS

The results for intra- and inter-assay imprecision are summarized in

iRBCs %

3.24
0.40
0.08
77.93
31.89
PNH

Table 2. The CV/SD for the large (>50%), small to medium (20%–50%)
0
0
and small (<20%) PNH iRBCs clones ranged from 1.31/0.54 to
3.99/1.05 for intra-assay imprecision and from 1.5/0.22 to 2.61/1.19
12 855 10 018
515
110

2
0
0
11
iRBCs
PNH

for inter-assay imprecision, respectively.

Results from spiking test of five replicates

The results from the spiking test for five replicates are summa-
1615
3390
2777
2417
2139
2284
iRBCs
Run 2

rized in Table 3. The results showed zero events for LOB defined as
the highest apparent signal detected in samples containing no
measurand and a mean of 10 events for LOD defined as the lowest
iRBCs %

level of target events, that can be reliably distinguished from LOB

3.82
0.35
0.04
79.39
30.31
PNH

with CV = 15.7% below the acceptable range of 30%.

0
0

The results from healthy volunteers and PNH patients showed

absolute values (100%) for both clinical specificity and sensitivity as
99
13 522 10 735
541

9
1
0
0
iRBCs
PNH

well as for both positive and negative predictive values.

The results from the comparative study are summarized in
1785
2590
2585
2407
2236
2142
iRBCs
Run 1

Figure 2. Comparison between PNH iRBC and PNH Ne clones

showed: 0.9436/0.9234/1.7 for coefficient of determination (r2),
1:100000
TABLE 3

1:10000

Pearson's correlation coefficient (r) and Bland–Altman mean bias. The

Normal
1:1000
1:100
Neat
1:10

total PNH iRBCs clone was apparently lower in 8/441 (1.8%) cases
and apparently higher in 12/441 (2.7%) cases compared to the total
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872 MARINOV ET AL.

(A) Correlation between % PNH iRBCs vs % PNH Ne (B) Correlation between % PNH iRBCs vs % PNH Mo

100 y=1.0855x 100 y=1.1133

r2=0.9436 r2=0.9553
% PNH Ne

% PNH Mo
50 50

0 0
0 50 100 0 50 100
% PNH iRBCs % PNH iRBCs

(C) Mean bias: 1.70 (D) Mean bias: 2.1

50

50

Difference in results
Difference in results

25
25

0 0
0 50 100 0 50 100
-25
-25
-50
-50
Mean of results % PNH iRBCs vs % PNH Ne Mean of results % PNH iRBCs vs % PNH Mo

FIGURE 2 Correlation (A,B) and agreement (C,D) between PNH iRBCs and Ne (A,C) and PNH iRBCs and Mo (B,D)

PNH Ne clone but comparable to the total PNH Mo clone in all cases.
The total PNH iRBCs clone was apparently lower in 19/441 (4.3%)
cases and apparently higher in 5/441 (1.1%) than both PNH Ne and
PNH Mo without any impact to clinical sensitivity and positive predic-
tive value. Comparison between PNH iRBC and PNH Mo clones
2
showed: 0.9533/0.9387/2.1 for coefficient of determination (r ),
Pearson's correlation coefficient (r) and Bland–Altman mean bias. The
total PNH iRBCs clone was apparently lower in 1/441 (0.2%) cases
and apparently higher in 1/441 (0.2%) cases compared to the total
PNH Mo clone but in all cases comparable to the total PNH Ne
clone (Figure 2).
4 | DISCUSSION
A variety of flow cytometry assays have been developed to detect
iRBCs (both nucleated RBCs and/or reticulocytes) based on nucleic
15,16
acid stain or more recently, antibodies targeting the transferrin
receptor CD7117,18 with potential contribution for the diagnosis and
follow up of PNH by flow cytometry. Sutherland et al. recently
demonstrated, that a simple modification by addition of CD71 to the
ICCS/ESCCA guidelines PNH RBC protocol significantly improves the
ability to analyse PNH clone sizes in the RBC lineage, regardless of
patient haemolytic and/or transfusion status and provides more
objective ability to delineate PNH Type III, Type II and Type I iRBCs
compared to that in mature RBCs.19
In the present study, we evaluated the performance characteris-
tics of this 3-colour PNH iRBC assay with respect to its potential role
for PNH screening by flow cytometry in laboratories with limited
experience of such analysis across both WBC and RBC lineages. Our
results confirmed excellent performance characteristics of the assay
with CVs <5% between replicates for all tested PNH clone sizes for
both intra- and inter-assay imprecision. All PNH patients were cor-
rectly identified by a positive screening result and all healthy individ-
uals were identified by a negative screening result, respectively.
Absolute probability of PNH given a positive screening test and
absence of PNH given a negative screening test were obtained.
A very important aspect of all screening tests with impact on both
clinical sensitivity and specificity is the analytical sensitivity deter-
mined by the highest apparent signal detected in samples containing

MARINOV ET AL.
no measurand (LOB) and the lowest level of measurand, that can be
reliably distinguished from the LOB (LOD). For all five replicates, we
demonstrated 0 events for LOB and a mean of 10 events for LOD
with CV = 15.7%, which is below the acceptable limit of 30%. 13
How-
ever, we support and recommend the generally accepted smallest
number of events required to reproducibly detect a PNH population
(LOD) to be 20 PNH events. The sensitivity of each assay is depen-
dent on the number of acquired events and the LOD (20 clustered
events). In another study, we confirmed acceptable CV below 10% for
limit of quantification (LOQ) ranging from 30 to 50 events, which is
the lowest level of measurand that can be reliably calculated (data not
shown).
Very good correlation (r2 = 0.9436, Pearson's r = 0.9234) and
agreement (Bland–Altman mean bias = 1.70) was observed between
PNH iRBC and PNH Ne clones in 441 PNH patients. In 1.8% of
cases, the total PNH iRBCs clone was present but lower than the
total PNH Ne clone, in 2.7% of cases, the total iRBCs clone was
higher than the total PNH Ne clone. Interestingly in all these cases,
the PNH iRBC clone was comparable in size to the total PNH Mo
clone and these percentual discrepancies did not have any impact
on the clinical sensitivity or specificity of the assay. In 4.3% of
cases, the total iRBCs clone was lower than both the total PNH Ne
and PNH Mo, and in 1.1% of cases, the total iRBCs clone was
higher than both the total PNH Ne and PNH Mo. However, we did
not observe any cases in which PNH WBCs were detected while
PNH iRBCs clones were not.
In another study, with patients who have been on complement-
inhibitory medications for some time, very rare PNH cases were
observed in which the mature RBC assay did not identify an obvious
PNH clone despite the presence of large PNH clones (over 95%) in
both the neutrophils and monocytes (example of this is shown in fig-
ure 7 of reference 19). The only unusual finding was the slightly
decreased level of CD59 expression on what was initially thought to
be Type I mRBCs. Looking at the iRBC assay, it demonstrated a pre-
dominant PNH type II population with very high CD59 expression,
which was not apparent from our initial assessment of the mRBCs.19
This clearly demonstrates the value of the iRBC assay in regards to
the sensitivity and specificity of the assay.
We also confirmed very good correlation (coefficient of determi-
nation r2 = 0.9553, Pearson's r = 0.9387) and agreement (Bland–
Altman mean bias = 2.1) between PNH iRBCs and PNH Mo. In 0.2%
of cases, the total PNH iRBCs clone was apparently lower and in 0.2%
of cases was apparently higher than the total PNH Mo clone but in all
cases comparable to the total PNH Ne clone, therefore with no
impact on the clinical sensitivity or specificity of the assay.
In a previous attempt at developing a PNH ‘screening assay’ for
non-expert flow laboratories, Gatti et al. published a simple 2-colour
WBC assay designed primarily to detect PNH granulocytes based only
on the use of CD15 conjugate in combination with FLAER.21 This
attempt was however somewhat controversial, since such a combina-
tion of only two reagents and gating strategy would not guarantee
sufficient compliance with other cell compartments and desired levels
for both clinical sensitivity and specificity.22
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873
Here, we have validated that the PNH iRBC assay meets most
criteria for a reliable PNH screening test for small, medium and large
PNH clones by flow cytometry. The acquisition of at least 1  106
CD235+ mRBCs usually allows the analysis of a sufficient number of
iRBCs to reach analytical sensitivity from 0.1% to 1.0% (LOD = 20
clustered events) and 100% clinical sensitivity for detection of small,
medium and large PNH clones in haemolytic PNH and related bone
marrow failure syndromes except for severe AA with low or absent
iRBCs, even though the presence of very low iRBC events in an
untransfused patient can be useful indication for bone marrow failure,
regardless of whether small PNH phenotypes are present or not. This
3-colour assay retains the ability to detect minor mRBC PNH clones
(<1.0%) and phenotypes (<0.1%) with very high sensitivity in severe
AA, in complete agreement with our previous publication, however
their absence does not exclude WBC PNH clones or phenotypes.19 In
such cases in which small numbers of PNH phenotypes can be
detected only among the mRBC subset, it is still mandatory to follow
up with the WBC assay to confirm the absence of two GPI-linked
antibodies for the neutrophil and monocyte lineages following the
2018 ICCS/ESCCA PNH Guidelines. In addition, any small decrease in
CD59 expression on putative Type I (normal) mRBC and iRBC should
be further investigated with reflexing to the WBC assay to exclude
the possibility of rare cases with PNH type II clone with high CD59
expression without any evidence of PNH Type III cells. This is espe-
cially important for labs with limited experience at high-sensitivity
testing and in cases with a detectable but not quantifiable PNH iRBC
clone. It has been shown that PNH shows a wide variety of different
and often very heterogeneous patterns and thus may require addi-
tional testing to confirm the presence or absence of a PNH clone
guiding the clinician in optimal patient management.
AUTHOR CONTRIBU TIONS
Iuri Marinov, Stephen J. Richards and D. Robert Sutherland designed
the study. Iuri Marinov, Adam Pešek and Stephen J. Richards prepared
and analysed the samples. Iuri Marinov performed statistical analysis
and wrote the first draft of the manuscript. All authors contributed to
the design, creativity, collection and editing of the manuscript. All
authors have read and approved the final version of the manuscript.
CONFLIC T OF INT ER E ST
The authors have no competing interests.
DATA AVAILABILITY STAT EMEN T
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
OR CID
Iuri Marinov https://orcid.org/0000-0003-0025-3131
Andrea J. Illingworth https://orcid.org/0000-0003-0237-3591
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How to cite this article: Marinov I, Richards SJ, Pešek A,
Illingworth AJ, Sutherland DR. Validation of a single tube 3-
colour immature red blood cell screening assay for the
detection and enumeration of small, medium and large
paroxysmal nocturnal haemoglobinuria clones by flow
cytometry. Int J Lab Hematol. 2022;44(5):868‐874. doi:10.
1111/ijlh.13877