Flow cytometry thresholds of myeloperoxidase detection todiscriminate between acute lymphoblastic or myeloblasticleukaemia

Julien Guy,1 Il�eana Antony-Debr�e,2,3

Emmanuel Benayoun,2 Isabelle Arnoux,4

Chantal Fossat,4 Magali Le Garff-

Tavernier,5 Anna Raimbault,5 Mich�ele

Imbert,2,3 Marc Maynadi�e,1 Francis

Lacombe,6 Marie C B�en�e7and Orianne

Wagner-Ballon2,3 on behalf of the GEIL

(Groupe d’Etude Immunologique des

Leuc�emies)1Haematology Laboratory, University Hospital of

Dijon, Dijon, 2Haematology Laboratory, Univer-

sity Hospital APHP Henri Mondor, 3UPEC,

Cr�eteil, 4Haematology Laboratory, University

Hospital AP-HM La Timone, Marseille,5Haematology Laboratory, University Hospital

APHP La Piti�e-Salp�etri�ere, Paris, 6Haematology

Laboratory, University Hospital Haut-L�eveque,

Bordeaux and 7Haematology Laboratory,

University Hospital of Nantes, Nantes, France

Received 2 December 2012; accepted for

publication 25 January 2013

Correspondence: Orianne Wagner-Ballon,

Haematology Laboratory, University Hospital

APHP Henri Mondor, UPEC, 51 Avenue du

Mar�echal de Lattre de, Tassigny 94000 Cr�eteil,

France.

E-mail: orianne.wagnerballon@hmn.aphp.fr

Summary

The World Health Organization 2008 Classification emphasizes myeloperoxidase

(MPO) detection as sufficient for assigning a blast population to the myeloid line-

age. Published MPO positivity thresholds are 10% for flow cytometry (FCM) but

3% for cytochemistry. Here we re-evaluated the FCM-MPO threshold by comparing

retrospectively 128 acute lymphoblastic leukaemias and 75 acute myeloid leukaemias

without maturation, all assessed by benzidine-based cytochemistry. A 13% threshold

was found to be relevant using an isotype control as background-reference (sensitiv-

ity 95�1%, specificity 91�7%). Residual normal lymphocytes proved to be an advan-

tageous alternative reference, a threshold of 28% yielding improved 97�4%sensitivity and 96�1% specificity.

Keywords: acute leukaemia, flow cytometry, myeloid leukaemia, myeloper-

oxidase, threshold.

The World Health Organization (WHO) 2008 Classification

considers the detection of myeloperoxidase (MPO) as suffi-

cient per se to assert that a blast population belongs to the

myeloid lineage (Borowitz et al, 2008), notably for the diag-

nosis of acute leukaemia (AL) of ambiguous lineage (Matutes

et al, 2011). Detection of MPO on bone marrow (BM) aspi-

rates or peripheral blood (PB) samples can be performed

either using enzyme cytochemistry, classically with a benzi-

dine- (Hattori, 1958) or benzidine dihydrochloride-based

stain (Kaplow, 1965), or flow cytometry (FCM; van der

Schoot et al, 1990), even if both are typically used as com-

plementary techniques. The French-American-British (FAB)

classification has defined AL as being of myeloid lineage

when 3% or more of blast cells show cytochemical reactivity

(Bennett et al, 1976). The European Group for Immunophe-

notypic Characterization of Leukaemias (EGIL) has estab-

lished guidelines for the immunological classification of AL

with a threshold of at least 10% of blast cells stained to con-

sider MPO positivity as for other intracellular markers such

as CD3, CD79a and TdT (Bene et al, 1995). Several series

have compared the detection of MPO by cytochemistry and

FCM, using different methods to assess positivity in FCM

(isotype external control or residual lymphocytes as negative

internal control) with cut-offs varying from 3 to 20% in vari-

ous subtypes of acute myeloid leukaemia (AML) (Nakase

et al, 1998; Nguyen et al, 1998; Peffault de Latour et al,

2003; Saravanan & Juneja, 2010) . In order to improve sensi-

tivity, it has been proposed to lower the FCM threshold

ª 2013 John Wiley & Sons Ltd First published online 25 February 2013British Journal of Haematology, 2013, 161, 551–555 doi:10.1111/bjh.12277

short report

down to 3% as per cytochemistry (Peffault de Latour et al,

2003). However, as no patients with acute lymphoblastic leu-

kaemia (ALL) were studied in the latter study, the specificity

of this threshold could not be determined.

Thus, as the WHO highlights FCM for the detection of

MPO, yet without proposing a threshold or establishing rec-

ommendations for interpretation, we decided to reevaluate

the MPO positivity threshold to be used in FCM. A multicen-

tre retrospective trial was initiated to compare primary AL

with homogenous blast populations defined as MPO-negative

(i.e. ALL) or MPO-positive (i.e., AML without maturation,

AML M1) by benzidine-based cytochemistry, the FAB ‘gold

standard’. Furthermore, as the use of isotype controls in mul-

tiparametric FCM is costly and therefore limited in everyday

practice, we decided to assess the relevance of using the resid-

ual normal lymphocyte population as an internal control.

This study proposes a method that was standardized

between four centres to interpret MPO staining by FCM

regardless of antibodies, permeabilization reagents or instru-

ments used. Moreover, we re-evaluated the more relevant

thresholds to discriminate between ALL and AML M1

according to the negative control used; these thresholds were

subsequently assessed on AML with minimal differentiation

(AML M0) classically defined by negative cytochemistry.

Materials and methods

Four French University Hospital laboratories participated in

this retrospective study. BM or PB from 221 patients with

AL assessed at diagnosis between 2004 and 2012 were

included. The patients’ age ranged from 2 months to

81 years old. Three subtypes of AL, classified according to

the WHO (Swerdlow, 2008)/FAB (Bennett et al, 1976) classi-

fications, were studied: 128 ALL (113 B-lineage, 15 T-line-

age), 75 AML M1 and 18 AML M0 (Table SI).

Smears were benzidine-stained at diagnosis as reported

elsewhere (Hattori, 1958; Kaplow, 1965) and cytochemistry

analysis was performed in each centre.

EDTA-anticoagulated samples were first labelled with at

least CD45-ECD, CD45-PC5, CD45-PC7 (Immunotech, Mar-

seille, France), CD45-PC5�5 or CD45-V500 (BD Biosciences,

Mountainview, CA, USA). Permeabilization was then per-

formed with commercial reagents (Fix and Perm Cell Per-

meabilization Kit, Caltag Labs, San Francisco, CA, USA;

Intrastain, Dako, Glostrup, Denmark; or Intraprep, Beckman

Coulter, Miami, FL, USA) according to the manufacturers’

recommended procedure. After permeabilization, the cells

were stained with 5 ll of anti-MPO- fluorescein isothio-

cyanate (FITC)-labelled monoclonal antibody (Dako or

Immunotech) or isotype controls (for 96/128 ALL and 40/75

AML M1 samples). Samples were analysed within 24 h of

collection on a flow cytometer (EPics XL, FC500, Beckman

Coulter or Canto, CantoII, BD Biosciences, Table SII).

Each centre provided flow cytometry standard listmode

data (fcs) for each of the samples generated on-site. The fcs

files were analysed centrally in a blind fashion using the Kal-

uza software (Beckman Coulter). Residual lymphocytes were

gated on a CD45 versus side-angle scatter (SSC) dot plot as

CD45high/SCClow cells whereas blast cells were defined by

low/intermediate light scatter and low to negative CD45

expression (Lacombe et al, 1997). Then, the expression of

MPO by blast cells was determined on a monoparametric

histogram. Two reference controls (isotype control and/or

residual lymphocytes) with five various cut-offs (2%, 1%,

0�75%, 0�5% and 0�25%) were assessed. Only samples that

contained at least 20% blast cells, more than 150 lympho-

cytes and with MPO-positive residual polymorphonuclears

were retained for data analysis.

All results were exported to a Microsoft Excel table. Recei-

ver Operating Characteristics (ROC) curves, Spearman corre-

lation coefficients, Bland and Altman and dot plots were

obtained using MedCalc for Windows, version 9.5.0.0 (Med-

Calc Software, Mariakerke, Belgium).

Results and discussion

A 1% cut-off allows for a harmonized interpretation ofMPO staining by FCM

This multicentre retrospective trial compared 128 ALL and

75 AML M1, respectively defined as MPO-negative or posi-

tive by benzidine-based cytochemistry. Blasts were consid-

ered MPO positive by flow cytometry when their mean

fluorescence intensity exceeded that of blast cells incubated

with an isotype control or that of residual lymphocytes,

used as internal control for fluorescence intensity (Ratei

et al, 2006), in the MPO-stained sample. In order to deter-

mine the best manner to assess MPO staining above refer-

ence controls, five different cut-offs were tested and

compared using ROC curves (Figure S1). The 1% cut-off

provided the best discrimination when residual lymphocytes

were used as reference while 0�75% and 1% cut-offs were

the best when using an irrelevant isotype. The percentages

of blast cells with fluorescence above that determined by

that 1% cut-off in each sample was then evaluated for the

203 patients.

Given that the Ratio of mean Fluorescence Intensity (RFI)

can be a useful parameter to analyse staining for markers

with unimodal distribution, the RFI of the MPO fluorescence

of blast cells relative to that of both controls was also evalu-

ated.

Different positivity thresholds discriminate ALL andAML according to the reference control used

Using this 1% cut-off, we then investigated which was the

more relevant threshold to apply in order to discriminate

between ALL and AML.

The optimal ROC-established threshold was 13% of

stained blasts with a sensitivity of 95�1% and specificity of

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552 ª 2013 John Wiley & Sons LtdBritish Journal of Haematology, 2013, 161, 551–555

91�7% (Figs 1A and 1E) using the isotype control as refer-

ence. Residual normal lymphocytes proved to be an advanta-

geous alternative, a ROC-established threshold of 28%

yielding improved 97�4% sensitivity and 96�1% specificity

(Figs 1B and 1E). An excellent correlation was observed

between both methods (Spearman coefficient (r) = 0�83).The percentage of positive blasts was higher using lympho-

cytes as reference, compared to the isotype control, since the

former bypass the higher non-specific binding of isotype

controls on blast cells (Bland-Altman test, data not shown).

The EGIL 10% threshold, using the isotype control as ref-

erence to assess positivity, provided 100% sensitivity and

85�4% specificity. This threshold is therefore pertinent,

although specificity can be improved by the newly-defined

thresholds reported above.

The cytochemistry 3% threshold proposed by Peffault de

Latour et al (2003) yielded an excellent sensitivity of 100%, but

an unacceptably low specificity (68�7% and 21�1% for the

isotype control or lymphocytes). This low threshold might

therefore wrongly lead to a conclusion of mixed-phenotype

acute leukaemia (MPAL) diagnosis according to the WHO

classification (Borowitz et al, 2008), in clinical practice (Fig 1E).

Two MPO RFI thresholds were also defined, at 3�4 and 5�1relative to the use of isotype control (Fig 1C) or residual

lymphocytes (Fig 1D), respectively, as reference. A better sen-

sitivity (90% vs. 86�5%) and specificity (95�8% vs. 88�5%) was

observed for the use of isotype controls versus lymphocytes.

The isotype control appeared to be the more relevant reference

to discriminate ALL against AML M1 (AUC: 0�980 vs. 0�934).Comparison of the four selected methods (i.e. percentages

or RFI with isotype or lymphocyte controls), therefore indi-

cates that the percentage of MPO-positive blasts, relative to

the internal control provided by lymphocytes, appears to be

the most pertinent to discriminate between ALL and AML

M1. These comparisons were performed on the whole

cohort, yet checked to demonstrate the absence of any

centre-effect (data not shown). This supports the fact that

this method of analysis was properly standardized between

the four participating centres. Its robustness suggests that it

can be applied in routine practice, whatever the laboratory

involved, regardless of antibodies, permeabilization reagents

or instruments used.

AML M0 assessment is improved using the lymphocyte-defined threshold

Finally, we assessed the relevance of this analysis method and

positivity thresholds on 18 cases of AML M0. These cases are

MPO-negative by definition in cytochemistry, yet liable to be

positive in FCM (Bene et al, 2001). FCM also allows

confirmation of their myeloid lineage through the expression

of myeloid markers. Interestingly, with the new appropriate

(A) (B)

(C)

(E)

(D)

Fig 1. Comparison between different analysis

methods of MPO staining interpretation and

usefulness of different thresholds used to dis-

criminate between ALL and AML according to

the reference control used. Isotype control used

as control (A). Percentages of blast cells show-

ing fluorescence above that of the negative

control with a 1% cut-off. Isotype control was

available for 96/128 ALL and for 40/75 AML

M1. Three thresholds are shown: EGIL 10%*,3%** and ROC-established 13%. Residual

lymphocytes used as control (B). Percentages

of blast cells showing fluorescence above that

of the negative control with a 1% cut-off (128

ALL and 75 AML M1). Three thresholds are

shown: EGIL 10%*, 3%** and ROC-estab-

lished 28%. Ratios of MPO mean fluorescence

intensity (RFI) for isotype control (C) or resid-

ual lymphocytes (D) used as control. Specific-

ity and sensitivity for the three thresholds with

both negative controls (E). Data obtained from

ROC curves for the three thresholds for isotype

control and residual lymphocytes used as

reference are presented.* (Bene et al, 1995);

**(Peffault de Latour et al, 2003).

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ª 2013 John Wiley & Sons Ltd 553British Journal of Haematology, 2013, 161, 551–555

thresholds as determined above, MPO staining was positive

for 10 of 18 AML M0 when using lymphocytes as reference

versus only 3 of 17 cases when using isotype controls (Fig 2).

Therefore, both cytochemistry and FCM methods may be

required to assess AML M0.

In conclusion, leukaemic cells may be assigned to the

myeloid lineage by FCM regardless of the reference control

used if an appropriate threshold for MPO positivity is

applied, 13% or 28% depending on the use of an isotype

control or residual lymphocytes, respectively. Moreover, the

analysis strategy developed here to evaluate MPO expression

is not only relevant to discriminate between ALL and AML

but can be useful for the diagnosis of AML M0, especially

using residual lymphocytes as reference. Of note, because the

diagnosis of MPAL relies on MPO detection for assignment

of the myeloid lineage, these new thresholds could also be

useful in this context.

Acknowledgements

The authors thank H�el�ene Jouault and Elodie Manuelein for

initiating this work.

Author contributions

J.G. contributed to the study design and to the manuscript

preparation and performed statistical analysis. I.A-D. helped

with data analysis and contributed to the manuscript prepa-

ration. I.A., C.F., J.R., M.L-T. and A.R. provided listmode

data, collected patient information and reviewed the manu-

script. E.B. collected information. F.L, M.M. and M.I.

reviewed the manuscript. MC.B. contributed to the manu-

script preparation and study design. O.W-B. designed the

study, analysed the data and wrote the manuscript.

Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig S1. Comparison between differents analysis methods

of MPO staining interpretation.

Table SI. Subtypes of acute leukaemia analysed (WHO/

FAB classifications).

Table SII. Different reagents and instruments used per

centre.

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