ImageStream Promyelocytic Leukemia Protein

Immunolocalization: In Search of Promyelocytic

Leukemia Cells

Peppino Mirabelli,1,2 Giulia Scalia,1 Caterina Pascariello,1,2 Francesca D’Alessio,1,2

Elisabetta Mariotti,1 Rosa Di Noto,1,2* Thaddeus C. George,3 Raymond Kong,3

Vidya Venkatachalam,3 David Basiji,3 Luigi Del Vecchio1,2

� AbstractAcute promyelocytic leukemia (APL) is a hematological emergency in which a rapid di-agnosis is essential for early administration of appropriate therapy, including all-transretinoic acid before the onset of fatal coagulopathy. Currently, the following methodol-ogies are widely used for rapid initial diagnosis of APL: 1) identification of hypergranu-lar leukemic promyelocytes by using classical morphology; 2) identification of cellswith diffuse promyelocytic leukemia (PML) protein distribution by immunofluores-cence microscopy; 3) evidence of aberrant promyelocyte surface immunophenotype byconventional flow cytometry (FCM). Here, we show a method for immunofluorescentdetection of PML localization using ImageStream FCM. This technique provides objec-tive per-cell quantitative image analysis for statistically large sample sizes, enabling pre-cise and operator-independent PML pattern recognition even in electronic and realdilution experiments up to 10% of APL cellular presence. Therefore, we evidence thatthis method could be helpful for rapid and objective initial diagnosis and the promptinitiation of APL treatment. ' 2012 International Society for Advancement of Cytometry

� Key termsImageStream; flow cytometry; acute promyelocytic leukemia; PML gene; all-trans reti-noic acid

INTRODUCTIONAcute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia

(AML) with specific biology and clinical presentation. The vast majority of cases are

characterized by a translocation that fuses the promyelocytic leukemia gene (PML) on

chromosome 15 with the gene-encoding retinoic acid receptor-alpha (RARa) on chro-

mosome 17 (1). Because APL can be rapidly fatal, with 5–10% of newly diagnosed

patients succumbing to hemorrhagic death, and because most patients can be cured

with early administration of therapy, sensitive and specific methods to screen AML cases

before the biomolecular confirmation of PML/RARa rearrangement are needed (2).

Morphologic evidence of hypergranular leukemic promyelocytes generally

implies the presence of APL cells and justifies immediate treatment initiation (2,3).

However, more robust diagnostic procedures are represented by cytogenetic, fluores-

cence in situ hybridization (FISH), and reverse transcriptase–polymerase chain reac-

tion (RT-PCR) in order to precisely identify the PML-RARa fusion gene, but they

can be time consuming and require specialized laboratories (3,4). Alternative

diagnostic procedures for APL diagnosis include PML pattern recognition by immu-

nofluorescence microscopy (4) and surface immunophenotypic analysis by conven-

tional flow cytometry (FCM) (5).

Immunofluorescence analysis is a rapid and specific diagnostic tool to distin-

guish APL blasts that exhibit multiple diffuse small PML bodies from cells with

1CEINGE-Biotecnologie Avanzate, Naples,Italy2Dipartimento di Biochimica eBiotecnologie Mediche, Universit�aFederico II, Naples, Italy3Amnis Corporation, Seattle, 2505 ThirdAvenue, Suite 210, Washington

Received 23 May 2011; Accepted 23December 2011

Additional Supporting Information(MIFlowCyt Item Location) can be foundin the online version of this article.

Grant sponsor: Associazione Italiana perla Ricerca sul Cancro (AIRC); Grantnumber: 10737; Grant sponsor: Ministerodell’Istruzione, dell’Universit�a e dellaRicerca��Programmi di Ricerca diRilevante Interesse Nazionale 2008(MIUR-PRIN 2008); Grant number:2008P8BLNF_004.

*Correspondence to: Rosa Di Noto,CEINGE-Biotecnologie Avanzate, ViaGaetano Salvatore 486, 80145 Naples, Italy

Email: rosa.dinoto@unina.it

Published online 20 January 2012 in WileyOnline Library (wileyonlinelibrary.com)

Conflict of interest: The authors PM, GS,CP, FD, EM, RDN, and LDV have no com-peting interests. The authors TG, RK, VVand DB are shareholders and employeesof Amnis Corporation.

DOI: 10.1002/cyto.a.22013

ª 2012 International Society forAdvancement of Cytometry

Original Article

Cytometry Part A � 81A: 232�237, 2012

wild-type distribution consisting of no more than 20 distinct

large PML bodies (4). However, this technique can involve op-

erator biases and has limited statistical power. Although con-

ventional FCM has shown documented efficacy for a rapid di-

agnosis of APL by the identification of APL blasts with patho-

logical immunophenotype, confirmation of the PML pattern

associated with APL is not possible by this technique (5,6).

Recently, Grimwade et al. (7) were able to quantify PML

protein pattern expression at diagnosis using the ImageStream

imaging FCM on a cohort of 18 AML patients, 4 of which

were APL with a blast range between 75 and 85%. In wake of

their work, we extended the strategy of ImageStream APL

identification by showing rare event detection in dilution

experiments. Our work was based on the fact that imaging

FCM combines high-speed image capture with image quantifi-

cation, enabling statistical and objective discrimination of cells

based on their appearance (8). In this way, we were able to: i)

identify single live cells in fixed/permeabilized cellular suspen-

sions; ii) determine ‘‘micro-’’ and ‘‘macrospeckled’’ PML

expression pattern in NB4 and HL60 cell lines as well as in

fresh APL and normal bone marrow (BM) cells, respectively;

iii) demonstrate the ImageStream ability to detect ‘‘micro-

speckled’’ pattern up to 10% APL cell presence in electronic

and real dilution experiments spiking NB4 in the midst of

HL60 as well as fresh APL blasts in normal BM cells.

MATERIALS AND METHODS

Cell Cultures

HL-60 (human AML FAB-M2) and NB4 (human APL FAB-

M3, PML/RARa1) cell lines were acquired from Deutsche

Sammlung von Mikroorganismen und Zellkulturen and main-

tained in continuous culture at CEINGE-Biotecnologie Avanzate

Cell Culture Facility (Naples, Italy) according to UKCCCR guide-

lines for the use of cell lines in cancer research (9). Particularly,

both cell lines were cultured in 24 well plates at 500,000 cells/mL

in RPMI (SIGMA-ALDRICH, St. Louis, MO) supplemented with

10% heat-inactivated fetal bovine serum (LONZA Basel, Switzer-

land) and 1% Ultraglutamine (LONZA Basel, Switzerland).

Patients

Normal BM sample was obtained from a patient with

non-Hodgkin lymphoma who underwent BM aspiration in

the context of routine clinical practice. APL BM was obtained

from a patient with 82% of leukemic promyelocytes at diagno-

sis. In both cases, BM sample was obtained following

informed consent.

Successively, APL and normal BM cells were obtained af-

ter red blood cell lysis with NH4CL. After lysis, cells were

counted with Invitrogen CountessTM automatic cell counter

(Life Technologies, MI, Italy), and cellular viability was

assessed by Trypan blue exclusion. Finally, cellular dilutions

were performed as described in Table 2, and a total of 500,000

cells were used for each ImageStream test.

Cellular Fixation, Permeabilization, and Staining

Cells were fixed and stained with BD Cytofix/Cytoperm

kit (BD Biosciences, San Jose, CA) according to the manufac-

turer’s instructions. Fixed/permeabilized cells were diluted in

100 lL of BD Perm/Wash solution containing 30 lL of Alexa-

Fluor488-conjugated anti-PML (clone PG-M3; Santa Cruz

Biotechnology, Santa Cruz, CA) monoclonal antibody and

incubated for 90 min at room temperature. Unstained fixed/

permeabilized cells were used as negative staining control. Af-

ter incubation, cells were washed twice with 1 mL 13 BD

perm/wash solution and stained with 50 nM of DRAQ5 (Bios-

tatus, UK), a cell permeable far-red fluorescent DNA dye.

All samples were run on the ImageStream100 flow cytom-

eter (Amnis Corp., Seattle), and 20,000 cells per sample were

collected with 150 mW 488-nm laser power with brightfield

set to channel 5. Only events with a minimum bright field

area of 12 lm2 or greater were included in the data file to

eliminate collection of small debris. Data were acquired using

the INSPIRE (Amnis Corp.) software and analyzed by the

IDEAS software (Amnis Corp.).

RESULTS

Single, nonapoptotic cells in BD Cytofix/Cytoperm fixed

cellular suspensions were identified as shown in Figure 1. First,

single cells included in the blue region (Fig. 1, panel A) with

normal DNA content and high-aspect (width/height) ratio

were discriminated from multicellular events (high-DRAQ5

intensity and low-aspect ratios) and debris (low-DNA con-

tent). Nonapoptotic cells were then selected in the yellow

region on the DRAQ5 threshold area versus SSC mean pixel

(Fig. 1, panel B). Condensed and fragmented nuclei of apo-

ptotic cells displayed lower nuclear areas compared to live

cells; apoptotic cells often had higher scatter as well. Finally,

within the live gate, the cells of best focus (cell in focus gate,

not shown) were gated for further analysis.

Qualitatively, two distinct ‘‘micro-’’ and ‘‘macrospeckled’’

PML expression patterns were seen in NB-4 and HL-60 cell

lines as well as in fresh APL and normal BM cells, respectively.

To quantitatively distinguish these patterns, two features were

used in the analysis: Spot Count and Modulation. The Spot

Count feature enumerates bright PML spots per cell. Using

this parameter, we found that HL-60 cells, which displayed a

predominantly ‘‘macrospeckled’’ phenotype, had much higher

mean spots per cell compared to NB4 cells, which displayed a

predominantly ‘‘microspeckeld’’ pattern (3.52 vs. 0.42 mean

spots/cell, Fig. 2, Panel A and B, respectively). We also used

the Modulation texture parameter to measure PML distribu-

tion. Modulation 5 (max pixel – min pixel)/(max pixel 1min pixel). The bigger the range in the per-cell PML stains,

the higher the modulation scores. Because of the high range in

pixel intensity associated with ‘‘macrospeckled’’ PML distribu-

tion, HL60 cells had significantly higher modulation values

than NB4 cells displaying a ‘‘microspeckled’’ pattern (Fig. 3).

Additionally, we evidenced the ‘‘microspeckled’’ pattern also

in case of fresh APL blasts using the modulation feature and

the ‘‘macrospeckled’’ one in case of normal BM cells (Fig. 4).

The second step of our work was to see if it was possible

to detect limiting numbers of APL cells within a larger cell

population, we performed preliminary electronic mixing

experiments of two cell lines such as NB-4 and HL-60. Briefly,

ORIGINAL ARTICLE

Cytometry Part A � 81A: 232�237, 2012 233

randomly selected events from the NB4 file were combined

with the HL60 file to form merged files that contained a speci-

fic ratio of HL60 and NB4 events. Triplicate files with 999/1

and 99/1 HL60/NB4 ratios, along with a single file with 9/1

and 1/1 HL60/NB4 ratios, were used (Table 1). These merged

files were then analyzed with the same template to obtain

PML modulation mean scores (Table 1). The relative percent-

age of NB4 cells detected within the merged files was esti-

mated by calculating the increase in the mean feature values

over negative control (F0 5 100% HL60 sample) and normal-

izing to the mean feature value range between negative and

positive controls (F100 5 100% NB4 sample). This normalized

percentage (Fn) was computed as follows: Fn 5 100 3 (Fm –

F0)/(F100 – F0), where Fm is the measured feature value. In this

way, the percentage of NB4 contamination was determined by

modulation normalization as follows: Fn (NB-4) 5 100 3 (Fm– 0.4479)/(0.2372 – 0.4479). Both the non-normalized and

normalized modulation scores were reported in Table I.

Figure 1. Panel A shows a representative dot-plot for selection of single HL-60 cells. Multicellular events have high-DRAQ5 intensity and low-

aspect ratios while debris exhibit low-DNA content. Representative bright field (BF) images show debris, single, and aggregate cells. Panel B

shows discrimination of live versus apoptotic HL60 cells based on threshold area of the DRAQ5 image and mean pixel of the SSC image. Ap-

optotic cells with condensed and fragmented nuclei have lower nuclear areas and higher SSC compared to live cells. Representative live

(upper) and apoptotic (lower) cells are shown. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE

234 PML Localization by Imaging FCM

Importantly, by the use of normalized modulation feature, we

were able to identify up to 10% of virtual contamination for

NB-4 cells (Table 1).

By virtue of these in silico tests, we evaluated the ability

of ImageStream FCM to detect ‘‘microspeckled’’ pattern in

real dilution experiments mixing NB4 in HL60 cells as well as

fresh APL blasts in normal BM cells at 50%, 10%, 1%, and

0.1% levels. As evidenced in Table 2, APL cellular contamina-

tions were detected up to 10% level using the mean modula-

tion feature in both NB4 mixed in HL60 as well as fresh APL

mixed in normal BM cells. Dilution at 1% and 0.1% levels was

nondetected, and the mean modulation was closely similar to

that evidenced for uncontaminated HL60 cell line and normal

BM cells (data not shown). Finally, using the mean modula-

tion normalization feature, we were able to find up to 10% of

APL-contaminating cells with respect to the performed dilu-

tions, as evidenced in Table 2.

DISCUSSION

APL is a medical emergency frequently presenting with an

abrupt onset (1). The high risk of early hemorrhagic mortality,

which still accounts for 5–10% of newly diagnosed patients,

and the potential for high-cure rate (>80%) highlight the im-

portance of immediate recognition and prompt initiation of

specific treatment (2). The identification of the APL-specific

genetic lesion in leukemic cells is feasible at chromosome,

DNA, RNA, and protein levels with the use of conventional

karyotyping, FISH, RT-PCR, and anti-PML monoclonal anti-

bodies, respectively. Compared to conventional karyotyping,

Figure 2. Panel A shows PML mean spot count in HL-60 cells.

Representative PML/DRAQ5 composite images from the indicated

histogram bins are shown. Note the macrospeckled nuclear distri-

bution of the PML stain. Panel B shows PML mean spot count in

NB4 cells. Note the diffuse pattern of PML staining that generally

does not overlap with nucleus. [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]

Figure 3. Panel A shows PML HL-60 pattern detection using the

modulation feature. Representative images of cells with low (left)

and high-(right) modulation values are shown. Panel B shows

PML NB4 pattern detection using the modulation feature. Impor-

tantly, NB4 cells had a dramatically lower mean modulation score

compared to HL-60 cells. Representative images of cells with low

(left) and high-(right) modulation values are shown. Panel C

shows modulation overlay between NB4 and HL60 cells evidenc-

ing the ImageStream ability to clearly discriminate ‘‘micro-

speckled’’ (green) and ‘‘macrospeckled’’ (red) pattern. [Color fig-

ure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]

ORIGINAL ARTICLE

Cytometry Part A � 81A: 232�237, 2012 235

RT-PCR and FISH do not require dividing cells for analysis,

and they allow results to be obtained in cases where the PML-

RARa fusion gene is formed as a result of cryptic or complex

rearrangements in the absence of the classic t(15;17) (3). How-

ever, because both these techniques are technically challenging

and RT-PCR is notoriously prone to contamination and arti-

facts (3), it is advisable that diagnostic and follow-up samples

are sent to reference laboratories where well-trained personnel

have specific experience with PML/RARa (3). Therefore, a

more rapid initial diagnosis procedure is required.

Rapid diagnostic techniques use microscopic and/or flow

cytometric analysis of patient blood and BM samples. The

morphologic appearance of hypergranular leukemic promye-

locytes allows typical cases to be identified and justifies imme-

diate treatment initiation, without waiting for diagnostic con-

firmation at the genetic level (3). In addition, cells with micro-

granular PML nuclear distribution associated with PML/

RARa can be readily distinguished from other leukemic and

normal hematopoietic cells having an aggregated PML nuclear

pattern by using immunofluorescence microscopy (4). In light

of its very convenient cost-benefit ratio, this assay is highly

recommended to rapidly confirm diagnosis of APL at the pro-

tein level, especially in small institutions not equipped and

experienced for genetic analyses (10). However, traditional

imaging techniques suffer from poor statistics and lack of

standard quantitative metrics, making definitive identification

of APL difficult in some cases.

Conventional nonimaging FCM also plays an important

role for the diagnosis and monitoring of APL cells (5,11).

Indeed, APL is characterized by a highly specific immunophe-

notype showing a consistent absence or very low expression of

CD15, CD11a, CD11b, CD11c, CD18, CD66b, and CD66c

molecules (5,12). At the same time, the blasts exhibit a wide

range of CD13 expression (broad histogram), whereas CD33

expression is homogeneous (sharp histogram). CD34 and

human leukocyte antigen (HLA)-DR are frequently absent (5).

This immunophenotype, in skillful hands, has been reported

to show high sensitivity and specificity for predicting APL mo-

lecular rearrangement (12,13).

The emergence of high-speed imaging FCM has allowed

exploration of new frontiers in clinical hematology, particu-

larly in case of APL diagnosis, as demonstrated in this work.

Indeed, ImageStream technology combines a precise method

of electronically tracking moving cells with a high-resolution

multispectral imaging system to acquire multiple images

of each cell in different imaging modes (8). The current

commercial embodiment simultaneously acquires up to 12

high-resolution images of each cell at high rates of capture,

with fluorescence sensitivity comparable to conventional

Figure 4. ImageStream analysis of fresh APL (mean 0.27) and nor-

mal BM (mean 0.47). Overlay evidence the ability of ImageStream

to distinguish the PML pattern between the two sample types.

[Color figure can be viewed in the online issue, which is available

at wileyonlinelibrary.com.]

Table 1. The non-normalized and normalized PML mean modula-

tion normalization scores in electronic dilution experiments

SAMPLE

PML MODULATION,

MEAN

% NB4,

MODULATION

NORMALIZED

100% NB4 0.2372 100

100% HL60 0.4479 0

99.9% HL60 1 0.1% NB4 #1 0.4473 0.2848

99.9% HL60 1 0.1% NB4 #2 0.4471 0.3797

99.9% HL60 1 0.1% NB4 #3 0.4471 0.3797

99% HL60 1 1% NB4 #1 0.4469 0.4746

99% HL60 1 1% NB4 #2 0.4470 0.4271

99% HL60 1 1% NB4 #3 0.4467 0.5695

90% HL60 110% NB4 0.4299 8.5430

50% HL60 1 50% NB4 0.3511 45.9421

ImageStream cytometer is able to identify NB-4 cells in 10%

virtual dilution experiments by normalized modulation, as evi-

denced in bold.

NB-4 modulation normalized 5 (Fm 20.4479)/(0.237220.4479) 3 100.

Table 2. Identification of ‘‘Microspeckled’’ pattern in real dilution

experiments

SAMPLE

PML MODULATION,

MEAN

MEAN

MODULATION

NORMALIZED (%)

100% NB4 0.2734 100

100% HL60 0.4718 0

50% HL60 150% NB4 0.376 48.29

90% HL60 1 10% NB4 0.4311 20.51

100% APL blasts 0.2716 100

100% normal-BM 0.476 0

50% normal-BM 1 50%

APL-blasts

0.3774 48.24

90% normal-BM 1 10%

APL-blasts

0.4226 26.13

PML mean modulation is able to evidence PML pattern

alteration in NB4 and fresh APL blasts. Real dilution experiments

evidenced APL leukemic presence up to 10% dilution. Mean mod-

ulation normalization values were useful to evaluate the percent-

age of contaminating APL cells.

NB-4 modulation normalized 5 (Fm 20.4718)/(0.273420.4718) 3 100.

Fresh APL blasts normalized 5 (Fm 2 0.4760)/(0.2716 20.4760) 3 100.

ORIGINAL ARTICLE

236 PML Localization by Imaging FCM

FCM (8). Thus, ImageStream cytometry can combine the

‘‘power of surface hematology’’ (5) with the quantitative

analysis of PML protein distribution.

In this work, we evaluated the feasibility of ImageStream

as a new potential diagnostic tool useful for a rapid, accurate,

and operator-independent APL diagnosis. Indeed, we were

able to differentiate the HL60 ‘‘macrospeckled’’ and NB4

‘‘microspeckled’’ PML pattern by ImageStream FCM using

Modulation parameter. Our finding extends the recent work

of Grimwade et al. (7), by demonstrating the feasibility of

using ImageStream cytometry for the detection of rare APL

events in electronic and real dilution experiments. For the first

time, by quantifying PML distribution using modulation pa-

rameter, we were able to clearly detect PML pathological pat-

tern even at 10% APL cell presence in both NB-4 mixed in

HL-60 cell lines as well as fresh APL blasts in normal BM cells.

Furthermore, because, as reported by Dimov et al. (14), the

APL blast percentage at diagnosis ranges from 21 to 97%, Ima-

geStream FCM could be particularly helpful for APL cases

where pathological promyelocytes are difficult to detect, and

the laboratory hematologist interest is to confirm a suspected

diagnosis.

Although future studies are needed and molecular geno-

typic analysis has the final say, our data importantly indicate

that imaging FCM may be a frontline test for rapid confirma-

tion of suspected APL increasing the laboratory hematologist

ability to rapidly identify even rare APL cells.

Ideally, combining surface immunophenotyping with

quantitative analysis of PML distribution pattern analysis in a

single platform analysis would be attractive for hematologists

desiring an operator-independent, prompt, and robust APL

diagnosis with 100% sensitivity and specificity.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the constant support

of the Biobank staff of CEINGE-Biotecnologie Avanzate di

Napoli. The authors Peppino Mirabelli, Giulia Scalia, Caterina

Pascariello, Francesca D’Alessio, Elisabetta Mariotti, and Rosa

Di Noto give special thanks to Professor Francesco Salvatore

for his constant guidance and help in professional growth.

AUTHORSHIP

PM, GS and RK prepared the cells, designed the experi-

ments and performed flow cytometry assays.

CP, FDA and EM participated in study design, data analy-

sis and revision of the manuscript.

TG and VV participated in data analysis and manuscript

preparation.

DB and LDV conceived the study, designed the experi-

ments and wrote the manuscript together with RDN.

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