automated quantification of apoptosis in b-cell chronic lymphoproliferative disorders: a prognostic...

Automated quantification of apoptosis in B-cell chronic

lymphoproliferative disorders: a prognostic variable obtained with

the Cell-Dyn Sapphire (Abbott) automated hematology analyzerM. FUMI, D. MARTINS, Y. PANCIONE, S. SALE, V. ROCCO

Clinical Pathology Laboratory,

A.O.R.N ‘G. Rummo’ di

Benevento, Benevento, Italy

Correspondence:

Dr Vincenzo Rocco, A.O.R.N.

‘G.Rummo’ di Benevento,

Laboratorio di Patologia Clinica,

via dell’Angelo n.1, 82100,

Benevento, Italy.

Tel.: +39082457256;

Fax: +39082457256;

E-mail: vincenzo.rocco1@

virgilio.it

doi:10.1111/ijlh.12198

Received 27 September 2013;

accepted for publication 13

November 2013

Keywords

Lymphoproliferative, Sapphire,

smudge cells, Gumprecht,

Iodide, propidium

SUMMARY

Introduction: B-chronic lymphocytic leukemia CLL, a neoplastic clo-

nal disorder with monomorphous small B lymphocytes with scanty

cytoplasm and clumped chromatin, can be morphologically differ-

entiated in typical and atypical forms with different prognosis:

Smudge cells (Gumprecht’s shadows) are one of the well-known

features of the typical CLL and are much less inconsistent in other

different types CLPD. Abbott Cell-Dyn Sapphire uses the fluores-

cence after staining with the DNA fluorochrome propidium iodide

for the measurement of nucleated red blood cells (NRBCs) and

nonviable cells (FL3+ cell fraction): We have studied the possible

correlation between presence and number of morphologically iden-

tifiable smudge cells on smears and the percentage of nonviable

cells produced by Cell-Dyn Sapphire.

Methods: 305 blood samples from 224 patients with B-cell lympho-

proliferative disorders and 40 healthy blood donors were analyzed

by CBC performed by Cell-Dyn Sapphire, peripheral blood smear,

and immunophenotype characterization.

Result: FL3+ fraction in CLPD directly correlated with the percent-

age of smudge cells and is significantly increased in patients with

typical B-CLL. This phenomenon is much less evident in patients

with atypical/mixed B-CLL and B-NHL.

Conclusion: In small laboratories without FCM and cytogenetic,

smudge cells%, can be utilized as a preliminary diagnostic and

prognostic tool in differential diagnosis of CLPD.

INTRODUCTION

Chronic lymphocytic leukemia (CLL) is a neoplastic

clonal disorder of B or T lymphocytes with intermediate

maturity. It is characterized, in its more common form,

according to the 2008 WHO Classification, by the

increase in peripheral blood of monomorphous small B

lymphocytes, with scanty cytoplasm and a round or

628 © 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 628–635

ORIGINAL ARTICLE INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

International Journal of Laboratory HematologyThe Official journal of the International Society for Laboratory Hematology

lightly indented nucleus with clumped chromatin.

B-cell CLL can be morphologically differentiated in typ-

ical an atypical forms, according to a number of features

(percentage of prolymphocytes, presence of large cells

or cleaved nuclei). Atypical B-cell CLL accounts for 15–

20% of the patients. Leukemic low-grade lymphomas

also share a number of features with CLL. The morpho-

logical subclassification of these chronic lymphoprolif-

erative disease correlates with immunophenotypic and

genetic features, as well as with prognosis and clinical

outcome [1].

The presence of smudge cells (also named Gumpr-

echt’s shadows) on the peripheral blood smear is one

of the well-known features of the CLL. Smudge cells

can be defined as broken cells with no intact cyto-

plasm and a disrupted nuclear membrane [2]. They

are an almost universal finding in peripheral blood

smears from B-CLL patients, with high interpatient

variability, but are much less common and inconsis-

tent in patients with different types of chronic lym-

phoproliferative diseases [3, 4]. Smudge cells are

formed at the time of smear preparation and were

considered for a long time as a mere mechanical arti-

fact, due to abnormal membrane characteristics of

CLL cells. More recently, however, several authors

have recognized that the percentage of smudge cells is

an individual characteristic of every single case of

B-cell CLL. It remains stable over time in each patient,

is independent of the absolute lymphocyte count and

the staining method [5], and is associated with

increased chromatin density, which could be an early

sign of apoptosis [6]. An inverse correlation has been

reported between the percentage of smudge cells on

the peripheral blood smear, the expression of the

cytoskeletal protein vimentin [2], and prognostic fac-

tors such as the proportion of CD38+ and Zap70+ cells

[7].

The automated blood cell counter Abbott Cell-Dyn

Sapphire (Abbott, Abbott Park, IL, USA) carries out

leukocyte differential count using a multiple angle

scatter separation (MAPSS) method, associated with a

measurement of fluorescence after staining with the

DNA fluorochrome propidium iodide for the measure-

ment of nucleated red blood cells (NRBCs) and nonvi-

able cells. Several studies have shown that this

fluorescence-based viability assessment is related to

the presence of apoptotic cells and correlates with the

flow cytometric determination of annexin V [8, 9].

The aim of the study

We have studied the possible correlation between the

presence and number of morphologically identifiable

smudge cells on smears and the percentage of nonvia-

ble cells stained with propidium iodide by the Cell-Dyn

Sapphire in two groups of patients, one with typical B-

CLL and the other with atypical B-CLL or leukemic

lymphomas. In consideration of occasional reports of an

influence of serum albumin level on the number of

smudge cells [10, 11], we have correlated the results

with patients’ serum albumin in both groups.

MATERIALS AND METHODS

Blood samples and patients

During a six-month period, from March to August

2011, we have analyzed 305 blood samples, obtained

from daily laboratory routine workload, from 224

patients with B-cell lymphoproliferative disorders

(LPD) and 40 healthy blood donors. They were subdi-

vided in two groups:

• A group: 149 samples from 85 patients with typical

B-CLL;

• group: 75 sample from patients with nontypical B-

CLL LPD: 47 samples from 27 patients with atypical/

mixed B-CLL and 29 samples from 18 patients with

different types of B-cell leukemic lymphomas (three

with hairy cell leukemia, eight with splenic

lymphoma with villous lymphocytes and four with

mantle cell lymphoma);

A third group included 40 samples from 40 healthy

blood donors (20 males and 20 females).

All cases of typical B-CLL and nontypical B-cell

LPD were classified according to the 2008 WHO diag-

nostic criteria, including peripheral blood cell count,

morphological analysis of peripheral blood and bone

marrow aspirate smear and immunophenotype of leu-

kemic cells by flow cytometry; bone marrow histopa-

thology and genetic studies were also performed on a

selected number of patients, according to the diagnos-

tic necessities. All samples had an absolute lympho-

cyte counts higher than 4.5 9 109/L.

Blood samples were collected in K3-EDTA and ana-

lyzed within five hours from venipuncture with the

Abbott CELL-DYN Sapphire. Peripheral blood smears

© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2014, 36, 628–635

M. FUMI ET AL. | AUTOMATED QUANTIFICATION OF CELL APOPTOSIS 629

were prepared manually and stained according to the

May-Gr€unwald-Giemsa method. Immunophenotype

studies were carried out using the following cell mark-

ers: PC5-conjugated anti-CD45; FITC-conjugated

anti-CD19; PE-conjugated anti-CD5, anti-CD10, anti-

CD20, anti-CD22, anti-CD23, anti-CD11c, anti-CD25,

anti-CD38, anti-CD103, anti-CD79b, anti-FMC7, anti-

IgS, anti-kappa, and anti-lambda light chains (Cytom-

ic FC-500 Beckman&Coulter).

Methods

Abbott CELL-DYN Sapphire: the FL3+ cell fraction

Abbott CELL-DYN Sapphire carries out automated

leukocyte cell differential analysis and counting, after

erythrocyte lysis, using an optical method based on a

combination of fluorescence and a solid-state blue

diode laser light (488 nm) scatter at different angles

(MAPPS), obtaining the following parameters: axial

light loss (ALL), that is size at 0 °; intermedium angle

scatter (IAS) measured as light scatter at 7 °and cor-

related with cellular complexity; polarized side scatter

(PSS), measured as light scatter at 90 ° and correlated

with cell lobularity and depolarized side scatter

(DSS), measured as capability to depolarize light of

90 °.

The different populations of leukocytes are divided

into polymorphonuclear and mononuclear cells on a

scatterplot obtained on the basis of cell complexity

IAS and PSS. Eosinophils are separated from neutro-

phil granulocytes by means of their capability to

depolarize polarized light on a scatterplot obtained on

the basis of PSS vs DSS. Mononuclear cells are further

subdivided into monocytes, lymphocytes, and basoph-

ils on a scatterplot obtained on the basis of ALL vs

IAS. Cell clusters are then located in the scatterplot

PSS vs ALL, where abnormal cell clusters are identi-

fied to obtain instrumental flags for the presence of

blasts, nucleated red blood cells (NRBCs), or immature

granulocytes.

Respect fluorescent there are three channels: FL1

that use fluorescein, FL2 for phycoerythrin, and FL3

that use iodide propidium.

The first fluorescent channel (FL1) was utilized to

reticulocyte analysis and CD61 platelet count. FL2 was

used for T lymphocyte subset analysis with CD3, CD4 and

CD8 mAb, but it can be utilized also with CD64 mAb.

Finally, a specific procedure with the DNA fluoro-

chrome propidium iodide is used to selectively stain

permeable cells, such as NRBCs and nonvital leuko-

cytes. The specific reagent containing propidium

iodide completely lyses mature erythrocytes and

leaves leukocytes intact. The lysis process permeates

the cell membrane, and the fluorochrome stains the

nuclei of NRBCs. The fragility of the membrane of

nonviable/apoptotic leukocytes also causes these cells

to be stained by propidium iodide, though not their

membrane completely lysed.

Red fluorescent cells can be identified on a scatter-

plot of size (0 ° light scatter) versus propidium iodide

red fluorescence (named FL3-DNA). Platelets, Howell-

Jolly bodies, and basophilic staining are also identified

as fluorescent particles, but not included in the count.

NRBCs, when present, are counted separately from

other leukocytes: the count is reported as NRBCs/100

leukocytes. Nonviable cells, on the other hand, are

not excluded from the differential leukocyte count,

but the proportion of viable cells is reported as a unit

fraction, named ‘white cell viable fraction’ (WVF)

[12].

FCS files analysis

For each sample, the Cell-Dyn Sapphire produces a

FCS file in digital format which can be analyzed using

a PC with a software for flow cytometric data analysis.

We have analyzed the 305 FCS obtained from our

study sample with the software WEASEL version 3.0.1

(Walter and Eliza Institute, Melbourne, Australia).

Positional parameters, including size on the ALL

axis, for the lymphocyte population have been mea-

sured as mean values and coefficient of variation (CV

%) using a gating strategy on the bivariate scatter-

grams log DSS vs ALL and ALL vs FL3, as well as on

the three-dimensional scattergram IAS vs ALL vs

logDSS.

After gating strategy, we refer to the value named

Ly-FL3+ as the cells whose nuclear DNA had been

stained by propidium iodide in the Sapphire and iden-

tified as separate cluster on the FL3 axis (Figure 1a,b).

Smudge cell count

Blinded duplicate microscope observation was sepa-

rately carried out by two pathologists on manually


630 M. FUMI ET AL. | AUTOMATED QUANTIFICATION OF CELL APOPTOSIS

smeared peripheral blood films stained using the May-

Gr€unwald-Giemsa method. Besides a 400-cell leuko-

cyte differential count, it included both the assessment

of lymphocyte morphology and the morphological

classification of the respective percentage of lympho-

cytes and smudge cells (on a total of 300 elements for

each of the two observers). The percentage of smudge

cells, valuated on 300 elements of lymphocyte count,

was estimated as the number of total smudge cell

divided by three [7].

Albumin concentration

Serum albumin concentration was measured in all

samples collected in BD SST II Advance tubes, on

ADVIA� 2400 Chemistry System (Siemens Healthcare

Diagnostic Inc., Deerfield, IL, USA).

Statistical analysis

Statistical analysis was carried out using a MICROSOFT

EXCEL 2007 (Microsoft Corporation, Redmond, WA,

USA) spreadsheet and the MEDCALC software version

11.4.2.0 (MedCalc Software, Oostende, Belgium).

Comparison between groups was carried out according

to the Box and Wiskers method. Groups correlation

and association was analyzed using the Spearman’s

test. The discriminant capability of the variables under

study has been tested using the area under the curve

from the corresponding ROC analysis.

RESULTS

Percentage of Ly-FL3+ leukemic cells

Ly-FL3+ cells were virtually absent in the control group

of healthy subjects (<1% in all cases). The percentage

of leukemic lymphocytes positively stained by the Sap-

phire propidium iodide was significantly higher in

patients with typical B-CLL in comparison with the

group of atypical CLL/leukemic lymphoma patients

(P < 0.0001) (Table 1 and Figure 2a,b).

Percentage of smudge cells

Smudge cells were extremely rare in the control

group of healthy subjects (<1% in 95% of cases). The

percentage of smudge cells was significantly higher in

group A patients with typical B-CLL (median 9.0%,

range = 1–26) than in group B patients with other

B-cell LPD (median 1.0%, range 0–14) (Table 1).

Correlation between Ly-FL3+ and smudge cells

The percentage of smudge cells counted on the

peripheral blood smear showed an excellent direct

correlation with the percentage of the Ly-FL3+ cells

obtained with the Sapphire (R2 = 0.97)(Figure 3).

Serum concentration of total protein and albumin

was moderately reduced in both group A and B of

patients, in comparison with healthy subjects. On the

(a) (b)

Figure 1. (a) Axial light loss vs FL3-DNA cytogram in patient with typical B-chronic lymphocytic leukemia t; (b)

Analysis of FCS file of the cytogram with WEASEL v3.0.1. software. R3 gate was referred to Ly-FL3+ after gating

strategy on lymphocyte Cluster.



other hand, there was no correlation between per-

centages of smudge cells and serum albumin concen-

tration; similarly, serum albumin was not correlated

with the proportion of Ly-FL3+ cells in our patients

(data not shown).

Discriminant efficiency of Ly-FL3+ cell percentage and

ALL positional parameter

The percentage of Ly-FL3+ cells demonstrates a good

efficiency in the preliminary differentiation of patients

with B-CLL compared with atypical/mixed B-CLL or

leukemic B-cell lymphoma. With a cutoff value of

Table 1. Mean of % of Ly-FL3+ cells and Smudge Cells in Chronic lymphocytic leukemia and aLLC/NHL

CTR Typical CLL a-LLC/NHL

% cells

Ly-FL3+

%Smudge

cells

% cells

Ly-FL3+

%Smudge

cells

% cells

Ly-FL3+

%Smudge

C.

Mean 0.55 � 0.3

*P < 0.0001

0.3 � 0.5

*P < 0.0001

32.4 � 24.8 11.1 � 6.9 4.07 � 7.6

**P < 0.0001

1.4 � 2.6

**P < 0.0001

Median 0.73 0.37 23.3 9 1.51 1

Data are given as mean and median (in bold) � SD (in italic) of FL3+ cell%; smudge C.% on peripheral blood smear.

*P-value and **P-value are referred to the comparison between the mean values of Ly-FL3+ cells% and % of smudge

cells in typical CLL and aCLL/NHL.

0

10

20

30

40

50

60

70

80

%_FL3_LyTot_CLL %_FL3_LyTot_aLLC\NHL

P < 0.0001

(b)

(a)

Figure 2. (a) Dot plot e (b) Box and whisker referred

to percentage of Ly-FL3+ cells in chronic lymphocytic

leukemia and aLLC/NHL.

Figure 3. Linear regression between percentage of

Ly-FL3+ cells and percentage of Shadows Gumprecht

found on peripheral smear.



7.54%, the receiver operator curve (ROC.) analysis

showed a AUC value of 0.887, with a sensitivity of

87.8% and a specificity of 83.3% for the diagnosis of

typical B-CLL versus patients of group B (Figure 4).

The positional parameter ‘ALL mean’ of the Sap-

phire reflects the white blood cell size as measured

through laser light absorbance measured at an angle

of 0 °. The morphological monomorphism in typical

B-CLL leukemic cell size was paralleled by a generally

lower ‘ALL mean’ value. With a discriminant value of

12.295 (in arbitrary units), the AUC was 0.898 with

76.1% sensitivity and 94.2% specificity for the diag-

nosis of typical B-CLL versus patients with atypical/

mixed B-CLL or leukemic B-cell lymphoma (group B)

(Table 2 and Figure 5).

DISCUSSION

We have found in our patients with B-cell LPD that

the count of the percentage of Ly-FL3+ leukemic cells

stained with propidium iodide in the automated blood

cell counter Abbott Sapphire is significantly increased

in patients with typical B-CLL with small lymphocyte

morphology (mean � SD: 32.4 � 24.8). This phenom-

enon is much less evident in patients with atypical/

mixed B-CLL and B-cell leukemic lymphoma (mean

� SD: 4.07 � 7.6), so that a threshold level of 7.54%

Ly-FL3+ cells can differentiate the two groups of dis-

ease with excellent sensitivity and specificity. Slightly

specificity respect Ly-FL3+ is given by the traditional

ALL parameter using a cutoff 12.295, but we obtained

low sensitivity to properly classify atypical/mixed B-

CLL and B-NHL, probably this is due to the different

intrinsic meaning of two parameters: The first is

%_Smudge cells LLC vs aLLC\NHL

0 20 40 60 80 100

0

20

40

60

80

100

100-Specificity

Sen

sitiv

ity

Sensitivity: 87.8 Specificity: 83.3 Criterion : <=7.54

Figure 4. Receiver operator curve for the percentage

value of the cluster Ly-FL3+ between typical and a-

LLC/NHL LLC. Sensitivity was 87.8% and the

specificity 83.3%, AUC = 0.887.

Table 2. Mean value in arbitrary units of axial light

loss in typical LLC and aLLC/NHL measured by

software WEASEL v3.1

ALL axis Typical CLL a-LLC/NHL

Mean 9.518 � 2.060 13.175 � 2.072

Median 9.085 12898

*P < 0.0001

Data are given as mean and median (in bold) � SD (in

italic) of ALL.

*P-value is referred to the comparison of ALL mean

value of cell in typical CLL aLLC/NHL.

Figure 5. Receiver operator curve for the mean axial

light loss of LLC-typical and a-=LLC/NHL. Sensitivitywas 76.1% and the specificity 94.2%, AUC = 0.898.



related to a physical parameter (cell size, ALL = 0 °),

while the second to a structural-functional properties

of the cell. In fact, Sapphire Ly-FL3+ leukemic cells

are permeable to the fluorochrome owing to mem-

brane damage, which can be similar to the biological

bases of the formation of smudge cells. The increase

in Ly-FL3+ cell in B-CLL, in effect, has shown in our

study a strong correlation with the percentage of

smudge cells morphologically counted on the periph-

eral blood smear (R2 = 0.97). Some studies report that

high level of serum albumin can protect CLL cells

from apoptosis by AKT signaling pathway [17]; inter-

estingly, bovine serum albumin (BSA) addition in a

blood smear results in the reduction in atypical lym-

phocyte and a decrease in a smudge cells [18]. On the

other hand, a low level of serum albumin has been

reported to be associated with an adverse prognosis in

lymphoproliferative disorders, but it can be usually

observed with latest stage of disease [19]. We did not

find a statistic association between serum albumin

level, smudge cells, and percentage of Ly-FL3+.

Automated blood cell counters can flag the pres-

ence of apoptotic lymphocytes in several conditions,

such as infectious mononucleosis [8, 13]. The Cell-

Dyn Sapphire, in particular, thanks to the use of pro-

pidium iodide and of a partial lyse method originally

developed to count circulating erythroblasts [14], is

able to distinguish them from viable WCB.

The Sapphire FL3-DNA method detects the pres-

ence of apoptotic cells and, in particular, cells with

membrane fragility which allows propidium iodide

(PI) to penetrate into the cell after treatment with a

mild lysing agent which leaves intact all cell mem-

branes, except erythroblasts, neutrophils after long

storage at room temperature and leukemic lympho-

cytes in B-CLL. Supravital exposure to PI, without

prior permeabilization, has been shown in research

laboratories to permit the identification of apoptotic

cells [9]. PI incorporation in experimental flow cyto-

metric studies is due to early alterations of the cell

membrane of unfixed cells [15]. Loss of membrane

integrity shown by supravital PI staining is correlated

with annexin V expression, another flow cytometric

method that detects the membrane exposure of phos-

phatidilserine (PS) as an index of apoptosis [9]. The

capability of the automated Abbott Cell-Dyn 4000,

which uses the same WBC method as the Abbott

Sapphire, of quantify apoptotic cells in LPD has been

demonstrated [8]: Leukemic cells were incubated in

presence of theophylline or fludarabine, known to be

apoptosis inducers, or in medium alone: After 36 h

of culture, the percentage of apoptotic cells deter-

mined from the FL3+ fraction showed an excellent

correlation with standard methods for apoptosis (in

situ detection of cell death on slides, or TUNEL test)

and flow cytometry with Annexin-5.

Matutes and Polliack (2000) [4] have reported

that the percentage of smudge cells in morphologi-

cally atypical CLL and in leukemic lymphomas is

considerably less than for typical B-CLL, although

higher than in normal samples. Subsequently, Matos

et al. (2009) [16] in the evaluation of chronic lym-

phoproliferative disorders of B lymphocyte reported

(using the cutoff of 10%, 20% and 30%), the pro-

portion of smudge cells was reduced in a statistically

significant manner in the morphologically atypical B-

CLL and leukemic NHL compared to typical B-CLL,

but have not found a relationship with the prognosis

(TTT and survival).

Nowakowski et al. [7] have shown that a cutoff

value of >30% smudge cells represents in their study

a morphological label of individual patients, which is

an independent indicator of low risk and predicts pro-

longed survival. It is also inversely correlated with the

content of the cytoskeletal protein vimentin, which is

responsible for cell membrane rigidity and integrity,

and could protect leukemic B-cell from rupture during

smear preparation, as well as an IgVH unmutated

state. Both high vimentin expression and IgVH unmu-

tated state also are negative prognostic factors in early

stage CLL.

In our study, we have shown that the Ly-FL3+ cell

fraction in LPD is directly correlated with the percent-

age of smudge cells, especially in patients with B-CLL.

Membrane changes due to apoptosis are probably the

cytological basis of both phenomena: increased per-

meability to PI and fragility on the smear. Then, the

Ly-FL3+ cell fraction provided by Cell-Dyn Sapphire

can be utilized as prognostic value in differential diag-

nosis of CLPD, in typical CLL and a-CLL/LNH. The

CELL-DYN Sapphire with% of nvWBC can give a first

aid in the screening of CLPD.

CONFLICT OF INTEREST DISCLOSURE

The authors have no competing interests.



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