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Received: 29 April 2018    Revised: 28 August 2018    Accepted: 13 September 2018

DOI: 10.1111/ijlh.12935

ORIGINAL ARTICLE

The diagnostic use of ADVIA 2120i Siemens and an “APL

criteria” can help to reduce the rate of early death in the APL

Vincenzo Rocco1  | Christian Castelli2 | Maurizio Fumi1 | Francesca Mancini3 | 

Ylenia Pancione1 | Michele Prisciandaro4 | Silvia Sale1 | Donatella Tanca5 | Davide Vagnoni6

1
Clinical Patology Laboratory, A.O.R.N. “G.
Rummo” di Benevento, Benevento, Italy Abstract
2
Department of Laboratory Medicine Introduction: Acute promyelocytic leukemia (APL) is a type of acute myeloid leuke-
EOLAB, Ente Ospedaliero Cantonale,
mia (AML) with a life-­threatening coagulopathy. Once it is suspected, ATRA should be
Bellinzona, Switzerland
3 started. Appreciation of APL details is critical, but an experienced hematopathologist
Department of Cellular Biotechnologies
and Hematology, Sapienza University, Rome, may not be available. We developed an algorithm, based on the parameters gener-
Italy
ated by automated blood cell counter ADVIA 2120i Siemens that can aid the diagno-
4
Department of Molecular
Medicine, Policlinico Umberto I, Sapienza sis of APL.
University, Rome, Italy Methods: All parameters in the algorithm were selected on the bases of the patho-
5
Department of Laboratory physiology of the APL and the analyzer’s technology. We used c1 cutoff:
Medicine, Azienda Sanitaria Locale Regione
Liguria, Lavagna, Italy PLT < 150 × 103; % Mono<10; % LUC<4; % hyperchromic cells > 2; %saturated cells ≥
6
Department of Laboratory Medicine, 1; % blasts > 4. Satisfying at least five of six cutoffs, we obtained an “APL criteria”. It
Civitanova Marche, Italy
was tested on 247.209 raw-data from routine and emergency samples and on 124
Correspondence raw-data from APL. We performed a multiparametric analyses and obtained defini-
Dr. Vincenzo Rocco, Clinical Patology
tive cutoffs (c2). It was validated on a new group of 51.002 raw-data from routine and
Laboratory—A.O.R.N. “G.Rummo” di
Benevento, Benevento, Italy. emergency. Finally, it was tested on AML and ALL, MDS, MPN, oncologic samples,
Email: vincenzorocco1@virgilio.it
and hemolytic and megaloblastic anemia.
Results: The algorithm provided a high sensitivity (86.30%) and high specificity
(99.83%) in APL with high or normal number of WBC and satisfactory results in APL
with cytopenia (80%). The specificity was very high also in groups of hematological
and nonhematological diseases. It can be used as an “APL criteria” to alert patholo-
gists of the possible presence of APL. It together with instrumental and classical mor-
phology may allow to reduce the time of diagnosis with further reduction in early
death.

1 |  I NTRO D U C TI O N
Acute promyelocytic leukemia (APL) is a subtype of acute myeloid
leukemia (AML) characterized by a specific balanced reciprocal
translocation t (15;17), which fuses the PML gene on chromosome
15 to the RAR-α (retinoic acid receptor-­α) gene on chromosome 17.1,2
Most patients exhibit a life-­
threatening coagulopathy. Since
the introduction of the differentiating agent all-­trans retinoic acid
(ATRA) in 1995, the disease has become the most curable subtype
of adult AML.3-5
Hence, once the APL is suspected on the basis of clinical findings
and the peripheral blood smear, bone marrow aspirate should be
highly recommended, but even without waiting for its examination,
and before the diagnosis is confirmed by cytogenetic or molecular
studies, ATRA should be started at the standard dose, to promptly
resolve the coagulopathy.6,7

Int J Lab Hematol. 2018;1–9. © 2018 John Wiley & Sons Ltd |  1
wileyonlinelibrary.com/journal/ijlh  
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2       ROCCO et al.

Morphologically, APL is not a homogeneous entity; the classical
hypergranular APL represents the great majority of all APL, and the
microgranular variant accounts for about 15%-­20% of all APL. In the
classical hypergranular APL, there are abnormal promyelocytes with
an abundant cytoplasm packed with large azurophilic granules. Auer
bodies are commonly found in many cells. The microgranular vari-
ant is characterized by the presence of leukemic cells with bilobed
or kidney-­shaped nuclei with very minute granules in the cytoplasm
that are not readily perceptible on light microscopy.8-10
Appreciation of these morphological details is critical; the short-
age of skilled and experienced morphologists in some setting means
that slides have to be transported to specialized laboratories for re-
view.6 This leads to unnecessary diagnostic delays and may compro-
mise patient’s outcome.

F I G U R E   1   Caracteristic pattern of RBC V/HC, Perox and BASO channel from ADVIA 2120i SIEMENS in A, normal sample, in B, APL
with hight WBC count, in C, cytopenic APL sample. %Bl, Blasts percentage; %Iper, Hyperchromic RBC; Ba, Basophils; E, Eosinophils; L,
Lymphocytes; LUC, Large Unstained Cells; M, Monocytes; N, Neutrophils; Perox % Sat, Percentage of Cells in perox saturation [Colour
figure can be viewed at wileyonlinelibrary.com]
ROCCO et al.
Many studies reported that parameters generated by auto-
mated blood cell counters have a possible role in aiding the diagnosis
and classification of acute leukemia.11-13 For example, Peroxidase
Activity and Nuclear Density Analysis (PANDA) classification, based
on the morphology of cell clusters on ADVIA 120 and 2120i, has
been proposed and confirmed as a useful tool for a premicroscopic
assessment of leukemic samples.10,14,15 In this context, APL provides
the most characteristic pattern among all leukemias as shown by
D’Onofrio, Gibbs, and Pillay, because the MPO activity in promy-
elocytes is maximum and they are arranged on the extreme right
of the perox channel and straddling the population of eosinophils
(Figure  1B-­C).16-20
Another study led to design of a diagnostic algorithm for APL
using as parameters the percentage of large unstained cells (LUC)
and delta neutrophil index (DNI), but this work has been done on
bone marrow aspiration fluid. 21
On the basis of these premises, and of the consideration that the
evaluation of complete blood count (CBC) is the first test that the pa-
tient performs, often in emergency situations (access to Emergency
Departments for hemorrhage), the purpose of our study was to build
an algorithm that allows to produce a “APL criteria,” on ADVIA 2120i
(Siemens), with high sensitivity and very high specificity, in order to
alert biotechnologists and pathologists of the possible presence of
the disease and not to burden on the loads working with the produc-
tion of a high number of false positives.
2 | M ATE R I A L A N D M E TH O DS
2.1 | Analyzer technology
The Siemens Advia 2120i (Siemens Healthcare Diagnostics,
Deerfield, Illinois, USA) is an automated hematology analyzer that
measures the total and differential WBC count using principles of
flow cytometry and a combination of reactions that occur within
the peroxidase and the basophil/nuclear lobularity channels. Cluster
analysis of the cells within each channel is used to generate a cyto-
gram and the pattern that emerges has been shown to assist with the
10,14-18
subtyping of hematological malignancies.
2.1.1 | WBC count
The peroxidase channel is used to measure peroxidase activ-
ity (PA). The white blood cells are fixed using formaldehyde
and in the presence of hydrogen peroxide and the chromogen
4-­chloro-­1-­naphthol, cells containing myeloperoxidase form a dark
precipitate and are characterized by their light scatter and light
absorption properties. This generates a scatter plot with the x-­a xis
representing increasing intensity of peroxidase staining and the y-­
axis representing increasing cell size. In this channel, the analyzer
also determines the percentage of saturation (Perox % Sat) that
expresses the percentage of granulocytes which having a higher
peroxidase activity and a population of white blood cells without
|
      3
peroxidase, called Large Unstained Cells (LUC). The basophil/
nuclear lobularity channel (ND) is used to derive nuclear density.
Phthalic acid strips the cytoplasm from white blood cells except
basophils. The WBCs pass through a laser flow cell and two-­angle
light scatter is then used to determine cell size and nuclear density
that are detected and recorded on the basophil cytogram in which
the x-­a xis reflects the nuclear complexity and the y-­a xis reflects
cell size.
In the basophil channel, the analyzer also provides a % value of
presumed blasts. In fact, the chromatin structure of blast cells is not
very dense and this feature causes a low signal of high-­angle scatter-
ing resulting in a location of the immature nuclei in the left proximal
part of the head of mononuclear (MN) cells. 21
2.1.2 | RBC count and classification
Erythrocytes are counted and classified on the bases of volume
and hemoglobin concentration. By applying Mie’s theory, 22 a soft-
ware is able to produce a single cytogram allowing to quantify the
different erythrocyte populations in a sample of peripheral blood
respect to their volume and hemoglobin concentration, provid-
ing, among others, the percentage of hyperchromatic red blood
cells (% Hyper) presumably due to spherocytes and microspheres.
(Figure 1A).
2.1.3 | PLT count
The ADVIA 2120i uses a two-­dimensional platelet analysis. This
method is based on the simultaneous measurement of laser light
scattered at two different angles. Both measurements are converted
into volume and refractive index. A cytogram is produced, where
platelets are identified in the region corresponding to a volume of
1-­60 fL and refractive index of 1.35-­1.40. 21,23-25
For each analyzed sample, the ADVIA 2120i (as well as its prede-
cessor ADVIA120) automatically saves all data, related to cell counts
and their respective positional parameters, in a single file, called
raw-­data. These data are tabulated and can subsequently be viewed
and processed using any spreadsheet such as Microsoft Excel®.
2.2 | Parameters used and construction of algorithm
All parameters entered in the algorithm were selected on the bases
both the pathophysiology of the APL and the analyzer’s technology.
They are represented by:
1. Thrombocytopenia: due to both DIC and failed production by
bone marrow;
2. Normal or reduced monocytes %: because the blasts, due to their
high peroxidase content, cannot be placed by the analyzer in the
monocyte box;
3. Normal or reduced LUC %: since the blasts, due to their very high
peroxidase content, can never be placed by the analyzer in the
LUC box;
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4       ROCCO et al.

TA B L E   1   Raw-­data and number of cases about groups of At this point, we kept the cutoff constant for five of the six pa-
hematological and nonhematological diseases rameters, varying time to time the sixth one and calculating sensitiv-

Raw-­data ity and specificity; those with higher sensitivity and specificity have
Diseases (n = 800) Cases (n = 650) been used as c2 cutoffs.
Then, to overcome the possible bias due to different calibrators
AML no APL 142 142
(Siemens ADVIA 2120i SETpoint™ Calibrator) and different analyz-
ALL 36 36
ers, standardization used in other laboratories, the specificity of the
MDS 90 90
algorithm for the APL-­Flag, was tested on further 51.002 raw-­data
MPN 47 47
of peripheral blood samples (from routine and emergency) from two
Megaloblastic anemia 30 30
laboratories participating in the study.
Acute and chronic hemolytic 39 39 To verify the specificity of this algorithm, with respect to inter-
anemia
fering factors deriving from particular conditions, it has been tested
Cytopenic cancer patients 34 34
on groups of patients affected from hematological and nonhemato-
Cancer patients with neutro- 134 134
logical diseases.
philic leukocytosis (by Growth
factors therapy)
APL 124 49 2.3 | Groups in study
Typical 112 45
Atypical 12 4
1. A total of 247.209 raw-data of CBC from routine (109.159)
and emergency (138.050) APL-negative
4. Increased % hyperchromic RBC: because the DIC, which is almost
always present in the APL, causes an increased percentage of This evaluation was necessary for the construction of the algo-
schizocytes and consequently of microspherocytes26,27; rithm. In fact, a too low specificity, and therefore with a high number of
5. Increased % Perox saturated cells: because the promyelocytic false positives, would lead to microscopic control of too many useless
blasts, due to their very high peroxidase content, are placed be- samples.
tween the saturated cells, leading to an increase in this It cannot be justifiable in these cases that almost all false posi-
parameter; tives are related to blood samples that should be lead to microscopic
6. Increased % blasts: because, due to the reduced chromatin den- review.
sity of the APL cells, a portion higher than 4% exceeds the prede- The actions resulting from the suspicion of APL are so peculiar
termined threshold and this causes these cells to be counted as and urgent that many false positives could lead to the renunciation
blasts by the analyzer. of the use of the flag;

Considering the normal values of the above-­mentioned parame- 2. A total of 51.002 raw-data of CBC from routine and emergency
ters, supplied in default by the analyzer, and their characteristic alter- assessed in a single group, coming from two others
ations, we have chosen, for each of them, the following cutoff (cutoffs laboratories.
c1) to insert in the algorithm: 3. Hematological and nonhematological diseases (Table 1) with cy-
tometric and morphological characteristics, which could produce
• PLT < 150 × 103/μL; false positives due to the possible presence of:
• % Mono < 10; a. Cells with high peroxidase content;
• % LUC < 4; b. Reduced LUC number;
• % hyperchromic cells > 2; c. Real or spurious increased blasts percentage;
• % saturated cells ≥ 1; d. Thrombocytopenia.
• % blasts > 4. e. Increased percentage of spherocytes.
f. Monocitopenia;
Firstly, for each of the c1 cutoffs, the statistical significance was g. Schizocytes and consequently of microspherocytes26-28
evaluated by comparing APL vs normal samples. 4. 127 raw-data from 127 donors group (normal samples)
Subsequently, this algorithm was built on 247.209 sample files
(raw-­data), deriving from the emergency and routine samples pro- All 124 raw-­data of 49 cases of APL (112 typical and 12 atypical)
vided by clinical laboratory of AORN “Rummo” (pilot laboratory). confirmed according to the WHO guidelines.29
With such cutoffs (c1), the algorithm A1 was constructed; the al- The files were collected in the years 2014-­17 in five laboratories
gorithm provided that at least five of six criteria must be verified in involved in the study.
order to trigger the “APL criterion.” With these c1 cutoffs, we have Clinical requests (CBC with differential formula, morphology,
obtained an initial sensitivity and specificity value. and cytofluorimetry) related to each case in the study were made
ROCCO et al.
by the clinicians as an integral part of the diagnostic process . No
samples were requested from the patient for this study.
2.4 | Statistical analysis
We used raw-­data provided by the ADVIA2120i (Siemens) for
each sample, to obtain a full list with all the parameters provided
by analyzer. Statistical analysis was performed using Microsoft
Excel (Microsoft, Redmond, WA, USA) and MedCalc software ver-
sion 11.4.2.0 (MedCalc, Ghent, Belgium). To test the distribution
of the data, we used the D’Agostino-­Pearson test because, for
each group, we observed a P-­value < 0.05 (data not fit a normal
distribution); data were presented as median, minimum and maxi-
mum, and interquartile range (IRQ), and a nonparametric method
was used . To display variation in groups, Box&Whishers distribu-
tion graphs were used; Mann-­W hitney test was used to analyze
association (a “P” value lower than 0.05 was considered statisti-
cally significant).
The main end-­point of the study was the construction of an al-
gorithm to predict the APL, so we used a group to build algorithm
(247.209 samples from routine and emergency), and a group to vali-
date it (51.002 samples from routine and emergency).
We used a univariate descriptive statistical analysis to compare
groups and parameters individually.
The diagnostic accuracy of all cutoff points and algorithm was
determined by calculating sensitivity and specificity tested on
the groups APL-­positive and on total routine and emergency APL-­
negative, respectively, toward individual groups of hematological
and nonhematological diseases ; positive and negative predictive
values (PPV and NPV) and likelihood ratios (LR+, LR−) have also been
calculated.
3 | R E S U LT S
3.1 | Normal samples vs APL: Comparative analysis
The comparison between normal samples and APL samples, re-
spect to the selected and above-­mentioned parameters, gave the
following results; as expected, both for the platelet count and for
TA B L E   2   Comparison between normal
Normal samples (N = 127)
samples and APL group
Parameters Median; (min-­max); IRQ
Platelets 221; (141-­389); 66.50
Monocytes
% LUC
% IPER
Perox %
saturation
% Blasts
Data are given as median; min-­max; IRQ.
P-­value is referred to comparison between normal samples and APL group, performed by Mann-­
Whitney U.
      5|
the percentage of hyperchromic cells, as well as for the percentage
of cells in saturation and the percentage of blasts, we found a sta-
tistically significant difference (Table 2). Monocyte count and per-
centage of LUC, although in the normal range, showed a statistically
significant trend (P < 0.05) at the lower limits (Figure 2 and Table 2).
3.2 | Diagnostic accuracy for parameters
in the study
Considering the statistically significant difference between normal
and APL samples found for each individual parameter, it was decided
to evaluate their diagnostic accuracy using cutoffs “c1” tested on
the groups routine and emergency APL-­negative and APL-­positive
(Table 3).
Except for the % Iper, the “c1” cutoff showed a good sensitivity
(>80%) for all parameters. About specificity, a low value is observed
for monocytes and LUC parameters (Table 3).
Then, these parameters with “c1” cutoffs were implemented in
an algorithm A1, according to which at least five of six criteria had
to be satisfied. This allowed to identify correctly the 89.16% of APL
raw-­data and the 99.65% of APL-­negative samples.
Thus, to improve these cutoffs, we analyzed the individual pa-
rameters as described in the paragraph “Parameters and construc-
tion of the algorithm” and we obtained cutoffs “c2” (Table 3).
Through these “c2” cutoffs, it was possible to obtain a sensitiv-
ity of 87.75% (43/49) patients at first diagnosis and a sensitivity of
86.30% (107/124) for all raw-­data of patients with APL; the specific-
ity was 99.83% (246.789/247.209) (VPP 18.77%, VPN 99.99%, LR+
507, LR− 0.002).
In particular, the algorithm correctly classified 93.4% (14 of 15
samples) of APL with normal count of WBC, 100% of the samples
with leukocytosis (42 of 42 samples) and surprisingly 80.6% of the
cytopenic APL (54 of 67 samples) (Figure 1B-­C and Table 4).
Analyzing the false positive (FP) from the total groups of rou-
tine and emergency APL-­negative (n = 247.209), it was found that
14.76% (n = 62) of them was represented by Megaloblastic Anemias
and transfused Megaloblastic Anemias, 9.52% (n = 40) by oncologic
samples, and 57.38% (n = 241) by “reactive” samples. As for the
18.34% (n = 77) remaining samples, it was represented by “reactive”
APL (N = 124) Median;
(min-­max); IRQ P-­value
42.5; (3-­158); 35.80 <0.05
6.3; (2.2-­9.9); 2.10 1.3; (0.0-­6.8); 1.63 <0.05
1.7; (0.6-­4.0); 0.80 0.9; (0.0-­6.5); 1.70 <0.05
0.6; (0.1-­3.2); 0.75 3.2; (0.40-­43.50); 5.33 <0.05
0.4; (0.1-­1.3); 0.20 18.60; (0.30-­61.9); 25.30 <0.05
1,0; (0.3-­2.8); 0.65 16.9; (0.30-­74.20); 27.80 <0.05
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6       ROCCO et al.

F I G U R E   2   Box & Whiskers plot of normal samples vs APL group. % LUC, percentage of Large Unstained Cells; % Mono, percentage
of monocytes; hyper_pcnt, percentage of hyperchromic cells; pcnt_blast (%), percentage of cells to the left of the BASO channel blast
threshold; pcnt_hight absorp (%), percentage of perox saturated cells; PLT, Platelets [Colour figure can be viewed at wileyonlinelibrary.com]

oncologic samples or “reactive with hemolysis” samples or sample

with glucose solution.
The algorithm, subsequently tested on the 51.002 raw-data deriv-
ing from the routine and from the emergency of two laboratories par-
ticipating in the study, however, provided a high specificity (99.88%).
3.3 | APL vs other pathological groups:
Comparative analysis
Then, it was evaluated A2 specificity in hematological and nonhe-
matological diseases of certain diagnosis, whose raw-data were
ROCCO et al.       7|
TA B L E   3   Evaluation of the diagnostic accuracy for parameters in study with two different cutoff (c1 e c2) alone and in the algorithm,
respectively, on the groups Routine and Emergency APL-­negative and APL-­positive

Parameters Cut-­off Sensitivity Specificity PPV NPV LR+ LR-­

c1 1 R1 1 1 1 1
Platelets < 150 98.33 83.10 0.64 100 5.81 0.17
c2 2 E1 2 2 2 2
< 100 95.8 82.35 0.48 100 5.57 0.18
R2 3 3 3 3
94.16 1.77 100 16.40 0.06
E2 4 4 4 4
93.07 1.19 99.99 13.82 0.07
c1 1 R1 1 1 1 1
Monocytes < 10 100 5.39 0.12 100 1.06 0.9
c2 2 E1 2 2 2 2
<6 99.2 4.96 0.09 100 1.05 0.95
R2 3 3 3 3
52.56 0.12 100 2.09 0.47
E2 4 4 4 4
43.92 0.15 100 1.76 0.56
c1 1 R1 1 1 1 1
% LUC <4 91.67 3.58 0.10 99.74 0.95 1.05
c2 2 E1 2 2 2 2
< 4.5 94.2 3.73 0.09 99.90 0.95 1.05
R2 3 3 3 3
2.60 0.11 99.75 0.96 1.03
E2 4 4 4 4
2.83 0.08 99.82 0.97 1.03
c1 1 R1 1 1 1 1
% IPER > 2.0 71.67 89.56 0.75 99.97 6.86 0.14
c2 2 E1 2 2 2 2
> 4.5 37.5 88.21 0.53 99.97 6.07 0.16
R2 3 3 3 3
98.22 2.27 99.93 21.06 0.04
E2 4 4 4 4
97.99 1.60 99.94 18.65 0.05
c1, c2 R 1 1 1 1
Perox % ≥1 92.50 94.98 1.99 99.99 18.42 0.05
saturation E
86.78 2
0.60 2
99.99 2
6.99 2
0.14
c1 1 R1 1 1 1 1
% Blasts >4 83.33 98.53 5.87 99.98 56.68 0.02
c2 2 E1 2 2 2 2
> 3.5 85.00 95.35 1.53 99.98 17.92 0.06
R2 3 3 3 3
97.78 4.04 99.98 38.28 0.03
E2 4 4 4 4
93.22 1.08 99.99 12.53 0.08
1 R1 1 1 1 1
Algorithm – 89.16 99.68 23.62 99.99 278.63 0.003
2 E1 2 2 2 2
86.30 99.03 7.40 99.99 91.92 0.01
R2 3 3 3 3
99.92 55.91 99.99 1078.3 0.001
E2 4 4 4 4
99.73 22.03 99.99 319.63 0.003
R1
: “Routine” samples with c1 cutoff; R2: “Routine” samples with c2 cutoff; E1: “Emergency” samples, with c1 cutoff; E2: “Emergency” samples, with c2
cutoff.

TA B L E   4   Evaluation of specificity and sensitivity of the A2 algorithm in APL group and hematological and nonhematological diseases,
respectively

GROUPS Specificity, % Sensitivity, % F.P. (n=) F.N. (n=)

Routine & emergency raw-­data to construction 99.83 – 420 –

(N = 247.209)
Routine & emergency raw-­data to validate 99.88 – – –
(N = 51.002)
APL (N = 124) – 86.30 – 17
AML & ALL (N = 178) 100 – 0 –
MDS (N = 90) 98.88 – 1 –
MPN (N = 47) 100 – 0 –
Oncologic (N = 168) 100 – 0 –
Hemolytic anemia (N = 39) 94.87 – 1 –
Megaloblastic anemia (N = 25) 96 – 1 –

available. It was found: one false positive both in the MDS group In the Megaloblastic Anemia group, the only false-­p ositive
and in Hemolytic Anemias in which five of six criteria were verified sample met five of six required criteria (except for the % Iper).
(except for the % blasts). (Table 4)
 |
8       ROCCO et al.
4 |  D I S CU S S I O N
Complete blood count, differential count and review of peripheral
blood smear usually are the first-­line investigations that alert the
clinician for a possible underlying leukemia, but further investiga-
tions are required to confirm the diagnosis and classify the leukemia
before appropriate therapy can be started.
Acute promyelocytic leukemia, although it is the most curable
of AML, is a hematological emergency since it requires prompt ini-
tiation of ATRA. Since the introduction of the drug in 1995, there
has been a dramatic reduction in mortality and even early death. In
reality, the rate of ED remains higher than estimated in clinical trials.
This, in part, can be attributed to delayed diagnosis or misdiagnoses.
Today diagnosis of haematological malignancy is an extremely
complex procedure that usually includes: cellular morphology and
immunophenotyping, cytogenetic, molecular studies.
Many studies have showed the ability of the WBC cytograms
of automatic analyzers, and in particular of the ADVIA 120/2120i
(Siemens), to offer an effective and simple tool that can be used by
less skilled morphologists to aid in the classification of the leuke-
mias. However, cytogram analysis also needs trained biotechnolo-
gists and pathologists.
On the basis of what suggested by Pillay about the possibility
of transform P6 cytographic pattern of PANDA classification in a
“diagnostic flag”,19 the purpose of this study was the construction
of an algorithm on the basis of the morphological characteristics of
the APL and on the characteristics of the ADVIA 2120i technology.
It could produce an “APL criteria” with high sensitivity (86.30%) and
very high specificity (99.83%) that can alert biotechnologists and
pathologists to the observation of either the cytograms, to eval-
uate adherence to the PANDA classification, or peripheral blood
smear.
The algorithm provided a high sensitivity, with excellent results
in APL with high or normal number of WBC and satisfactory results
in APL with cytopenia, because it correctly classified 80.6% of the
cytopenic APL. Another advantage of this algorithm, and of the con-
sequent Flag, is that of a very high specificity (and therefore a high
negative predictive value 99.99%) verified on 247.209 samples from
routine and from emergency department. Moreover, the specificity
of the algorithm was very high also in groups of hematological and
nonhematological diseases, whose characteristics could have pre-
dicted a high number of false positives.
Nevertheless, the first CBC from four patients with APL, that
was not “flagged” for APL, was correctly classified in third or
fourth day (data not shown). Meanwhile in two cytopenic patients,
neither the first CBC nor the following ones would have flagged
(diagnosis obtained only on morphological evaluation of bone mar-
row aspirate).
The very high specificity with a limited number of false positives
allows to reduce the workloads, sparing the revision of unnecessary
peripheral blood smears.
However, since it does not allow to alarm all the APL samples, we
think that classical morphology is still mandatory.
In conclusion, the integration of this flag, instrumental and classi-
cal morphology, may allow to reduce the time of diagnosis in patients
with Acute Promyelocytic Leukemia.
Since the algorithm was built retrospectively, it would be neces-
sary to be tested prospectively in APL cases of new diagnosis.
ORCID
Vincenzo Rocco  http://orcid.org/0000-0001-8713-9897
REFERENCES
1. Rowley JD, Golomb HM, Dougherty C. 15/17 translocation, a con-
sistent chromosomal change in acute promyelocytic leukaemia.
Lancet. 1977;1(8010):549‐550.
2. Grignani F, Ferrucci PF, Testa U, et al. The acute promyelocytic
leukemia-­specific PML-­R ARα fusion protein inhibits differen-
tiation and promotes survival of myeloid precursor cells. Cell.
1993;74(3):423‐431.
3. Lo Coco F, Cicconi L. History of acute promyelocytic leukemia.
Mediterr J Hematol Infect Dis. 2011;3(1):e2011067.
4. Wang ZY, Chen Z. Acute promyelocytic leukemia: from highly fatal
to highly curable. Blood. 2008;111(5):2505‐2515.
5. Tallman MS, Altman JK. How I treat acute promyelocytic leukemia.
Blood. 2009;114(25):5126‐5135.
6. Park JH, Qiao B, Panageas KS, et al. Early death rate in acute promy-
elocytic leukemia remains high despite all-­trans retinoic acid. Blood.
2011;118(5):1248‐1254.
7. McClellan JS, Kohrt HE, Coutre S, et al. Treatment advances have
not improved the early death rate in acute promyelocytic leukemia.
Haematologica. 2012;97(1):133‐136.
8. Awisati G, Lo Coco F, Mandelli F. Acute promyelocytic leukemia:
clinical and morphologic features and prognostic factors. Semin
Hematol. 2001;38(1):4‐12.
9. D’Onofrio G, Zini G. Morfologia delle Malattie del Sangue. Rome,
Italy: Verduci Ed. Press; 2013.
10. Bain BJ. Blood Cells: A Practical Guide, 4th edn. Carlton, Vic: Wiley
Blackwell; 2006.
11. Siekmeier R, Bierlich A, Jaross W. The white blood cell differential:
three methods compared. Clin Chem Lab Med. 2001;39(5):432‐445.
12. Jung S, Chae H, Lim J, et al. Differential blast counts obtained by au-
tomated blood cell analyzers. Korean J Lab Med. 2010;30(6):540‐546.
13. Bruno A, Del Poeta G, Venditti A, et al. Diagnosis of acute
myeloid leukemia and system Coulter VCS. Haematologica.
1994;79(5):420‐428.
14. D’Onofrio G. PANDA -­Innovative classification of hematopoietic
malignancies. Bloodline Rev. 2001;2-R:3‐6.
15. D’Onofrio G, Mancini S, Leone G, Bizzi B, Mango G. Identification
of blast cells in peripheral blood through automatic assessment of
nuclear density: a new tool for monitoring patients with acute leu-
kaemia. Br J Haematol. 1987;66(4):473‐477.
16. Gibbs GJ. ADVIA 120/2120i: A Guide to Cytogram Interpretation.
Lancashire, UK: Rossendale Books; 2011.
17. Gibbs GJ. Peroxidase activity and nuclear density analysis (PANDA)
in the diagnosis of haematological malignancy. Br J Biomed Sci.
2005;62(3):142‐144.
18. Gibbs G . Diagnosis of acute promyelocytic leukaemia: the PANDA
approach. Biomedical Scientist Gazette; 2012:105‐107.
19. Pillay D. Diagnosis of haematological malignancies in the era of
total laboratory automation: comparision of the ADVIA 2120 to im-
munophenotyping and morphology A research report submitted to
the Faculty of Health Sciences, University of the Witwatersrand,
ROCCO et al.
Johannesburg March 2015. Link to: https://pdfs.semanticscholar.
org/21bd/171eba483e26184cee28929f514aecc79f78.pdf
20. D’Onofrio G, Zini G. Automated cytochemistry of acute promyelo-
cytic leukemia: there’s more than numbers. Int J Lab Hematol. 2015
Feb;37(1):137‐138.
21. Jang MJ, Choi HW, Lee SY, et al. Application of bone marrow sam-
ples for discrimination of acute promyelocytic leukemia from other
types of acute leukemia using the routine automated hematology
analyzer. Int J Lab Hematol. 2014;36:531‐540.
22. ADVIA. 2120: White blood cell technology. Available from: www.
medical.siemens.com
23. Mie G. Beiträge zur Optik trüber Medien, speziell kolloidaler
Metallösungen”. Ann Phys. 1908;330(3):377‐445.
24. Fisher G, Zelmanovic D, Shapiro P, Greenbaum A, Kunicka JE.
Evaluation of the Two Dimensional Platelet Method on the ADVIA
120TM Hematology System. In First International Bayer Diagnostics
Central Laboratory Symposium. Wilton, Conn: Chase Medical
Publishers; 1997:46.
25. Giacomini A, Legovini P, Antico F, et al. Evaluation of platelet
analysis on the ADVIA 120 hematology system. Lab Hematol.
2001;7:180‐185.
|
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26. Zini G, D’Onofrio G, Briggs C, et al. ICSH recomandations for iden-
tification, diagnostic value and quantitation of schizocytes. Int J Lab
Hematol. 2012;34:107‐116.
27. Bain JB. Blood cell morphology in health and disease. Dacie and
Lewis Practical Hematology, Vol. 5. Philadelphia, PA:Churchill
Livingstone; 2006:79‐113.
28. Bain BJ. Schistocytes in megaloblastic anemia. Am J Hematol.
2010;85(8):599.
29. Swerdlow SH, Campo E, Harris N, et al. WHO Classification of
Tumours of Haematopoietic and Lymphoid Tissues, 4th edn. Lyon,
France: IARC; 2008.
How to cite this article: Rocco V, Castelli C, Fumi M, et al.
The diagnostic use of ADVIA 2120i Siemens and an “APL
criteria” can help to reduce the rate of early death in the APL.
Int J Lab Hematol. 2018;00:1–9. https://doi.org/10.1111/
ijlh.12935