0196-4763/S3/0306-0435$00.

00/0 Pages 435-438

CYTOMETRY Vol. 3, No. 6, 1983
Copyright 0by the Society for Analytical Cytology Printed in U S A .

Classification of Immature and Mature Cells

of the Neutrophil Series using
Morphometrical Parameters
M. Bins, L. H. van Montfort, T. Timmers, G. H. Landeweerd, E. S. Gelsema, M. R. Halie
From the Division of Haematology, Department of Medicine (M.B., M.R.H.) and the Department of Medical Physics
(L.H.vM.), University of Groningen, The Netherlands; and Department of Medical Informatics (T.T., G.H.L., E.S.G.)Free
University, Amsterdam, The Netherlands
Received for publication May 18,1982; January 12,1983

Quantitative morphological data of six
classes of immature and mature cells of the
neutrophil series of the bone marrow of nor-
mal persons were used for statistical classi-
fication experiments (myeloblasts, promye-
locytes, myelocytes, metamyelocytes, bands
and segments). On each cell, parameters
were measured directly from the image or
calculated from the shape of the density his-
togram or the counting densitogram using a
Texture Analysis System (E. Leitz, Wetzlar,
Germany). The parameters were analyzed
with the interactive statistical pattern rec-
ognition system ISPAHAN. One half of the
data were used as a learning set and the
other half as the test set. The parameters
were compared according to their perform-
ance in discrimination between the classes,
alone and in combinations. Parameters not
contributing to an improvement of the dis-
crimination were disregarded. Eleven pa-
rameters were selected and used for classi-
fication by two different methods: a stepwise
and a “one-shot” method. Stepwise classifi-
cation resulted in a 79% correct classifica-
tion rate. Most errors occurred between cell
classes in neighboring stages of maturation.
In 96% of all cases the computer classifica-
tion was either in accordance with that of
the technician or with a cell class of a neigh-
boring maturation stage, One step classifi-
cation by the computer was in agreement
with the technicians in 82% of the cases. For
98% of the cells the computer classification
was either in accordance with that of the
technician or with a cell class of a neighbor-
ing maturation stage.
The data set was collected by two techni-
cians, operating independently. Differences
in their interpretation of the maturation
stage were found by comparing the perform-
ance of classifiers based on both cell sam-
ples. Since the images of the cells were not
available €or reexamination, the causes of
disagreement in classification between the
technicians and between computer and tech-
nicians could not be evaluated.
Key terms: Image analysis, mature and im-
mature neutrophil leukocytes

In a previous study it was shown that discrimination be-
tween 5 normal mature white blood cell classes is possible
with a high degree of accuracy (98.5%, (4)).However, when
more than 5 normal white blood cell classes are taken into
account, discrimination becomes more difficult, both for the
technician and for the image analysis computer (1).So far
only a few papers have reported on the classification of
immature white blood cells based on morphometric criteria
’ This study was supported by a grant from the Dutch Cancer
Fund, Koningin Wilhelmina Fonds.
Presented a t the Conference on Cytometry in the Clinical Labo-
ratory, Santa Barbara, California, April 25, 1982.
435
(5, 7 , 8). The present study was performed to investigate the
possibility of discrimination among the various classes of
immature white blood cells by using parameters obtained with
a standard commercially available image analysis computer,
and also to identify the parameters which contributed most
to this discrimination.
Materials and Methods
Bone marrow was aspirated by sternal puncture from 10 healthy
volunteers including 6 males and 4 females. A leukocyte enriched
suspension was prepared (10) and the cells were deposited onto a
slide as a monolayer using a Larc cell-spinner (Corning Medical
Instruments, Medfield, MA). The slides were stained with May-Grun-
436 BINS ET AL.

wald-Giemsa in different batches in an automated staining machine A L L DATA

(Shandon Southern Instruments, Inc., Sewickley, PA) and studied
with a Leitz Orthoplan microscope using a x 800 magnification (X
100 oil immersion objective, N.A. 1.32) and unfdtered light (12 V 100
W Halogen). A Plumbicon television camera generated images with
a resolution of 0.15 pn/pixel, which were used for the measurements
by a Texture Analysis System (TAS, E. Leitz). The images of touching
cells were separated on demand by an algorithm which frst shrinks
the images to separation and then restores them to the original SEGMENTS DECISION 2
boundaries by multiple dilations and comparisons to the original
image, not allowing dilating particles to merge. If the separation was

-7-T-
not correct, the cells were disregarded. The cells to be measured were
classified by the operating technician according to the examples in an
atlas of haematology (2). Only cells that could be classified unambig-
uously were measured. The data set was collected by two technicians,
operating independently from one another. For the classification
experiments a training set and a test set were derived from the
combination of the data. In a control experiment both sets were split
into two halves, a training set and a test set. Two classifiers were
calculated based on each training set, and the performance of each
classifier was determined using both test sets, yielding four independ- META-
MYELOCYTES
H W DECISION

MYELoCYTES
4 DECISION

PRO-
MYELOCYTES
5

MYELoBLASTS
ent evaluations.
Morphometric parameters: The TAS was used for the measure- FIG.2. Hierarchical decision tree used for stepwise classification.
ment and calculation of the morphometric parameters. Measuring
programs and data were stored in a PDP 11/34 computer (Digital
Equipment Company, Ma-ward, MA). The parameters measured Results
were essentially the same as described earlier for normal white blood
cells (4). The TAS hardware allowed the grey scale to be divided into
Parameter selection: The interactive pattern recognition
100 intervals which were used to create the density histogram and system ISPAHAN (6) was used for evaluation.
the counting densitogram (3). The mean values of 3 histograms were 1. The measured cells were divided into two groups to serve
used to calculate the parameters. Since the shape of the counting as a learning set and a test set, respectively.
densitogram may be very irregular, features were extracted from the 2. In each parameter group (a. geometric; b. derived from
original histogram as well as from a smoothed version. The smoothing
was accomplished applying the transformation x ( n )= x ( n - 1 ) + 2x(n)
the densitogram; c. derived from the counting densitogram)
+ x ( n + l ) , where x ( n ) represents the contents of the nth bin. The the parameters were ranked in the order of decreasing classi-
cells measured per class are listed in Table 1. fication power, taking into account correlations within each
group.
3. In each parameter group the highest ranking parameter
Table 1
Number o f Cells Studiedper Class was used in combination with an increasing number of lower
ranking ones, and the classification results were determined.
Myeloblasts 97
Promyelocytes 143 In each group the percentage of correctly classified cells
Myelocytes 270 increased initially, but the improvement of the classification
Metamyelocytes 29 1 result decreased when an increasing number of parameters
Bands 274 was used, apparently because no more independent informa-
Segments 286
tion was introduced. Figure l shows the results of the 3 groups
separately. All curves have reached a plateau when 7 param-
eters are entered.
4. By using 21 parameters (the best 7 from each group)
CORRECT
selected as described, a hierarchical decision tree (Figure 2)
CLASSIFICATION was designed by the agglom-option of ISPAHAN (6). The
( 3 90 design of this decision tree is based on Bhattacharyya dis-

8ot tances between classes in a multidimensional parameter space.

COMBINATION

3
j
The a priori classes with the centers nearest to each other are
considered to be the most difficult to be separated and the

:
GEOMETRY
decision between them is positioned far from the root in the
hierarchical decision tree.
I
,"..
_.--_ _--
DENSITY H I S T O G R M
_-- 5. At each branching point of this decision tree the contri-
bution of each of the 21 selected parameters to the correct
COUIITING D E N S I T O G R M classification in the resulting subgroups was determined, and
50 the best performing parameters were selected by determining
I I , L I O I ,
the ranking order and combining an increasing number of
5 10 15 parameters until a plateau was reached, as described above.
NUMBER OF P A R A M E T E R S A t each branching point a combination of up to five param-
FIG. 1. Percent of correct classification obtained by using an in- eters appeared to be sufficient to reach the optimal classifi-
creasing number of parameters. cation result. These optimal combinations of parameters are
 MORPHOMETRY OF IMMATURE WHITE BLOOD CELLS 437

listed in Table 2. This selection used 11parameters out of the Table 4

21 initially selected. These 11 parameters were used for the ConfusionMatrix of the Classification by Technician and
Computer of Mature and Immature Cells of the Neutrophil Series"
final classification experiments.
Computer
Classification: Classification was performed using the pa-
rameters listed in Table 2 following two different procedures,
(a) a stepwise and b) a "one shot" method. A control classifi-
cation experiment (c) was performed by comparing the data -J --
sets of the two technicians with one another. Segment 125 15 3
a. Stepwise classification according to the decision tree (Fig. Band 5 113 17 2
2) using the linear maximum likelihood classifier (6). A t each Metamy elocyte 2 133 10
Mve1ocvte 2 26 98 9
branching point different subsets of the parameters were used 12 50 9
Pr&nyilocyte
(Table 2). The classification result is shown in the confusion Mveioblast 1 2 6 39
matrix of Table 3. Agreement between computer and techni-
a One shot, Fischer classifier.
cian was present in 79% of the cases. Most of the errors (116
out of 140) originated from confusion between neighboring number of errors between nonneighboring classes was 10 on
classes. The number of errors between nonneighboring classes a total of 679 classifications (1%).
was 24 out of 679 classifications. c. By separation of the data into the sets obtained by each
b. One-shot classification method combines the 11 param- technician and by classifying both test sets using two classi-
eters (Table 2) to calculate a classifying equation (pairwise fiers based on both training sets, differences in classification
Fischer classifier, (6)).The classification result is shown in the results were obtained (Table 5 ) . For classification the param-
confusion matrix of Table 4. Agreement between technician eters shown in Table 2 were used in a one-shot procedure.
and computer was present in 82%. Most of the errors were
found between neighboring classes (111 out of 121). The Discussion
Table 2 The problem of classification is essentially different in im-
Parameters Selected for Classification" mature and mature white blood cells. Mature cells represent
-
end stages of a development and no transitional forms between
~~ ~

Decision Number
1 2 3 4 5 classes are present. Only the discrimination between lympho-
Geometric cytes and monocytes may occasionally be difficult. Immature
Shape factor nucleus x x X cells develop gradually from class to class and each cell class
Area nucleus x x can be seen as the intermediate between its neighbors, Dis-
Nucleus/ceIl ratio X X crimination between cell classes which show a continuous
Light area nucleusb X
Cell area X
change in morphology is unavoidably arbitrary when transi-
Densitogram tional forms between the classes are concerned. Another dif-
Range between maxima of ference between mature and immature white blood cells is
Nucleus and cytoplasm X X their availability for study. Not only is marrow puncture more
Minimum between peaks x x unpleasant than venipuncture, and accordingly not attractive
Range cytoplasm peak X
Counting densitogram for healthy volunteers, but also the adaptation of a bone
Sum neg counts cytoplasm x x marrow preparation to the needs of morphometry is more
Range neg counts nucleus X demanding. Overlapping and clumping of cells is highly prev-
Max pos count cytoplasm X alent in the routine bone marrow smears which makes a
a Five geometric, three from the densitogram, three from the count- reliable selection for analysis of single cells difficult or even
ing densitogram. impossible. More complicated and highly standardized tech-
'This parameter indicates the area of the nucleus having an optical niques are essential for the preparation of slides for morpho-
density in the cytoplasmic range.
metric analysis (10). To diminish subjectivity in classification
by the technician in this study a haematological atlas ( 2 ) was
Table 3
ConfusionMatrix of Technician and Computer Classification of used for reference. Cells presenting difficultiesin classification
Immature and Mature Cells of the Neutrophil Series" because of insufficient resemblance with any of the reference
Computer pictures, were not measured. Nonetheless, a subjective factor
in the interpretation of the morphology can not be avoided.
Technician Seg- Mye- Mye- The confusion matrices of Tables 3 and 4 reflect the transition
ment Band mye1o- locyte mye1o- loblast in morphology from one type to another. Most of the errors
cyte cyte
(116 out of 140 and 111 out of 121) occurred between neigh-
Segment 130 13 boring classes.
Band 17 115 4 1
Metamyelocyte 10 123 12
The parameters with the best classifying properties (Table
Myelocyte 7 28 92 7 1 2) were found to be distributed over the three parameter
Promyelocyte 3 9 12 38 9 groups, i.e., geometric parameters, parameters derived from
Myeloblast 1 2 4 41 the densitogram and those derived from the counting densi-
" Stepwise classification, linear maximum likelihood classifier. togram. The segmented and band shaped neutrophils were
438 BINS ET AL.

Table 5 classification results when judged by decision rules derived

Classification of the Data Sets Collected by Two Technicians from their own training set. The decision rules derived from
Using Decision Rules Derived from a Learning Set of One of Them
the training set of technician A caused a considerable number

Exp.
Techni- Myelob-
cian last
z:i0Myel'-
-cyte Meta-
myelo- Band ment
Seg- of misclassifications of promyelocytes of technician B, which
cgte cyte were assigned to the myeloblast class. A similar misclassifi-
cation occurred to a smaller extent between bands and seg-
A 82.6 71.8 68.6 86.6 92.0 92.2
I" ments. Similar discrepancies were found in a study on imma-
B 92.0 25.0 72.6 82.1 97.3 84.8
A 69.6 51.3 68.6 89.6 78.0 98.4 ture blood cells (7) where cellular images were available for
'Ib B 76.0 50.0 70.2 87.2 89.2 89.9 reexamination. Cells which lead to a discrepancy in classifi-
Learning set of technician A. cation between man and computer were reclassified. In about
Learning set of technician B. one third of the cases the reclassifications were in accordance
with the first one. In the remaining cases the classification
characterized by simple parameters, 2 geometric ones and 2 was either in accordance with that of the computer or different
derived from the densitogram. Parameters describing texture from both classifications. This also demonstrates the subjec-
appeared to be useful for discriminating between the imma- tivity of immature white cell classification. The problem of
ture classes farther down the hierarchical tree (decisions 4 the classification of bands and segmented neutrophils was
and 5). This is in agreement with what are thought to be discussed by Mui et al. (9).
valuable features for the visual discrimination between these In conclusion it appeared that morphometric parameters of
cell classes. mature and immature cells of the neutrophil series could be
Only a few studies have been reported in literature on the used with an acceptable degree of accuracy for classification.
classification of immature white blood cells using morpho- T o study the causes of disagreement in classification between
metric methods. The results of Brenner et al. (5) are only man and computer the image of the cells would have to have
partly comparable because in their study 17 cell classes were been available for reexamination.
considered. They reached an overall correct classification rate
of 67.3%.It was also found that the majority of the errors were Acknowledgments
made between neighboring classes. Landeweerd (7) studied 7
The expert technical assistance of W. Huiges, R. Bloem and J.
classes of the neutrophil myeloid series from the peripheral Pietens is gratefully acknowledged.
blood. The groups are comparable to the present study, except
for an additional group consisting of hypersegmented neutro- Literature Cited
phils. Corrected for this seventh group a correct classification
was reached in 269 of 348 cases (77.3%). Confusion between 1. Bacus JW, Gose EE: Leucocyte pattern recognition. IEEE Trans
Man Cyb, SM-2, 513-526,1972
neighboring classes was present in 72 on a total of 79 errors. 2. Begemann H, Rastetter J: Atlas der Klinischen Hamatologie.
In his study other essentially comparable parameters were Zweite Auflage (1972). Springer Verlag, Berlin
used with the main difference being the use of color filters. 3. Bins M, Landeweerd GH, Gelsema ES, van Montfort LH, Halie
Results of differential classification of immature cells by com- MR: Texture of white blood cells expressed by the counting
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4. Bins M, Landeweerd GH, Gelsema ES, van Montfort LH, Halie
a cell-by-cell basis are not available for comparison. MR: Relevance of morphometrical parameters for the classifca-
Since the images of the cells could not be reexamined after tion of normal white blood cells. Clin Lab Haematol4 45-53,1982
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studied. It is conceivable that in some cells, on reconsideration, Vastola E Automated classification of normal and abnormal
leucocytes. J Histochem Cytochem 22: 697-706,1974
the morphology would be found in agreement with the com- 6. Gelsema ES: Ispahan, an interactive system for pattern analysis;
puter classification rather than with the human classification. structure and capabilities. In: Pattern Recognition in Practice.
The errors between nonneighboring classes can hardly be Gelsema ES, Kanal LN (eds.). North Holland Publishing Com-
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DJ: Pattern recognition of nucleated cells from the peripheral
cytoplasm in the nuclear image caused by an overlap of parts blood. Pattern Recognition, 16131,1983
of the segmented nucleus. By using half of the data sets of 8. Lipkin BS, Lipkin CE: Textural parameters related to the nuclear
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study was possible although no second opinion on the cellular Cytochem 22: 583-590, 1974
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neutrophils. J Histochem Cytochem 2 2 633-640,1977
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The test sets of both technicians showed the best average Acta Suppl. 4: 96-101, 1980