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ORIGINAL ARTICLE

An analytical study on peripheral blood smears

in anemia and correlation with cell counter
generated red cell parameters
Ashutosh Kumar, Rashmi Kushwaha, Chani Gupta, U. S. Singh
Department of Pathology, King Georges Medical University, Lucknow, Uttar Pradesh, India
Address for correspondence: Dr. Rashmi Kushwaha, Flat No. 504, T.G Hostel Khadra, Lucknow - 226 005, Uttar Pradesh, India.
E-mail: docrashmi27@yahoo.co.in

ABSTRACT
Context: Manual examina on of peripheral blood smear in diagnosis of anemia has taken a backseat with the advent of automated
counters. Though a lot of studies have been done to assess the ecacy and significance of red blood cell parameters in dierent
hematological condi ons fewer eorts have been made to standardize the visual examina on of peripheral blood smears for diagnosing
anemias.
Aims: Standardiza on and grading of abnormal red cell morphology in peripheral blood smear and counter based red cell indices in cases
of anemia of various e ologies.
Sengs and Design: Cross-sec onal study of one year dura on conducted in the Hematology laboratory, in a ter ary care hospital in North India.
Materials and Methods: In 60 anemic pa ents, automated counts and peripheral blood smear were prepared and evaluated by three
observers, according to a red cell morphology grading guide.
Stascal Analysis Used: ANOVA, Tukey post hoc test were used.
Results: Objec ve grading of peripheral blood smears in cases of anemia have a good inter observer correla on and hence have reduced
subjec ve varia on. Manual parameters like microcytosis, macrocytosis and hypochromia expressed as a percentage, have shown significant
correla on, with their corresponding automated parameters, and the regression model so generated may provide a novel way for quality
control of automated counters, if calculated for dierent models.
Conclusions: Even in the age of molecular analysis, the blood smear remains an important diagnos c tool and sophis cated modern
inves ga ons of hematologic disorders should be interpreted in the light of peripheral blood features as well as the clinical context.
Key words: Anisocytosis, automated parameters, microcytosis, peripheral blood smear

Along the years there have been studies from time to

time assessing the utility and accuracy of most of the
automated generated parameters in general as well as
with respect to specific types of anemia, but fewer efforts
have been made in the direction of devising methods to
objectively assess peripheral blood smear manually, and
its practical utility in leading to a diagnosis.

INTRODUCTION
Peripheral blood examination has been a window
for hematological ongoings since decades. Analyzing
blood films routinely has facilitated interpretation of
various hematological disorders and has been a major
diagnostic tool especially for etiopathological work up
of anemias.

This study is an attempt to standardize further few

automated red cell parameters, and also objective
grading of RBC morphology on peripheral smear and
interpreting its utility in indicating a diagnosis. We hope
to get a significant correlation between inter-observer
assessments, get a degree of correlation between
automated and manual parameters, and hope to assess
possible causes of common discrepancies and establish
utility of manual grading in making a diagnosis.

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DOI:
10.4103/1658-5127.127896

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Kumar, et al.: Peripheral blood smear evaluation

MATERIAL AND METHODS

OBSERVATIONS AND RESULTS

This was a cross-sectional study of one year duration

conducted in a tertiary care hospital in North India. It
was carried out with the objectives of standardization
and grading of abnormal red cell morphology in
peripheral blood smear, counter based red cell indices
in cases of anemia of various etiologies and comparative
evaluation of peripheral blood smear examination
and automated red cell indices including RDW, MCV,
MCHC, MCHC, fragmented RBC and others, for
anisopoikilocytosis in diagnosing anaemia.

The present study evaluates peripheral blood smears

in anemia and correlates it with cell counter generated
red cell parameters of 60 anemic patients.

Patient Characteristics
The basic characteristics of all anemic patients
are summarized in Table 2. The age of all anemic
patients ranged from 60 yrs with mean ( SD)
Table 1: The following grading criterion was
applied[1]

All patients with non neoplastic anemias were screened

and selected on the basis of clinical evaluation and
hemoglobin values.

Cell type

Hematological studies included complete blood count

by automation and comparing it to the general blood
picture.
Sixty patients identified by hemoglobin values lower
than 9 g/dl, with chief complaints, along with clinical
correlation were identified.
All peripheral blood films were prepared manually
and were stained by a single trained person to
minimize variation in smear spreading and staining
due to interpersonal differences in technique.
Two practicing hematologists and one second year
medical junior resident examined each peripheral
blood film independently. Each peripheral blood film
was visually graded for RBC morphology following
guidelines given in Blood cell morphology Grading
guide written by Gene Gulati and published in
2009.[1] [Table 1].

1+

2+

3+

4+

Table 2: Basic characteristics of anemic patients

Characteristics

The EDTA sample of each patient was run through

automated counters.

Age (yrs)
10 yrs
11-20 yrs
21-30 yrs
>30 yrs
Gender
Females
Males
Anemia
Mild (Hb 6 mg/dl)
Moderate (Hb < 6 mg/dl)
Diagnosis
IDA
MA
HA
Miscellaneous/others

Statistical Analysis

Continuous data were summarized as Mean SD,

while discrete (categorical) data was summarized
in percent. The continuous groups were compared
by one-way analysis of variance (ANOVA) and the
significance of mean difference between the groups
was done by Tukey post hoc test after ascertaining the
normality by Shapiro-Wilk test and the homogeneity
of variance by Levenes test. Groups were also
compared by one way ANOVA followed by Tukeys
post hoc test. The categorical variables were compared
by Chi-square ( 2) test. Concordance correlation
coefficient was used to asses inter observer reliability
and reproducibility. A two-sided ( =2) P < 0.05 was
considered statistically significant. All analysis were
performed on STATISTICA (window version 6.0).
Journal of Applied Hematology

Occasional

Anisocytosis
Occasional <2
2-3
3-4
>4
Poikilocytosis %
NA
<25
25-50
50-75
>75
Microcytosis %
NA
<25
25-50
50-75
>75
Size
>3/4 -3/4 -3/4 <1/2
Macrocytosis %
NA
<25
25-50
50-75
>75
Size
<2
2-3
3-4
>4
Hypochromia %
NA
<25
25-50
50-75
>75
Central pallor
0.4
0.5-0.6 0.6-0.7 >0.7
Target cells %
<5
5-10
10-30
30-60
>60
Tear drop cells %
<1
1-3
3-6
6-12
>12
Schistocytes %
<1
1-3
3-6
6-12
>12
Spherocytes %
<1
1-3
3-6
6-12
>12
Sickle cell %
<5
5-10
10-30
30-60
>60
Acanthocytes%
<5
5-10
10-30
30-60
>60
Echinocytes %
<10
10-25 25-50
50-75
>75
Elliptocytes %
<6
6-20
20-50
50-75
>75
Ovalocytes, %
<6
6-20
20-50
50-75
>75
Blister cells %
<1
1-5
5-10
10-15
>15
Stomatocytes %
<5
5-10
10-30
30-60
>60

No. of subjects
(n=60)

No. of subjects
(%)

19
16
13
12

31.7
26.7
21.7
20.0

24
36

40.0
60.0

32
28

53.3
46.7

20
19
13
8

33.3
31.7
21.7
13.3

IDA=Iron deficiency Anemia, MA=Megaloblastic anemia,

HA=Hemolytic anemia

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Kumar, et al.: Peripheral blood smear evaluation

20.47 17.12 yrs and median 15 yrs. Most of the

anemic patients were below 10 yrs (31.7%). Out of the
total patients, 33.3% patients were diagnosed with Iron
deficiency Anemia (IDA), 31.7% with Megaloblastic
anemia (MA), 21.7% with Hemolytic anemia (HA)
and 13.3% belong to other anemic group.

coefficient (r value) method and summarized in Table 4.

Pearson correlation revealed not a significant correlation
between Microcytosis and MCV (r = 0.004, P > 0.05),
Hypochromia and MCH (r = 0.026, P > 0.05), and
Hypochromia and MCHC (r = 0.010, P > 0.05),
Macrocytosis and MCV (r = 0.003, P > 0.05). In
other words, manual reporting in terms of grades did
not show statistically significant reliability for assessing
cell counter generated red cell parameters in peripheral
blood smears in anemia.

Hemolytic anemias were grouped together under a

single category, with majority being comprised of
Thalassemia. Thalassemia Intermedia and Thalassemia
Major constituted 4 cases each, Thalessemia minor
3 cases, hereditary spheroytosis and thrombotic
thrombocytopenic purpura 1 case each.

The correlation (reliability) of manual reporting

(final grades in %) with automated parameters
(MCV, MCH and MCHC) was assessed by Pearson
correlation coefficient (r value) method and summarized
in Table 5. Pearson correlation revealed a significant and
negative (inverse) correlation between Microcytosis
and MCV (r = 0.51, P < 0.001), Hypochromia and
MCH (r = 0.56, P < 0.001), and Hypochromia
and MCHC (r = 0.58, P < 0.001) while significant
and positive (direct) correlation between Macrocytosis
and MCV (r = 0.54, P < 0.001). In other words,
manual reporting had a high reliability for assessing
cell counter generated red cell parameters on peripheral
blood smears in anemia.

Manually Graded Red Cell Parameters

Reproducibility- Inter-rater agreement

The reproducibility (inter-rater agreement) of grades

given by three independent observers on manual
reporting was assessed using concordance correlation
coefficient (r value) method and summarized in
Table 3. Concordance correlation revealed significantly
(P < 0.01 or P < 0.001) high inter-rater agreement on
all manual parameters with highest being for Tear drop
cells and least for Elliptocytes. In other words, manual
reporting had a high reproducibility for assessing cell
counter generated red cell parameters on peripheral
blood smears in anemia.

RELIABILITY-CORRELATION OF MANUAL
REPORTING WITH DIAGNOSIS

RELIABILITY-CORRELATION OF MANUAL
REPORTING WITH AUTOMATED
PARAMETERS

The correlation (reliability) of manual reporting

(in grades) with Diagnosis (1: IDA, 2: MA, 3: HA,
4: Others) were assessed by Pearson correlation
coefficient (r value) method [Table 6]. Pearson
correlation revealed there was no significant correlation
between Microcytosis, Hypochromia and Anisocytosis.
However, Macrocytosis did show borderline significant
association with the diagnosis (r = 0.25, P < 0.05).

The correlation (reliability) of manual reporting

(in grades) with automated parameters (MCV, MCH
and MCHC) was assessed by Pearson correlation
Table 3: Summary of inter-rater agreement
(n=60) of three observers on manual reporting
on different parameters
Parameters

Anisocytosis (%)
Poikilocytosis (%)
Microcytosis (%)
Macrocytosis (%)
Hypochromia (%)
Target cells
Tea drop cells
Schistocytes
Spherocytes
Ellipocytes
Ovalocytes

The correlation (reliability) of manual reporting (final

grades in %) with diagnosis (1: IDA, 2: MA, 3: HA,
4: Others) was assessed by Pearson correlation coefficient
(r value) method [Table 7]. Pearson correlation revealed
a significant and negative correlation of Microcytosis
(r = 0.41, P < 0.001) and Hypochromia (r = 0.26,

Concordance correlation coefficient

(r value)
Observer 1
Observer 1
Observer 2
vs.
vs.
vs.
Observer 2
Observer 3
Observer 3
0.76***
0.88***
0.70***
0.77***
0.84***
0.91***
0.95***
0.85***
0.82***
0.58***
0.86***

0.73***
0.67***
0.79***
0.70***
0.80***
0.68***
0.89***
0.85***
0.76***
0.36**
0.77***

0.76***
0.70***
0.73***
0.69***
0.88***
0.71***
0.90***
0.78***
0.85***
0.44***
0.84***

Table 4: Manual red cell parameters vs. automated

Correlation
Anisocytosis vs. RDW
Microcytosis vs. MCV
Macrocytosis vs. MCV
Hypochromia vs. MCH
Hypochromia vs. MCHC

0.011ns
0.004ns
0.003ns
0.026ns
0.010ns

ns
-P>0.05. RDW=Red cell distribution width; MCV=Mean corpuscular
volume; MCH=Mean corpuscular hemoglobin; MCHC=Mean corpuscular
hemoglobin concentration

**P<0.01, ***P<0.001

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Concordance correlation coefficient

(r value)

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Kumar, et al.: Peripheral blood smear evaluation

Table 5: Correlation (n=60) of manual reporting

parameters with automated parameters

Table 6: Manual red cell parameters (grades) vs.

diagnosis

Correlations

Correlation

Pearson correlation coefficient (r value)

Microcytosis vs. MCV

Macrocytosis vs. MCV
Hypochromia vs. MCH
Hypochromia vs. MCHC

0.51***
0.54***
0.56***
0.58***

Anisocytosis
Microcytosis
Macrocytosis
Hypochromia
Hypochromia

**P<0.01, ***P<0.001. RDW=Red cell distribution width; MCV=Mean

corpuscular volume; MCH=Mean corpuscular hemoglobin; MCHC=Mean
corpuscular hemoglobin concentration

ns

Pearson correlation coefficient (r value)

Poikilocytosis
Microcytosis
Macrocytosis
Hypochromia

0.28*
0.41***
0.21ns
0.26*

ns

-P>0.05, *P<0.05

A similar trend was seen with MCH. Mean and standard

deviation for each group were calculated and it showed a
significant test result on applying ANOVA. As expected
from previous studies, MCH showed a significant
difference between MA group, and IDA group as well as
between MA group and HA. The mean MCH for IDA
and HA was not found to be statistically significant,
probably because reduced hemoglobin is a feature of
both IDA as well as Thalassemias.

-P>0.05, *P<0.05, ***P<0.001

P < 0.05) with the diagnosis while Poikilocytosis

showed significant and positive correlation with the
diagnosis (r = 0.28, P < 0.05). In other words, manual
reporting had a high reliability for assessing diagnosis red
cell parameters on peripheral blood smears in anemia.
The mean percentage of Target cells (19 cases), Tear
drop cells (23 cases), elliptocytes (14 cases) and
Ovalocytes (17 cases) in various anemias is depicted
in Figures 1-4 respectively.

MCHC though an indicator of reduced hemoglobin,

did not give as consistent a result as MCH, though
overall it was just significant on ANOVA, was helpful in
differentiating mainly MA from IDA. The above pattern
indicates that though the MCH was decreased in our
cases of HA, the MCHC was not markedly reduced as
compared to IDA.

DISCUSSION
The mean hemoglobin as per the different diagnostic
groups, showed minor variations with IDA group
having a mean hemoglobin of 5.85 g/dl, MA 5.25 g/dl,
HA 6.88 g/dl and others 5.03 g/dl. On applying ANOVA,
the difference was found to be statistically insignificant,
indicating that severity of anemia is not a function of
the type of anemia but instead depends on the severity
which can be seen at any grade.

Along the years there have been many studies on

RDW still it has not been standardized. In our study
also ANOVA gave a statistically insignificant result,
it being significant only in differentiating IDA from
HA. In 2008 Mauro Buttarello et al., concluded that,
a continued effort still needed to be made for some
parameters like RDW, IRF, MCVr, and MPV for which
results provided were still too different when produced
by different analyzers.[3]

For all the parameters, a mean value was calculated with

respect to the different diagnostic groups. MCV showed
a mean of 68.83 fl for IDA, 99.4fl for MA, 78.85 fl for
HA and 87.5fl for others. On applying ANOVA, the
P value was found to be statistically significant hence
consolidating the fact that MCV was an expected
reliable parameter, in particular for differentiating MA
from IDA group, and MA from HA group which in our
study were comprised predominantly of Thalassemias.
Although in our study there was a difference in the
mean MCV of IDA and HA, it was not found to be
statistically significant, probably because our HA
group had predominantly Thalassemias, and also a
Journal of Applied Hematology

0.22ns
0.15ns
0.25*
0.08ns
0.16ns

case of Hereditary Spherocytosis, both of which have

a microcytic picture. The finding was in concordance
with a study in 2002 by Melo MR et al., on the use of
erythrocyte (RBC) indices in the differential diagnosis
of microcytic anemias to evaluate prospectively RBC
indices as a diagnostic tool.[2]

Table 7: Correlation (n=60) of manual reporting

parameters (%) with diagnosis
Correlations

Concordance correlation coefficient (r value)

It has been a common dictum that increased RDW is one

of the first signs of identifying IDA and in some studies
has also been helpful in differentiating Thalassemia
minor from IDA. In our study also we had three cases
of Thalassemia Minor, too less to form a separate
group, so we had them under HA. We calculated the
mean RDW for each type of Thalassemia. The mean
RDW for Thalassemia minor was 14.5 as compared to
that for IDA which was 16.23 years. Ours was a very
small sample of Thalassemia minor, but in other studies
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Kumar, et al.: Peripheral blood smear evaluation

Figure 2: Comparative mean of tear drop cells with diagnosis

Figure 1: Comparative mean of target cells with diagnosis

Figure 3: Comparative mean of elliptocytes with diagnosis

Figure 4: Comparative mean of ovalocytes% with diagnosis

RDW has not proven its worth, as in a study in 2010,

Ferrara et al., evaluated the reliability of red blood
cell indices and formulas to discriminate between
Thalassemia trait and iron deficiency in children. They
concluded that none of RBC indices or formulas
appeared reliable to discriminate between the two.[4]

significant correlation for each observer with the

other two [Table 3]. In case of Elliptocytosis, a lesser
degree of concordance was seen among observers, still
it was also statistically significant. The probable reason
could be subjective variation in calling a cell elliptocyte
or ovalocyte by different observers.

One of our aims was to assess the reliability and

applicability of objectively grading the peripheral blood
smears. For this we had graded all the smears according
to blood cell morphology grading guide by G. Gulati.
All the 60 smears were observed by our three observers
and grades were given to each parameter.[1]

One of the most important reasons for variability

of whatever degree could be the field chosen by the
different observers. Our observers counted a minimum
of 200 cells; such an error could probably be minimized
by counting cells in more number of fields and counting
more number of cells, thereby further improving the
inter-observer correlation.

One of the statistical aims of our study was to assess

interobserver correlation, to assess the reproducibility
of the method and to assess the degree of subjective
variation if such a system of manual grading to assess
RBC morphology objectively was brought into practice;
i.e., we wished to see how much the subjective difference
in opinion will hamper this method.

Our next step was to assess how far the manual grades
could be correlated with the parameters generated on
automated cell counters. For doing this we first took
a median grade of four manually graded parameters,
in each case, for comparison with their respective
counterparts on automated generated red cell indices.
For those parameters, in a case where a median
value could not be reached, the grade given by the
most experienced observer was considered as final.
When we applied concordant correlation coefficient
for anisocytosis with RDW, microcytosis with MCV,
macrocytosis with MCV, hypochromia with MCH and

A concordance correlation coefficient was applied,

comparing the observations of each observer from
the other two. Correlation Coefficient for parameters
anisocytosis, poikilocytosis microcytosis, macrocytosis,
hypochromia, target cells, tear drop calls schistocytes,
spherocytes, and ovalocytes showed statistically
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Kumar, et al.: Peripheral blood smear evaluation

hypochromia with MCHC, the results were found to

be statistically insignificant [Table 4]. In contrast, in
a study by David Simel in 1988 on visual inspection
of blood films as against automated analysis of
RDW in which they did semi quantitative analysis
of anisocytosis using the following ordinal scale: 0,
no anisocytosis; 1+, mild anisocytosis; 2+, moderate
anisocytosis; 3+, prominent anisocytosis; and 4+,
marked anisocytosis it was found that semi quantitative
assessment of anisocytosis correlated fairly well with
automated counter generated values of RDW.[5]

red cell parameters on peripheral blood smears in

anaemia. The regression models so generated could
open doors for a novel way of quality control of
automated counters through manual assessment.
After seeing that the method had reproducibility as well
as good correlation with the automated counts, our next
aim was to see if these manual grades correlated with
the diagnosis. For this, Pearson correlation coefficient
was applied first for final grades with the diagnosis.
Though a level of correlation was reached it was found
to be statistically insignificant [Table 6].

The probable reason for our result could be that the

automated parameters were a continuous data and had
a much wider range than the grades which were discrete
and had a range from 1 to 4 only. Moreover the grading
system we followed had grades for percent of the cell type
and the size, in case of microcytosis and macrocytosis,
and central pale area in case of hypochromia. So, when
percent denoted a different grade and size denoted a
different grade, we chose percent as the dominating
factor. Probably this inconsistency in choosing the final
grade, and ignoring the other parameter, could have been
one of the causes, for statistically insignificant results.

Again as in the case of correlation with automated

parameters, correlation of diagnostic groups with
percentage given to each parameter was also done. The
correlation (reliability) of manual reporting in percent
with Diagnosis (1: IDA, 2: MA, 3: HA, 4: Others)
were assessed by Pearson correlation coefficient
(r value) [Table 7]. Pearson correlation revealed a
significant and negative correlation of Microcytosis
(r = 0.41, P < 0.001) and Hypochromia (r = 0.26,
P < 0.05) with the Diagnosis while Poikilocytosis
showed significant and positive correlation with the
Diagnosis (r = 0.28, P < 0.05). In other words, objective
manual reporting in percentage had a high reliability for
assessing diagnosis on peripheral blood smears in anemia.
However, Macrocytosis did not show a significant
association with the diagnosis (r = 0.21, P > 0.05) but
had a positive and borderline significance. In our study,
MCV showed a significant association in differentiating
MA from both IDA as well as HA, and also showed a
significant concordant correlation coefficient (0.54) with
manually graded macrocytosis in percent. However, the
manual grading in percent was found insignificant in
association with the diagnosis, in concordance with the
study in 1978, by RJL Davidson and P Hamilton.[6] In
contrast, in 2006 in an article by Florence Aslina et al., on
MA and other causes of macrocytosis it was stated that
the peripheral blood smear was more sensitive than RBC

Therefore we thought that instead of comparing the

automated parameters with overall grade, maybe we
should take the net percentage given in each case for
each parameter, according to the final grade and the
grade given by the most experienced observer.
On calculating correlation coefficient for each
manual parameter in percent and its corresponding
automated parameter, Pearson correlation revealed a
significant and negative (inverse) correlation between
microcytosis and MCV (r = 0.51, P < 0.001),
hypochromia and MCH (r = 0.56, P < 0.001), and
hypochromia and MCHC (r = 0.58, P < 0.001) while
significant and positive (direct) correlation between
macrocytosis and MCV (r = 0.54, P < 0.001) [Table 5].
The regression models were as follows:
Microcytosis and MCV: MCV = 109.8 0.710
(% Microcytosis); [Figure 5].
Macrocytosis and MCV: MCV = 71.6 + 0.615
(% Macrocytosis); [Figure 6].
Hypochromia and MCH: MCH = 30.19 0.142
(% Hypochromia); [Figure 7].
Hypochromia and MCHC = 32.19 0.074
(% Hypochromia); [Figure 8].

Figure 5: Scatter plot showing correlation of microcytosis % with

MCV. The regression model: MCV = 0.710 (% microcytosis) +
109.8.99 Correlation coefficient r = 0.51

In other words, manual reporting in terms of percentage

had a high reliability for assessing cell counter generated
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Kumar, et al.: Peripheral blood smear evaluation

Of the 60 samples, target cells were reported in 19 cases

including cases of Thalassemia Major, Thalassemia
Minor, Thalassemia Intermedia, IDA, MA and Anemia
of Chronic Disease, thus covering almost the entire
spectrum of anemia diagnosis which we had in our
study. Next we compared the mean in each diagnosis
and found it highest at 32%, for Thalassemia minor,
followed by 19% for Thalassemia intermedia, and
6.16%, for Thalassemia major [Figure 1].
Tear drop cells were reported in around 23 cases
and gave the maximum mean of 22.67 in patients
with Thalassemia major followed by around 6% in
Thalassemia intermedia, and about 4.6 in Thalassemia
minor, which was only marginally higher than that
in IDA and MA. Though tear drop cells are seen in
whole range of diagnosis, the number was significantly
higher for Thalassemia Major, and partially higher
for Thalassemia Intermedia, indicating that besides
presence, percentage of tear drop cells and target cells
can go a long way in, if not establishing but making a
provisional diagnosis of Thalassemia Major, Minor and
Intermedia [Figure 2].

Figure 6: Scatter plot showing correlation of macrocytosis % with

MCV. The regression model: MCV = 0.615 (% macrocytosis) +
71.76 Correlation coefficient r = 0.54

Similarly, ovalocytes showed a clear-cut peak for MA

with 15.5%, significantly different from those of other
diagnostic groups whose mean values lied in the range
of 2 to 6% [Figure 4].

Figure 7: Scatter plot showing correlation of hypochromia % with

MCH. The regression model: MCH = 0.142 (% hypochromia) +
30.99 Correlation coefficient r = 0.56

In 1993 Wisconsin medical technologists, Pat

Garrity and Jeri Walters developed a two tiered
system for grading morphology which recognized the
fact that some abnormal forms were significant in
low numbers (i.e., spherocytes, schistocytes, sickle
cells, Howell jolly bodies, etc.) and others were only
significant when present in high numbers (i.e., target
cells, ovalocytes, microcytes, macrocytes, etc.). They
called them splitters and lumpers.[8] In our findings
also, though target cells, tear drop cells and ovalocytes
were present in low numbers across all the diagnostic
groups, a higher percentage signified Thalassemia types.
As stated above, numerous studies have found red
cell indices, inadequate alone to discriminate between
different diagnoses.

Figure 8: Scatter plot showing correlation of hypochromia % with

MCHC. The regression model: MCH = 0.074 (% hypochromia)
+ 32.19 Correlation coefficient r = 0.58

Barbara J. Bain in her review on the place of

peripheral blood smear examination in the age of
automation in 2005 had said that even in the age
of molecular analysis, the blood smear remains an
important diagnostic tool and sophisticated modern
investigations of hematologic disorders should be
interpreted in the light of peripheral-blood features
as well as the clinical context.[9] To add further, if we
could analyze the peripheral blood smears objectively, it

indices for identifying early macrocytic changes because

the MCV represented the mean of the distribution curve
and was insensitive to the presence of small numbers of
macrocytes.[7]
Some parameters which we assessed manually did not
have a corresponding parameter on automated counts.
For these parameters we calculated the mean percentage,
and compared them with our final diagnosis.
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Journal of Applied Hematology

[Downloadedfreefromhttp://www.jahjournal.orgonTuesday,April08,2014,IP:190.237.88.115]||ClickheretodownloadfreeAndroidapplicationforthisjournal

Kumar, et al.: Peripheral blood smear evaluation

well be instrumental in not only reducing interobserver

variation, and in making the diagnosis but may also
serve as a quality control for automated counters
using the regression models we got for different
parameters. For the application of these regression
formulas on different types of automated counters and
their applicability remains to be seen. Further studies
with larger sample size, targeting one parameter and
automated counter at a time are needed.

3.
4.

5.

6.

Not many studies have been taken up in the past to

assess the validity and applicability of manual objective
assessment of the peripheral smears for red cell parameters.
Our study therefore is an attempt for the same, and to
provide a cost effective method to consolidate, though not
replace the automated cell counters, and also to provide
a proposed method for quality control of automated
counters for red cell parameters.

7.
8.
9.

How to cite this article: Kumar A, Kushwaha R, Gupta C,

Singh US. An analytical study on peripheral blood smears in
anemia and correlation with cell counter generated red cell
parameters. J Appl Hematol 2013;4:137-44.

REFERENCES
1.
2.

of microcytic anemias: Is it an approach to be adopted? Rev

Assoc Med Bras 2002;48:222-4.
Buttarello M, Plebani M. Automated blood cell counts: State of
the art. Am J Clin Pathol 2008;130:104-16.
Ferrara M, Capozzi L, Russo R, Bertocco F, Ferrara D. Reliability
of red blood cell indices and formulas to discriminate between
thalassemia trait and iron deficiency in children. Hematology
2010;15:112-5.
Simel DL, DeLong ER, Feussner JR, Weinberg JB, Crawford J.
Erythrocyte anisocytosis. Visual inspection of blood films vs
automated analysis of red blood cell distribution width. Arch
Intern Med 1988;148:822-4.
Davidson R.J.l. Hamilton P.J. high mean red cell volume: Its
incidence and significance in routine haematology. J Clin Pathol
1978;31:493-8.
Aslinia F, Mazza JJ, Yale SH. Megaloblastic anemia and other
causes of macrocytosis. Clin Med Res 2006;4:236-41.
Garrity P, Walters G. Standardization of red cell morphology reporting
(video), Clinical Laboratory Education. Milwaukee, WI; 1993.
Bain BJ. Diagnosis from the blood smear. N Engl J Med
2005;353:498-507.

Gulati G. Blood cell morphology grading guide. Hongkong:

American Society for Clinical Pathology; 2009.
Melo MR, Purini MC, Cancado RD, Kooro F, Chiattone CS.
The use of erythrocyte (RBC) indices in the differential diagnosis

Source of Support: Nil, Conflict of Interest: None declared.

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