STUDY OF RBC HISTOGRAM IN

VARIOUS ANEMIAS

FATHIMA RAFNA CP
2020 MBBS
 INTRODUCTION
The RBC histogram, a graphic representation of particle size distribution, is now routinely
available on automated cell analyzers as a standard part of automated complete blood count
(CBC) analysis. Over the past few years complete blood count (CBC) by the automated
haematology analyzers and microscopic examination of peripheral smear have complimented
each other to provide a comprehensive report on patient’s blood sample. The histogram in
association with other CBC parameters such as RBC distribution width (RDW) and mean
corpuscular volume (MCV),has been found abnormal in various haematological disorders. In
addition, it is frequently used, along with the peripheral smear as an aid in monitoring and
interpreting abnormal morphological changes, particularly dimorphic cells. Shapes of
histogram can identify pathology to some extent and give us hint, before the blood smear
could be examined.
The normal red cell distribution curve is Gaussian (bell-shaped)and the peak of the
curve should fall within the normal MCV range 80.0 – 100.0 fL. The red cell distribution
curve will get wider as the red cells vary more in size. Thus a narrow distribution curve
indicates a homogenous population and a wider distribution curve indicates a heterogenous
population. An increase in RDW is observed when the size of the RBC vary within the red
cell population. This is physiologically a mix of different cell sizes and is known as
anisocytosis. RDW is not a determinant of “normal” red cell size since it may falls in
reference range even if the cell population is mostly large or small cells.
If the RBCs are larger than normal (macrocytic) as in megaloblastic anaemia, the
curve will shift towards right. If the RBCs are smaller than normal (microcytic) as in
untreated iron deficiency anaemia, the curve will shift towards left. The extension of the
lower end of the scale allows for the detection of RBC fragments, WBC fragments, large
platelets. If the histogram curve is bimodal (camel humps) then there are two population of
red blood cells as might be seen when a patient received a blood transfusion. Other conditions
that will cause a bimodal distribution curve are cold agglutinin disease, haemolytic anaemia
with presence of schistocytes or anaemias with different size cell populations.
A study of histogram can become a new parameter along with red cell indices in the
diagnosis of anaemias. Histograms can be useful tool for technologists to prioritize the cases
to be studied in detail and thus help in speedy disposal of samples in the laboratory. However
automation has also created problems relating to maintenance of expertise especially in the
field of microscopic examination.
A fair knowledge of histogram pattern can help to differentiate subcilinical
anaemias. Patients with iron deficiency anaemia, sequential histograms can show the
progressive appearance of a new well hemoglobinized erythrocyte population well ahead of
that indicated by the conventional parameters. Automated histogram have also been proven to
identify red cell fragments more effectively than routine peripheral smear examinations.
 OBJECTIVES
PRIMARY OBJECTIVES
1. To study the RBC indices in anaemia
2. To study the automated histogram patterns along the morphological features noticed
on peripheral smear.

SECONDARY OBJECTIVES
1. To identify various types of anaemias such as microcytic, macrocytic, normocytic,
dimorphic anaemia.
 LITERATURE REVIEW
Study titled “ Study of RBC histogram in various anaemias” consisting of a total of 100
anaemia cases having haemoglobin less than 12 gm/dL conducted in patients of Central
Diagnostic Laboratory of AIIMS. All cases were studied by comparing results of peripheral
smear. The cases consisted of Normocytic normochromic anaemia, Microcytic hypochromic
anaemia, dimorphic anaemia, Pancytopenia & Thalassemia, as diagnosed by peripheral
smear. This study incudes predominantly females(53%) more than males(47%). The age of
patients ranged from 1 day to 79 years. Maximum number of patients was in 30-40 years of
age range. The anaemia subjects had different types of anaemia. Microcytic hypochromic
anemia was the most common (61%), 17% of the cases are Normocytic normochromic
anaemia, 15% of cases were dimorphic anaemia. Macrocytic anaemia and Pancytopenia seen
in 3% of the cases and Thalassemia is seen in 1%. Representing the histogram variation in
various anaemias. Out of the 17% of Normocytic normochromic anaemia 8% showed normal
histogram and 9% showed mild broad base curve histogram. Out of the 61% microcytic
hypochromic anaemia, 4% were normal histogram, 27% were left shift histogram, 26% were
broad base curve histogram, 2% short peak histogram and 2% abnormal (bimodal) histogram.
Out of Macrocytic anaemia 2% shoed right shift with broad base curve histogram and 1%
showed short peak histogram where haemoglobin was 5.3g/dL and RBCs count was
1.3*10^6/uL. Out of 15% of dimorphic anaemia 3% were of normal histogram, 3% were
broad base histogram, 3% were normal histogram, 4% right and 3% left shift histogram and
1% each of short peak & abnormal histogram. Right shift curves correlated well with
increased MCV, MCH, and RDW. Short peak correlated well with low Hb and red cell count.
Out of 3% of the Pancytopenia with a 2% were broad based, and 1% were of short peak of
normal MCV with low Hb (4.6g/dL), red cell count(1.37*10^6/uL) and increased RDW. The
1% of Thalassemia showed abnormal histogram with left shift, bimodal broad base curve,
correlating well with low MCV, MCH, HGB (2.5g/dL) and red cell count (1.18*10^6/uL)
with normal MCHC and increased RDW.

Study titled “ Study of RBC histograms in various anaemia: A six months prospective study”
conducted from January 2017 to June 2017 in Prathima Institute of Medical Sciences on 100
patients of >1 year age who were anaemic. The results were Microcytic hypochromic
anaemia was the most common (68%). Representing the histogram variation in various
anaemia: Out of the 7% cases of normocytic normochromic anaemia, 5% showed RBC
histogram with short peak and 2% showed mild broad base curve histogram. Out of 7% cases
of Macrocytic anaemia , 5% showed right shift with broad base curve , 1% showed short peak
histogram , 1% showed bimodal curve. Out of 6% cases of dimorphic anaemia 1% showed
normal histogram, 1% showed broad base curve , 1% right and 1% left shift histogram and
2% showed bimodal histogram. Out of the 6% cases of Pancytopenia , 3% showed right shift
and 3% showed short peak. Among 4% cases of thalassemia ,2% showed abnormal histogram
which was not starting at the baseline with left shift bimodal , broad base curve, 1% showed
bimodal curve. 2% cases of sickle cell anaemia showed broad based curves with short peak.

METHODOLOGY

 Study design: Case series study

 Study Setting: Government Medical College Trivandrum, Clinical Pathology Lab

 Study subjects: Patients whose blood samples received in Clinical Pathology Lab
during the study period.

Inclusion Criteria:
Females having haemoglobin concentration less than 12mg%
Males having haemoglobin concentration less than 13mg%

Exclusion Criteria:
Patients whose blood smear is defective or unable to read.

 Study period: 2 weeks

  Sample size:
25 cases

 Study variables:

RBC count, Hb concentration, Mean Cell Volume (MCV), Mean Cell Haemoglobin
(MCH), Mean Cell Haemoglobin Concentration (MCHC), Red Cell Distribution
(RDW), RBC histograms.

 Data collection tool:

Histogram charts and Lab reports including haematological indices of peripheral
smear positive for anaemia cases are collected.

 Data collection technique:

Data of blood samples including haematological indices and histogram charts are
collected directly from the Clinical Pathology Lab by automatic analyzer

DATA ANALYSIS
Data analyzed manually.

ETHICAL CONSIDERATIONS

Data collection is done after taking permission from Pathology Department. It is not
used for other purposes than study. Names of the cases are not made public
 RESULTS
5.1 DESCRIPTIVE ANALYSIS

The total number of cases 25

5.1.1. DISTRIBUTION OF GENDER OF THE STUDY PARTICIPANTS

Table 5.1.1. Distribution of gender of the study participants

Gender Frequency Percentage

Male 7 28

Female 18 72

Total 25 100

Chart Title
20
18
18

16

14

12

10

8 7

0
Male Female

There were 28% males and 72% females in the study.

 5.1.2. DISTRIBUTION OF AGE OF THE STUDY PARTICIPANTS

Table 5.1.2. Distribution of age of the study participants

Age group Frequency Percentage

<20 years 0 0
20-39 years 8 32
40-59 years 10 40
60-79 years 6 24
80-99 years 1 4
Total 25 100

Chart Title
12

10
10

8
8

6
6

2
1
0
0
<20 years 20-39 years 40-59 years 60-79 years 80-99 years

The majority of the cases (40%) belonged to age intervel 40-59 years.
 5.1.3. DISTRIBUTION OF CASES BASED ON HEMOGLOBIN
PERCENTAGE.

Table 5.1.3. Distribution of cases based on hemoglobin percentage.

Mild= 9-12gm%
Moderate=7-9gm%
Severe=<7gm%

Degree of anaemia Frequency Percentage of cases

Mild 12 48

Moderate 6 24

Severe 7 28

Total 25 100

Frequency
14

12

10

0
Mild Moderate Severe

The cases were 48% mild anaemic, 24% moderate anaemia, 28% severe anaemic.
 5.1.3. DISTRIBUTION OF CASES FOR PERIPHERAL SMEAR
DIAGNOSIS

Table 5.1.3. Distribution of cases for peripheral smear diagnosis

Types of anaemia Frequency Percentage

Normocytic Normochromic 12 48
Microcytic Hypochromic 5 20

Dimorphic 6 24
Pancytopenia 2 8
Total 25 100

Frequency
Normocytic Normochromic Microcytic Hypochromic
Dimorphic Pancytopenia

8%

24%
48%

20%

Out of 25 cases, Majority (48%) cases were Normocytic Normochromic Anaemia.20%

Microcytic Hypochromic Anaemia and 24% Dimorphic Anaemia and 8% Pancytopenia.
 5.1.3. DISTRIBUTION OF RBC HISTOGRAM IN PRESENT STUDY

Table 5.1.3. Distribution of RBC histogram in present study

Type of histogram Frequency Percentage

Normal curve 7 28
Left shift 15 60
Right shift 0 0
Broad base curve 2 8
Bimodal curve 1 4
Total 25 100

Frequency
1
2

15

Normal curve Left shift Right shift Broad base curve Bimodal curve

Majority cases were left shift curve(60%), 28% normal curve, 8% broad base curve and 4%
bimodal curve.
 5.1.5. HISTOGRAM VARIATION IN VARIOUS ANAEMIAS

Table 5.1.5. Histogram variation in various anaemias.

Frequency Normal Left Right Broad Bimodal

Curve shift shift base
curve

Normocytic 12 7 5 0 0 0
Normochromic (58.3%) (41.6%)
Microcytic 5 0 5 0 0 0
Hypochromic (100%)
Dimorphic 6 0 3 (50%) 0 2(33.3% 1(16.6)
)
Pancytopenia 2 0 2(100%) 0 0 0

Total 25 7 15 0 2 1

Chart Title

100%

90%

80%

70%
Bimodal
60% Broad base curve
Right shift
50% Left shift
Normal Curve
40%

30%

20%

10%

0%
Normocytic Microcytic Hypochromic Dimorphic Pancytopenia
Normochromic
Among 12 Normocytic normochromic anaemia, 7 (58.3%)cases were normal curve and 5
(41.6%) were left shift. All 5 cases of Microcytic hypochromic anaemias were left shift curve.
Among 6 cases of dimorphic anaemia, half of them were left shift, 2(33.3) cases were broad
base and 1 case(16.6%) was bimodal. Both cases of pancytopenia were left shifted.
 DISCUSSION
Histogram can be used for screening but not considered diagnostic for any pathological
condition6. A CBC report, if abnormal should always be correlated with clinical features and blood
smear findings for meaningful conclusion3.

THE ERYTHROCYTE HISTOGRAM: The analyzer counts those cells as red cells, volume of which
ranges between 36 fL and 360 fL. Causes of lower discriminator flag include red cell fragments,
microspherocytes, nucleated RBCs, non lyzed RBCs, elliptocytosis, giant platelets, platelet clumps and
causes of upper discriminator flag are interfering substances such as cryoglobulin, cold agglutinin,
and roulaex formation8. MCV is perpendicular line from peak of curve to base.

ANALYSIS AND INTERPRETATIONS OF RBC HISTOGRAM: Homogenous population of RBC give narrow
distribution curve while if more anisopoikilocytosis, then curve will be broad. The curve is said to be
symmetric if both sides of the curve coincide when folded in half or are mirror images to one
another. A histogram distribution that is bimodal can be seen in various situations, are usually
associated with therapeutic transfusion and/or hematinic agent response to microcytic and
macrocytic anaemia, but they may also indicate other hematological disorders like cold agglutinin
disease, in the presence of erythrocyte fragments, in IDA (microcytic) with recent blood transfusion,
in sideroblastic anemia especially in acquired forms, and megaloblastic anemia (macrocytic anemia)
with recent blood transfusion

NORMOCYTIC ANEMIA: Out of the 48% of normocytic normochromic anemia 28% showed normal
histogram and 20% showed left shift. The population of the cells would be variable in size.

MICROCYTIC ANEMIA: Out of the 20% of microcytic hypochromic anemia, 20% were left shift
histogram.

DIMORPHIC ANEMIA: In a dimorphic picture, the histogram may have two or more (multiple) red cell
populations. Out of 24% cases of dimorphic anemia , 8% showed broad base histogram, 1% , 12% left
shift histogram and 4% showed bimodal curve .

PANCYTOPENIA : All the 8% cases of pancytopenia were left shift histogram.

 CONCLUSION
Histogram can also be used in differentiating various types of anemia. Histogram changes correlated
well with peripheral smear findings in majority of the cases. Histogram analysis is often a neglected
part of the automated haemogram which if interpreted well, has significant potential to provide
diagnostically relevant information even before higher level investigations are ordered. The speed
and the reliability of the modern analyzers allow technologists, time to evaluate abnormal blood
films, consider diagnostic clues and correlate clinical findings to histograms and other hematologic
parameters with greater confidence and efficiency, all of which produce high returns in terms of
patient health care