“ROLE OF RETICULOCYTE COUNT IN THE DIFFERENTIAL DIAGNOSIS OF MACROCYTIC ANEMIA” By RIJI. M. GEORGE Dissertation Submitted to the Rajiv Gandhi University Of Health Sciences, Karnataka, Bangalore In partial fulfillment of the requirements for the degree of MSc. MLT IN HAEMATOLOGY AND TRANSFUSION MEDICINE Under the guidance of Dr KARUNA RAMESHKUMAR Department of Clinical Pathology St Johns Medical College Bangalore 2010
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“ROLE OF RETICULOCYTE COUNT IN THE DIFFERENTIAL
DIAGNOSIS OF MACROCYTIC ANEMIA”
By
RIJI. M. GEORGE
Dissertation Submitted to the
Rajiv Gandhi University Of Health Sciences, Karnataka, Bangalore
In partial fulfillment
of the requirements for the degree of
MSc. MLT IN HAEMATOLOGY AND TRANSFUSION MEDICINE
Under the guidance of
Dr KARUNA RAMESHKUMAR
Department of Clinical Pathology St Johns Medical College
Bangalore 2010
ACKNOWLEDGEMENT��
������������������I am thankful to Rev. Fr. Lawrence D’souza, Director, St. John’s National
Academy of Health Sciences, Dr. Prem Pais, Dean, St. John’s Medical College for
giving me the opportunity to pursue my postgraduate studies.
I express my deep sense of gratitude to my beloved guide Dr.Karuna Ramesh
kumar, Professor and Head, Department of Clinical Pathology, who provided constant
guidance and advise through out the study and without whose initiative and
enthusiasm this study would not have been completed.
Many thanks to all my teachers, colleagues, technical and non-technical staff of
the Department of Clinical Pathology, for their help, assistance and co-operation.
I wish to thank all the staffs of Molecular biology, Clinical Biochemistry,and
Blood Bank for their co-operation and help directly and indirectly for the completion of
the study.
I am very much thankful to Mr.John .S ,Librarian and all the staffs of Zablocki
library, SJMCH for their heartfull co-operation in every walks of typing works of thesis .
I would like to express my indebtedness to the patients whose contribution in this
process of learning was invaluable.
On a personal note, I thank my family members, my friends for their endless
patience, encouragement and constant moral support in this process of learning.
Above all my thanks to Almighty for making this study a reality.
Date: Riji M George
Place: Bangalore MSc.MLT student
Hematology &
Transfusion medicine
St. John’s Medical College
Bangalore-560034.
ABBREVATIONS
AIHA Auto Immune Hemolytic Anemia
BCB Brilliant Cresyl Blue
CBC Complete Blood Count
CHr Mean Reticulocyte Fraction
CHCr Mean reticulocyte hemoglobin concentration
DNA Deoxyribonucleic acid
EDTA Ethylene Diamene Tetra Aceticacid
G-6 PD Glucose 6 phosphate dehydrogenase
HIV Human Immunodeficiency Virus
Hb Hemoglobin
HFR High Fluorescence Ratio
HELLP Hemolysis elevated liver enymes and low platelets
HCT Hematocrit
IRF Immature Reticulocyte Fraction
MCV Mean Corpuscular Volume
MCH Mean Corpuscular Hemoglobin
MCHC Mean Corpuscular Hemoglobin Concentration
MFR Middle Fluorescence Ratio
MRV Mean Reticulocyte Volume
MSCV Mean Sphered Cell Volume
MSRV Mean Spherical Reticulocyte Volume
MDS Myelodysplastic Syndrome
PMNs Polymorphonuclear leucocytes
RBC Red Blood Cells
RDW Red Cell Distribution Width
RNA Ribonucleic acid
Ret –Y Mean channel value of the forward scatter histogram within
the reticulocyte fraction
SLS-Hb Sodium Lauryl Sulphate Hemoglobin
VIT B12 Vitamin B12
LIST OF TABLES
TABLE NO: TITLE PAGE NO:
1. Common pathologic causes of macrocytosis 05
2. Reticulocyte parameters 15
3. Correlation of mean Hb and retic in group 1 30
4. Correlation of mean Hb and retic in group 2 30
5. Correlation of mean Hb and retic in group 3 33
6. Correlation of mean Hb and retic in group 4 33
7. Comparison of manual and machine retic count 34
LIST OF FIGURES
FIGURE NO: TITLE PAGE NO:
1. Macrocytic blood picture (Leishman’s stain X 1000) 04
2. Reticulocytes (Brilliant cresyl blue and Leishman’s stain
X 1000)
12
3. Reticulocyte scattergram 14
4. Scheme for investigating patients with macrocytic anemia 20
5. Clinical suspicion of anemia 26
6. Distribution of Hb and MCV in study subjects 27
7. Macrocytic blood picture (Leishman’s stain X 1000) 27
8. Male female ratio 28
9. Age wise case distibution 28
10. Immature reticulocytes with abundant reticulum and
mature reticulocytes with remnants of reticulum
(Leishman’s stain X 1000)
29
11. � thalassemia blood picture (Leishman’s stain X 1000 31
12. Auto immune hemolytic anemia( Leishman’s stain X
1000)
31
13. Hemolytic anemia with high reticulocyte count( Brilliant
cresyl blue and leishman’s stain X 1000)
32
14. Erythroid hyperplasia seen in bonemarrow in hemolytic
anemia( Leishman’s stainX 1000)
32
15. Comparison of manual and machine retic count 34
ABSTRACT
BACKGROUND: Macrocytosis is a common finding in clinical settings in 1.7 to 3.6%
of cases involving patients seeking medical care. In most surveys , the most common
cause of macrocytosis is megaloblastic anaemia.
OBJECTIVES: Primary objective was to evaluate the utility of reticulocyte count in the
differential diagnosis of macrocytic anemias and to compare the reticulocyte count in
patients with macrocytic blood picture in the peripheral smear to differentiate between
macrocytic blood picture due to B12 and / or folic acid deficiency and hemolytic anemia .
The secondary was to compare the reticulocyte count by manual method and automated
method.
MATERIALS AND METHODS: The study was prospective in nature and was done
over a period of one year from Jan 1 2009- December 31, 2009. The patients who have
been clinically suspected with anemia were further investigated with hemoglobin
estimation and red cell parameters. All patients who had <10 gm hemoglobin and MCV
>100 fl were included for the study irrespective of the age and gender. The clinical details
were retrieved from the records. In these patients peripheral smear analysis and
reticulocyte count were done for further classification.
RESULTS: In the present study out of 75 patients , 31 had low reticulocyte count, of
which 19 patients, megaloblastic marrow was observed. The mean reticulocyte count
was 1.9%. In the rest, five of them were diagnosed as MDS and seven were diagnosed as
HIV.
In 8 patients the reticulocyte count was high ( the maximum – 54.4% )and they
were further evaluated with hemolytic workup. Among them, six had auto immune
hemolytic anemia and two had inherited hemolytic anemia (1- � thalassemia major, 1-
Hereditary spherocytosis). The mean reticulocyte count was 24.2%.
In 13 patients, the reticulocyte count was higher than 2%, but was less than 6%.
These patients on further evaluation were found to have liver disease.
In 23 patients, the reticulocyte count was in the borderline ( range 0.7 -12%)
who could not be classified as hemolytic or megaloblastic.
CONCLUSION: Low reticulocyte count was the commonest finding in megaloblastic
anaemia where as in the case of hemolytic anaemia there was high reticulocyte count.
Hence, in the present study, in the era of cell counters where MCV is easily available as
an objective parameter, reticulocyte count and peripheral smear examination give
directions for further investigations in cases of anemia.
CHCr : Mean reticulocyte hemoglobin concentration.
CHr : Mean reticulocyte hemoglobin content
IRF : Immature Reticulocyte Fraction
MSRV: Mean spherical reticulocyte volume
Ret-Y: Mean channel value of the forward scatter histogram within the reticulocyte
Population.
2.4.5. Bone marrow examination
Macrocytosis associated with a megaloblastic marrow is usually accompanied by
anemia due to ineffective erythropoiesis. The bone marrow is hypercellular, showing
evidence of abnormal proliferation and maturation of multiple myeloid cell lines. These
abnormalities are most evident in the erythroid precursors with large megaloblastic
erythroblasts present in increased numbers throughout the marrow. Similar morphologic
abnormalities can be seen in the other myeloid elements, e.g., large or giant
metamyelocytes and other granulocytic precursors. This ineffective erythropoiesis is
16
accompanied by intramedullary hemolysis causing an elevated lactate dehydrogenase and
indirect bilirubin in the serum. (27) However, the reticulocyte count is low due to the
abnormal maturation process. More severe degrees of abnormal proliferation and
maturation are seen with myelodysplasia and myeloid leukemias. It is imperative that a
hematologist or hematopathologist examine the marrow in order to appreciate these
important, subtle, hematopoietic abnormalities. Patients with macrocytosis who are not
anemic and have no other abnormalities noted on the peripheral blood smear do not
usually need a bone marrow examination.
Serum B12 levels
Vitamin B12 levels may be reported as normal or elevated in myeloproliferative
disorders, liver disease, congenital transcobalamin II deficiency, intestinal bacterial
overgrowth and antecedent administration of vitamin B12. Moreover, there are reports of
falsely low vitamin B12 levels with folate deficiency, pregnancy, use of oral
contraceptives, congenital deficiency of serum haptocorrins and multiple myeloma.(28)
The prevalence of vitamin B12 deficiency among the elderly ranges from 1.5% to
4.6%(29) and was reported to be as high as 15% in the population over the age of 60
years.(30) The deficiency in many cases is associated with gastric achlorhydria, resulting in
decreased synthesis and availability of intrinsic factor, a necessary binding protein that
facilitates vitamin B12 absorption in the ileum. This constellation of events eventually
leads to pernicious anemia and requires prompt intervention with exogenous vitamin B12
preparations. The diagnosis of pernicious anemia can be confirmed by identifying and
measuring intrinsic antibody levels in the serum. Parietal cell antibodies, although not
17
specific, are also commonly present. However, these tests are expensive and not always
available to the practicing clinician.
Serum Folate levels
Folic acid deficiency in the United States is extremely rare because of the
fortification of foods.(31) Although tissue stores may be normal, serum folate levels can
decrease within a few days of dietary folate restriction. Thus, patients should fast prior to
testing for serum folate levels, as serum folate levels increase with feeding. Because of
the high concentration of folate within the RBC, mild degrees of hemolysis can falsely
elevate serum folate levels.(27)Pregnancy, certain anticonvulsant drugs, and alcohol intake
may also cause a decrease in serum levels despite adequate tissue stores. Serum folate
levels tend to be increased in patients with vitamin B12 deficiency, presumably because
of impairment of the methionine synthase pathway and accumulation of
methyltetrahydrofolate, the principal form of folate in the serum.(32)
Clinical presentation of megaloblastic anemia
A number of nonhematologic manifestations of vit B12 and folic acid deficiency may
appear clinically. These include effects on epithelial tissues, such as the characteristic
beefy, red, smooth tongue (vit B12 and folic acid deficiency) and neuropsychatric
manifestations. ( vit B12 deficiency only)
18
Hematologic manifestations
1) CBC and peripheral blood smear examination.
a) Erythrocytes demonstrate an increased MCV with anisocytosis and
poikilocytosis.
b) Polymorphonuclrar neutrophils (PMNs) may demonstrate nuclear
hypersegmentation, defined as 5% of PMNs with five lobes or one PMN
with 5 lobes or one PMN with six lobes. A finding of 3 or more PMNs
with five lobes is highly suggestive of vit B12 or folic acid deficiency.
c) Mild to moderate leucopenia and thrombocytopenia may be present.
2) Bone marrow aspiration.
Bone marrow aspirate typically reveals hypercellurarity with hyperplasia of all three
major hematopoietic cell lines and abnormal appearance of the hematopoietic cells.11
The marrow of patients with severe megaloblastic anemia is intensely hypercellular with
a preponderance of early red cell precussors.(33)
Other causes of megaloblastic anemia
� Nutritional megaloblastic anemia due to lack of folic acid,vit B12and other
essential food factors.
� Megaloblastic anemia of infancy.
� Megaloblastic anemia of pregnancy.(34)
19
2.5 Hemolytic anemia :
In hemolytic anemias, persist reticulocytosis is the result of a constant demand for
new erythrocytes and acute episodes are generally followed by a sudden rise in
reticulocyte to even higher levels. A persistently elevated reticulocyte count is the rule in
chronic hemolytic anemia. (17) The reticulocyte count is increased in the majority of
patients with hemolytic diseases. The reticulocyte count may be used as an index of red
cell production in hemolytic anemia provided allowances are made for the reduction in
total red cell count and the presence of shift reticulocytes in the peripheral blood.7
Because of erythroid hyperplasia in the marrow there is rise in reticulocyte count,
however, the degree of reticulocytosis is variable, being mild (5 – 10%) in
hemoglobinopathies, and moderate to marked (10 -60%) in immune haemolytic anemia,
spherocytosisand hemolytic attack in in G-6 PD deficiency cases. In hemoglobinopathies
reticulocyte count is slightly elevated because of ineffective erythropoiesis.(35)
20
Figure 4. Scheme for investigating patients with macrocytic anemia.(36)
Laboratory test Interpretation Peripheral Smear Macrocytic Anaemia Bonemarrow Examination Megaloblastic Non megaloblastic Changes changes Recticulocyte count Low High Low Therapeutic Respond to Respond to Possible possible response Vit B12 Folic acid hemolytic liver diseases anaemia Vit B12 Folic acid deficiency deficiency
21
3. OBJECTIVES
Primary
To evaluate the utility of reticulocyte count in the differential diagnosis of
macrocytic anemias
To compare the reticulocyte count in patients with macrocytic blood picture in the
peripheral smear to differentiate between macrocytic blood picture due to B12 and / or
folic acid deficiency and hemolytic anemia
Secondary
To compare the reticulocyte count by manual method and automated method
22
4. MATERIALS AND METHODS
The study was prospective in nature and was done over a period of one year
from Jan 1 2009- December 31, 2009. The patients who have been clinically suspected
with anemia were further investigated with hemoglobin estimation and red cell
parameters. All patients who had <10 gm hemoglobin and MCV >100 fl were included
for the study irrespective of the age and gender. The clinical details were retrieved from
the records. In these patients peripheral smear analysis and reticulocyte count were done
for further classification.
4.1 Hemoglobin estimation and red cell parameters
The analysis of Hb and MCV were performed in automated hematology analysers
Sysmex XT 1800i and Sysmex XT 2000i, using EDTA anticoagulated blood fresh
venous blood sample.
Haemoglobin principle
Sulfolyser (SLS) is added to hemolyze the red blood cells and the Hb is convereted
into SLS-Hb. The concentration of SLS-Hb is measured as light absorbance, and is
calculated by comparison with the absorbance of the diluent measured before the sample
was added.
23
4.2 Peripheral blood Smear preparation and staining
Principle
Romanowsky stains are a combination of acidic and basic dyes, which stains the
acidic component of the cell blue and basic component pink. The property of these dyes
to make subtle distinctions in shades of staining and of staining granules differentially is
made use of to stain blood and bone marrow cells.
Specimen
EDTA anticoagulated whole blood.
Reagents
1. Leishman stain
Leishman powder -1.5 gm
Acetone free methanol -1000ml
Keep it for 15-20 days in room temperature.
2. Buffer solution (PH 6.8)
Solution A:
Sodium hydroxide – 8.0 gm
Distilled water -1000 ml
Solution B:
Potassium dihydrogen phosphate - 27.2 gm
Distilled water - 1000 ml
Stock buffer
Solution A -23.7 ml , Solution B - 50 ml
For use, dilute 20 ml of stock to 1000 ml distilled water.
24
Procedure
• Thin smears were made on glass slide.
• The smears were air dried.
• The air dried smears were kept in a staining rack.
• The smears were covered with leishman stain and kept for 4 minutes.
• The smears were diluted with phosphate buffer (PH 6.8) and for 8
minutes.
• The smears were washed under running tap water.
• The smears were air dried and examined under microscope.
Stained peripheral smear was used to study the morphology of the RBC’s and classify as
microcytic, normocytic and macrocytic.
4.3 Reticulocyte count
Reticulocyte count was done by manual method using supravital staining
technique and the result was expressed in percentage.
Principle
Reticulocytes are juvenile red cells; they contain remnants of the ribosomal
ribonucleic acid (RNA) that was present in the larger amounts in the cytoplasm of the
nucleated precussors from which they were derived. Reticulocytes are stained supra
vitally with Brilliant cresyl blue (BCB). The filamentous network of RNA is precipitated
by stain which can be examined under microscope.
25
Reagents
Diluting fluid
Brilliant cresyl blue -1 gm
Normal saline -100 ml
Method
• Add 2 drops of dye into plastic tube.
• Add 2 to 4 volumes of anticoagulated blood to the dye solution and mix.
• Keep the mixture at 37 c for 15 to 20 minutes.
• Resuspend the red cells by gentle mixing.
• Select films on glass slide.
• Counter stain with Leishman stain.
Manual count
The reticulocytes were counted in 10 oil immersion fields where RBC’s were just
touching each other and the number of RBCs was taken as 100. The mean value of 10
fields was taken and was expressed as reticulocyte percentage.
Automated count
An automated reticulocyte count was performed by using flow cytometry in which
fluorochrome combined with RNA of reticulocytes.
The manual reticulocyte count was compared with machine value whenever possible.
The following algorithum was used in this study
26
Following algorithm was used in this study
FIGURE 5. Clinical suspicion of anemia
Hemoglobin estimation
< 10gm > 10gm
Based on MCV
Microcytic macrocytic normocytic
Based on reticulocyte count
Decreased Increased
Megaloblastic or nonmegaloblastic Hemolytic
Further investigations
bonemarrow examination peripheral smear and other
B12 and folic acid assays workup for hemolytic anemia
27
5. RESULTS
In the present study a total case of 75 cases were included based on the inclusion
criteria of Hemoglobin < 10gms and MCV above 100 femtoliters to analyse the utility of
reticulocyte count in classifying and monitoring anemias.
FIGURE 6. Distribution of Hb and MCV in study subjects.