“role of reticulocyte count in the differential …
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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.
KEYWORDS: Macrocytosis, Megaloblastic anaemia, Hemolytic anaemia, Reticulocyte
count.
INDEX
1. INTRODUCTION
1.1. Macrocytosis 01
1.2. Macrocytosis and anemia 02
1.3. Reticulocytes 02
2. REVIEW OF LITERATURE
2.1. General 04
2.2. Macrocytosis with anemia 05
2.3. Macrocytosis due to nutritional anemia 06
2.4. Investigation of macrocytosis 07
2.4.1. History and physical examination 07
2.4.2. Red cell indices 08
2.4.3. Peripheral smear 10
2.4.4. Reticulocyte count 11
2.4.5. Bone marrow examination 15
2.5. Hemolytic anemia 19
3. OBJECTIVES 21
4. MATERIALS AND METHODS
4.1. Hemoglobin estimation and red cell parameters 22
4.2. Peripheral blood smear preparation 23
4.3. Reticulocyte count 24
5. RESULTS 27
5.1. Demographic details 28
6. DISCUSSION 6.1. General 36
6.2. Reticulocyte count as an indicator of erythropoiesis 36
6.3. Reticulocyte in hemolytic anemia 37
6.4. Automated versus manual count –problems and pitfalls 38
6.5. Macrocuytosis- A broad term requiring direction 39
7. SUMMARY 40
8. CONCLUSION 41
9. BIBLIOGRAPHY 42
10. APPENDIX
10.1. Appendix 1 48
ST. JOHN’S MEDICAL COLLEGE BANGALORE – 560034
Ph (080) 22065050 Telegrams: “SAINJOHNS”
DECLARATION
I, RIJI.M.GEORGE, hereby declare that this dissertation
entitled, ‘ROLE OF RETICULOCYTE COUNT IN THE DIFFERENTIAL
DIAGNOSIS OF MACROCYTIC ANEMIA’ has been prepared by me
under the guidance and direct supervision of Dr.KARUNA
RAMESHKUMAR Professer and Head, Department of Clinical Pathology,
St. John’s Medical College. This dissertation is submitted in partial
fulfillment of the regulations of Rajiv Gandhi University of Health Sciences
and has not formed the basis of a degree or diploma to me by any other
university before.
Place: Bangalore
Date: RIJI.M.GEORGE,
MSc MLT student,
(Haematology
& Transfusion medicine),
SJMC, Bangalore.
ST. JOHN’S MEDICAL COLLEGE
BANGALORE – 560034
Ph (080) 22065050 Telegrams: “SAINJOHNS”
COPYRIGHT
DECLARATION
I hereby declare that the Rajiv Gandhi University of Health
Sciences, Karnataka shall have the rights to preserve, use and disseminate
this dissertation in print or electronic format for academic / research
purpose.
Place: Bangalore
Date:
RIJI.M.GEORGE,
MSc MLT student,
(Haematology & Transfusion medicine),
SJMC, Bangalore.
1
1. INTRODUCTION
1.1Macrocytosis.
In approximately 2 to 4 percent of patients, laboratory evidence of macrocytosis is
found. The term macrocytosis refers to a blood condition in which red blood cells (RBC)
are larger than normal. Macrocytosis is reported in terms of mean corpuscular volume
(MCV). Normal MCV values range from 80 to 100 femtoliters (fl) and vary by age and
reference laboratory. (1) MCV is calculated according to the following formula:
Macrocytosis can be identified by reviewing peripheral blood smears by
automated RBC indices. The peripheral blood smear is more sensitive than RBC indices
for identifying early macrocytic changes because the MCV represents the mean of the
distribution curve and is insensitive to the presence of small numbers of macrocytes. (2)
Compared to the peripheral blood smear, MCV may underestimate macrocytosis in over
30% of cases.(3) Although determination of the MCV by automated blood cell counter is
rarely inaccurate, hyperglycemia, marked leukocytosis and cold agglutinins may result in
false elevations of the MCV.(4) Moreover, partial occlusion of the instrument aperture
and/or leaving the blood sample at room temperature for several hours may also result in
false elevations of the MCV value. Macrocytosis is a relatively common finding in the
era of automated blood cell counters, with prevalence estimates ranging from 1.7% to
3.6%.(5)
2
1.2.Macrocytois and anemias
Macrocytic anemias are classified as those resulting from disorders of DNA
synthesis of erythrocyte precursors in bone marrow (megaloblastic anemias)(6) or those
caused primarily by alcoholism, liver disease and hypothyroidism (non megaloblastic
anemias). A blood smear should be performed to differentiate the two forms.
Hemolytic anemias occur due to destruction of RBCs due to various
mechanisms, which can be classified basically as inherited and acquired. The inherited
can be further classified as due to intracorpuscular and extracorpuscular and the acquired
can be further classified as immune mediated and non immune mediated. The anemia in
hemolytic disease may be either normocytic and normochromic or slightly macrocytic; in
some cases macrocytosis may be a very pronounced for in severe destruction there may
be a very marked reticulocytosis.(7)
1.3 Reticulocytes
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 production of in total red cell count and the
presence of shift reticulocytes in the peripheral blood. Nucleated red cells are commonly
present in the peripheral blood in hemolytic anemia.In general ,the higher the retic count
and the more anemia the patient the more numerous are the normoblasts.(8)
3
The presence of increased polychromasia of the macrocytes on the peripheral
smear and a reticulocyte count of >10% should raise suspicion of hemolysis or an acute
bleed. These large polychromatophilic erythrocytes noted on the peripheral smear
represent reticulocytes, immature RBCs that are larger than mature RBCs, and are
indicative of increased erythropoiesis or RBC production and, if present in increased
number, can raise the MCV. Additionally, the reticulocyte maturation parameters
performed on the peripheral blood may also be helpful to differentiate megaloblastic from
hemolytic causes of the macrocytosis.(9) A persistently elevated reticulocyte count is the
rule in chronic hemolytic anemia.
The present study is aimed at utilizing the reticulocyte parameters in macrocytic
blood picture to differentiate between macrocytic anemias and hemolytic anemias, which
will give further directions for evaluation of anemia.
4
2. REVIEW OF LITERATURE
2.1 General
Large circulating erythrocytes are not always associated with a pathologic process or
condition. In fact, RBCs of newborns and infants tend to be larger (mean MCV = 108 fl)
than normal adult RBCs,(10) and large erythrocytes can be seen during pregnancy in the
absence of an obvious etiology. Macrocytosis without anemia may be a normal variant
and is only noted as a result of repeated peripheral RBC indices in the absence of any
known or existing clinical problems. In some instances this variation from normal can be
found in other family members, which suggests a genetic predisposition, and requires no
therapeutic intervention or further investigation.(11)
FIGURE 1. Macrocytic blood picture Leishman’s stain X 1000
5
2.2. Macrocytosis with anemia
Macrocytic anemia describes an anemic state characterized by the presence of
abnormally large RBCs in the peripheral blood. The cause of macrocytic anemia may be
due to a variety of illnesses and demands further clinical and laboratory assessment.
Macrocytic anemia can usually be divided into two categories, megaloblastic and
nonmegaloblastic, (12 ) based on the examination of the bone marrow. This categorization
is important and frequently aids in determining the etiology of the anemia.
The spectrum of etiologies associated with macrocytic anemia includes nutritional
deficiencies (e.g., vitamin B12 and folate), drugs, primary bone marrow disorders (e.g.,
myelodysplasia and leukemia) and other chronic illnesses.
Table I Common pathologic causes of macrocytosis. (14)
1. Vitamin B12 deficiency
2. Folate deficiency
3. Drugs
4. Alcoholism
5. Nonalcoholic and alcoholic liver disease
6.Hypothyroidism
7. Multiple myeloma
8. Myelodysplastic syndromes
9. Myelodysplastic syndromes
10. Acute leukemia
6
2.3 Macrocytosis due to nutritional anemias
Macrocytosis due to vitamin B12 or folate deficiency is a direct result of
ineffective or dysplastic erythropoiesis. These important vitamins and cofactors are
required for normal maturation of all cells. Marrow erythroblasts are no exception. When
either of these two factors is deficient, RBC proliferation and maturation result in large
erythroblasts with nuclear/cytoplasmic asynchrony. These abnormalities are caused by a
defect in DNA synthesis that interferes with cellular proliferation and maturation. RNA
synthesis and cytoplasmic components remain relatively unaffected. The marrow is
hypercellular with all forms of the myeloid cell line being increased and erythroid
elements being dominant on the marrow aspirate smear preparations. The erythroblasts
become large, oval shaped and contain a characteristic immature, lacy nucleus. These
bone marrow features are called "megaloblastic" and are highly suspicious of a vitamin
B12 or folate deficiency. Megaloblastoid (megaloblastic-like) abnormalities of the
marrow are frequently seen in other hematologic disorders not associated with vitamin
B12 or folate deficiency, (e.g., myelodysplasia and leukemia) and a careful examination
of the bone marrow is necessary to make this distinction. Macrocytosis is frequently
linked to alcoholism, with or without liver disease. In fact, it is purported to be one of the
most common causes of nonmegaloblastic macrocytosis. (13 )
7
2.4 Investigation of Macrocytosis
The evaluation of the patient with macrocytosis requires a systemic approach. It
should begin with a comprehensive history and physical examination followed by
appropriate laboratory studies that include a complete blood count, a peripheral blood
smear and reticulocyte count. In some cases, a bone marrow examination may be
necessary. Determining the underlying cause of the macrocytosis can be particularly
challenging when thalassemia trait or iron deficiency or other nutritional deficiencies
coexist with a vitamin B12 or folate deficiency. In these instances the peripheral blood
smear may show a mixed population of microcytic and macrocytic RBCs with an
elevated distribution width. (14 )
2.4.1 History and Physical examination
Evaluation of macrocytosis begins with a complete history and physical
examination to search for signs and symptoms related to an acute or chronic underlying
illness that may be obvious or occult in nature. Medications such as antimicrobial,
chemotherapeutic and anticonvulsant agents can account for a significant number of cases
of macrocytosis, with or without anemia (table 1), emphasizing the importance of taking a
careful inventory of the patient’s medications. In some instances, macrocytosis may serve
as a surrogate marker indicating the patient’s compliance in taking his/her medications.
(15)similar degree of importance applies to the patient’s dietary history and his/her use of
alcohol.
8
2.4.2. Red cell indices
RBC count, Mean corpuscular volume (MCV), mean corpuscular hemoglobin
(MCH), and mean corpuscular hemoglobin concentration (MCHC) were first introduced
by Wintrobe in 1929 to define the size (MCV) and hemoglobin content (MCH, MCHC)
of red blood cells. Termed red cell indices, these values are useful in elucidating the
etiology of anemias .The erythrocytes indices is extremely helpful in classifying the
erythrocytes as to their size and Hb content. Hb, HCT, erythrocyte count are used to
calculate the three indices: MCV, MCH, MCHC. (16 )
With the general availability of electronic cell counters, red cell indices are now
automatically measured in all blood count determinations .Variation in the size of red
cells (anisocytosis) can be quantified and expressed as red cell distribution width (RDW)
or as red cell morphology index. The size distribution of a population of cells is
graphically represented by the red cell histograms.( 17 )
MCV defines the size of the red blood cells and is expressed as femtoliters (10 -15;
fl) or as cubic microns (�m 3). The normal values for MCV are 87 ± 7 fl.
MCH quantifies the amount of hemoglobin per red blood cell. The normal
values for MCH are 29 ± 2 picograms (pg) per cell .High MCH values are obtained in
uncomplicated in macrocytic anemia. At times macrocytic anemia will be accompanied
by deficient hemoglobin synthesis, in which case normal,or even low values for MCH
may be obtained. MCH is higher in newborn and other infants, since there MCV is
generally higher than adults. (18 )
MCHC indicates the amount of hemoglobin per unit content with the volume
of the cell. It is expressed as g/dl of red blood cells or as a percentage value. The normal
9
values for MCHC are 34 ± 2 g/dl. This index indicates whether the general cell
population is normochromic, hypochromic, or hyperchromic.(15 )
RDW represents the coefficient of variation of the red blood cell volume
distribution (size) and is expressed as a percentage. The normal value for RDW is 13 ± 1
.5%. In macrocytic anemias, the MCV is is increased above 100 fl, the MCH is increased
and the MCHC is within the normal range. Mean Copuscular Volume (MCV) has
been used to guide the diagnostic work up in patients with nutritional anemia to
differentiate between microcytic anemia due to iron deficiency and macrocytic anemia
due to vitamin B12 or folic acid deficiency. ( 17 )
Clinical laboratories now use automated machines to perform blood counts
(commonly called CBC) that include red cell indices as part of the profile. Two types of
automated machines are generally used. Instruments like the Coulter S model employ the
principle of electric impedance; others, like the Hemalog System Analyzer, use optical
methods in performing cell counts. Most of the automated machines give the following
values: white cell count, red cell count, platelet count, hemoglobin, hematocrit, MCV,
MCH, and MCHC. In red cell agglutination, double erythrocytes are counted as one, and
larger clumps are not counted as red blood cells at all. This leads to a "decrease" in red
cell count and a falsely elevated MCV .(16 ) Determination of the hemoglobin value is not
affected prewarming the sample eliminates these spurious values. In hyperglycemia, red
cells are transiently hypertonic in relation to the isotonic diluting fluid, resulting in
swollen cells and an elevated MCV. This can be avoided if some time is allowed for
equilibration after dilution. Increased release of reticulocytes from the bone marrow can
10
raise the MCV. Reticulocytes are about 20% larger than the mature erythrocytes.in
stressful conditions with accelerated bone marrow production of RBC precussors ,cell
division may be skipped during the maturation process,resuting in the release of
macroreticulocytes which can be twice the size normal RBCs. This mechanism accounts
for the macrocytosis seen in severe hemolytic anemia, including thalassemia; where the
marked reticulocytosis may result in normal or even high MCV.(19 )
When the values of hemoglobin, red cell count, and MCV are affected, MCH and
MCHC also become abnormal, since these indices are calculated and are not directly
measured. The MCV, since it is an average value, can be normal in the presence of two
different cell populations (eg. dimorphic anemias, red cell fragmentation with
reticulocyte response). It is, therefore, important to examine the peripheral smear.
2.4.3 Peripheral smear
A review of the peripheral smear is imperative in determining the etiology of
macrocytosis.21 The presence of macro-ovalocytes having an MCV >115 fl, anisocytosis,
poikilocytosis and hypersegmented neutrophils suggests a megaloblastic disorder
associated with a nutritional deficiency, i.e., vitamin B12 or folate deficiency. Round
macrocytes are commonly seen in a variety of chronic illnesses, and round target-
appearing macrocytes are characteristic of liver disease such as hepatitis, obstructive
jaundice, and acute and chronic alcoholism with liver disease. Inspection of the blood
smear for the presence of macro ovalocytes and hypersegmented neutrophils remains
necessary in the interpretation of MCV elevations. (20) or patients who present with
disordered immaturity, hypogranulated or hyposegmented neutrophils, and cytopenias, a
11
bone marrow examination is necessary to rule out or confirm a primary bone marrow
disorder such as a myelodysplastic syndrome or leukemia.
2.4.4 Reticulocyte count
Normally about 0.5 to 1.5%of all erythrocytes in adults are reticulocytes. Normal
values at birth range from 2.5 to 6.5%, falling to the normal adult level by the end of the
next week. (21 )
RBC enters the circulation from the bone marrow as reticulocytes. Reticulocytes
are immature RBCs witch contain remnants of RNA in the cytoplasm and hence are seen
as polychromatophilic cells in the peripheral smear. Polychromatophilic RBCs
corresponds to young reticulocytes with high RNA content. Late reticulocytes are
indistinguishable from mature RBCs on Romanovsky films, except by virtue of their
large size. Supra vital staining with methylene blue allows better visualization of
reticulocytes at various stages.
Significant polychromasia is a reflection of an increased percentage of
reticulocytes, but not necessarily an increased absolute reticulocyte. The
polychromatophilic
Erythrocyte visible on a blood film corresponds to young reticulocytrs with high amount
of residual RNA. Polychromasia can be seen in
1) Neonates
2) As a response to hemorrhage or hemolysis
3) In response to iron or B12/folate theraphy
4) During recovery from bone marrow failure
5) Bone marrow infiltration. (21 )
12
A reticulocyte count should be obtained if there is evidence of hemolysis on the
peripheral smear, i.e, increased polychromasia, nucleated RBCs, spherocytes or
schistocytes.(22)The presence of increased polychromasia of the macrocytes on the
peripheral smear and a reticulocyte count of >10% should raise suspicion of hemolysis or
an acute bleed. These large polychromatophilic erythrocytes noted on the peripheral
smear represent reticulocytes, immature RBCs that are larger than mature RBCs, and are
indicative of increased erythropoiesis or RBC production and, if present in increased
number, can raise the MCV. Additionally, the reticulocyte maturation parameters
performed on the peripheral blood may also be helpful to differentiate megaloblastic from
nonmegaloblastic causes of the macrocytosis. (9)An elevated reticulocyte maturation value
is more suggestive of a megaloblastic rather than a non-megaloblastic anemia.
FIGURE 2. Reticulocytes ( shown by arrow) Brilliant cresyl blue and Leishman’s stain
X1000
13
The peripheral blood reticulocyte count indicates the degree of
effective bonemarrow activity and is one of the most useful and cost effective laboratory
test in classifying the pathophysiology of anemia. (15 ) Reticulocyte count is a relatively
accurate index of effective red cell production when it is expressed in absolute number or
corrected for anemia by relating it to the hematocrit or hemoglobin. (23) Reticulocyte
counting requires the preparation of thin smears on which reticulocyte containing a blue
precipitate can be detected. The amount of reticulum or precipitate varies in reticulocytes
of different maturation states.
Reticulocytes may also be enumerated by flow cytometry using fluorescent dyes
that bind to RNA.This automated counting technique allows evaluation of a larger
number of red cells resulting in accuracy and precision.
.
A common test to determine the concentration of reticulocytes in peripheral
blood uses supravital stain to precipitate intracellular RNA, the distinguishing feature of
reticulocytes. Supravital staining of RNA has been added to several automated
hematology analyzer to enumerate reticulocytes. (24)
Using automated cell counter, the following parameters can be analyzed and
compared.
• Immature Reticulocyte Fraction(IRF)
• Mean Reticulocyte Volume(MRV)
• Mean sphered cell Volume(MSCV)(9)
14
Reticulocyte fractions are separated based on the RNA content with the more
immature cells containing the highest amount of reticulum. The immature reticulocytes
quantitatively describe the youngest reticulocytes with the greatest staining intensity.
This parameters allows early detection of an increased erythropoetic response, which is
important in determining the bone marrow recovering from chemotheraphy or transplant
or in response to erythropoietin therapy.(25)
FIGURE 3 Reticulocyte scattergram of a condition with high retic count.
In this scattergram , the X axis represent the intensity of the lateral fluorescent light, and
the Y axis the intensity of the forward scattered light. Here the scattergram is divided into
three RET zones based on the intensity of the fluorescent light, and the ratio of the
reticulocytes in each zone to the total number of reticulocytes is calculated.(48)
15
Table II: RETICULOCYTE PARAMETERS (26)
Reticulocyte immaturity fractions Reticulocyte cellular indices
IRF -
3- population/ IRF MCVr
3- population/ IRF MCVr, CHr, CHCr, dispersion indices
IRF MSRV
3- population/ IRF* Ret-Y
* Analysed in Sysmex XE 2100.
Rest of the parameters are analysed in various analyzers like Abbott Cell Dyn, ABX
Pentra 120 Retic, Bayer ADVIA 120 and Beckman Coulter LH 750.
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.
10 20 30 40 50 60 700
20
40
60
80
100
120
140
Hb(
g/dL
) an
d M
CV
(f/L
)
Total Patients
Hb MCV
FIGURE 7. Macrocytic blood picture Leishman’s stain X1000
28
5.1 Demographic details
The number of males was more compared to females and the male: female ratio was 7:8.
FIGURE 8.
MALE:FEMALE RATIO
35, 47%40, 53%
MALE FEMALE
0
5
10
15
20
25
1 to15 16 to 30 31 to 45 46 to 60 >60
AGE WISE CASE DISTRIBUTION
cases
FIGURE 9. Bar chart illustrating the number of cases were more in the third and
fourth decade.
The number of cases were more in the third and fourth decade. The number of
cases in the pediatric and geriatric group was less compared to the general population.
29
Based on the reticulocyte count with a cut off 2%, the cases were broadly
classified into megaloblastic and hemolytic anemias. Peripheral smear examination was
done and correlated with reticulocyte count.
FIGURE 10. Immature reticulocyte with abundant reticulum and mature reticulocyte with
remnants of reticulum are seen. Brilliant cresyl blue and Leishman’s stain x1000
Table no III, 1V, V , V1 shows the correlation of mean hemoglobin and reticulocyte
count. It was observed in classic megaloblastic and hemolytic cases, reticulocyte count
gave directions for further evaluation. But in other cases of macrocytic anemias, only
hemolytic causes can be ruled out with correlation of history and peripheral smear.
30
In 31 patients, reticulocyte count was low and these patients were further
evaluated by bone marrow examination and / or vitamin B12 or folic acid estimation. In
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. The
mean reticulocyte count in each group was respectively 3.2 % and 4.5%.
Table III
CAUSE Cases Mean
Hb(gm/dL)
Mean retic
(%)
Megaloblastic 19 7.3 1.9
MDS 05 5.2 3.2
HIV 07 7.7 4.5
In 8 patients the reticulocyte count was high ( the maximum – 54.4% )and these
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%.
Table IV
CAUSE Cases Mean Hb (gm/dL)
Mean retic (%)
Hemolytic anemia
08 6.5 24.2
31
FIGURE 11. Beta thalassemia blood picture Leishman’s stain X1000
FIGURE 12. Auto immune hemolytic anemia. Observe the marked polychromasia.
32
FIGURE 13. Hemolytic anemia with high reticulocyte count Brilliant cresyl .
. blue and Leishman’s stain X1000
FIGURE 14. Erythroid hyperplasia seen in bone marrow in hemolytic anemia .
. Leishman’s X1000
33
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.
Table V
CAUSE Cases Mean Hb
(gm/dL)
Mean retic
(%)
Liver disease 09 7.2 5.4
Alcoholic syndrome 03 6.3 8.1
Wilson disease 01 7.8 6.7
In 23 patients, the reticulocyte count was in the borderline (range 0.7-12%) who could
not be classified as hemolytic or megaloblastic. The etiology was varied in this group as
shown by table no V1.
Table VI
CAUSE
Cases Mean Hb(gm/dL)
Mean retic (%)
Hypothyroidism 01 9.9 2.7
Hypertension 01 8.7 4.3
Nephropathy 01 6.2 12
Aplastic anemia
06 5.8 1.7
Renal disease
09 7.3 1.8
Venous malformation 01 5.6 0.7
Dengue encephalitis
01 8.1 2.1
HELLP Syndrome
01 5.8 7
Ab. tuberculosis
01 9.2 1.3
Malaria
01 7.9 0.8
34
FIGURE 15. Comparison of manual and machine reticulocyte count.
0
5
10
15
20
25
30
35
Ret
ic c
ount
(%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Patients
manualmachine
Table: V11 Comparison of manual and machine reticulocyte count.
NO. Cases Manual retic count (%)
Machine retic count(%)
1 Megaloblastic anemia 0.3 1.04 2 Megaloblastic anemia 3.3 3.23 3 Megaloblastic anemia 2.71 4 4 Megaloblastic anemia 2.9 2.6 5 Megaloblastic anemia 1.4 1.2 6 AIHA 5.1 4.67 7 AIHA 33 15.1 8 Aplastic anemia 3 3.8 9 Aplastic anemia 1.9 2.1 10 Aplastic anemia 1.7 2.3 11 Renal failure 1.1 1.1 12 Liver disease 10 15.2 13 Liver disease 0.1 1 14 Liver disease 7.8 9.28 15 Hypothyroidism 2.7 2.9 16 Nephropathy 12 13.2 17 Alcoholic syndrome 4.8 5.6 18 Venous malformation 0.7 1.34 19 HELLP syndrome 7 7.9 20 HIV 2 1.06 21 HIV 19.8 15.4 22 Malaria 0.8 1.02
35
Pearson’s correlation coefficient test was applied to analyse the difference
between manual and automated retic count. It was observed , the difference was not
significant (p = 0.859 ) indicating the manual count still holds a place . When the manual
count values were plotted against machine count, it was observed that at the lower limit,
the difference was not much. However, when the retic count was high , the dispersion
was more. This was probably due to high sensitivity of the automated count..
36
6. DISCUSSION
6.1. General
The pathological conditions associated with macrocytic anemia are much more
diverse than is often appreciated and macrocytosis is not to be equated with
megaloblastosis, since there are varied conditions associated with non megaloblastic
macrocytosis.(5) The polychromasia observed in hemolytic anemia may also show
increased MCV in the automated cell counter and an examination of peripheral smear
will help to identify the etiology as hemolytic. The diversity and complexity of factors
leading to macrocytic anemias preclude a single or uniform method of investigation. The
investigative pattern must be tailored to the individual patient, giving importance to the
clinical presentation. The present study was aimed at evaluating the utility of reticulocyte
count to facilitate in the differential diagnosis of patients with macrocytic anemia. 75
patients were identified during the study period with increased MCV. Among them, 15
had follow up and had repeated hemogram which helped in follow up of the patients
6.2. Reticulocyte count as an indicatorof erythropoiesis
Crosby et al and Baldini et al had suggested in 1960s that reticulocyte count is not
an accurate indicator of marrow response to erythropoietin stimulation.(37,38) .Later
Crouch and Kaplow in 1985 suggested based on periodic follow up of anemia patients
suggested that the shift reticulocyte count is a more useful index of marrow response to
anemia than the reticulocyte count.(39) With the advent of automated cell counters, where
reticulocyte maturity can also be quantitated, it has been proposed as an indicator of
37
qualitative abnormality of erythropoiesis (40). In the present study, in 31 patients, who
were later diagnosed with vitamin b12 or folic acid deficiency, the low reticulocyte count
indicated ineffective erythropoiesis. In hemolytic anemia, marked increase was observed
indicating stimulated erythropoiesis. This finding is consistent with previous reports. (41)
This increase may be produced by the enhanced stimulation of bone marrow
erythropoietin. Wells et al has shown that the mean fluorescence intensity of reticulocytes
correlated with the serum total iron binding capacity and ferritin concentrations,
suggesting that the reticulocyte immaturity is influenced by a patient’s iron status.(42 )As
the present study was restricted to macrocytic anemias, no such correlation was made.
6.3. Reticulocyte count in hemolytic anemia
In patients with hemolytic anemia, the reticulocyte count increases.(6) In a
case report by Anthony V. Pisciotta in autoimmune hemolytic anemia, reticulocytosis
along with clinical details were taken to the study and they got a retic count as high as
29%.(43) A similar observation of increased retic count with a mean 24% was found in
case of hemolytic anemia in the present study.
In a study by Paul A Volberdins etal on Anemia in HIV infection they
described that an increased or premature RBC destruction in the spleen or circulatory
system may occur in patients with HIV infection. In the present study a case with
increased retic count of 19.8% was found in a case of HIV infection. But the rest of 6
cases of HIV infection this increase of reticulocyte count was not found which can be due
to medications to resolve anemia in such patients.(44)
38
6.4. Automated versus manual count – problems and pitfalls
The introduction of flowcytometric methods using stains that selectively bind
RNA and therefore produce signals that are proportionate to the RNA content has
allowed for the classification of individual reticulocytes based on their maturity in a
reproducible way. (45) The term immature reticulocyte fraction was proposed to indicate
the least mature fraction of reticulocytes.(46) In the present study, the automated counter
values were available only in 22 patients. Comparison of the manual count and
automated count showed, the manual count was always more except in two patients. This
was probably due to very small amount of RNA present in the RBCs corresponding to
Group IV reticulocyte of Heilmyer, which could have been missed in the manual count.
When using the IRF in the differential diagnosis between marrow aplasia and
early erythropoietic response, both conditions characterized by reduced reticulocyte
count, methods using fluorescence and argon laser showed greater sensitivity and are
more robust than methods using a helium neon laser or diode laser which measure light
scattering absorbance.(47) Hence there is a need to use the same method in the sequential
monitoring of marrow aplasia and caution to be exercised in the use of bivariate graphs
produced by some analysers for differential diagnosis between marrow aplasia and early
erythropoietic response.
39
6.5. Macrocytosis – A broad term requiring direction
Red cell size as related to mean corpuscular volume has for many years been used
to classify anemias. With the advent of the electronic cell counters, MCV has become an
integral and useful feature of red cell profile. Macrocytosis is seen in 1.7-3.6% of patients
seeking medical care and is a common finding in any clinical setting , often in the
absence of anemia.(5) In the present study, as the inclusion criteria was decreased
hemoglobin and increased MCV, such a prevalence could not be calculated. However,
among the 75 patients, 19 patients were confirmed to be due to vitamin B12 deficiency
and/or folic acid deficiency. Among them 6 belonged to 5th and 6th decade, the prevalence
of which is consistent with other reports. (29)
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.
40
7. SUMMARY
Macrocytosis is a common finding in clinical settings in 1.7 to 3.6% of cases
involving patients seeking medical care.The total reticulocyte count was used to assess
the cause of anaemia. It was found to be useful in the differential diagnosis of macrocytic
blood picture.
The objective of the study 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
and also to compare the reticulocyte count by manual method and automated method.
Out of the 75 patients studied, 31 had low reticulocyte count ,of which 19 had
megaloblastic marrow and 8 had hemolytic anaemia.Those with reticulocyte count
between 2-6% were identified as liver disease cases.Rest 23 cases were neither
megaloblastic nor hemolytic.
Comparison of manual and automated retic count in limited number of patients
showed, the difference was not significant. (p =0.859). Due to the high sensitivity of the
automated count, grade IV type of reticulocytes , which may be missed in a manual
count, will be identified by automated method.
In conclusion reticulocyte count and peripheral smear examination were useful
in the differential diagnosis of macrocytic blood picture and plays an important role in the
diagnosis and management of macrocytic anaemia and also avoid extensive workup
required for hemolytic anaemia. Further measurement of reticculocyte maturation
parameters will be useful for monitoring of anemia if automated counters are avialable.
41
8. CONCLUSION
In conclusion reticulocyte count and peripheral smear examination were useful
in the differential diagnosis of macrocytic blood picture and plays an important role in the
diagnosis and management of macrocytic anaemia and also avoid extensive workup
required for hemolytic anaemia. Further measurement of reticculocyte maturation
parameters will be useful for monitoring of anemia if automated counters are avialable.
Comparison of manual and automated retic count in limited number of patients
showed, the difference was not significant (p = 0.859).
42
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48
10. APPENDIX
10.1 Appendix 1 RETICULOCYTE COUNT (AUTOMATEDC METHOD) PRINCIPLE: A sample volume of a whole blood specimen is is introduced into the
analyzer where a portion of it is aitomatically diluted into a 1:200 dilution with RET-
SEARCH (11) DILUENT. RET-SEARCH (11) DILUENT DYE is then and the entire
dilution is maintained at a constant temperature for a defined time period in order stain
the reticulocytes present in the sample. The stained sample is then introduced into the
sheath flow detector where forward light scatter and side fluorescent emission are
measured allowing the reticulocyte count (RET#), the reticulocyte percent (RET %), and
the RBCcount to be computed.
Reticulocyte scattergram of a condition with high retic count.
In this scattergram , the X axis represent the intensity of the lateral fluorescent
light, and the Y axis the intensity of the forward scattered light. Here the scattergram is
divided into three RET zones based on the intensity of the fluorescent light, and the ratio
of the reticulocytes in each zone to the total number of reticulocytes is calculated.(48)
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