“ROLE OF RETICULOCYTE COUNT IN THE DIFFERENTIAL …

“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




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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Documents

“ROLE OF RETICULOCYTE COUNT IN THE DIFFERENTIAL …