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CE update [hematology | blood banking]

Reticulocyte Enumeration: Past & Present


Roger S. Riley, MD, PhD, Jonathan M. Ben-Ezra, MD, Ann Tidwell, MT (ASCP), SH
Department of Pathology, Medical College of Virginia Hospitals of Virginia Commonwealth University, Richmond, VA

After reading this article, the reader should be able to discuss the biology of the reticulocyte, list the present laboratory techniques of
reticulocyte enumeration and the advantages/disadvantages of each technique, identify artifacts that can interfere with automated reticulo-
cyte counts, discuss the recent technological advances on reticulocyte enumeration, and discuss the utilization of reticulocyte enumeration
data in patients with anemia, chronic renal failure, bone marrow transplantation, and other clinical conditions.
Hematology 0103 exam questions and corresponding answer form are located after the “Your Lab Focus” section, p 617.

왘 Erythrocyte formation and physiology effective than manual counting and are in- the peripheral blood and undergo final
왘 Principles of manual and automated creasingly being performed in the clinical maturation. The progression from a
reticulocyte enumeration laboratory. In addition, the newer pronormoblast to a non-nucleated ma-
왘 Factors influencing reticulocyte techniques provide a variety of reticulo- ture red blood cell requires 3-5 days.
enumeration cyte-related parameters, such as the reticu- The production of red blood cells is very
왘 Etiology of reticulocytosis and locyte maturation index and immature tightly regulated by erythropoietin and
reticulocytopenia reticulocyte fraction, which are not avail- other factors in order to maintain a
왘 Clinical interpretation of the able with light microscopy and appear hematocrit of 40 to 45%, the concentra-
reticulocyte count valuable in the clinical diagnosis and moni- tion at which optimal oxygen delivery to
toring of anemia and other diseases.1 the tissues occurs.
The enumeration of peripheral blood The term “reticulocyte” originated
reticulocytes is often performed to obtain Physiology of the Reticulocyte from the deep blue precipitate seen in
information about the functional integrity Red blood cells are continuously these cells after staining with new meth-
of the bone marrow. Reticulocytosis (an renewed in the bone marrow from the ylene blue and other tricyclic, heterochro-
increased number of peripheral blood retic- hematopoietic stem cell. The pronorm- matic, cationic dyes that bind and
ulocytes) occurs in anemic patients with oblast is the first morphologically identi- cross-link RNA and aggregate other or-
functional bone marrow while anemic pa- fiable red blood cell. Subsequent cellular ganelles. The “age” of a reticulocyte is
tients with dysfunctional bone marrow pro- stages in erythropoiesis include the ba- reflected by its relative RNA content.
duce decreased numbers of reticulocytes, sophilic normoblast, polychromatophilic Very young reticulocytes have a dense,
and have decreased numbers of peripheral normoblast, orthochromatic normoblast, coherent mass of RNA and other
blood reticulocytes (i.e., reticulocytopenia) reticulocyte, and mature red blood cell.2- organelles. The RNA becomes less dense
in the absence of extramedullary 4 Reticulocytes undergo an initial period with further maturation and a reticular
hematopiesis and other factors. In addition of maturation in the bone marrow and network appears in the region of the orig-
to the evaluation of anemic patients, reticu- are then released into the peripheral inal mass. The RNA later scatters and
locyte enumeration is also of value in mon- blood where further differentiation into decreases in amount, and only scattered
itoring bone marrow regenerative activity the mature red blood cell (RBC) occurs. RNA remnants remain in the most mature
after chemotherapy or bone marrow trans- The reticulocyte is an anucleate red reticulocytes. This RNA is lost when the
plantation. In the laboratory, the differentia- blood cell, which is slightly larger than reticulocyte differentiates into a mature
tion of the reticulocyte from the mature red the mature red blood cell (10-15 µm ver- red blood cell.
blood cell is based on the presence of RNA sus 6-8 µm). Early reticulocytes continue
and other substances in the reticulocyte, to synthesize hemoglobin, and approxi- Reticulocyte Enumeration
which are lost during differentiation into mately 20-30% of the total hemoglobin Clinical laboratory techniques of
the mature red blood cell. Manual counting of the red blood cell is synthesized at reticulocyte enumeration are based on 599
of reticulocytes by light microscopy with this stage of red blood cell development. the detection of RNA in the cytoplasm of
supravital dyes for RNA was developed in However, hemoglobin synthesis gradu- the reticulocyte. From the late 1940s
the 1940s and remains the standard method ally decreases as cellular organelles are until the early 1980s, reticulocyte enu-
of reticulocyte enumeration. However, au- progressively lost and the reticulocyte meration was performed entirely by mi-
tomated methods of reticulocyte enumera- becomes a mature red blood cell. After croscopic examination of peripheral
tion developed during the past decade are about two days in the bone marrow, blood smears stained with supravital
much more accurate, precise, and cost- reticulocytes are normally released into dyes, even though this technique is

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Present Laboratory Techniques of Reticulocyte Enumeration*

Instrument/Company Dye Technique Present


T1
Utilization

Light microscopy New methylene blue Manual counting 30.4%


Light microscopy with New methylene Manual counting 25.2%
Miller ocular blue
Flow cytometry Thiazole orange Fluorescence detection 1.2%
(Becton Dickinson,
Beckman Coulter M)
ABX PENTRA 120 Retic, Thiazole orange Fluorescence detection <1%
ABX (Montpellier, France) M
Cell-Dyn 4000 CD4K530 Fluorescence detection 4.0%
Hematology Analyzer
(Abbott Diagnostics,
Santa Clara, CA) M
Sysmex R Series Auramine O Fluorescence detection 4.1%
(TOA Medical Electronics,
Kobe, Japan)
XL Hematology Analyzer Coriphosphine-O Fluorescence detection <1%
(Beckman Coulter Inc.,
Fullerton, CA)
Bayer /Miles ADVIA 120 Oxazine Optical light 4.0%
Hematology System scatter detection
(Bayer/Miles Diagnostics Division,
Tarrytown, NY) M
Bayer /MilesTechnicon Ho3 Oxazine Optical light <1%
Hematology Analyzer scatter detection
(Bayer/Miles Diagnostics Division,
Tarrytown, NY)
Cell-Dyn 3500 New methylene Optical light < 1%
Hematology Analyzer blue scatter detection
(Abbott Diagnostics,
Santa Clara, CA)
Coulter GEN.S System New methylene Optical light 16.1%
(Beckman Coulter Inc., blue scatter detection
Fullerton, CA)
STKS/MAXM New methylene Optical light 17.8%
Hematology Analyzers blue scatter detection
(Beckman Coulter Inc.,
Fullerton, CA)

*Data from College of American Pathologists Survey RT, RT-04, 2000. Data from 3443 participating laboratories is represented.

relatively slow, imprecise, and labor inten- recent incorporation of flow cytometric standard method for reticulocyte enumer-
sive. Rapid, precise, automated reticulo- technology (i.e., light scatter and immuno- ation.5,6 In this technique, a few drops of
cyte enumeration utilizing RNA-specific fluorescence detection) into the hematol- the supravital dye solution (1.0% w/v of
fluorescent dyes became possible in the ogy analyzer, accurate reticulocyte counts new methylene blue or brilliant cresyl
1980s with the development of the flow can be a routine part of the hematologic blue) are mixed with an equal volume of
600 cytometer. The later development of flow evaluation. A summary of the present lab- EDTA-anticoagulated peripheral blood
cytometers dedicated to reticulocyte enu- oratory practices for reticulocyte enumera- and incubated for 10 minutes. A thin
meration proved a popular alternative to tion are summarized in T1. smear of the stained blood preparation is
light microscopy for laboratories perform- made on a microscope slide, a Wright
ing high-volume reticulocyte enumeration. Manual Reticulocyte counterstain is applied, and the slide is
Flow cytometric analysis was also found Enumeration examined by light microscopy. An ade-
to provide clinically useful information not The supravital staining technique quate number of erythrocytes (usually
available by light microscopy. With the described by Brecher in 1949 remains the 1000) in a well-stained area are

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examined, and the proportion of reticulo-


cytes is determined. Reticulocytes are
recognized by a blue intracytoplasmic
precipitate, which can vary from individ-
ual small blue granules to a network of
blue reticular material [I1]. Many reticu-
locytes also have a faint, diffuse
basophilic hue (polychromasia).
The reticulocyte count is reported
as a percentage (i.e. reticulocytes per
total red blood cells examined). The
normal mean percentage reticulocyte
count by light microscopy is 1.0% to
1.5%, with 3% being the upper limit of
normal. However, the relative reticulo-
cyte count is misleading when the red
blood cell count is abnormal and there
is significant erythropoietic stimulation
to the bone marrow, such as in severe [I1] Photomicrograph of a new methylene blue-stained peripheral blood smear (1000x).
anemia. Under these circumstances, Reticulocytes are differentiated from mature red blood cells by the presence of brown-black
mathematical corrections must be ap- intracytoplasmic precipitate (reticulin).
plied to the relative count. The packed
cell volume (PCV) correction (reticulo- 32.4%.7,8 The situation improved with using an RNA/DNA-specific
cyte index) is necessary for specimens adoption of the recommendations of the fluorochrome (acridine orange).11 Fluo-
from anemic patients to compensate for National Committee for Clinical Labora- rescence techniques offered few practi-
the decrease in mature red blood cells tory Standards (NCCLS) for reticulocyte cal advantages over light microscopy,
while a “shift correction” is applied to enumeration, a national program of and were not widely used. In contrast to
percentage reticulocyte counts from pa- whole blood proficiency testing, and fluorescence microscopy, automated
tients undergoing intense erythropoietic other measures. The 1994-1995 CAP reticulocyte enumeration by flow
stimulation. A more accurate method to Reticulocyte Survey revealed much bet- cytometry is rapid, objective, semiauto-
correct for the effect of anemia is to ter precision for the automated methods, mated, technically easy to perform, and
calculate the absolute number of reticu- with CVs at or below 15%.9 requires less technical labor than man-
locytes. The absolute reticulocyte count Cytoplasmic particles other than ual slide-based techniques. Automated
is normally between 25 and 125 × RNA (i.e., debris, Heinz bodies, Howell- analysis also provides accurate informa-
109/L. Jolly bodies, nuclear remnants, siderotic tion on the age distribution of the reticu-
A number of factors besides patho- granules, etc.), are another source of locyte population, eliminates subjective
physiologic phenomena may compro- counting inaccuracy, since they can be technical inconsistencies and counting
mise the accuracy of reticulocyte counts stained by supravital dyes, and confused imprecision of manual analysis, and is
performed by manual counting.6 In addi- with reticulum granules.10 Heinz bodies cost efficiency for the analysis of large
tion to problems in specimen collection, are particles of denatured hemoglobin numbers of specimens. The major disad-
transportation, and storage, common which occur in patients with a variety of vantages of this technique are
sources of laboratory variation include hematological disorders (i.e., hemolytic spuriously high flow cytometric reticu-
interobserver variability in morphologi- anemia, thalassemia major, congenital locyte counts caused by interferents,
cal identification, sample size (total Heinz-body anemia, postsplenectomy such as nucleated red blood cells and
number of cells counted), type and qual- disorders, etc.). Although adequate tech- Howell-Jolly bodies, and the necessity
ity of blood film (distributional variabil- nologist training can usually alleviate for expensive, complex instrumentation
ity of reticulocytes), staining variations, problems caused by the misidentification and highly-trained technologists.12
and the use of an ocular counting aids of these particles, special stains are some- A new era in reticulocyte enumera- 601
for standardized area reduction Inter- times used to confirm their presence. tion began with the recent development of
and intralaboratory variability are the methods for automated reticulocyte enu-
most important of these factors.6,7 For Automated Reticulocyte meration using existing hematology ana-
example, in 1984, a national study con- Enumeration lyzers. This has enormous advantages for
ducted by the College of American Reticulocyte enumeration by fluo- most laboratories, since it appears to offer
Pathologists (CAP) Reticulocyte Project rescence microscopy was first reported the accuracy and sensitivity of flow cyto-
found CVs ranging from 26.2% to by Kozenow and Mai in the early 1950s, metric reticulocyte enumeration without

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[F1] Schematic diagram of a single parametric flow cytometric histogram (fluorescence intensity versus cell number) of a TO-stained peripheral blood
sample illustrating the concept of reticulocyte age. The youngest reticulocytes have the largest amount of RNA and exhibit the greatest fluorescence
intensity (high fluorescent reticulocytes, HFR). Reticulocytes of intermediate RNA content show intermediate fluorescence intensity (medium fluorescence
intensity, MFR) while the oldest reticulocytes have the least amount of RNA and the lowest fluorescence intensity (low fluorescence intensity, LFR). The
RMI is the mean fluorescence of the entire reticulocyte population, while the IFR is the sum of the proportion of reticulocytes with medium and high
fluorescence intensity (i.e. MFR + HFR).

the need for separate instrumentation. In (AO) propidium iodide (PI), ethidium of the spectrum (488 nm). In addition,
addition, this technology has the potential bromide (EB), thioflavin T, and auramine commercial, FDA-approved TO-based
to eliminate the need for a technical staff were initially used for reticulocyte enu- reagent kits became available for clinical
trained in flow cytometry to perform meration by flow cytometry. However, in laboratories.
reticulocyte enumeration, to greatly re- the mid-1980s the development of a Reticulocyte analysis by flow cytom-
duce laboratory expenses through elimi- thioflavin T analogue (thiazole orange, etry is performed by mixing an aliquot of
nation of manual reticulocyte counts, and TO) specifically for reticulocyte enumera- fresh, anticoagulated blood with a solu-
to provide more rapid turnaround time. In tion greatly improved counting accuracy tion of thiazole orange dye, incubating
addition, flow cytometric facilities oper- and made flow cytometric reticulocyte the mixture in the dark at room tempera-
602 ate only during weekdays in many institu- enumeration practical for clinical labora- ture for a brief period of time, and then
tions, which precludes the use of this tories with commercial clinical flow cy- performing flow cytometric analysis. Red
instrumentation for reticulocyte counts, tometers with relatively low-powered blood cells are accurately discriminated
which are required for patient care at visible lasers.13 This was largely due to from platelets and white blood cells by
nights and on weekends and holidays. the fact that, unlike thioflavin T and other forward angle light scatter (FALS), or by
Fluorescence with RNA/DNA-Spe- fluorochromes which require ultraviolet a combination of FALS and right angle
cific Fluorochromes. A wide variety of excitation from large, expensive lasers, light scatter (RALS), and gated analysis
fluorochromes, including acridine orange TO excitation occurs in the visible region is then performed on the red blood cell

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population to differentiate mature red cytometric facilities in the 1980s and absolute values for reticulocyte enumera-
blood cells from reticulocytes by the level early 1990s, but it has been supplanted in tion in comparison to light microscopy.
of green fluorescence intensity. Young, most clinical laboratories by the Possible explanations include the higher
immature reticulocytes are brightly fluo- dedicated reticulocyte analyzer and sensitivity of fluorescence detection and
rescent (high RNA content), while matur- hematology analyzer. Dedicated, fully differences in the binding of NMB and
ing reticulocytes show an intermediate automated flow cytometers specifically thiazole orange to RNA.25 Another con-
fluorescence intensity (intermediate RNA designed for reticulocyte enumeration are cern is the effect of red blood cell inclu-
content), and older reticulocytes show produced two companies, TOA and ABX sions (i.e. Howell-Jolly bodies, Heinz
dim fluorescence (low RNA content).14 M.19 The TOA instruments (Sysmex R- bodies, etc.) and other artifacts on flow
The mean fluorescence of the TO-stained 1000, Sysmex R-3000) are mechanically cytometric reticulocyte enumeration.12, 26-
reticulocyte population, the reticulocyte similar to conventional bench-top analyt- 28 Although the significance of red cell

maturation index (RMI), is an indicator ical flow cytometers and utilize inclusions is controversial, and may be
of the relative amount of reticulocyte in- auramine-O as the fluorochrome for nu- instrument and software dependent, man-
tracellular RNA, and proved a useful clin- cleic acid detection. Reticulocyte analy- ual reticulocyte enumeration is best per-
ical indicator of erythropoietic activity sis is performed automatically, with formed on these specimens at the present
and a valuable supplement to the conven- minimal operator intervention. Platelets time. Potential sources of interference
tional reticulocyte count.15 The RMI is and nucleated cells are excluded from with automated reticulocyte enumeration
greatly increased in patients with many consideration by gating (forward scatter are listed in T2.
“young” reticulocytes, including bone vs. side scatter), and both the red blood Optical Light Scatter. The develop-
marrow regeneration post-chemotherapy cell count and the absolute reticulocyte ment of technology to perform reticulo-
or bone marrow transplantation, count (109/L) are reported. In addition, cyte enumeration by optical light scatter
iatrogenic erythropoietic stimulation, or the reticulocyte population is automati- was a major breakthrough for the clinical
rapid erythrocyte turnover from red cell cally divided into fractions showing low, laboratory, since it could be incorporated
hemolysis of other causes. Unfortunately, intermediate, and high fluorescence, and into existing hematology analyzers with
the RMI was found to be affected by iron the percentage of cells in each fraction is little additional cost. Beckman Coulter
stores and other factors, and was later calculated. The ABX PENTRA 120 Inc.M first provided reticulocyte enumer-
replaced by a more accurate parameter, RETIC (VEGA RETIC) is a similar in- ation technology on their STKS™,
the immature reticulocyte fraction (IRF). strument that utilizes thiazole orange as a MAXM™, and MAXM A/L hematology
The IRF is the sum of reticulocyte frac- reticulocyte detection agent. One hema- analyzers. The Coulter technique utilizes
tions with medium and high fluorescence tology analyzer, the Cell-Dyn 4000 (Ab- a new methylene blue stain and differenti-
[F1]. Several investigators identified an bott Diagnostika GmbH, M), provides a ates reticulocytes from mature red blood
elevation in the IRF as the first sign of fluorescent measurement of the reticulo- cells, white blood cells, and platelets
hematologic recovery in the majority of cyte count and reticulocyte quantitative through the measurement of impedance,
patients receiving remission-induction maturational data in whole blood using a radio frequency, and laser light scatter
chemotherapy and first sign of engraft- proprietary fluorescent dye (VCS technology). Whole blood is first
ment in those undergoing bone marrow (CD4K530).20-24 incubated with a new methylene blue dye
transplantation.16-18 The comparative accuracy of flow stain. An aliquot of the stained sample is
The conventional flow cytometer cytometric and manual reticulocyte enu- then diluted with a hypotonic acid solu-
was widely utilized for reticulocyte enu- meration has been a subject of continuing tion to clear hemoglobin from the cells.
meration by researchers and large clini- interest. Most, but not all flow cytometric The instrument determines the volume,
cal laboratories with dedicated flow studies have demonstrated slightly higher conductivity and laser light scatter char-

Potential Sources of Interference with Automated Methods of Reticulocyte Analysis*

Cellular Elements Cellular Inclusions Miscellaneous


T2
Platelet clumps Howell-Jolly bodies Autofluorescence (porphyria, drugs) 603
Giant platelets Heinz bodies Paraproteins
Leukocytes Pappenheimer bodies Cold agglutinins
Leukocyte fragments Parasites (malaria, babesia) Platelet/RBC coincidence
Nucleated RBCs Basophilic stippling Hemolysis
Hemoglobin H inclusion bodies

*Updated from 14.

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Clinical Significance of the Reticulocyte Count

Clinical Disease Mechanism Cause


T3
Reticulocytopenia
Hypochromic anemias Impaired hemoglobin synthesis Iron deficiency, anemia of chronic disease,
thalassemia, sideroblastic anemia
Aplastic anemias Impaired erythropoiesis Idiopathic, renal disease, metastatic infiltration
of bone marrow, viral infection. mmunologic-,
drug-, or radiation -induced red cell aplasia
Megaloblastic anemia Impaired DNA synthesis Vitamin B12 deficiency, folate deficiency
Aplastic crisis in Variable Variable
hemolytic anemia
Reticulocytosis
Blood loss Increased erythropoiesis Covert blood loss,
subacute blood loss
Hemolytic anemias Increased RBC destruction Hemoglobulinopathies,
disorders of the red cell membrane,
immune hemolytic disease,
hypersplenism

acteristics of each cell and plots the re- The CELL-DYN 3500 (Abbott Di- globin concentration (CHCMr), reticulo-
sults within a three-dimensional matrix. agnostika GmbH) Hematology Analyzer cyte cell hemoglobin content (CHr) and
The position of the reticulocytes within has been recently utilized for reticulocyte their respective distribution widths
the matrix indicates their relative matu- enumeration. This instrument incorpo- (RDWr, HDWr, and CHDWr).35
rity. Less mature reticulocytes have more rates a 633 nm neon helium laser with Image analysis. The MICRO21
residual RNA, scatter more laser light, optics and electronics for flow cytometric system (Intelligent Medical Imaging,
and are larger than the more mature single cell analysis by electrical resistiv- Inc. M) uses a form of artificial intelli-
reticulocytes. With maturation, the retic- ity (impedance) and multi-angle light gence to locate and identify reticulo-
ulocyte loses RNA and moves toward the scatter at 0°, 10°, 90° polarized, and 90° cytes on conventional peripheral blood
mature erythrocyte population, until fi- unpolarized.34 With this technology, smears stained with New Methylene
nally it has no RNA, absorbs no stain whole blood is stained in a single step Blue. The images of up to 5,000 reticu-
and merges with other mature red blood dilution procedure with a supra-vital dye locytes/slide are taken and stored for
cells. Both percent and absolute reticulo- solution, incubated for five minutes, and display on a high-resolution color mon-
cyte counts are clinically reportable. Sev- analyzed using 0°, 10°, and 90° light itor for operator review, verification,
eral published studies show excellent scatter. Software-based data management and archiving. The system can process
correlation of the Beckman Coulter opti- provides the absolute and relative reticu- up to 30 bar-coded slides/hour and can
cal light scatter analysis with manual and locyte count, together with reticulocyte store images from 180 slides. A white
flow cytometric methods of reticulocyte maturity information. The Bayer/Miles blood cell differential count can also be
enumeration, although there is an overes- Technicon H*3 blood analyzer performed. A comparative study of this
timation of reticulocytes at low concen- (Bayer/Miles, Diagnostics Division) uti- method of reticulocyte enumeration has
trations and a moderate underestimation lizes the nucleic acid-binding dye not been published at the time this
at normal and high reticulocyte concen- oxazine 750 for reticulocyte enumeration manuscript was completed.
trations.29-31 The Beckman Coulter by helium-laser light absorption and light
GEN S Hematology system is a top-of- scatter at low angle (2° to 3°) and high Clinical Applications of
the-line hematology analyzer (GEN S angle (5° to 15°). An absorption thresh- Reticulocyte Data
604 Hematology System) that utilizes nonlin- old is used to separate stained reticulo- The erythropoietic activity of the
ear separation techniques, multiresolu- cytes from unstained red blood cells. In bone marrow and the rate of delivery of
tion analysis, and adaptive gating to addition to the absolute and relative retic- cells from the bone marrow into the pe-
accurately differentiate small red cell ulocyte counts, this technology permits ripheral blood determine the number of
populations with overlapping features. As direct measurement or calculation of reticulocytes in the peripheral blood.
a result, the instrument has a linear range reticulocyte cellular indices, including Since reticulocyte enumeration provides
of 0-30%, or 0 – 750 reticulocytes × the reticulocyte mean cell volume information about the bone marrow ac-
109/L.32,33 (MCVr), reticulocyte mean cell hemo- tivity and the effectiveness of red blood

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cell production, it is crucial in the diag- Clinical Utilization of Reticulocyte Enumeration


nosis of anemic patients, and for moni-
toring bone marrow transplantation Hematologic Diagnosis
T4
patients, patients undergoing therapy
with marrow toxic drugs, and patients Classification of anemic patients
being treated for anemia.36 Common
Diagnosis and severity of hemolytic anemia
causes of reticulocytopenia and reticulo-
cytosis are shown in T3. Assessment of bone marrow function
Aplastic crisis in hemolytic anemia
General Considerations Myelodysplasia
Reticulocytosis (an increased number
Occult or compensated hemorrhage or hemolysis
of circulating reticulocytes) normally oc-
curs in anemic patients with functional Sickle cell crises and other complications in sickle cell anemia
bone marrows. This includes patients Treatment Monitoring
with blood loss or hemolytic anemias
(sickle cell anemia, thalassemia, sphero- EPO therapy in ESRD, AIDS, MPO, infants, etc.
cytosis, glucose-6-phosphate dehydroge- Bone marrow regeneration post BMT or chemoRx
nase deficiency, immune hemolytic
Renal transplantation engraftment
disease, and hypersplenism), and patients
who have been successfully treated for Anemia therapy (Fe, ruEPO, B12, folate, etc.)
other types of anemia. In contrast, Bone marrow toxic insults
patients with marrow ablative disorders, Hydroxyurea therapy in sickle cell anemia
impaired erythropoiesis, or decreased ery-
Neonatal transfusion requirements
thropoietin production may show a nor-
mal or decreased reticulocyte count in Other Applications
spite of severe anemia. Such patients in-
clude those with iron, folate, or vitamin Timing of stem cell harvest
B12 deficiency anemias, pernicious ane- Erythropoietin abuse in sport contestants
mia, immunologic or drug-induced red
cell aplasia, leukemia or metastatic carci-
noma, renal failure, idiopathic myelofi- elective surgical procedures can facili- and very low immature fractions (< 10%)
brosis, and other disorders. tate preoperative autologous blood col- were characteristic of patients with aplas-
Accurate reticulocyte enumeration lection and minimize the need for tic or megaloblastic anemias, while pa-
is critical for the diagnosis of many allogeneic blood transfusions. Lastly, tients with marrow infiltrative diseases
hematologic diseases and for the classifi- rhEPO and other hematologic growth had nearly normal reticulocyte counts,
cation of patients with anemia. In addi- factors are essential to promote bone but high immature fractions (> 30%). In
tion to its diagnostic value, the marrow regeneration in patients receiv- patients with pancytopenia resulting from
reticulocyte count is also playing an in- ing chemotherapy or bone marrow trans- aplastic anemia, infiltrative marrow dis-
creasingly important role in monitoring plantation. The major clinical order, hypersplenism, or megaloblastic
the progress of patients receiving con- applications of the reticulocyte count anemia, the absolute reticulocyte counts
ventional or experimental therapy for a and reticulocyte indices are listed in T4. were lowest in patients with aplastic ane-
variety of diseases. In this regard, a new mia or megaloblastic anemia and highest
era in clinical hematology began with Anemia in patients with hypersplenism. The mar-
the use of recombinant human erythro- Anemias secondary to bone marrow row reticulocyte counts and shift ratio to
poietin (rhEPO) and other hematologic aplasia, nutritional disorders, and bone circulating blood did not add additional
growth factors to stimulate erythron pro- marrow infiltration are characterized by useful information for the classification
duction. rhEPO, used in conjunction very low reticulocyte counts (< 2% cor- of these anemias. The reticulocyte mean
with oral or parenteral iron administra- rected). In these patients, reticulocyte channel fluorescence has been reported 605
tion, has been recently utilized to stimu- enumeration by manual supravital stain- to show a significant correlation with
late erythropoiesis in anemia subjects, ing confirms the low reticulocyte count, total iron-binding capacity (P < 0.0001, r
including those refractory anemia asso- but does not provide further diagnostic = 0.62) and ferritin (P < 0.0001, r =
ciated with various malignant disorders, information. Lin and collaborators evalu- 0.40).39 The clinical value of the flow
preterm infants, chronic renal failure, ated automated reticulocyte evaluation in cytometrically determined reticulocyte
and other patients. In addition, the use of a large group of these patients.37,38 Very count and RMI was also proven for renal
rhEPO in patients scheduled for major low reticulocyte counts (< 0.03 x 1012/L) transplantation patients and patients with

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anemia from a variety of causes.15,40 For plantation, or malaria infestation. Much of anemia in HIV-infected patients, sickle
example, in children, they found CHr the interest in the clinical utilization of auto- cell anemia, and other diseases. Since
and Hgb levels the only significant pre- mated reticulocyte technology has focused ruEPO-stimulated erythropoiesis depends
dictors of iron deficiency, while the CHr on patients with chemotherapy-induced on an adequate and continuous supply or
was the only significant multivariate pre- marrow aplasia, including patients undergo- iron, close laboratory monitoring of iron
dictor of iron deficiency anemia. A CHr ing bone marrow transplantation. In these status is critical to achieving an optimal
value of 26 pg provided the optimal cut- patients, early prediction of marrow engraft- therapeutic response, and ruEPO treat-
off for iron deficiency based on sensitiv- ment is essential for the efficient utilization ment is often accompanied by
ity and specificity analysis.41 of blood products, as well as identifying intravenous (IV) iron therapy. Of the con-
Hemolytic anemia without reticulocy- patients with delayed or failed marrow re- ventional parameters, the reticulocyte
tosis is characteristic of pernicious anemia generation. During chemotherapy induction, count are the most useful laboratory mon-
(PA), where the reticulocyte count remains the reticulocyte count reaches an extreme itors. More recently, several other param-
relatively low (2-3%) in spite of a florid nadir and consists only of cells with a low eters have been shown to provide a more
hemolytic anemia, short red blood cell life fluorescent ratio (LFR). The reticulocyte accurate measure of the marrow iron sup-
span, and marked marrow erythroblastic fraction with medium fluorescence ratio ply. These parameters are the CHr, per-
hyperplasia. In PA this occurs because the (MFR) begins to rise approximately two centage of hypochromic erythrocytes, and
severe nuclear-cytoplasmic dyssynchrony weeks post-chemo- therapy, followed by red blood cell ferritin (RBCFer).56,57,59-61
results in the loss of RNA prior to nuclear HFR reticulocytes a day or so later and
extrusion.42 This phenomenon also occurs granulocytes several days later.50 Many stud- Other Applications
in patients with folic acid deficiency or ies of patients undergoing chemoradiother- Miscellaneous uses of flow cyto-
iron deficiency anemia with superimposed apy-induced aplasia and autologous or metric reticulocyte enumeration include
hemolytic disease.43 A different situation allogeneic bone marrow transplantation have investigations of the persistence of
occurs in autoimmune hemolytic anemia, identified the RMI and the proportion of reticulocytes in refrigerated blood stor-
where life-threatening aplastic crises may highly fluorescent reticulocytes (HFR%) to age and in vivo post-transfusion, the
occur in spite of a striking erythroblastic be significant predictors of marrow engraft- relationship between CD36 expression
response because of immune destruction ment.16,51-53 In patients mobilized with and stress reticulocytosis in patients
of erythroblasts.44 Aplastic crises with chemotherapy and growth factors for pe- with sickle cell anemia and other
reteiculocytoopenia are also seen in viral ripheral blood stem cell harvest, an increase chronic hemolytic disorders, and the
infections, hereditary spherocytosis, de- in immature reticulocyte fractions was re- presence of reticulocytes reacting with
layed hemolytic transfusion reactions, and ported to precede the presence of circulating the IgG fraction of an antiserum against
chronic hemolytic diseases such as sickle CD34+ cells by about two days, suggesting cord red blood cell (RBC) membranes
cell anemia.45-47 A high IRF with an in- that flow cytometric reticulocyte enumera- (F-IgG reactive RBCs).62-64
creased Absolute reticulocyte count (ARC) tion might be helpful in monitoring the tim- A recent novel application of retic-
was characteristic of an enhanced erythro- ing of stem cell harvesting.54 In patients ulocyte enumeration is to detect
poietic state, such as an acquired with end stage renal disease on hemodialy- erythropoietin abuse in sports.65 Under
hemolytic anemia or acute blood loss, sis, reticulocyte enumeration has been uti- these circumstances, precise, accurate
while an increased IRF with a normal or lized in the diagnosis of iron deficiency determinations of the reticulocyte count
reduced ARC was found in patients with anemia and to follow the efficacy of treat- are needed.
dyserythropoietic or ineffective erythropoi- ment with oral iron supplementation, ery-
etic conditions, such as acute myeloid thropoietin, and other agents.55-58 Conclusions
leukemia, myelodysplastic syndrome, The final stage of red blood cell
aplastic anemia, or megaloblastic anemia. Chronic Renal Failure differentiation occurs in the peripheral
Reticulocyte maturity was normal in pa- The recent introduction of recombi- blood. The immature red blood cells
tients with reduced erythropoiesis, includ- nant human erythropoietin (ruEPO, epo- (reticulocytes) that are released by the
ing chronic renal failure and iron etin beta) revolutionized the treatment of bone marrow still contain RNA and
deficiency anemia.48,49 anemia associated with chronic renal fail- some cellular organelles. Reticulocytes
ure. In addition, ruEPO therapy is under gradually lose their protein synthesizing
606 Bone Marrow Regeneration evaluation for anemia associated with machinery, and normally become ma-
In addition to the diagnosis of hemato- many other diseases associated with rela- ture red blood cells (erythrocytes) after
logical diseases, reticulocyte counting also tive erythropoietin deficiency or bone about three days in the peripheral
provides useful information regarding the marrow suppression, including cancer, blood. Since the number and character-
degree of regenerative activity that takes cancer chemotherapy and bone marrow istics of the reticulocytes in the periph-
place after treatment of iron deficiency or transplantation, myelodysplasia, prematu- eral blood reflect the activity of the
vitamin B12/folate deficiency anemias, a rity, perioperative state and preparation bone marrow, reticulocyte counting has
course of chemotherapy, bone marrow trans- for elective surgery, zidovudine-induced become a fundamental part of the eval-

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