General Chemistry
Technical Information
Immunodiagnostics
Centrifugation
Disease Management
Molecular Diagnostics
Hematology
The LH Reticulocyte Count
and Associated Parameters
Hemostasis
Lab Automation
Information Systems
Flow Cytometry
Primary Care
The Emergence of the
Automated Reticulocyte
The reticulocyte count has historically been used as a tool for providing information
regarding red cell production, or erythropoiesis. Typically, the most common clinical
indicator for performing a reticulocyte count is to diagnose or monitor the treatment
of anemia. The emergence of flow cytometric methods of reticulocyte counting has
resulted in improvements in accuracy and precision, as well as provide non-traditional
parameters associated with reticulocyte maturation and size. Independent clinical studies have begun to establish clinical utility for these non-traditional parameters as
potentially useful tools in the diagnosis and treatment of many anemic states.
The traditional method of manual reticulocyte counting can be limited in its usefulness
as a monitoring test because of inherent imprecision,2,6 especially in the low and high
ends of the counting range. Blood smear variability, staining differences, the limited
number of cells evaluated, and inter-technologist bias have all been cited as contributors
to coefficients of variation ranging from 25% to over 50%.1,7,8,9,10
In recent years, automated reticulocyte analysis has been incorporated into many of
the medium and high volume routine hematology analyzers. What originally began as
an outside the instrument pre-preparation method has now developed into second
generation hematology instrumentation that aspirate, prepare, and analyze reticulocyte
information automatically. This total automation of the reticulocyte analysis process
has removed the variables associated with manual blood smears, staining, and intertechnologist bias, resulting in more standardization within the laboratory. In addition,
the 30,000+ cells analyzed with the typical automated reticulocyte method, as opposed
to the 1000 cells analyzed manually, lends the automated method to improved precision and reproducibility.6,13
Beckman Coulter
Reticulocyte Technology
Bulletin 9231
The Reagent System
The Beckman Coulter procedure of reticulocyte analysis uses new methylene blue
stain, a non-fluorochrome dye, to precipitate the residual RNA within the reticulocytes. The function of the reticulocyte stain is to identify and delineate the reticulocytes from mature red cells.
Blood is mixed with the new methylene blue stain and allowed to incubate for a
short period of time. An acidic, hypo-osmotic, ghosting solution is then introduced,
clearing the hemoglobin while preserving the stained RNA within the reticulocytes.
The function of the ghosting solution is two-fold – to permit hemoglobin to leak from
the red cells and to swell the red cells to a spherical shape without bursting them.
The irregular shapes of mature red cells and reticulocytes produce unpredictable light
scatter information when subjected to a laser light beam at angles from 0° to 90°. The
sphering of the red cells provides reproducible light scatter information that forms the
basis for determining the reticulocytes in the sample. Therefore, the ghosting solution
also has a fixative property so that the cells maintain the resulting spherical shape
caused by the swelling. The removal of the hemoglobin from the sphered red cell is
essential as the decrease in the hemoglobin content enhances the definition of the
reticulum and the consequent determination of the reticulocytes.
VCS Technology
Once the cells have been stained and cleared, the VCS Technology method of
reticulocyte analysis is employed, utilizing a unique flow cytometric means of cell
interrogation to count and classify reticulocytes. Cells are identified and classified by
simultaneous three-dimensional analysis using Volume, Conductivity, and Light
Scatter.
Volume, as measured by direct current, is used to identify the size of the cell.
Reticulocyte analysis is highly dependent on size information, as immature red cells,
by definition, are larger than mature red cells. Volume is displayed on the y-axis of
the DataPlot.
Conductivity, or radio frequency measurements, provide information about the
internal characteristics of the cell. Conductivity is displayed on the z-axis of the
DataPlot, but is only seen in the rotating three-dimensional mode.
Light scatter measurements, obtained as cells pass through the helium-neon laser
beam, provide information about cell surface characteristics and cell granularity.
Reticulocyte analysis is also highly dependent on light scatter information as the red
cells containing residual RNA scatter more light than mature red cells. Light scatter
is displayed on the x-axis of the DataPlot.
Volume
Conductivity
Light Scatter
DataPlot Development and Contour Analysis
The raw data events collected from volume, conductivity and light scatter are plotted
three-dimensionally on a DataPlot.
Using the valleys that separate the populations, the platelets and white cells are segregated, leaving only mature red cells and reticulocytes for further analysis.
White Cells
Reticulocytes
Mature Red
Contouring
Contour analysis involves a series of steps designed to separate the reticulocyte from
the mature red cells in a non-linear mode that follows the contour of the populations,
as opposed to a straight line.
Reticulocyte Positional Parameters
The reticulocyte positional parameters are displayed at the Workstation as
troubleshooting tools and to aid in DataPlot interpretation.
Based on 256 channels of histogram data for each measurement of volume,
conductivity, and light scatter, the mean channel of each measurement for each
population is established. The mean channels determine the actual position of the
subpopulations on the DataPlot. Additionally, the standard deviation is calculated
for each population to determine the degree of variability for each measurement.
Both the mean channel and the standard deviation can be affected by instrument
optimization, gain adjustments, reagents, or may be related to a specific patient
sample.
Additional Reticulocyte Information
In addition to the positional parameters, the Workstation displays a series of
temperature, count, and time parameters associated with the reticulocyte count.
The displayed temperature refers to the ambient temperature inside the instrument, at
the flow cell. Extremes in ambient temperature due to fluctuations in the laboratory
environment trigger changes in the incubation time of the reagents. The displayed
setting indicates the temperature level currently set by the service representative. The
settings can be low, normal, or high. The typical recommendation is normal.
The total counts refers to the total
number of events that pass through
the flow cell. The maximum total
count is 32,767. The analyzed count
refers to the number of mature red cell
events, reticulocyte events, and white
cell events. The displayed count refers
to all mature red cell and reticulocyte
events. The actual time is the length
of time required by the instrument to
count 32,767 events. Whereas the low
and high time are calculated by the instrument as minimum and maximum limits.
Typical Reticulocyte DataPlot
The figure at the left illustrates a typical reticulocyte DataPlot. The positional,
temperature, count, and time parameters for this DataPlot are displayed above.
Reticulocyte Flagging
Using complex algorithms, the positional
parameters are used in combination with
other population statistics to ensure integrity
of results and to trigger reticulocyte flagging.
Certain clinical conditions can generate
DataPlots that are inconsistent with known
mature red cell and reticulocyte patterns.
These atypical patterns will then generate
either a Verify Retic or Abnormal Retic
Pattern message to alert the laboratory that
further review is necessary. All reticulocyte
flags, codes, and messages are listed and
defined at the end of the monograph.
Examples of the Abnormal Retic Pattern Message
During the process of contour gating, a subpopulation of high light scatter events
is observed with a volume mean more consistent with mature red cells than with
reticulocytes. This pattern is consistent with the high light scatter properties of
incompletely lysed (ghosted) red cells commonly seen with thalassemia. This
pattern may be subtle or pronounced based on the heterogeneity of the disease
and the condition of the patient.
Sickle cell samples containing multiple shapes and sizes of red cells will present
with an increased volume mean and increased volume standard deviation for both
reticulocytes and mature red cells. Again, the pattern may be subtle or pronounced
based on the heterogeneity of the disease and the condition of the patient.
During the process of DataPlot development, a subpopulation of high volume
events is observed with light scatter mean more consistent with white cells than
with reticulocytes. This population has been found to be a low volume continuum
of the white cell population, perhaps due to fragile or deteriorating white cells.
Reticulocyte Associated
Parameters
Automation of reticulocyte analysis and the use of flow cytometric light scatter measurements have enabled Beckman Coulter to provide more detail about the reticulocyte
population. The reticulocyte population is further divided into ten regions of maturity.
Those reticulocytes with the most RNA are found in the highest light scatter regions,
three through ten.
In addition to the Reticulocyte Percent (RET%) and Reticulocyte Absolute Number
(RET#), the Immature Reticulocyte Fraction (IRF), Mean Reticulocyte Volume
(MRV), Mean Sphered Cell Volume (MSCV), and High Light Scatter Reticulocyte
parameters (HLR% and #) are products of VCS Technology reticulocytes.
The following table defines the reticulocyte parameters available on the COULTER®
LH 700 Series Hematology Systems. It should be noted that reference ranges may
differ significantly between the various technologies used for reticulocyte analysis.
Reference Range Reference Range
Parameter
Derivation
Unit
Retic %
All Retic Events
All Red Cell Events
Retic #
Beckman Coulter
Fourcade29
%
0.45 – 2.28
0.5 – 2.0
Retic % x CBC Red Count
100
106 cells/µL
0.02 – 0.11
0.02 – 0.10
Retic Events (3-10)
All Retic Events
Ratio
0.163 – 0.362
0.20 – 0.40
Average of All Retic Events
FL
102.73 – 124.89
100 – 125
@ MSCV
Average of all Red Cell Events
FL
84 – 104
@ HLR %
Retic Events (3-10)
All Red Cell Events
%
0.07 – 0.71
@ HLR #
HLR % x CBC Red Count
100
106 cells/µL
0.003 – 0.050
IRF
MRV
@ Research Use Only
While all manufacturers have a common goal of defining reticulocytes and their subpopulations of immature reticulocytes, methodologies and algorithms vary. It is always
advisable to establish individual laboratory reference ranges.
Immature Reticulocyte
Fraction (IRF)
The Immature Reticulocyte Fraction (IRF) is defined as the ratio of young or
immature reticulocytes to the total number of reticulocytes. Immature reticulocytes
are larger and are classified by light scatter methods as those reticulocytes having
the greatest light scatter properties, therefore, the highest level of RNA.
Clinical Utility of IRF
Immature reticulocytes, also referred to as shift or stress cells, normally constitute less
than five percent (5%) of the total number of reticulocytes. Immature reticulocytes are
released into the peripheral blood during periods of intense erythropoietic stimulation,
such as hemorrhage, certain anemias, or in response to therapy designed to stimulate
bone marrow production.2 The IRF may be called the red cell equivalent of the “left
shift” typically associated with neutrophilic white cells, providing additional red cell
information that may shorten the time from diagnosis to therapy or shorten therapy
itself.
The literature suggests that the IRF, in combination with the reticulocyte count,
might be useful in improving the classification of anemias, monitoring bone marrow
recovery, and monitoring anemia therapies. 3,4,5,10,11,12,18-22
Further and more specific clinical uses of the IRF from these references
include: 3,4,5,10,11,12,18-22
•
•
•
•
•
•
•
•
Monitoring stem cell regeneration after bone marrow transplant
Monitoring bone marrow regeneration after intensive chemotherapy
Monitoring iron, vitamin B12, or folate therapy
Monitoring affects of toxic drugs on the bone marrow
Monitoring kidney transplant engraftment via erythropoiesis
Classifying and evaluating anemias
Monitoring neonatal transfusion requirements
Detection of aplastic crisis
Improving the Classification, Diagnosis, and Treatment of Anemias
Davis, et al 2,12 suggests that an improvement in the classification of anemias can
be accomplished when the IRF is used in combination with the reticulocyte count.
The following table provides a summary of the interaction between the IRF and the
reticulocyte count in different clinical conditions.
Clinical Condition
IRF
Reticulocyte Count
Bone Marrow Engraftment
Elevated
Decreased
Hemolytic Anemia
Elevated
Elevated
Iron Deficiency Anemia
Elevated
Normal to Decreased
B12 or Folate Deficiency
Elevated
Normal to Decreased
Recent Hemorrhage
Elevated
Normal to Elevated
Thalassemia
Normal to Elevated
Elevated
Myelodysplastic Syndromes
Normal to Elevated
Normal to Decreased
Hypoplastic Anamia
Normal to Elevated
Decreased
Aplastic Crisis
Normal to Decreased Decreased
Myeloid-Lymphoid Malignancies
Decreased
Normal
Aplastic Anemia
Decreased
Decreased
Monitoring Bone Marrow Recovery Following Bone Marrow Transplant
Bone marrow transplants are widely performed and generally accepted for some
hematologic disorders and solid tumor therapy. Prior to the marrow transplant, the
patient receives high doses of chemotherapy and radiation in an effort to irradiate the
bone marrow. This process leaves the patient with little or no red cell, white cell, or
platelet production, requiring extensive monitoring to determine the amount and
duration of administration of prophylactic antibiotics and blood products.15
After the transplant, the CBC, differential and reticulocyte count are used to monitor
the patient for early signs of bone marrow recovery. The absolute neutrophil count is
generally accepted as a primary indicator of returning bone marrow production of
myeloid cells and a successful bone marrow engraftment. A post transplantation
increase in neutrophils and immature myeloid cells to 0.5 x109/L or greater typically
defines successful myeloid engraftment.5,15,16
Platelet counts, although useful indicators of megakaryocyte production, are less
frequently monitored. Since patients require frequent platelet transfusions, an
increase in the platelet count may be due to the transfusions and may not reflect
actual engraftment.3,5
An increase in the reticulocyte percent of greater than 1% or an increase in the
absolute reticulocyte count of 50 x 109/L or greater is used as an indicator of
erythroid engraftment.3,15 Although increased reticulocyte counts represent marrow
regeneration, they are less sensitive and later indicators than the absolute neutrophil
count. Davis performed a study of twelve bone marrow transplant patients that
showed a one to seven week lag in reticulocyte count response when compared to
the response of absolute neutrophil counts.15
Davis and others have also evaluated the IRF in cases of bone marrow transplants.3,17
In contrast to the reticulocyte count, they found that the IRF response closely tracks,
and often precedes the absolute neutrophil count response to returning bone marrow
activity. A substantial benefit of the IRF is that it is less likely to be affected by events
that add variability to the absolute neutrophil counts, such as clinical or subclinical
infections. An increase of the IRF of more than 20% from the post marrow transplant
suggests successful erythroid engraftment.
The IRF has been shown by Davis and others to indicate successful engraftment in
half the time from transplantation when compared to the reticulocyte count. Davis
has concluded that the IRF is the most sensitive indicator of successful bone marrow
engraftment since a rise in the IRF is the earliest response detectable by currently
available laboratory methods.14
Monitoring Erythropoiesis Following Kidney Transplant
Erythropoietin (EPO) is a hormone that regulates red cell production in the bone
marrow. During erythropoietic stress generated by an anemic state, erythropoietin
levels rise, red cell production increases, and reticulocytes are released from the bone
marrow into circulation.23
Since the kidneys are the primary source of EPO, chronic renal failure results in a
chronic anemia associated with the lack of erythropoietin production and release.
While traditional therapy may involve blood transfusions to maintain hemoglobin
levels, kidney transplant is the most effective physiological treatment of anemia
caused by chronic renal failure.
Moulin et al.27 followed 74 renal transplant patients using serial measurements of
serum erythropoietin, hematocrit, reticulocyte values, and serum creatinine. After
transplant, the IRF increased by 25% over the pre-transplant values and at an average
of seven days before an increase in the absolute reticulocyte count. Moulin concluded
that the IRF was a more sensitive, early indicator of erythropoiesis after renal transplant than the absolute reticulocyte count.
Monitoring EPO Therapy in Patients with Chronic Renal Failure
As all patients with chronic renal failure are not candidates for kidney transplant,
recombinant human erythropoietin (r-HuEPO), an alternative to blood transfusions,
has become available that reduces the risks associated with continuous transfusions.24
R-HuEPO is an effective treatment in patients with anemia associated with chronic
renal failure, compensating for the kidney’s lack of erythropoietin production and
maintaining hemoglobin levels. R-HuEPO has also proved to be effective in treating
anemia caused by an insufficient response to EPO that may be associated with
acquired immune deficiency syndrome, cancer, prematurity, or in patients that deny
blood transfusions.24
As the costs related to treating chronic renal failure are rising, there is a significant
potential for an increase in cost savings by monitoring the effectiveness of
r-HuEPO.25,26 The IRF is likely to be an important and sensitive parameter for
evaluating a patient’s response to EPO therapy.12,19
Mean Reticulocyte
Volume and Mean
Sphered Cell Volume
After staining with new methylene blue, the red cells are treated with an acidic,
hypo-osmotic, ghosting solution that spheres the cells and clears the hemoglobin. The
resulting sphered cells are then classified as either mature red cells or reticulocytes
using VCS Technology. The average volume of all events classified as reticulocytes is
referred to as the Mean Reticulocyte Volume (MRV). The average volume of all red
cell events - both mature red cells and reticulocytes, is referred to as the Mean Sphered
Cell Volume (MSCV).
Clinical Utility of MSCV
As part of an instrument evaluation, Chiron, et al.28 assessed the correlation between
the Mean Corpuscular Volume (MCV) from the CBC and the MSCV of 286 samples.
As expected with the biconcave structure of the red cell, the overall correlation indicated a positive bias of the MSCV. There was, however, an unexpected subpopulation
of 48 samples with the MSCV less than the MCV. This subpopulation contained the
entire group of 31 samples from patients with hereditary spherocytosis (HS).
HS is a relatively common disorder related to red cell structural protein defects. The
actual red cells of HS exhibit a decreased surface to volume ratio in the absence of the
biconcave structure that leads to an increased osmotic fragility. Based on testing,
Chiron discusses the possibility that the red cells of HS reach a critical level of osmotic volume expansion and then fragment, resulting in a lower than expected MSCV.
When using a simple cutoff of MSCV < MCV, Chiron obtained a sensitivity of 100%
and specificity of 93.3% for HS. The conclusion to the evaluation suggests that the
relationship of the reticulocyte MSCV to the CBC-MCV may be a highly indicative
test of hereditary spherocytosis.
Conclusion
Recent advances in technology have provided clinicians with more precise reticulocyte
counts and new reticulocyte parameters that may prove to be of great value. One of
the most promising new parameters is the immature reticulocyte fraction which has
practical implications in the practice of medicine.25 Additionally, the relationship
between the MSCV and the MCV has generated much interest as an indicator of
hereditary spherocytosis.
Possible clinical relevance makes it reasonable to assume that research involving reticulocyte associated parameters will increase. Beckman Coulter currently provides a
number of reticulocyte associated parameters and will continue to support research
using these parameters for the future.
Reticulocyte Flags, Codes, and Messages
Definitive Messages
Reticulocytosis %
Laboratory-defined as RET % > high action limit
Reticylocytosis #
Laboratoroy-defined as RET # > high action limit
Suspect Messages
Verify Retic
• Triggered by a number of internal algorithm checks.
Abnormal Retic Pattern
The Abnormal Retic Pattern message is triggered by any one
of the following Research Suspect Messages:
• RET Interference
• Sickle
• Thalassemia
• Low Volume WBC
Research Suspect Messages
RET Interference
• Both the mature red cell population and the reticulocyte
population exhibit high light scatter and high volume
means.
Sickle
• Both the mature red cell population and the reticulocyte
population exhibit high volume means and standard
deviations.
Thalassemia
• A populatlion of events with high light scatter and low
volume characterics.
DDD
• Data Discontinuity Detector: Indicator that flow rate
abnormalities due to flow cell blockage were detected during analysis.
Low Vollume WBC
• A populaltion of high volume events with light scatter
consistent with white cells.
Instrument Flags and Codes
.....
(incomplete computation)
The Incomplete Computation (.....) code is triggered by any
one of the following conditions:
• If RBC incomplete (.....), then RET# and HLR# will
display (.....)
• If RBC voteout (-----_, then RET# and HLR# will display
(.....)
• If RET# >999.9 x 106 cells/_L, then all reticulocyte
parameters except RET# will display (.....)
• If RET% >100% then alal reticulocyte parameters except
RET% will display (.....)
• If RET% =0.00, then IRF will display (.....)
+
(exceeds reportable range)
The Exceeds Reportable Range code (+) is triggered by one
of the following conditions:
• If with RET%, then the RET% > 100%
• If with RET#, then the RET# > 0.7500 x 106 cells/_L
Reticulocyte Flags, Codes, and Messages (continued)
Instrument Flags and Codes (continued)
+++++
triggered by
(exceeds printable range)
The Exceeds Printable Range code (+++++) is
one of the following conditions:
• If with RET%, then the RET% > 100%
• If with RET#, then the RET# > 999.9 x 106 cells/_L
R
(review results)
The R flag is triggered by any one of the following
conditions:
•
•
•
•
•
References
WBC exceeds linearity
RBC exceeds linearity
Platelet exceeds linearity
RET% > 30%
Verify Retic message
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