Methods of Reticulocyte Quantification & Reporting
Melinda S. Camus, DVM, DACVP
University of Georgia College of Veterinary Medicine
mscamus@uga.edu
Lecture Outline:
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What are reticulocytes?
Why are reticulocytes important?
How are reticulocytes measured?
How are reticulocytes reported?
Terminology:
“After the nucleus has been extruded, the cell (erythrocyte) is known as a reticulocyte” 1
“The stage of maturation of erythroid cells between the metarubricytes and mature RBCs” 2
“Polychromatophilic erythrocytes are reticulocytes that stain bluish red due to the combined
presence of hemoglobin (red staining) and individual ribosomes and polyribosomes (blue
staining)”3
Reticulocyte Characteristics:
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Up to 20% larger by volume than mature erythrocytes
Contain numerous organelles: ribosomes, mitochondria, Golgi4
Supravital stainsprecipitation of ribosomal RNA into “reticulum”
o Appearance affected by numerous factors, including:
 Dye concentration
 Drying method
 Stain pH
 Age of sample
Should “contain more than 2 dots of ribosomal material”2
In non-mammals, aggregated RNA should encircle the nucleus5,6
Importance of Reticulocytes:
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Primary means of assessing bone marrow regeneration
Help classify anemias as “regenerative” or “nonregenerative”
Must be interpreted in light of clinical presentation
Quantification Methods:
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Manual
Automated
Immunostaining
Manual Methods
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Standard Method
o Count reticulocytes on two blood smears
RBCs counted
Reticulocytes seen
Smear 1
Smear 2
Total
500
4
500
8
1000
12
o Calculate the reticulocyte percentage
% Retics = # retics counted X 100
#RBC counted
% Retics = 12 X 100
1000
% Retics = 12
10
% Retics = 1.2
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Miller’s Ocular
Smear 1
Smear 2
RBCs counted (B)
126
144
Retics counted (A&B) 16
28
Calculate the totals:
Retics counted: 16 + 28 = 44
Total RBCs counted: 126 + 144 = 270
Calculate the percentage of reticulocytes:
% Retics = # retics counted X 100
Total RBCs X 9
% Retics = 44
X 100
270 X 9
% Retics = 44 _ X 100
2430
% Retics = 1.8
*Examples adapted from Basic Clinical Laboratory Technique7
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Problems with Manual Methods
o Time
o Decreased precision and accuracy v. automated methods
o CV 8-23%
o Error can be minimized using an “area reduction device”—WHO8
Automated Methods
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Numerous analyzers: Sysmex®, ADVIA®, IDEXX LaserCyte®, Oxford Science ForCyte®2,9
Use flow cytometry
Most correlate well with manual counts in dogs and cats
Analyzer specific dyes, stain requirements, and available parameters (rMCV, rHC, rCH)
Problems with automated methods
o Expensive
o Specialized equipment
o Require increased sample volume v. manual
Immunostaining10
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Utilizes anti-CD71 (transferrin receptor) antibody
Can use with flow cytometry or confocal microscopy
Problems with immunostaining methods
o Expensive equipment
o Potentially undercounts “mature” reticulocytes
o Species specificity
Reporting Reticulocytes
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% is counted with all methods
Multiply by RBC count to get absolute
Corrected Reticulocyte Percentage (CRP)4
o Designed to “correct” for red cell mass
o Retic % X (patient HCT/“normal HCT”)
Reticulocyte Production Index (RPI)4
o Designed to “correct” for overzealous marrow response with severe anemia
o CRP (or absolute #)/maturation factor
o Based on premise that circulating maturation time increases 0.5 days per 10%
HCT drop
o Example:
Dog has HCT of 25% and CRP of 3%
RPI = CRP/maturation factor
Determination of maturation factor: At 35% a retic takes 1 day to mature
and takes 0.5 extra days for each 10% drop below 35%, therefore: at
25%, maturation time should be 1.5 days
RPI = 3/1.5
RPI = 1.5%
References
1. Dessypris EN. Erythropoiesis. In: Lee GR et al. Wintrobe’s Clinical Hematology, 10th ed.
Baltimore, MD: Williams & Wilkins. 1999: 169-192.
2. Tvedten H and Mortiz A. Reticulocyte and Heinz body staining and enumeration. In:
Weiss DJ & Wardrop KJ, eds. Schalm’s Veterinary Hematolog. 6th ed. Ames, IA: WileyBlackwell. 2010: 1067-1073.
3. Harvey, JW. Veterinary Hematology: A Diagnostic Guide and Color Atlas. St. Louis, MO:
Elsiever Saunders. 2012: 21-24.
4. Christian, JA. Erythrokinetics and erythrocyte destruction. In: Weiss DJ & Wardrop KJ,
eds. Schalm’s Veterinary Hematology. 6th ed. Ames, IA: Wiley Blackwell. 2010: 136143.
5. Weiser G. Laboratory technology for veterinary medicine. In: Thrall MA, et al.
Veterinary Hematology and Clinical Chemistry. 2nd ed. Ames, IA: Wiley-Blackwell ; 2012:
16-18.
6. Mitchell EB and Johns J. Avian hematology and related disorders. Vet Clin Exot Anim.
2008. 11: 501-522.
7. Estridge BH and Reynolds AP. Basic Clinical Laboratory Technique. 6th ed. Clifton Park,
NY: Delmar Cengage Learning. 2012: 341-354.
8. England, JM et al. ICSH guidelines for reticulocyte counting by microscopy on
supravitally stained preparations. World Health Organization. Prepared September 4,
1992.
9. Moritz A and Becker M. Automated hematology systems. In: Weiss DJ & Wardrop KJ,
eds. Schalm’s Veterinary Hematology. 6th ed. Ames, IA: Wiley-Blackwell. 2010:
1054-1066.
10. Kono M, et al. Morphological definition of CD71 positive reticulocytes by various
staining techniques and electron microscopy compared to reticulocytes detected by an
automated hematology analyzer. Clinica Chemica Acta. 2009. 404: 105-110.