Reticulocyte Count
Date of SC final approval
About the Measure
Domain:
Measure:
Definition:
Sickle Cell Disease: Cardiovascular, Pulmonary, and Renal
Reticulocyte Count
A bioassay to measure the number of reticulocytes (immature red blood cells).
Purpose:
Reticulocyte count measures how fast red blood cells are made. Elevated reticulocyte
counts are associated with blood loss, hemolysis and hemolytic anemia, and in
response to the anemia of clinical conditions such as sickle cell disease. Reticulocyte
counts can also be used to measure efficacy of treatments (e.g., iron replacement
therapy).
About the Protocol
Description of
Protocol:
Selection
Rationale:
Specific
Instructions:
This protocol provides instructions for drawing, processing and storing blood
according to the National Health and Nutrition Examination Survey (NHANES)
methods. Because there are many comparable assays and instruments for measuring
reticulocyte count, the protocol also provides basic guidelines to aid comparability
among different studies.
The Sickle Cell Disease Working Group 1 (Cardiovascular, Pulmonary, and Renal)
selected the National Health and Nutrition Examination Survey 2011-2012 protocol as
the best standardized methodology for blood collection, processing and storage.
 The National Health and Nutrition Examination Survey (NHANES) instructions
for drawing, processing, and storing blood provide a standard methodology
used successfully for many years to ensure comparable results across study
sites. However, the Sickle Cell Disease Working Group 1 (Cardiovascular,
Pulmonary, and Renal) notes that certain aspects (e.g., exclusion criteria) of
the NHANES protocol are study specific and might not be applicable to all
types of studies (e.g., sickle cell disease).

Reticulocyte count can be combined with other indirect markers of hemolysis
(haptoglobin level, bilirubin level, red blood cell aspartate aminotransferase
level, and lactate dehydrogenase level) to derive a hemolytic component for
sickle cell disease patients.

Collection of blood samples for the measurement of analytes requires a
general determination of whether to use serum or plasma for the assay and
also a determination of the type of collection tube to be obtained. For example,
if serum is to be used a determination needs to be made as to whether red top
or serum gel separator collection tubes are used. While comparable values are
obtained for many analytes from either serum or plasma, there may be
situations where differences are more pronounced and serum or plasma
specific norms will be needed for references. The NHANES protocol presented
here uses red top/serum separator tubes. At times it may be possible to collect
both, but other considerations such as participant burden may be the deciding
factor. It is important to match assay type with sample type. Some automated
devices may preclude the use of serum, for example, while others may be
optimized for it. Investigators should choose methods of collection that match
the methods of analysis. This will best be done by communicating with the
laboratory where the proposed assays will be performed. They will become an
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Reticulocyte Count
Protocol Text:
Date of SC final approval
important partner with you in ensuring that there is compatibility from collection
to assays to interpretation and reporting of levels and results
The following is a summary version of the full National Health and Nutrition
Examination Survey 2011-2012 protocol.
Exclusion Criteria
Persons will be excluded from this component if they:
• Report that they have hemophilia; or
• Report that they have received cancer chemotherapy in the last 4 weeks
SP = Sample Person.
1. Do you have hemophilia?
1[]
2[]
7[]
9[]
Yes
No
Refused
Don’t Know
If the SP answers "Yes," the SP is excluded from the blood draw.
If SP answers "No" or "Don’t Know," blood is drawn from the SP.
2. Have you received cancer chemotherapy in the past four weeks or do you
anticipate such therapy in the next four weeks?
1 [ ] Yes
2 [ ] No
7 [ ] Refused
9 [ ] Don’t Know
If the SP answers "Yes," the SP is excluded from the blood draw.
If SP answers "No" or "Don’t Know," blood is drawn from the SP.
Venipuncture Procedures
Editor’s Note: Please review chapter 4 of the Laboratory Procedures Manual from the
2011-2012 National Health and Nutrition Examination Survey (NHANES) for a full
description of phlebotomy procedures. This manual is posted here, and is also
available at the NHANES website: 2011-2012 NHANES Laboratory Procedures
Manual
Venipuncture should generally be performed using the median cubital, cephalic, or
basilic veins in the left arm unless this arm is unsuitable. If the veins in the left arm are
unsuitable, look for suitable veins on the right arm. If the veins in the antecubital
space on both arms are not suitable, then look for veins in the forearm or dorsal side
of the hand on the left arm/hand and then the right arm/hand.
Recording the Results of the Venipuncture Procedure
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Reticulocyte Count
Date of SC final approval
Immediately after completing the venipuncture, record the results of the blood draw,
the reasons for a tube not being drawn according to the protocol, and any comments
about the venipuncture.
Blood Processing
Please review chapter 8 of the Laboratory Procedures Manual from the National
Health and Nutrition Examination Survey 2011-2012 for a full description of blood
processing procedures: 2011-2012 NHANES Laboratory Procedures Manual
•
•
•
Allow the blood to clot by setting aside for 30 to 45 minutes at room
temperature. Do not clot for more than an hour.
Centrifuge the tube at room temperature to separate the serum and aliquot
into an appropriate storage tube.
Determine if the serum is hemolyzed, turbid, lipemic, or icteric. If so, enter a
comment to describe the serum.
Laboratory Assay for Reticulocyte Count
The Sickle Cell Disease Working Group 1 (Cardiovascular, Pulmonary, and Renal)
notes that there are a number of different assays and instruments that are appropriate
to measure the reticulocyte count from serum. Once an assay is chosen for a
particular study, the Working Group recommends that no changes in the protocol be
made over the course of the study. To aid comparability, the Working Group
recommends that the investigator record the make and manufacturer of equipment
used and the repeatability and coefficients of variation for the assay.
Reference Ranges for Reticulocyte Count:
Children: 3 - 6%
Adults: 0.5 - 1.5%
Percentage of reticulocytes and absolute reticulocyte counts (ARC) are given by the
following formulas:
Reticulocyte Percentage = [Number of Reticulocytes / Number of total Red Blood
Cells] X 100
Participant:
Source:
Language of
Source:
Absolute reticulocyte count (thousands/µL) = reticulocyte % x RBC count (millions/µL)
x 10
Individuals age 1 year and older.
Centers for Disease Control and Prevention (CDC). (2011). National Health and
Nutrition Examination Survey Questionnaire, Laboratory Procedures Manual.
Hyattsville, MD: U.S. Department of Health and Human Services, Centers for Disease
Control and Prevention.
English
Personnel and
Training Required:
Equipment Needs:
Phlebotomist
Protocol Type:
Bioassay
Laboratory with the ability to perform the reticulocyte count assay.
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Reticulocyte Count
Date of SC final approval
Requirements:
Requirements Category
Common Data
Elements:
General
References:
Required (Yes/No):
Major equipment
No
Specialized training
No
Specialized requirements for biospecimen
collection
Average time of greater than 15 minutes in an
unaffected individual
TBD by PhenX Team
No
No
Potoka, K. P., & Gladwin, M. T. (2015). Vasculopathy and pulmonary hypertension in
sickle cell disease. American Journal of Physiology, Lung Cellular and Molecular
Physiology, 308, L314-L324/
Nouraie, M., Lee, J. S., Zhang, Y., Kanias, T., Zhao, X., Xiong, Z., Oriss, T. B., Zeng,
Q., Kato, G. J., Gibbs, J. S., Hildesheim, M. E., Sachdev, V., Barst, R. J., Machado, R.
F., Hassell, K. L., Little, J. A., Schraufnagel, D. E., Krishnamurti, L., Novelli, E., Girgis,
R. E., Morris, C.R., Rosenzweig, E. B., Badesch, D. B., Lanzkron, S., Castro, O. L.,
Goldsmith, J. C., Gordeuk, V. R., Galdwin, M. T., & Walk-PHASST Investigators and
Patients. (2013). The relationship between the severity of hemolysis, clinical
manifestations and risk of death in 415 patients with sickle cell anemia in the US and
Europe. Haematologica, 98(3), 464-472. doi: 10.3324/haematol.2012.068965
Sachdev, V., Kato, G. J., Gibbs, J. S., Barst, R.J., Machado, R.F., Nouraie, M.,
Hassell, K. L., Little, J. A., Schraufnagel, D. E., Krishnamurti, L., Novelli, E. M., Girgis,
R. E., Morris, C. R., Rosenzweig, E. B., Badesch, D. B., Lanzkron, S., Castro, O. L.,
Taylor, J. G. 6th, Hannoush, H., Goldsmith, J. C., Gladwin, M. T., Gordeuk, V. R., &
Walk-PHASST Investigators. (2011). Echocardiographic markers of elevated
pulmonary pressure and left ventricular diastolic dysfunction are associated with
exercise intolerance in adults and adolescents with homozygous sickle cell anemia in
the United States and United Kingdom. Circulation, 124(13), 1452-1460.
Additional Information About the Measure
Essential Data:
Current age, Race, Ethnicity
Related PhenX
Measures:
Liver Function Assay, Cell Free Hemoglobin, Red Blood Cell Microparticles,
Haptoglobin, Lactate dehydrogenase, Red Blood Cell Aspartate Aminotransferase,
Bilirubin
Hemolytic Component:
Derived Variables:
Reticulocyte count can be combined with other indirect markers of hemolysis
(haptoglobin levels, bilirubin levels, red blood cell aspartate aminotransferase levels,
and lactate dehydrogenase levels) by principle component analysis (PCA) to derive
a hemolytic component for sickle cell disease patients.
Version 10 – 10/21/09
Reticulocyte Count
Date of SC final approval
Nouraie, M., Lee, J.S., Zhang, Y., Kanias, T., Zhao, X., Xiong, Z., Oriss, T.B., Zeng,
Q., Kato, G. J., Gibbs, J. S., Hildesheim, M. E., Sachdev, V., Barst, R. J., Machado,
R. F., Hassell, K. L., Little, J. A., Schraufnagel, D. E., Krishnamurti, L., Novelli, E.,
Girgis, R. E., Morris, C.R., Rosenzweig, E. B., Badesch, D. B., Lanzkron, S., Castro,
O. L., Goldsmith, J. C., Gordeuk, V. R., Galdwin, M. T., & Walk-PHASST
Investigators and Patients. (2013). The relationship between the severity of
hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell
anemia in the US and Europe. Haematologica, 98(3), 464-472.
Keywords/Related Reticulocyte count, hemolysis, hemoglobin, sickle cell disease, SCD, anemia,
Concepts:
hypoxemia, hemolytic anemia, pulmonary hypertension, chronic kidney disease,
CKD, vasculopathy, stroke, rare genetic conditions, hemolytic component
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