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Protocol - Reticulocyte Count

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Description

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.

Specific Instructions

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 Cardiovascular, Pulmonary, and Renal Working Group 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). Investigators who want to include participants that have hemophilia or have received cancer chemotherapy in the last 4 weeks will need to implement special venipuncture procedures.

Reticulocyte count can be combined with other indirect markers of hemolysis (Aspartate Aminotransferase Level, Haptoglobin Level, Lactate Dehydrogenase Level, and Bilirubin Level) to derive a hemolytic component for sickle cell disease patients.

Reticulocyte count analysis is performed on anticoagulated blood, collected in an EDTA tube.

The Sickle Cell Disease Curative Therapy Working Group recommends that reticulocyte count be determined in sickle cell patients undergoing hematopoietic cell transplant at one time point pre-transplant (baseline) and 100 days, six months, and annually post-transplant.

Availability

Available

Protocol

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 [ ] 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.

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

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

Fill a 3 or 4 ml K3 EDTA tube with blood.

Laboratory Assay for Reticulocyte Count

The Sickle Cell Disease Cardiovascular, Pulmonary, and Renal Working Group 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 (thousands/µL) x RBC count (millions/µL)] X 100

Absolute reticulocyte count (thousands/µL) = reticulocyte % x RBC count (millions/µL) x 10

Personnel and Training Required

Phlebotomist

Equipment Needs

Laboratory with the ability to perform the reticulocyte count assay.

Requirements
Requirement CategoryRequired
Major equipment No
Specialized training No
Specialized requirements for biospecimen collection No
Average time of greater than 15 minutes in an unaffected individual No
Mode of Administration

Bioassay

Lifestage

Toddler, Child, Adolescent, Adult, Senior, Pregnancy

Participants

Individuals age 1 year and older.

Selection Rationale

The Sickle Cell Disease Cardiovascular, Pulmonary, and Renal Working Group selected the National Health and Nutrition Examination Survey 2011-2012 protocol as the best standardized methodology for blood collection, processing and storage.

Language

English

Standards
StandardNameIDSource
Human Phenotype Ontology Reticulocytosis HP:0001923 HPO
Human Phenotype Ontology Sickle Cell Anemia ORPHA:232 HPO
Human Phenotype Ontology Anemia OMIM:603903 HPO
caDSR Form PhenX PX810601 - Reticulocyte Count 6252732 caDSR Form
Derived Variables

Hemolytic Component:

Reticulocyte count can be combined with other indirect markers of hemolysis (Haptoglobin Level, Bilirubin Level, Aspartate Aminotransferase Level, and Lactate Dehydrogenase Level) by principle component analysis (PCA) to derive a hemolytic component for sickle cell disease patients.

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.

Process and Review

Not applicable.

Protocol Name from Source

National Health and Nutrition Examination Survey Questionnaire (NHANES), Laboratory Procedures Manual, 2011

Source

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.

General References

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

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

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.

Protocol ID

810601

Variables
Export Variables
Variable Name Variable IDVariable DescriptiondbGaP Mapping
PX810601_Reticulocyte_Age
PX810601030000 Age Variable Mapping
PX810601_Reticulocyte_Amount
PX810601070000 Number of reticulocytes N/A
PX810601_Reticulocyte_Assay_Repeatability
PX810601100000 Repeatability of the reticulocyte count assay. N/A
PX810601_Reticulocyte_Blood_Draw_Results
PX810601040000 Record the results of the blood draw. N/A
PX810601_Reticulocyte_Blood_Draw_Tube_Deviation
PX810601050000 Record reasons for a tube not being drawn more
according to the protocol. show less
N/A
PX810601_Reticulocyte_Chemotheraphy_4weeks
PX810601020000 Have you received cancer chemotherapy in the more
past four weeks or do you anticipate such therapy in the next four weeks? show less
Variable Mapping
PX810601_Reticulocyte_Coefficients_Variation_Assay
PX810601110000 Coefficients of variation of the more
reticulocyte count assay. show less
N/A
PX810601_Reticulocyte_Equipment_Make
PX810601080000 Make of the equipment used to perform the more
reticulocyte count assay. show less
N/A
PX810601_Reticulocyte_Equipment_Manufacturer
PX810601090000 Manufacturer of the equipment used to more
perform the reticulocyte count assay. show less
N/A
PX810601_Reticulocyte_Hemophilia
PX810601010000 Do you have hemophilia? Variable Mapping
PX810601_Reticulocyte_Venipuncture_Comments
PX810601060000 Record any comments about the venipuncture. N/A
SCD Cardiovascular, Pulmonary, and Renal
Measure Name

Reticulocyte Count

Release Date

July 30, 2015

Definition

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).

Keywords

Reticulocyte count, hemolysis, hemoglobin, sickle cell disease, SCD, anemia, hypoxemia, hemolytic anemia, pulmonary hypertension, pH, chronic kidney disease, CKD, vasculopathy, stroke, rare genetic conditions, hemolytic component, "Cardiovascular, Pulmonary, and Renal"

Measure Protocols
Protocol ID Protocol Name
810601 Reticulocyte Count
Publications

There are no publications listed for this protocol.