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Protocol - Bilirubin Level

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Protocol Name from Source:

This section will be completed when reviewed by an Expert Review Panel.

Availability:

Publicly available

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 for measuring total bilirubin, the protocol also provides basic guidelines to aid comparability among different studies.

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 [link[www.phenx.org/Portals/0/phenx-content/Supplements/SickleCell/WG1/DocumentsForReview/Documents_for_Additional_Information/2011-12_Laboratory_Procedures_Manual.pdf|here]], and is also available at the NHANES website: [link[www.cdc.gov/nchs/data/nhanes/nhanes_11_12/2011-12_Laboratory_Procedures_Manual.pdf|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: [link[www.cdc.gov/nchs/data/nhanes/nhanes_11_12/2011-12_Laboratory_Procedures_Manual.pdf|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.

There are many methods and recommended blood volumes for neonatal and pediatric bilirubin measurements.

  • Neonatal blood collection - volumes may change depending on the laboratory but volumes as low as 0.1mL in a green or red top tube have been used.
  • Pediatric blood collection - volumes may change depending on the laboratory but volumes as low as 0.5mL in a green or red top tube have been used.

Laboratory Assay for Total Bilirubin

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 concentration of total bilirubin. 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 Total Bilirubin:

Neonatal

Age of Participant (days)

mg/dL

0-1

2.0-6.0

1-2

6.0-10.0

3-5

4.0-8.0

>5

0.2-1.3

Personnel and Training Required

Phlebotomist

Equipment Needs

Laboratory with the ability to perform the bilirubin assay.

Requirements

Requirement CategoryRequired
Average time of greater than 15 minutes in an unaffected individualNo
Major equipmentNo
Specialized requirements for biospecimen collectionNo
Specialized trainingNo

Mode of Administration

Self-administered

Life Stage:

Infant, Toddler, Child, Adolescent, Adult, Senior, All Ages, Pregnancy

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.

The Working Group notes that while total bilirubin levels can be an overall assessment of morbidity from sickle cell disease, these levels are extensively influenced by liver function.

Calculation of indirect bilirubin levels can be used to determine the rate of hemolysis. Indirect bilirubin levels can also be elevated with ineffective erythropoiesis, heart failure, and with genetic polymorphisms common in the African American population. Total and Indirect bilirubin can be misleading as an indicator of hemolysis when direct bilirubin is elevated. In this situation an elevated level of indirect bilirubin would be a complex result of red cell breakdown, abnormal liver function, biliary drainage, and hepatic blood flow.

Total bilirubin levels can be combined with other indirect markers of hemolysis ([mlink[811200|Aspartate Aminotransferase Level]], [mlink[810600|Reticulocyte Count]], [mlink[811000|Lactate Dehydrogenase Level]], and [mlink[810800|Haptoglobin Level]]) to derive a hemolytic component for sickle cell disease patients.

Determining bilirubin levels via a serum sample is preferred. However, the Working Group acknowledges that 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 important partner with you in ensuring that there is compatibility from collection to assays to interpretation and reporting of levels and results.

Research Domain Information

Release Date:

July 30, 2015

Definition

A bioassay to measure levels of total bilirubin, a yellow substance found in bile produced when the liver breaks down old red blood cells.

Purpose

Abnormal bilirubin levels are associated with liver disease, gallstones, pancreatic cancer, anemia, and hemolytic diseases, including sickle cell disease.

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
Common Data Elements (CDE)Laboratory Procedure Total Bilirubin Result Unspecified Value2181337CDE Browser

Process and Review

This section will be completed when reviewed by an Expert Review Panel.

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.

Burtis, C. A., Ashwood, E. R., & Bruns, D. E. (2006). Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th ed. St. Louis, MO: Elsevier Saunders.

General References

Potoka, K. P., and 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.

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:

810901

Variables:

Export Variables
Variable NameVariable IDVariable DescriptionVersiondbGaP Mapping
PX810901_Billirubin_HemophiliaPX810901010000Do you have hemophilia?4N/A
PX810901_Billirubin_Chemotheraphy_4weeksPX810901020000Have you received cancer chemotherapy in the past four weeks or do you anticipate such therapy in the next four weeks?4N/A
PX810901_Billirubin_AgePX810901030000Age4N/A
PX810901_Billirubin_Blood_Draw_ResultsPX810901040000Record the results of the blood draw.4N/A
PX810901_Billirubin_Blood_Draw_Tube_DeviationPX810901050000Record reasons for a tube not being drawn according to the protocol.4N/A
PX810901_Billirubin_Venipuncture_CommentsPX810901060000Record any comments about the venipuncture.4N/A
PX810901_Billirubin_Serum_DeterminationPX810901070100Determine if the serum is hemolyzed, turbid, lipemic, or icteric.4N/A
PX810901_Billirubin_Serum_DescriptionPX810901070200If serum is hemolyzed, turbid, lipemic, or iteric, then describe.4N/A
PX810901_Billirubin_Total_Billirubin_ConcentrationPX810901080000Total billirubin concentration4N/A
PX810901_Billirubin_Equipment_MakePX810901090000Make of the equipment used to perform the total billirubin assay.4N/A
PX810901_Billirubin_Equipment_ManufacturerPX810901100000Manufacturer of the equipment used to perform the total billirubin assay.4N/A
PX810901_Billirubin_Total_Billirubin_Assay_RepeatabilityPX810901110000Repeatability of the total billirubin assay.4N/A
PX810901_Billirubin_CoefficientsVariation_TotalBillirubin_AssayPX810901120000Coefficients of variation of the total billirubin assay.4N/A