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Protocol - Glycosylated Hemoglobin - Assay

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Description:

This protocol provides instructions for drawing, processing, and storing blood for the hemoglobin A1c assay according to the National Health and Nutrition Examination Survey (NHANES) methods. Toolkit users are referred to the National Glycohemoglobin Standardization Program (NGSP) for standardized laboratory assays.

Protocol:

The following is a summary version of the full National Health and Nutrition Examination Survey 2007-2008 protocol.

The full National Health and Nutrition Examination Survey 2007-2008 Glycohemoglobin (HbA1C) test is part of the Laboratory Procedures Manual which can be found here: 2007-2008 NHANES Lab Manual.

The National Health and Nutrition Examination Survey protocol lists the following specifications for the Glycohemoglobin (HbA1C) test on page 8-3 of the Laboratory Manual:

Sample (mL): 0.4

Sample type: Whole Blood

Collection type: 4 mL EDTA

Vessel type: 2 mL

Remarks: Refrigerate

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 the SP answers "No" or "Don’t Know," blood is drawn from the SP.

2. Have you received cancer chemotherapy in the past 4 weeks or do you anticipate such therapy in the next 4 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 the SP answers "No" or "Don’t Know," blood is drawn from the SP.

Venipuncture

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.

Editor’s Note: Please review chapter 4 of the Laboratory Procedures Manual from the National Health and Nutrition Examination Survey for a full description of Phlebotomy procedures. 2007-2008 NHANES Lab Manual.

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.

Process the Sample for the Glycosylated Hemoglobin Assay

Note: If the EDTA tube is clotted, do not process the HbA1C vessel.

• Invert the appropriate EDTA tube several times to remix contents thoroughly.

• Use trace-metal-free pipette tips and the 50-1,000 μL Eppendorf pipette to aliquot 0.4 mL whole blood for vessel 4 (Glycohem) on primary SPs aged 12+ into a 2 mL vessel.

• Securely close all vessels to prevent leakage and evaporation.

• Store specimen in a refrigerator at 4°C until the sample is tested.

Laboratory Assay for Glycosylated Hemoglobin

There are now many laboratory assays that have been certified by the National Glycohemoglobin Standardization Program (NGSP). The Diabetes Working Group (WG) recommends that Toolkit users review the National Glycohemoglobin Standardization Program (NGSP) website to select an appropriate assay that is standardized to the Diabetes Control and Complications Trial (DCCT). Once an assay is chosen for a particular study, the WG recommends that no changes in the protocol be made over the course of the study.

Reference Ranges

Glycosylated hemoglobin ranges between 4.4% and 6.0% normally

Protocol Name from Source:

2007-2008 National Health and Nutrition Examination Survey (NHANES) Oral Glucose Tolerance Test Manual

Availability:

Publicly available

Personnel and Training Required

Phlebotomist and laboratory that can perform the glycosylated hemoglobin test. Staff trained to use point-of-care devices

Equipment Needs

Phlebotomy supplies; point-of-care devices

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

Life Stage:

Child, Adolescent, Adult, Senior

Participants:

Participant aged 12 years and older

(The lower age limit in National Health and Nutrition Examination Survey [NHANES] was determined based on logistical considerations. The Expert Review Panel said that the assay may be used for ages less than 12 years.)

Specific Instructions:

Note from the Diabetes Working Group (WG): If more bioassays are being performed, the Diabetes WG recommends using a 10 mL EDTA tube.

Note from the Diabetes WG: The Diabetes WG recommends that the investigator record whether the blood was drawn and whether the full amount was obtained.

Note from the Diabetes WG: The sample should be stored at 4°C until testing and shipped with refrigerated packs.

Note from the Diabetes WG: The investigator should record the reason a sample person is excluded from the blood draw.

To aid comparability, the Diabetes WG recommends that the investigator record the make and manufacturer of equipment used and the repeatability and coefficients of variation for the assay.

Note from the Expert Review Panel (ERP): Plasma or Serum

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 plasma is to be used, a determination needs to be made as to whether EDTA- or heparin-containing 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 protocol presented here uses EDTA plasma. 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 EDTA plasma, 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 assuring that there is compatibility from collection to assays to interpretation and reporting of levels and results.

Note from the ERP: Hemoglobinopathies (e.g., sickle cell anemia) and other disorders which affect the half-life of red blood cells can alter the level of hemoglobin A1c, thus reducing its utility in these settings.

Note from the ERP: Hemoglobin A1C Point of Care

Numerous point-of-care devices are now available for determining hemoglobin A1c in the clinic at the time of examination, and some have been certified by the NGSP (formerly the National Glycohemoglobin Standardization Program). The immediate feedback offers numerous advantages, although it may come at a cost of precision. Using such devices may facilitate studies. If a point-of-care device is used, it is recommended that it be a device certified by the NGSP, which is supported by the National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK) (www.ngsp.org), and that all manufacturer recommendations be followed. Point-of-care devices, however, would generally not serve as a substitute for a centralized laboratory in the conduct of multi-centered studies.

Selection Rationale

The National Health and Nutrition Examination Survey (NHANES) 2007-2008 protocol was selected as the best practice standardized methodology for blood collection, processing, and storage.

Language

English, Spanish

Standards
StandardNameIDSource
Common Data Elements (CDE) Hematology Glycosylated Hemoglobin Measurement Lab Percentage Value 3049798 CDE Browser
Logical Observation Identifiers Names and Codes (LOINC) Glycosylated hemoglobin assay 62854-5 LOINC
Derived Variables

The threshold Glycosylated hemoglobin values for the scoring (+/-) of prediabetes and diabetes are updated yearly by the American Diabetes Association and can be found in the journal Diabetes Care. Prediabetes: Glycosylated hemoglobin between 5.7% and 6.4% Diabetes: Glycosylated hemoglobin greater than or equal to 6.5% American Diabetes Association. (2014). Diagnosis and classification of diabetes mellitus. Diabetes Care, 37(Suppl. 1), S81-S90.

Process and Review

The Expert Review Panel #1 reviewed the measures in the Anthropometrics, Diabetes, Physical Activity and Physical Fitness, and Nutrition and Dietary Supplements domains.

Guidance from the ERP includes:

• Changed name of measure

• Added recommendations on use of serum or plasma

Back-compatible: no changes to Data Dictionary

Previous version in Toolkit archive (link)

Source

Centers for Disease Control and Prevention (CDC), National Center for Health Statistics (NCHS). (2007). 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

American Diabetes Association (ADA). (2014a). Diagnosis and classification of diabetes mellitus. Diabetes Care, 37(Suppl. 1), S81-S90.

American Diabetes Association (ADA). (2014b). Standards of medical care in diabetes-2014. Diabetes Care, 37(Suppl. 1), S14-S80.

Behan, K. J., & Mershen, J. (2011). HbA1c does not always estimate average glucose. Clinical Laboratory Science, 24(2), 71-77.

Miles, R. R., Roberts, R. F., Putnam, A. R., & Roberts, W. L. (2004). Comparison of serum and heparinized plasma samples for measurement of chemistry analytes [Letter to the Editor]. Clinical Chemistry, 50(9), 1704-1705.

Protocol ID:

140901

Variables:
Export Variables
Variable NameVariable IDVariable DescriptionVersiondbGaP Mapping
PX140901_Assay_Repeatability PX140901080000 Repeatability of the assay 4 N/A
PX140901_Assay_Type PX140901060000 Record the type of assay used to perform the determination of glycosylated hemoglobin: 4 N/A
PX140901_Blood_Draw_Comments PX140901040200 Record any comments about the blood draw, including any reasons for the tube not being drawn according to the protocol. 4 Variable Mapping
PX140901_Blood_Draw_Done PX140901040000 Was blood drawn? 4 Variable Mapping
PX140901_Blood_Draw_Sample PX140901040100 Was full sample obtained? 4 Variable Mapping
PX140901_Chemotherapy PX140901020000 Have you received cancer chemotherapy in the past four weeks or do you anticipate such therapy in the next four weeks? (exclusion from Phlebotomy) 4 N/A
PX140901_Coefficient_Of_Variation PX140901090000 Coefficient of variation for the assay 4 N/A
PX140901_Equipment_Make_Manufacturer PX140901070000 Make and manufacturer of the equipment used to perform the determination of glycosylated hemoglobin: 4 N/A
PX140901_Exclusion_Criteria PX140901030000 Exclusion Criteria 4 N/A
PX140901_Hemophilia PX140901010000 Do you have hemophilia? (exclusion from Phlebotomy) 4 Variable Mapping
PX140901_Percent_Glycosylated_Hemoglobin PX140901100000 Percent Glycosylated Hemoglobin 4 N/A
PX140901_Sample_Comments PX140901050000 Record any comments about the blood during processing, such as whether or not the sample was clotted. 4 Variable Mapping
Research Domain Information
Measure Name:

Glycosylated Hemoglobin - Assay

Release Date:

October 1, 2015

Definition

A bioassay to measure percent glycosylated hemoglobin (Glycohemoglobin, Hemoglobin A1C, A1C), or the amount of glucose attached to the hemoglobin in red blood cells (ADA, 2014a).

Purpose

Glycosylated hemoglobin is a well-recognized assay of blood glucose concentration over the preceding several months, and results are predictive of diabetes complications and outcomes. The glycosylated hemoglobin assay can also be used as a diagnostic criterion for diabetes (ADA, 2014a).

Keywords

Diabetes, hyperglycemia, glycohemoglobin, glycosylated hemoglobin, hemoglobin, A1C, metabolic syndrome, NHANES