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Protocol - Hemoglobin Characterization

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Description

This protocol provides basic instructions for drawing and storing blood and performing the bioassay for hemoglobin characterization. Because there are many assays that may be required to characterize hemoglobin abnormalities, the protocol also provides basic guidelines to increase comparability among different studies.

Specific Instructions

The Sickle Cell Disease Research and Scientific Panel (SRSP) recommends that investigators also capture Complete Blood Count and Reticulocyte Count.

The SRSP recommends that results from Hemoglobin Characterization be interpreted in the context of age-appropriate normal values and history of blood transfusions within the last 120 days.

The results of Hemoglobin Characterization can be interpreted using the algorithms in the Association of Public Health Laboratories and Center for Disease Control and Prevention document: " Hemoglobinopathies: Current Practices for Screening, Confirmation and Follow-up". Characterization is based on the results of hemoglobin electrophoresis, isoelectric focusing, high performance liquid chromatography, capillary zone electrophoresis, immunoassays, and DNA genotype. More definitive diagnosis may require complete blood count (CBC) with mean corpuscular volume (MCV), reticulocyte count, and family studies.

Published guidelines on hemoglobinopathy interpretation all focus on newborn screening results, however, the Sickle Cell Disease Research and Scientific Panel (SRSP) suggests that they can be used in adult populations by modifying the algorithms by removing fetal hemoglobin if it is not the dominant hemoglobin (e.g., "FS" becomes "S", "FSC" becomes "SC", "FSA" becomes "SA", "FAS" becomes "AS" etc.).

Protocol

Hemoglobin Characterization

Blood Draw

Blood should be drawn into an appropriate EDTA tube.

Laboratory Assay for Hemoglobin Characterization

The Sickle Cell Disease Scientific and Research Panel (SRSP) notes that there are numerous different assays and instruments (e.g., hemoglobin electrophoresis, isoelectric focusing, high performance liquid chromatography, capillary zone electrophoresis, DNA sequencing analysis) that are appropriate to characterize hemoglobin variants. Once an assay is chosen for a particular study, the SRSP recommends that the same protocol be used over the course of the study. To aid comparability, the SRSP recommends that the investigator record the make and manufacturer of equipment used and the repeatability and coefficients of variation for the assay.

The SRSP notes that investigators should record the assay results for the following hemoglobins (the exact list of hemoglobins that can be distinguished will depend on the assay being used). The hemoglobins are listed in order of the amount present with the most prevalent coming first.

  • A
  • F
  • S
  • C
  • A2
  • E
  • D
  • G
  • O
Availability

Available

Personnel and Training Required

Phlebotomist

Equipment Needs

CLIA certified laboratory with the capability to perform the hemoglobin 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

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

Participants

All ages

Selection Rationale

The Association of Public Health Laboratories and Center for Disease Control and Prevention document: "Hemoglobinopathies: Current Practices for Screening, Confirmation and Follow-up" was compared to other protocols and selected by the Sickle Cell Disease Research and Scientific Panel (SRSP) because it discusses advantages and disadvantages of the various techniques as well as the ability of the assays to separate the various hemoglobin variants.

Language

English

Standards
StandardNameIDSource
Derived Variables

None

Process and Review

The PhenX Sickle Cell Disease Research and Scientific Panel (SRSP) reviewed this protocol in May 2022. Guidance from the SRSP includes:

  • Updated protocol

Protocol Name from Source

Association of Public Health Laboratories (APHL)

Source

Association of Public Health Laboratories (APHL). Hemoglobinopathies: Current Practices for Screening, Confirmation and Follow-up. https://www.aphl.org/aboutAPHL/publications/Documents/NBS_HemoglobinopathyTesting_122015.pdf. Accessed April 26, 2022

General References

CLSI. Newborn Screening for Hemoglobinopathies. 1st ed. CLSI guideline NBS08. Wayne, PA: Clinical and Laboratory Standards Institute; 2019.

The Laboratory Diagnosis of Haemoglobinopathies. (1998). British Journal of Haematology, 101(4), 783-792. doi: 10.1046/j.1365-2141.1998.00809.x

Kutlar, F. (2007). Diagnostic approach to hemoglobinopathies. Hemoglobin, 31(2), 243-250.

Kutlar, A., & Huisman, T. (1996). Detection of hemoglobinopathies. In F. Hommes (Ed.), Techniques in Diagnostic Human Biochemical Genetics: A Laboratory Manual (pp. 519-560). New York, New York: J. Wiley & Sons.

Protocol ID

830302

Variables
Export Variables
Variable Name Variable IDVariable DescriptiondbGaP Mapping
PX830302_Hemoglobin_Characterization_Assay_Equipment_Coefficients
PX830302040000 What are the coefficients of variation for more
the assay? show less
N/A
PX830302_Hemoglobin_Characterization_Assay_Equipment_Make
PX830302030000 What is the make of the equipment used? N/A
PX830302_Hemoglobin_Characterization_Assay_Equipment_Manufacturer
PX830302020000 Who is the manufacturer of the equipment used? N/A
PX830302_Hemoglobin_Characterization_Assay_Equipment_Repeatability
PX830302050000 Describe the repeatability of the assay: N/A
PX830302_Hemoglobin_Characterization_Assay_Results
PX830302060000 Were the assay results recorded for the more
following hemoglobins? A, F, S, C, A2, E, D, G, O show less
N/A
PX830302_Hemoglobin_Characterization_Assay_Results_List
PX830302070000 What were the assay results for the more
following hemoglobins? A, F, S, C, A2, E, D, G, O show less
N/A
PX830302_Hemoglobin_Characterization_Blood_Draw
PX830302010000 Was the blood drawn into an appropriate EDTA tube? N/A
Sickle Cell Disease Core: Tier 1
Measure Name

Hemoglobin Characterization

Release Date

October 30, 2023

Definition

A bioassay for hemoglobin classification.

Purpose

This protocol can be used to identify and characterize the different variants in structure and synthesis of hemoglobin that cause sickle cell disease.

Keywords

hemoglobin characterization, sickle cell disease, SCD, SC disease, anemia, thalassemia, hemoglobin, hemoglobinopathy, high-performance liquid chromatography, HPLC, isoelectric focusing, IEF, immunoassay

Measure Protocols
Protocol ID Protocol Name
830302 Hemoglobin Characterization
Publications

There are no publications listed for this protocol.