Download ES1_Acid Hemoglobin

Acid Hemoglobin for Hb S and Hb C
1.
Purpose
1.1
1.2
Methodology
1.1.1 The HYDRAGEL 7 Acid (E) Hemoglobin kit is designed for separation of the
normal hemoglobin A, abnormal hemoglobins S and C and fetal hemoglobin F,
by electrophoresis on acidic agarose gels. They are used in conjunction with the
semi-automated HYDRASYS system. The resulting electrophoregrams are
evaluated visually for pattern abnormalities. Electrophoresis on HYDRAGEL 7
ACID (E) is essential to confirm the identification of hemoglobin variants
previously detected on alkaline gels, in particular to differentiate hemoglobins S
from D and E from C. This procedure will be run if the patient does not have a
previously confirmed hemoglobin abnormality.
Clinical Implications
Most clinically significant hemoglobinopathies are inherited defects of the 
globin chain of adult hemoglobin. While some  globin is present at birth, red
blood cells of newborns have a predominance of fetal hemoglobin, which does
not contain  globin. For this reason, clinical signs and symptoms of  globin
abnormalities are usually not apparent at birth but become evident later after
adult hemoglobin replaces fetal hemoglobin.
Historical Background
1.2.1
1.3
1.3.1
2.
Specimen
2.1
3.
Individuals with one abnormal  globin gene (heterozygous) are said to have a
hemoglobin trait and are carriers for the disease. Persons with two abnormal 
globin genes, homozygous have disease. Therefore, confirmatory testing is
always necessary to adequately diagnose a hemoglobin disease or trait.
Collection Instructions
2.1.1 Fresh anticoagulated blood samples are recommended for analysis. EDTA,
citrate and heparin are acceptable. (Stored at 2-8C for 5 days).
2.1.2 Let red blood cells precipitate for several hours at 2-8°C or centrifuge the blood
sample at 3,500 rpm for 10minutes. Discard carefully the maximum volume of
plasma and vortex for 5 seconds.
2.1.3 Container volume: One 5 mL EDTA lavender/black tube.
2.1.3.1 Wash the red blood cells 2 times with 10 volumes of saline.
2.1.3.2 Discard any excess of saline over the red cells pellet and vortex them
before taking 10 µL to hemolyze.
2.1.3.3 Hemolyze 10 µL of packed red cells with 130 µL of hemolyzing
solution.
2.1.3.4 Vortex 10 seconds and incubate 5 minutes at room temperature.
2.1.4 Dilute the hemolysate 1:2 with distilled water by adding 50 µL hemolysate + 50
µL of distilled water. The assay is performed on the hemolysate from the washed
red blood cells.
Reagents
3.1
HYDRAGEL 7 ACID (E) Kit
3.1.1 Agarose Gels (ready to use). Each gel contains; agarose, 1.5 g/dL; acidic buffer
pH 6.0 0.1 and serves as the support medium for hemoglobin electrophoresis.
3.1.2 Buffered strips (ready to use). Each contains: acidic buffer pH 6.0  0.2. Buffered
strips function as electrophoresis buffer reservoirs and ensure contact between
the gel and the electrodes.
3.1.3 Staining solution diluent used in the preparation of the Amidoblack stain.
3.1.3.1 Add 15 mL of stain diluent to the concentrated amidoblack vial.
Carefully close the vial and shake vigorously for 5 seconds.
3.1.3.2 Pour this solution in the container for staining solution processing.
Acid Hemoglobin for Hb S and Hb C
3.1.3.3
3.1.4
3.1.5
3.1.6
3.1.7
3.1.8
3.1.9
4.
Materials
4.1
4.2
5.
Consumables:
4.1.1 Micropipettor; either manual or automated
4.1.2 Wet storage chamber supplied with HYDRASYS
4.1.3 Container kit supplied with HYDRASYS
4.1.4 Pipettes: 10 µL and 200 µL
4.1.5 Gel holder for half gels, Sebia, PN 1278.
Equipment
4.2.1 HYDRASYS System Sebia
Quality
5.1
Control
5.1.1
5.1.2
5.1.3
5.1.4
5.1.5
5.1.6
6.
Repeat this process twice, three times if necessary. Then pour the
remaining diluent in the container and complete the volume to 300 mL
with distilled water.
3.1.3.4 Mix contents of stain cubitainer well for 5 to 10 minutes.
3.1.3.5 The staining solution is ready for use. After dilution, the working
staining solution contains: acid solution pH=2; amidoblack, 0.4 g/dL;
ethylene-glycol, 6.7%; additives, nonhazardous at concentrations used,
necessary for optimum performance.
Hemolysing Solution (ready to use). It is a buffer with additives and used to
hemolyze red cells. Store hemolyzing solution in refrigerator. It is stable until the
expiration date indicated on the kit package or hemolyzing vial label.
Applicators are precut and ready for single use for sample application.
Filter papers are precut, single use, thin absorbent paper pads for blotting
excessive moisture off the gel surface prior to sample application. Store the thin
filters in a dry place at refrigerated temperature.
Destaining solution Sebia PN 4540, 10 vials, 100 mL each to be diluted up to 100
liters with distilled water. It is best to dilute 5 mL of the stock solution to 5 liters,
which is the volume of the destaining solution container. This is used to remove
excess and background stain from the gels.
Hydrasys wash solution. Sebia, PN 4541, 10 vials, 80 mL each. To be diluted up
to 5 liters with distilled water. It serves to clean the HYDRASYS staining
compartment.
Saline 0.9 g/dL for washing red cells and for dilution of the hemolysates.
Controls: AFSC Control, Sebia PN 4792.
Preparation and Storage: Reconstitute with 1.0 mL distilled water.
Method and Frequency: The AFSC control should be included in each analysis.
Acceptance Limits:
5.1.4.1 Hemoglobin C migrates behind the application point
5.1.4.2 Hemoglobin S remains at the application point.
5.1.4.3 Hemoglobins D, E and A are superimposed
5.1.4.4 Hemoglobins F and A1 are superimposed.
Corrective actions: Repeat procedure if Quality Control is not acceptable. If
repeat fails; Call Technical Service providing the instruction for the preparation
and storage of materials, and the procedure were carefully followed.
Documentation of control acceptability should be made in the LIS.
Procedure
6.1
Migration Set Up
6.1.1 Switch on HYDRASYS instrument.
6.1.2 Place one applicator on a flat surface with the well numbers in the right-side-up
position.
Acid Hemoglobin for Hb S and Hb C
6.1.3
6.2
Apply 10 µL of hemolyzed sample (from sample prep instructions) into the
appropriate wells being careful to load within 2 minutes.
6.1.4 Place the applicator into the wet storage chamber with the teeth up (handle it by
the plastic tooth protection frame).
6.1.5 Let the samples diffuse into the teeth for 5 minutes after the last sample
application.
6.1.6 Open the lid of the Migration Module and raise the electrode and applicator
carriers.
6.1.7 Select 7 ACID (E) migration program.
6.1.8 Remove buffered strips from the package and place the flat side against the back
with the strip’s plastic backing to the pins on the electrode carrier.
6.1.9 Unpack the HYDRAGEL plate and roll quickly and uniformly one thin filter paper
onto the gel surface to absorb the excess liquid. Remove the paper immediately.
6.1.10 Pool 120 µL of distilled water on the lower third of the plate printed on the
Temperature Control plate of the migration module.
6.1.11 Place the gel plate (with gel side up) against the bottom of the printed frame.
Bend the gel down easing it onto the pooled water avoiding bubbles. Water
should now be under the entire gel.
6.1.12 Lower both carriers down. Do not force them down.
6.1.13 Remove the applicator from the wet carrier handling it by the protection frame.
6.1.14 Snap off the applicator’s teeth protection frame and place the applicator into
position 9 on the carrier. The numbers on the applicator must face the operator.
6.1.15 Close the lid of the migration module.
6.1.16 Start the procedure immediately by pressing the green arrow<Start> key on the
left side of the keyboard.
Gel processing Set-Up
6.2.1 Open the lid.
6.2.2 Remove the applicator and discard.
6.2.3 Raise both carriers; remove the buffered strips by their plastic ends and discard.
6.2.4 Remove the dried film for further processing.
6.2.5 Wipe the electrodes and temperature control plate gently with a soft wet tissue.
6.2.6 Open the Gel holder. Lay it flat and position the dried gel with the gel facing up
into the grooves of the two rods and close the holder.
6.2.7 Place the gel holder into the Gel Processing/Staining Module.
6.2.8 Always check the stain volumes before proceeding.
6.2.8.1 The staining container is filled with 300 mL of staining solution.
6.2.8.2 The destaining container contains at least 1 liter of destaining solution.
6.2.9
6.3
7.
Select ACID (E) Hb staining program from the instrument menu and start the run
by pressing the START key.
Gel Processing Completion
6.3.1 Remove the holder and gel from the compartment.
6.3.2 Interpret gel visually against the AFSC control.
Results
7.1
7.2
7.3
Most hemoglobinopathies are due to substitution by mutation of a single amino acid in
one of the four-polypeptide chains. The clinical significance of such a change depends on
the type of amino acid and the site involved. In clinically significant disease, either the chain or the -chain is affected.
Electrophoresis on HYDRAGEL 7 ACID (E) serves to identify hemoglobin variants
previously detected on alkaline gels primarily hemoglobins S from D and E from C.
The migration order is the following, from the anode to cathode.
7.3.1 Hemoglobin C, migrates behind the application point
7.3.2 Hemoglobin S, remains at the application point
Acid Hemoglobin for Hb S and Hb C
7.3.3
7.3.4
7.3.5
8.
Method Parameters
8.1
8.2
8.3
8.4
8.5
9.
Hemoglobins D, E and A0 are superimposed
Hemoglobins F and A1 are superimposed
A weak band may also be seen slightly anodic to the hemoglobin A1/F band, due
to denaturation during storage.
Reference Range: Negative
Critical Value: Not applicable
Reportable Range: Confirmation of Hemoglobin S or C.
Analytical Sensitivity/Specificity: 100%
Interfering Substances: Do not use hemolyzed samples.
References
9.1
Sebia Hydragel 7 (Hemoglobin E) Ref.4106