The use of capillary electrophoresis for hemoglobinopathies diagnosis:

preliminary analytical performances of CAPILLARYS 3 TERA, SEBIA

Florence HOLOGNE, Armelle GARCIA, Denis SIMONIN and Frederic ROBERT
SEBIA, R&D department, Lisses, France
BACKGROUND
Hemoglobinopathies are the most common monogenic diseases in human. Approximately 7% of world population is carrier and 2.7 conceptions per 1000 are affected by these pathologies. Hemoglobinopathies are qualitative (Hemoglobin
variants) and/or quantitative disorders (thalassemias) of the hemoglobin. They can lead to several clinical symptoms, from benign (mild microcytosis) to the most severe (sickle cell disease, hydrops fetalis) with multiple end-organ damages.
According to different national and international guidelines, diagnosis of hemoglobinopathies is mainly based on Complete Blood Count and on the separation and quantification of the different hemoglobin fractions. These last points can be
achieved by different separative techniques such as HPLC, IEF or more recently, Capillary Electrophoresis. Here we describe the analytical performances of the new hemoglobin diagnosis kit CAPI 3 HEMOGLOBIN(E) on CAPILLARYS 3 TERA, the
high-throughput and high-volume capillary electrophoresis instrument from SEBIA.

MATERIALS & METHODS

Hemoglobin testing by capillary electrophoresis:
The CAPILLARYS 3 TERA instrument uses the principle of capillary electrophoresis (electrokinetic separation technique carried out in a tube of internal diameter lower than 100 μm filled with a buffer composed of electrolytes) in free solution.
With this technique, charged molecules are separated by their electrophoretic mobility in an alkaline buffer (pH 9.4). Separation also occurs according to the electrolyte pH and electroosmotic flow.
The CAPILLARYS 3 TERA instrument has silica capillaries functioning in parallel allowing 12 simultaneous analyses for hemoglobin quantification in a whole blood sample. Minimum sample volume required is 100 µL. A sample dilution with
hemolysing solution is prepared and injected by aspiration at the anodic end of the capillary. A high voltage protein separation is then performed and direct detection of the hemoglobin fractions is made at the cathodic end of the capillary at
415 nm (hemoglobin absorbance wavelength). Direct detection provides accurate relative quantification of individual hemoglobin fraction.

Resolution:
Resolution (Hb variants detection capability) of the CAPI 3 HEMOGLOBIN(E) method was assessed by analyzing samples with Hb variants (Hb S, F, C, D, E, other Hb variants), all at heterozygous state.

Precision study:
The precision of the method was evaluated in a study based on the CLSI EP05-A3 guideline. This encompassed reproducibility within the same capillary from the same instrument, reproducibility between capillaries from the same instrument
and reproducibility between lots and between instruments, obtained by analyzing 12 blood samples on CAPILLARYS 3 TERA and the CAPI 3 HEMOGLOBIN(E) procedure. The coefficients of variation (CV %) were calculated for percentage (%) of
hemoglobin fractions for each sample.

Limit of detection:
Limit of detection of the CAPI 3 HEMOGLOBIN(E) procedure was evaluated in a study based on CLSI EP17-A2 guideline. For each hemoglobin fraction, the limit of detection was determined using 5 different blood samples.

Linearity studies:
Linearity studies for Hb A, A2, F, S, C, D and E were evaluated according to the CLSI EP06-A guideline. Two blood samples (one with low level of the fraction of interest and the other one with high level of the same fraction) were mixed within
different proportions and analyzed on CAPILLARYS 3 TERA and the CAPI 3 HEMOGLOBIN(E) procedure.
The CAPILLARYS 3 TERA, a fully
Correlation study: automated capillary electrophoresis
Correlation study was carried out according the CLSI EP09-A3 protocol, with 153 blood samples, including 64 samples with Hb variants. Samples were analyzed on both CAPILLARYS 3 TERA with CAPI 3 HEMOGLOBIN(E) procedure and on instrument performing Hemoglobin testing
CAPILLARYS 2 Flex Piercing with HEMOGLOBIN(E) procedure (reference method). The measured values of Hb fractions were analyzed by a linear regression statistical procedure. Sensibility and specificity of the CAPI 3 HEMOGLOBIN(E) procedure
compared to the reference procedure have been calculated.

RESULTS
The CAPILLARYS 3 TERA is able to pick up all major common Hb variants (Hb S, F, C, D, E, G-Philadelphia, Constant Spring, etc) with no interference with physiological Hb fractions such as Hb A, Hb A2 or Hb F (Figure 1). Adducts of the hemoglobin
are not separated by design, leading to clear patterns. Profiles are divided into 15 zones, indicating the potential positions of the different hemoglobin fractions. A drop-down library of variants found in each zone (348 in total) is available. This
feature facilitates the interpretation of the profiles, when used in conjunction with the Sebia Hemoglobins Atlas. Hemoglobin fractions are automatically quantified.

Hb A/S Hb A/E Hb A/Lepore

Overall imprecision is good for all Hb fractions considered, with CV lower than 6%. Results of reproducibility studies are summarized in the Table 1.

Reproducibility within the same capillary reproducibility between capillaries reproducibility between lots Figure 1: Hemoglobin profiles obtained
Fractions with the HEMOGLOBIN(E) method
from the same instrument (CV %) from the same instrument (CV %) and between instruments (CV %)
Hb A 0.0 – 1.4 % 0.0 – 1.2 % 0.0 – 0.9 % on CAPILLARYS 3 TERA
Hb A2 0.0 – 5.1 % 0.6 – 5.1 % 1.1 – 5.0 %
Hb F 0.0 – 2.4 % 0.3 – 3.1 % 0.4 – 2.3 %
Hb S 0.3 – 1.4 % 0.5 – 1.0 % 0.8%
Hb C 0.0 – 2.4 % 0.8 – 2.2 % 1.1 – 1.2 % Table 1: Summary of the reproducibility studies
Hb D 0.2 – 0.8 % 0.3 – 0.6 % 0.4 %
for the different Hb fractions considered.
Hb E 0.4 – 3.1 % 0.5 – 1.8 % 1.0 %

The limit of detection measured for the Hb A fraction, Hb A2, Hb F, Hb S, Hb C, Hb D and Hb E were 0.9%, 0.2%, 0.5%, 0.8%, 0.3%, 0.7% and 0.2% respectively.
The CAPILLARYS 3 TERA and the CAPI 3 HEMOGLOBIN(E) method appeared to be linear within the entire range studied for Hb A fraction (between 0.9 and 97.3 % Hb A), Hb A2 (0.2 - 9.1%), Hb F (0.5 - 83.1%), Hb S (0.8 - 89.7%), Hb C (0.3 - 82.0%),
Hb D (0.7 - 43.5%) and Hb E (0.2 - 86.9%).

The correlation between CAPILLARYS 3 TERA and CAPILLARYS 2 Flex Piercing is excellent (r > 0.990), showing perfect agreement in measuring Hb A, Hb A2, Hb F and major common Hb variants (Hb S, C, D and E) (Figure 2).

Hb A correlation Hb A2 correlation Hb S correlation

Figure 2: Correlation studies
of the different hemoglobin fractions
between the CAPILLARYS 3 TERA
and the CAPILLARYS 2 Flex Piercing
(reference method).
Correlation studies for Hb F, Hb C, Hb D
and Hb E not shown (r > 0.990)

For the detection of hemoglobin variants (Hb S, C, D and E), the CAPI 3 HEMOGLOBIN(E) procedure showed 100.0 % sensibility and a 100.0 % specificity compared to the reference procedure.
In addition, no carry-over for Hb S has been detected. No influence of high levels of triglycerides and bilirubin was noticed.

CONCLUSION
CAPILLARYS 3 TERA from SEBIA is a multiparametric capillary electrophoresis instrument which can analyze serum proteins and perform their immunotyping. It is also able to manage whole blood on
capped tube at a high throughput (up to 70 samples/hour), allowing the measurement of Hb A1c. In addition, the CAPILLARYS 3 TERA and HEMOGLOBIN(E) method allows an accurate separation and
quantification of the hemoglobin fractions. Its good overall analytical performances make it a solution of choice for any type of laboratories that would like to perform hemoglobinopathies diagnosis at high
degree of confidence.

Parc Technologique Léonard de Vinci

CP 8010 Lisses – 91008 EVRY Cedex France