CAPILLARYS HEMOGLOBIN(E)

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CAPILLARYS HEMOGLOBIN(E) - 2010/10 All abnormal hemoglobins or abnormal levels of normal hemoglobins detected with the CAPILLARYS HEMOGLOBIN(E) procedure were in agreement with the comparative HPLC system and clinical diagnosis. There was no case observed of false positive, i.e., detection of an abnormal band or abnormal level of a normal band where no such abnormality existed. In USA, the levels of hemoglobin fractions were measured in 59 blood samples, including 19 samples with hemoglobin variants such as hemoglobins S, C and other variants, by electrophoretic separations obtained with the CAPILLARYS HEMOGLOBIN(E) procedure with the CAPILLARYS 2 FLEX PIERCING system and 2 commercially available systems for hemoglobins analysis by capillary electrophoresis (CE) and HPLC. The measured values of hemoglobin fractions from all 3 procedures were analyzed by a linear regression statistical procedure. The results of linear regression analysis for Hb A, Hb A2, Hb F and Hb S are tabulated below (y = CAPILLARYS HEMOGLOBIN(E)) : Correlation coefficient y-Intercept Slope Range of Hb % values Hb fraction Number of samples CAPILLARYS CE HPLC CE HPLC CE HPLC HEMOGLOBIN(E) Hb A 59 0.999 0.996 -0.340 3.145 1.003 0.965 43.7 – 98.7 Hb A2 57 0.990 0.880 -0.087 0.515 1.026 0.773 1.3 – 5.6 Hb F 22 0.999 0.999 0.033 0.304 1.009 1.019 0.0 – 31.8 Hb S 13 0.996 0.995 1.087 -0.546 0.983 1.047 14.3 – 49.0 All abnormal hemoglobins (S, C and other variants) or abnormal levels of normal hemoglobins detected with the CAPILLARYS HEMOGLOBIN(E) procedure were in agreement with the 2 comparative capillary electrophoresis and HPLC systems and clinical diagnosis. There was no case observed of false positive, i.e., detection of an abnormal band or abnormal level of a normal band where no such abnormality existed. - 56 -
CAPILLARYS HEMOGLOBIN(E) - 2010/10 CAPILLARYS CORD BLOOD PROCEDURE WITH THE CAPILLARYS SYSTEM INTENDED USE The CAPILLARYS CORD BLOOD procedure is designed for the separation of the normal hemoglobins (F and A) in umbilical cord blood samples from human new-borns, and for the detection of the major hemoglobin variants (especially S, C, E, D and Bart’s), by electrophoresis in alkaline buffer (pH 9.4) with the CAPILLARYS System. The CAPILLARYS performs all sequences automatically to obtain a complete hemoglobin profile for the qualitative or quantitative analysis of hemoglobins. The assay is performed on sedimented, centrifuged or washed red blood cells; washing red blood cells is not essential to perform the analysis. This procedure must be performed with the components of the CAPILLARYS HEMOGLOBIN(E) kit. The hemoglobins, separated in silica capillaries, are directly and specifically detected at an absorbance wave length of 415 nm which is specific to hemoglobins. The resulting electrophoregrams are evaluated automatically for pattern abnormalities with identification of normal and pathological patterns. Direct detection provides accurate relative quantification of individual hemoglobin fraction. The sickle cell disease diagnosis is improved due to the separation of the different hemoglobin fractions which enables the differentiation of hemoglobin S from other hemoglobin variants. For In Vitro Diagnostic Use. PRINCIPLE OF THE TEST (1-19) Hemoglobin is a complex molecule composed of two pairs of polypeptide chains. Each chain is linked to the heme, a tetrapyrrolic nucleus (porphyrin) which chelates an iron atom. The heme part is common to all hemoglobins and their variants. The type of hemoglobin is determined by the protein part called globin. Polypeptide chains α, ß, δ and γ constitute the normal human hemoglobins : • hemoglobin A............................................... = α2 ß2 • hemoglobin A2............................................. = α2 δ2 • fetal hemoglobin F....................................... = α2 γ2 The α-chain is common to these three hemoglobins. The hemoglobin spatial structure and other molecular properties (like that of all proteins) depend on the nature and the sequence of the amino acids constituting the chains. Substitution of amino acids by mutation is responsible for formation of hemoglobin variants which have different surface charge and consequently different electrophoretic mobilities, which also depend on the pH and ionic strength of the buffer. The resulting qualitative (or structural) abnormalities are called hemoglobinopathies (5, 10, 13) . Decreased synthesis of one of the hemoglobin chains leads to quantitative (or regulation) abnormalities, called thalassemias (5, 16) . Hemoglobin electrophoresis is a well established technique routinely used in clinical laboratories for screening samples for hemoglobin abnormalities (1, 2, 3, 4, 12) . The CAPILLARYS System has been developed to provide complete automation of this testing with fast separation and good resolution. In many respects, the methodology can be considered as an intermediary type of technique between classical zone electrophoresis and liquid chromatography (6, 9) . The CAPILLARYS System uses the principle of capillary electrophoresis in free solution. With this technique, charged molecules are separated by their electrophoretic mobility in an alkaline buffer with a specific pH. Separation also occurs according to the electrolyte pH and electroosmotic flow (5) . The CAPILLARYS System has capillaries functioning in parallel allowing 7 simultaneous analyses for hemoglobin quantification. A sample dilution with hemolyzing 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 hemoglobins is made at 415 nm at the cathodic end of the capillary. Before each run, the capillaries are washed with a Wash Solution and prepared for the next analysis with buffer. The hemoglobins, separated in silica capillaries, are directly and specifically detected at an absorbance wave length of 415 nm which is specific to hemoglobins. The resulting electrophoregrams are evaluated visually for pattern abnormalities. By using alkaline pH buffer, normal and abnormal (or variant) hemoglobins are detected in the following order, from cathode to anode : C, A2/O-Arab, F-Ouled Rabah, E, S, D, G-Philadelphia, Korle-Bu, F, degraded F, A, degraded A and Bart’s. Variants generated by the mutation of the gamma chain may appear in different zones of the electrophoretic pattern. The carbonic anhydrase is not visualized on the hemoglobin electrophoretic patterns with capillary electrophoresis. REAGENTS AND MATERIALS SUPPLIED IN THE CAPILLARYS HEMOGLOBIN(E) KIT See the previous part "CAPILLARYS HEMOGLOBIN(E) PROCEDURE WITH THE CAPILLARYS SYSTEM". REAGENTS REQUIRED BUT NOT SUPPLIED 1. Hb AF CONTROL Composition The Hb AF Control (SEBIA, PN 4777) is obtained from a pool of normal blood samples from human adults and from new-borns umbilical cord blood samples. The Hb AF Control is in a stabilized lyophilized form. Intended use The Hb AF Control is designed for the migration control before starting a new analysis sequence, and for the quality control of the human hemoglobin F detection by electrophoresis with the CAPILLARYS CORD BLOOD procedure. Reconstitute the Hb AF Control vial with the exact volume of distilled or deionized water, as indicated in the package insert of the Hb AF Control. Allow to stand for 30 minutes and mix gently (avoid formation of foam). - 57 -
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CAPILLARYS HEMOGLOBIN(E)

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