CAPILLARYS HEMOGLOBIN(E)

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CAPILLARYS HEMOGLOBIN(E) - 2010/10 Patterns are automatically adjusted with regard to Hb F fraction to facilitate their interpretation: - when Hb F fraction is not detected on an electrophoretic pattern, a yellow warning signal appears, the adjustment is then performed using the position of the Hb F fraction on the two previous patterns obtained with the same capillary ; - when the adjustment is not possible, a red warning signal appears, Hb A, Hb F and Hb A2 fractions are then not identified (Call SEBIA). In those two cases, the different variant zones (C1 to C12) do not appear neither on the screen of the system, nor on the ticket result. PLEASE CAREFULLY READ THE CAPILLARYS INSTRUCTION MANUAL. III. END OF ANALYSIS SEQUENCE At the end of each analysis sequence, the operator must initiate the "stand by" or "shut down" procedure of the CAPILLARYS system in order to store capillaries in optimal conditions. IV. FILLING OF REAGENT CONTAINERS The CAPILLARYS system has a reagent automatic control. IMPORTANT: Please refer to the instructions for replacement of reagent containers respecting color code for vials and connectors. A message will be displayed when it is necessary to perform one of the following tasks: • Place a new buffer container and / or; • Fill the container with working wash solution and / or; • Fill the container with filtered distilled or deionized water for rinsing capillaries and / or; • Empty the waste container. WARNING: Do not use marketed deionized water, such as water for ironing for example (risk of important capillaries damage). Use only water with ultrapure quality, such as injection grade water. IMPORTANT: Before filling the rinse container, it is recommended to wash it with plenty of distilled or deionized water. PLEASE CAREFULLY READ THE CAPILLARYS INSTRUCTION MANUAL. QUALITY CONTROL After having changed the analysis buffer vial (even if the lot number is unchanged) or the technique, after a capillary cleaning sequence with CAPICLEAN or after capillaries activation and before starting a new analysis sequence, it is necessary to run two analysis sequences with the Hb AF Control, SEBIA, PN 4777, and the sample rack No. 0 intended for control blood sample (see paragraph REAGENTS REQUIRED BUT NOT SUPPLIED). It is also advised to include into each run of samples, an assayed control blood (for example, a blood sample containing hemoglobins A, F, C and S, such as Hb AFSC Control, SEBIA, PN 4792, or the Hb AF Control, SEBIA, PN 4777). * US customers: Follow federal, state and local guidelines for quality control. RESULTS Values Direct detection at 415 nm in capillaries yields relative concentrations (percentages) of individual hemoglobin zones. Interpretation See ELECTROPHORETIC PATTERNS, figures 1 - 8. The different migration zones of hemoglobin variants (called C1 to C12) are shown on the screen of the system and on the result ticket. Passing the mouse cursor over a zone name displays icon information containing possible hemoglobin variants that could be seen in this zone. For each fraction, the maximum position defines the migration zone. See the table showing the potential variants located in each zone. WARNING: The graduation of the horizontal axis does not allow, in any case, the identification of an hemoglobin variant. 1. Qualitative abnormalities: Hemoglobinopathies Most hemoglobinopathies are due to substitution by mutation of a single amino acid in one of the four types of polypeptide chains (1, 2, 4, 9, 12) . The clinical significance of such a change depends on the type of amino acid and the site involved (13) . In clinically significant disease, either the α-chain or the ß-chain is affected. More than 900 variants of adult hemoglobin have been described (6, 14) . The first abnormal hemoglobins studied and the most frequent have an altered net electric charge, leading to an easy detection by electrophoresis. There are five main abnormal hemoglobins which present a particular clinical interest: S, C, E, O-Arab and D. The CAPILLARYS CORD BLOOD procedure is intended for the identification of hemoglobinopathies and alpha-thalassemias. Hemoglobin S Hemoglobin S is the most frequent. It is due to the substitution of one glutamic acid (an acidic amino acid No. 6) of the ß-chain by a valine (a neutral amino acid): when compared to Hb A, its isoelectric point is elevated and its total negative charge decreased with the analysis pH. Its electrophoretic mobility is therefore increased in the capillary and this hemoglobin is faster than A and F fractions. With the alkaline buffered CAPILLARYS CORD BLOOD procedure, hemoglobin S migrates before A and F fractions. - 62 -
CAPILLARYS HEMOGLOBIN(E) - 2010/10 Hemoglobin C One glutamic acid of the ß-chain is substituted by a lysine (a basic amino acid No. 6): its mobility is strongly reduced. When compared to Hb A, its isoelectric point is highly elevated and its total negative charge decreased with the analysis pH. Its electrophoretic mobility is therefore increased in the capillary and this hemoglobin is faster than A, F and S fractions. Hemoglobins C, E and O-Arab are not superimposed on the electrophoretic pattern and can easily be identified. Hemoglobin E One glutamic acid of the ß-chain (No. 26) is substituted by a lysine. With the CAPILLARYS CORD BLOOD procedure, hemoglobin E migrates anodically behind hemoglobin C and is totally separated from it. Then, it is possible to diagnose a heterozygous C / E patient. Hemoglobin O-Arab One glutamic acid of the ß-chain (No. 121) is substituted by a lysine. With the CAPILLARYS CORD BLOOD procedure, hemoglobin O-Arab migrates separately between E and C fractions. When the blood sample contains Hb A2, Hb O-Arab migrates exactly like this hemoglobin. The analysis being performed on new-borns blood samples, with very low Hb A2 concentration, in such a case, Hb A2 does not interfere on Hb O-Arab detection. Hemoglobin D (-Los Angeles) One glutamic acid of the ß-chain (No. 121) is substituted by a glutamine. With the CAPILLARYS CORD BLOOD procedure, hemoglobin D (called D-Punjab, D-Los Angeles, D-Chicago or D-Portugal) migrates behind hemoglobin S, this property allows for the differentiation of S and D hemoglobins. 2. Quantitative abnormalities: Thalassemias Thalassemias constitute a quite heterogeneous group of genetic disorders characterized by decreased synthesis of one type of the polypeptide chains. The molecular mechanism of this decrease has not been fully described. There are two types of thalassemia syndromes: Alpha-thalassemias They are characterized by the decrease of synthesis of the α-chains, consequently affecting the synthesis of all normal hemoglobins. The excess of synthesis of the ß- and γ-chains in relation to α-chains induces the formation of tetramers without any α-chain: • hemoglobin Bart’s = γ4, • hemoglobin H = ß4. Hemoglobin Bart’s, which presents a low isoelectric point, is in blood samples from new-borns but also, less frequently, in blood samples from adults. Hemoglobin H is not detected in blood sample from new-borns. With the CAPILLARYS CORD BLOOD procedure, hemoglobin Bart’s migrates more anodic than hemoglobin F (and may appear as a wide fraction not focused). Beta-thalassemias They are characterized by the decrease of synthesis of the ß-chains. Only hemoglobin A synthesis is affected. Since the analysis is made on umbilical cord blood samples from new-borns, the amount of hemoglobin A2 in the sample does not allow the diagnostic of beta-thalassemias. The CAPILLARYS CORD BLOOD procedure is not intended to the diagnostic of beta-thalassemias in new-borns. 3. Particular cases • Hemoglobin A2 is generally absent or in low concentration in umbilical cord blood samples from new-borns, according to the term of pregnancy. In a normal new-born blood sample, hemoglobin F is the major fraction, hemoglobin A is the minor one and hemoglobin A2 is generally absent or in low concentration. When a larger fraction appears in the Hb A2 migration zone, a variant may be suspected. In this case, the fraction appeared in the Hb A2 migration zone will be detected by the CAPILLARYS system but it is necessary to perform complementary analyses in order to confirm the variant. • When there is no hemoglobin A in the sample or present at a low level (blood sample from premature baby, beta chain deficiency, etc), or in aged samples, a fraction may be observed in the C9 migration zone before the C10 migration zone of Hb A. This fraction is acetylated F hemoglobin and degraded F hemoglobin. The CAPILLARYS system can identify this acetylated hemoglobin separately from the hemoglobin A without any confusion. • When analysing blood samples from newborn babies, Hb A from samples containing Hb F at high concentrations may be disturbed, especially due to the presence of degraded Hb F in its migration zone. The Hb A percentage indicated by the software may be overvalued. In addition, when hemoglobin variants (> 4 %, such as Hb S, Hb C, Hb E or Hb D-Punjab) are present in blood samples containing high Hb F levels (> 60 %), it is necessary to perform complementary analyses in order to confirm the presence of Hb A. IMPORTANT: When analyzing blood samples without hemoglobin A but with one or many abnormal X variants, it is possible to consider the level of abnormal X fractions and that of the hemoglobin fraction located in the degraded Hb F migration zone to analyze the electrophoretic pattern: a low fraction located in C9 and C10 migration zones can not be considered as Hb A if abnormal fractions are in large proportion. In most cases, A /X heterozygous samples have Hb A and variants X fractions at the same intensity, sometimes with Hb A fraction higher than variant X. • Some hemoglobin variants (such as Hb Porto-Alegre and Hb Q-Thailand, for example) migrate close to Hb F and may be partially separated from it. The CAPILLARYS system can identify Hb F without any confusion due to the large proportion of Hb F in new-born blood sample. • Alpha-thalassemia (deletion of one to three α chains) may be detected on the electrophoretic pattern due to the presence of Hb Bart’s. The deletion of only one chain induces silent alpha-thalassemia because it is asymptomatic. The deletion of 2 or 3 α chains induces quantities of Hb Bart’s on the electrophoretic pattern. • When analysing cord blood samples from transfused new-borns with high Hb A level, the electrophoretic pattern may appear disturbed. Sickle cell disease may be hidden and confused with A / S heterozygous diagnosis. It is necessary to analyze the hematologic state and to perform complementary studies in order to confirm the diagnosis. • Gamma chain mutations may lead to abnormal hemoglobins which appear in different zones of the electrophoretic pattern. These hemoglobins have been less studied than hemoglobins due to mutations of other chains. Interference and Limitations • See SAMPLES FOR ANALYSIS. • Do not use unsedimented blood samples. • Avoid aged, improperly stored blood samples; degradation products (or artefacts) may affect the electrophoretic pattern after 7 days storage. - 63 -
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