CAPILLARYS HEMOGLOBIN(E)

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CAPILLARYS HEMOGLOBIN(E) - 2010/10 • When an abnormal hemoglobin is detected, use other means of identification (e.g., globin chain electrophoresis), or consult or send sample to a specialized laboratory. IMPORTANT: It is also necessary to analyze the hematologic state, as complementary results. • The migration of a hemoglobin variant close to Hb A involves an underestimation of Hb A fraction and that of the variant and consequently, an overestimation of Hb A2 fraction. In order to quantify Hb A2 with precision, it is necessary to delete the separate integration of both variants and Hb A, and to quantify these fractions together. • Some homozygous "S" subjects receive a "Hydrea"® (hydroxyurea) treatment that can induce synthesis of foetal hemoglobin. With CAPILLARYS HEMOGLOBIN(E) procedure, the mobility of the induced hemoglobin F is not different from the physiological hemoglobin F. • Due to the resolution and sensitivity limits of zone electrophoresis, it is possible that some hemoglobin variants may not be detected with this method. Troubleshooting Call SEBIA Technical Service of the supplier when the test fails to perform while the instruction for the preparation and storage of materials, and for the procedure were carefully followed. Kit reagent Safety Data Sheets and information on waste products elimination are available from the Technical Service of the supplier. PERFORMANCE DATA Results obtained using the CAPILLARYS HEMOGLOBIN(E) procedure with the CAPILLARYS 2 FLEX PIERCING system indicate a very good reproducibility for quantitative analysis with a mean CV % of about 0.9 % for each hemoglobin component. All electrophoregrams were also interpreted visually. Results presented below have been obtained using the standard parameters of the CAPILLARYS software (smoothing 0 and hemoglobin fractions automatic quantification with HEMOGLOBIN(E) analysis program). Reproducibility within run Six (6) different blood samples (normal bloods A and E, blood F with increased Hb A2, blood B with Hb S, blood C with increased Hb F and blood G with Hb F, S and C components) were run using the CAPILLARYS HEMOGLOBIN(E) procedure in 8 capillaries of 3 CAPILLARYS 2 FLEX PIERCING systems and with 3 lots of CAPILLARYS HEMOGLOBIN(E) kits. The mean, SD and CV (n = 8) were calculated for each hemoglobin component, each sample, each lot of kit and each system. The table shows the values for the 6 tested samples for each hemoglobin component calculated with the 3 lots of kit and the 3 CAPILLARYS 2 FLEX PIERCING systems. Hemoglobin component Hb A Hb A2 Hb F Hb S Hb C Blood sample A : lot No. 1 & system No. 1 - lot No. 2 & system No. 2 - lot No. 3 & system No. 3 MEAN (%) 97.4 - 97.35 - 97.4 2.6 - 2.65 - 2.6 / / / SD 0.03 - 0.05 - 0.05 0.03 - 0.05 - 0.05 / / / CV (%) 0.03 - 0.1 - 0.05 1.3 - 1.9 - 1.8 / / / Blood sample B : lot No. 1 & system No. 1 - lot No. 2 & system No. 2 - lot No. 3 & system No. 3 MEAN (%) 58.1 - 58.1 - 58.0 3.0 - 3.0 - 3.0 / 38.9 - 38.9 - 39.0 / SD 0.10 - 0.16 - 0.06 0.05 - 0.03 - 0.03 / 0.12 - 0.17 - 0.05 / CV (%) 0.2 - 0.3 - 0.1 1.7 - 1.1 - 1.1 / 0.3 - 0.4 - 0.1 / Blood sample C : lot No. 1 & system No. 1 - lot No. 2 & system No. 2 - lot No. 3 & system No. 3 MEAN (%) 83.0 - 83.0 - 83.14 2.3 - 2.3 - 2.32 14.7 - 14.7 - 14.54 / / SD 0.08 - 0.13 - 0.10 0.03 - 0.03 - 0.04 0.05 - 0.10 - 0.10 / / CV (%) 0.1 - 0.2 - 0.1 1.4 - 1.4 - 1.9 0.3 - 0.7 - 0.7 / / Blood sample E : lot No. 1 & system No. 1 - lot No. 2 & system No. 2 - lot No. 3 & system No. 3 MEAN (%) 97.5 - 97.4 - 97.3 2.5 - 2.6 - 2.7 / / / SD 0.04 - 0.04 - 0.07 0.04 - 0.04 - 0.07 / / / CV (%) 0.04 - 0.04 - 0.1 1.7 - 1.7 - 2.5 / / / Blood sample F : lot No. 1 & system No. 1 - lot No. 2 & system No. 2 - lot No. 3 & system No. 3 MEAN (%) 93.6 - 93.6 - 93.25 6.4 - 6.4 - 6.75 / / / SD 0.07 - 0.16 - 0.11 0.07 - 0.16 - 0.11 / / / CV (%) 0.1 - 0.2 - 0.1 1.1 - 2.5 - 1.7 / / / Blood sample G : lot No. 1 & system No. 1 - lot No. 2 & system No. 2 - lot No. 3 & system No. 3 MEAN (%) 50.1 - 50.05 - 48.85 2.2 - 2.25 - 2.45 24.5 - 24.57 - 24.26 18.7 - 18.59 - 19.66 4.5 - 4.54 - 4.78 SD 0.25 - 0.19 - 0.21 0.04 - 0.05 - 0.09 0.08 - 0.11 - 0.10 0.19 - 0.11 - 0.11 0.08 - 0.05 - 0.04 CV (%) 0.5 - 0.4 - 0.4 1.9 - 2.2 - 3.5 0.3 - 0.4 - 0.4 1.0 - 0.6 - 0.6 1.8 - 1.1 - 0.9 In addition, none of the repeats showed false positive or false negative values. - 52 -
CAPILLARYS HEMOGLOBIN(E) - 2010/10 Reproducibility between runs Eight (8) different blood samples were run 10 times using the CAPILLARYS HEMOGLOBIN(E) procedure on 3 CAPILLARYS 2 FLEX PIERCING systems and with 3 lots of CAPILLARYS HEMOGLOBIN(E) kits (1 lot per system). The analyzed samples included 3 samples with normal Hb A2 level, 1 sample with elevated Hb A2 level, 1 sample with elevated Hb F level and 3 samples with abnormal hemoglobins (Hb S or Hb C or Hb E). The mean, SD and CV (n = 10) were calculated for each sample, each hemoglobin component, each lot of kit and each system. The table shows the ranges of hemoglobins values for the 8 tested blood samples on the 3 CAPILLARYS 2 FLEX PIERCING systems with the 3 lots of kits and a mean CV calculated from the pooled CV's for each hemoglobin component (n = 24 for Hb A and Hb A2, n = 6 for Hb F and Hb S, n = 3 for Hb C and Hb E). Hb COMPONENT MEAN (%) SD CV (%) MEAN CV (%) Hb A 49.0 - 97.5 0.03 - 0.22 0.03 - 0.3 0.1% Hb A2 2.2 - 6.6 0.00 - 0.22 0.0 - 3.4 1.7% Hb F 7.5 - 24.5 0.05 - 0.11 0.3 - 1.2 0.6% Hb S 18.4 - 37.0 0.05 - 0.14 0.2 - 0.6 0.3% Hb C 4.5 - 4.7 0.03 - 0.05 0.7 - 1.1 0.9% Hb E 26.1 - 26.4 0.08 - 0.13 0.3 - 0.5 0.4% Reproducibility between lots and between systems Eight (8) different blood samples (identical as those assayed for the "Reproducibility between runs" study) were run 10 times using the CAPILLARYS HEMOGLOBIN(E) procedure on 3 CAPILLARYS 2 FLEX PIERCING systems and with 3 lots of CAPILLARYS HEMOGLOBIN(E) kits (1 lot per system). The mean, SD and CV (n = 30) were calculated for each sample, for each hemoglobin component with the 3 lots of kits and the 3 CAPILLARYS 2 FLEX PIERCING systems. The table shows the ranges of hemoglobins values for the 8 samples tested on the 3 CAPILLARYS 2 FLEX PIERCING systems and with the 3 lots and a mean CV calculated from the pooled CV's for all samples (n = 8 for Hb A and Hb A2, n = 2 for Hb F and Hb S, n = 1 for Hb C and Hb E). Hb COMPONENT MEAN (%) SD CV (%) MEAN CV (%) Hb A 49.7 - 97.4 0.05 - 0.57 0.1 - 1.1 0.3% Hb A2 2.3 - 6.5 0.03 - 0.18 1.2 - 4.9 2.5% Hb F 7.7 - 24.5 0.12 - 0.13 0.5 - 1.5 1.0% Hb S 18.9 - 36.7 0.26 - 0.46 0.7 - 2.4 1.6% Hb C 4.6 0.10 2.2 2.2% Hb E 26.2 0.15 0.6 0.6% In addition, none of the repeats (reproducibility between runs, systems and lots) showed false positive or false negative values. Linearity Different characteristic blood samples were mixed by pairs within different proportions and the dilutions were electrophoresed with the CAPILLARYS HEMOGLOBIN(E) procedure. The tests were determined to be linear within the entire ranges studied for each of the hemoglobin fraction (Hb A, Hb F, Hb S and Hb A2). In addition, three different characteristic blood samples were also serially diluted in saline and electrophoresed with the CAPILLARYS HEMOGLOBIN(E) procedure. The tests were determined to be linear within the entire ranges studied from 1.1 to 36.0 g/dL total hemoglobin and hemoglobin fractions percentages were not affected by the hemoglobin concentration of the samples. Hb A2 linearity Two different blood samples (one sample containing 13.4 g/dL hemoglobin with 0.0 % Hb A2 and another containing 13.2 g/dL with 9.8 % Hb A2) were mixed within different proportions and the dilutions were electrophoresed with the CAPILLARYS HEMOGLOBIN(E) procedure. The tests gave a good linearity for Hb A and Hb A2 hemoglobin fractions within the entire range studied, with a maximum of about 1.3 g/dL for Hb A2 (between 0.0 and 9.8 % of Hb A2). In addition, a normal blood sample (containing 13.3 g/dL hemoglobin with 2.7 % Hb A2) was concentrated twice and serially diluted in saline and electrophoresed with the CAPILLARYS HEMOGLOBIN(E) procedure. The tests were determined to be linear within the entire range studied from 26.6 to 1.3 g/dL total hemoglobin and Hb A2 fraction percentages were not affected by the hemoglobin concentration of the samples. Hb F linearity One umbilical cord blood sample (containing 18.0 g/dL hemoglobin with 86.0 % Hb F) was mixed with a normal blood sample (containing 13.4 g/dL hemoglobin with 0.0 % Hb F) within different proportions and the dilutions were electrophoresed with the CAPILLARYS HEMOGLOBIN(E) procedure. The tests gave a good linearity for Hb A, Hb A2 and Hb F hemoglobin fractions within the entire range studied. The CAPILLARYS HEMOGLOBIN(E) tests gave a good linearity for Hb F hemoglobin fraction within the entire range studied, with a maximum of about 15.5 g/dL for Hb F (between 0.0 and 86.0 % of Hb F). In addition, this same umbilical cord blood sample (containing 18.0 g/dL hemoglobin with 86.0 % Hb F) was concentrated twice and serially diluted in saline and electrophoresed with the CAPILLARYS HEMOGLOBIN(E) procedure. The tests were determined to be linear within the entire range studied from 36.0 to 1.8 g/dL total hemoglobin and Hb F fraction percentages were not affected by the hemoglobin concentration of the samples. Hb S linearity One blood sample with Hb S (containing 11.5 g/dL hemoglobin with 39.8 % Hb S) was mixed with a normal blood sample without Hb S (containing 13.4 g/dL hemoglobin) within different proportions and the dilutions were electrophoresed with the CAPILLARYS HEMOGLOBIN(E) procedure. The tests gave a good linearity for Hb A, Hb S and Hb A2 hemoglobin fractions within the entire range studied, with a maximum of about 4.6 g/dL for Hb S (between 0.0 and 39.8 % of Hb S). - 53 -
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CAPILLARYS HEMOGLOBIN(E)

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