CAPILLARYS HEMOGLOBIN(E)

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CAPILLARYS HEMOGLOBIN(E) - 2010/10 Quality control among a series of samples to analyze : The Hb AFSC Control should be used as a normal human blood. After reconstitution, use directly the Hb AFSC Control as a blood sample to analyze or as a migration control (with the sample rack No. 0, see the paragraph before). It will be automatically diluted with hemolyzing solution. It is recommended to include the Hb AFSC Control into each series of analysis. IMPORTANT: For optimal use of the Hb AFSC Control with the CAPILLARYS System, it is necessary to use one bar code label intended to identify the hemolyzing tube holding the microtube which contains the Hb AFSC Control (cut the cap of the microtube before using it). Storage, stability and signs of deterioration Before reconstitution, store the lyophilized Hb AFSC Control refrigerated (2 to 8 °C). It is stable until the expiration date indicated on the box or vial labels. Store the reconstituted Hb AFSC Control at 2 - 8 °C. Due to the risk of microbial contamination and denaturation, use it within one week. The reconstituted Control may also be frozen (in aliquots) and stored at - 20 °C for 6 months maximum. IMPORTANT: After storage at 2 - 8 °C or at - 20 °C, homogenize the reconstituted Hb AFSC Control before the analysis with the CAPILLARYS system. NOTE: For optimal use with the CAPILLARYS system, it is recommended to split the Control into aliquots in microtubes before freezing. Before use, store the thawed Hb AFSC Control at 2 - 8 °C and use it within the day, after homogeneization. Do not freeze and thaw the Control more than 10 times. No test method can provide an absolute assurance of the absence of HIV, hepatitis B and C or other infectious agents. Therefore, handle the Hb AFSC Control as a hazardous biological material. This control was found negative on assays approved by FDA or EU equivalent regulatory agency : - against hepatitis B surface antigen ; - for antibody to HCV ; - for antibody to HIV1 and HIV2. NOTE: During transportation, the lyophilized Hb AFSC Control can be kept without refrigeration (15 to 30 °C) for 15 days without any adverse effects on performance. EQUIPMENT AND ACCESSORIES REQUIRED 1. CAPILLARYS System SEBIA, PN 1222. 2. Sample racks supplied with CAPILLARYS. 3. Container Kit supplied with CAPILLARYS: Rinse (to fill with distilled or deionized water), wash solution and waste container. SAMPLES FOR ANALYSIS Sample collection and storage Fresh umbilical cord blood samples from new-borns, anticoagulated with EDTA, are recommended for analysis. Avoid anticoagulants containing iodoacetate. Blood must be collected according to established procedures used in clinical laboratory testing. IMPORTANT: The volume of collected blood sample must not exceed the recommended volume of the collection tube. In the opposite case, if the volume of anticoagulant is insufficient for the collected blood sample, viscous aggregates in red blood cells may be observed. It is necessary to discard them before the analysis. Samples may be stored for up to 7 days between 2 and 8 °C. For longer storage, samples can be frozen at - 80 °C within 8 hours of collection after having washed the red blood cells according to the following procedure: Centrifuge anticoagulated blood at 5 000 rpm for 5 minutes; discard the plasma; wash the red blood cells (RBC) 2 times with 10 volumes of saline (centrifuge after each washing step); discard the excess of saline over the red blood cells pellet and vortex them before freezing. Frozen blood samples are stable for 3 months maximum at - 80 °C. IMPORTANT: For optimal storage of blood samples, store them at - 80 °C. Do not store at - 20 °C (see BIBLIOGRAPHY, J. Bardakdjian-Michau et al, 2003). NOTE: Samples should not be stored at room temperature! Progressive hemoglobins (Hb) degradation may occur for samples stored between 2 to 8 °C. When the blood sample is stored for more than 7 days at 2 - 8 °C: • Degraded Hb F becomes more visible and appears as a shoulder on the cathodic side of the Hb A (C9 zone, see the table in paragraph "Interpretation") due to the sample degradation. The Hb A percentage is then overvalued. • When Hb A is present, a spread fraction corresponding to degraded Hb A ("aging fraction" of Hb A) appears more anodic (C11 zone, see the table in paragraph "Interpretation"). • Due to the sample degradation, a weak fraction, corresponding to methemoglobin, may appear in the Hb E migration zone (C3 zone, see the table in paragraph "Interpretation"). When stored for more than 10 days, viscous aggregates in red blood cells are observed; it is necessary to discard them before the analysis. Sample preparation • Let red blood cells precipitate for several hours at 2 - 8 °C or centrifuge the blood sample at 5 000 rpm for 5 minutes. • Discard carefully the maximum volume of plasma. • Vortex for 5 seconds. IMPORTANT: Do not use blood samples containing 3 mm maximum residual plasma over red blood cells; when more than 3 mm plasma is present in the tube, the analysis should be affected. - 60 -
CAPILLARYS HEMOGLOBIN(E) - 2010/10 Samples to avoid • Do not use unsedimented blood samples. • Avoid aged, improperly stored blood samples; the automated hemolysis of samples may be disturbed by viscous aggregates in red blood cells. Then, degradation products (as artefacts) may affect the electrophoretic pattern. PROCEDURE The CAPILLARYS system is a multiparameter instrument for hemoglobins analysis on parallel capillaries. The hemoglobins assay uses 7 of the total 8 instrument capillaries to run the samples. The sequence of automated steps is as follows: • Bar code reading of sample tubes (for up to 7 tubes) and samples-racks; • Sample hemolysis and dilution from primary tubes (without any plasma) into dilution segments; • Capillary washing; • Injection of hemolyzed samples; • Hemoglobin separation and direct detection of the separated hemoglobins on capillaries. The manual steps include: • Placement of opened sample tubes in sample-racks in positions 1 to 7; • Placement of hemolysing solution tube in sample-racks in position 8; • Placement of new dilution segments in sample-racks; • Placement of racks on the CAPILLARYS instrument; • Removal of sample-racks after analysis. PLEASE CAREFULLY READ THE CAPILLARYS INSTRUCTION MANUAL. I. PREPARATION OF CAPILLARYS ANALYSIS 1. Switch on CAPILLARYS instrument and computer. 2. Set up the software, enter and the instrument automatically starts. 3. For the CAPILLARYS CORD BLOOD procedure, the CAPILLARYS HEMOGLOBIN(E) kit is used with the "CORD BLOOD" analysis program from the CAPILLARYS instrument. To select "CORD BLOOD" analysis program and place the CAPILLARYS HEMOGLOBIN(E) buffer vial in the instrument, please read carefully the CAPILLARYS instruction manual. 4. The sample rack contains eight positions for sample tubes. Place seven opened sample tubes without any plasma on each sample rack (positions 1 to 7) ; the bar code of each tube must be visible in the openings of the sample rack. IMPORTANT: If the number of tubes to analyze is less than 7, complete the sample rack with tubes containing distilled or deionized water. 5. Pour 4 mL CAPILLARYS HEMOGLOBIN(E) hemolysing solution in a tube without introducing air bubbles and place it in position No. 8 on the sample rack. IMPORTANT: Ensure the absence of foam in the tube before placing it on the sample rack. 6. Position a new dilution segment on each sample rack. A message will be displayed if the segment is missing. 7. Slide a sample rack No. 0 with the Hb AF Control: See the previous paragraph "Hb AF Control" when using it with the CAPILLARYS CORD BLOOD procedure, part "Migration control". 8. Slide the complete sample carrier(s) into the CAPILLARYS system through the opening in the middle of the instrument. Up to 13 sample racks (including the sample rack No. 0 intended for the control blood sample) can be introduced successively and continuously into the system. 9. Remove analyzed sample racks from the plate on the left side of the instrument. 10. Take off carefully used dilution segments from the sample rack and discard them. WARNING: Dilution segments with biological samples have to be handled with care. DILUTION - MIGRATION - DESCRIPTION OF THE AUTOMATED STEPS 1. Bar codes are read on both sample tubes and sample racks. 2. Samples are diluted in hemolysing solution and the sample probe is rinsed after each sample. 3. Capillaries are washed. 4. Diluted samples are injected into capillaries. 5. Migration is carried out under constant voltage for about 8 minutes and the temperature is controlled by Peltier effect. 6. Hemoglobins are detected directly by scanning at 415 nm and an electrophoretic profile appears on the screen of the system. NOTE: These automated steps described above are applied to the first introduced sample rack. The electrophoretic patterns appear after about 20 minutes from the start of the analysis. For the following sample rack, the first two steps (bar code reading and sample dilution) are performed during analysis of the previous sample rack. II. RESULT ANALYSIS At the end of the analysis, relative quantification of individual hemoglobin fractions is performed automatically and profiles can be analyzed. The hemoglobin fractions, Hb A, Hb F and Hb A2 are automatically identified and the Hb F hemoglobin fraction is adjusted at a well-defined position. The resulting electrophoregrams are evaluated visually for pattern abnormalities. The potential positions of the different hemoglobin variants (identified in zones called C1 to C12) are shown on the screen of the system and indicated on the result ticket. The table in paragraph "Interpretation" shows known variants which may be present in each corresponding zone. When the software identifies a hemoglobin fraction in a defined zone, the name of this zone is framed. The identification of normal blood samples and of pathological blood samples are automatically performed and the profiles can be distinguished in the curve review window of patterns by a blue color for normal samples and a red color for pathological samples. This identification is based on the detection of abnormal fractions of hemoglobin on a pattern or on the absence of Hb F or Hb A hemoglobin fractions. The software is optimized to limit the interferences due to degraded hemoglobins on the analysis. Moreover, the software allows for the detection of low Bart’s hemoglobin concentrations which may alert the user on a probable alpha-thalassemia diagnostic. - 61 -
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CAPILLARYS HEMOGLOBIN(E)

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