CAPILLARYS HEMOGLOBIN

Transcription

1 CAPILLARYS HEMOGLOBIN TRAINING MANUAL Sebia, Inc Copyright 2005 Sebia, Inc. All rights reserved. The content of this manual may change without notice. This manual may not be reprinted or copied in whole or in part without the written consent of Sebia Inc. Version 5.41 June 2006 Sebia, Inc Corporate Drive Norcross, Georgia (770) Fax: (770) (800)

2 Capillarys Hemoglobin Assay Learning Objectives After reading this chapter you should be able to: Understand the principles of the Hgb Assay Be able to run the Hgb Assay Use the appropriate Quality Control Interpret basic Capillarys Hgb results Perform weekly maintenance 2

3 Capillarys Hemoglobin Assay Sample Type: Fresh anticoagulated blood samples are recommended for analysis. Common anticoagulants such as those containing EDTA, citrate or heparin are acceptable ; avoid those with iodoacetate. Blood must be collected according to established procedures used in clinical laboratory testing. Sample preparation: Let red blood cells precipitate for hours (optimum: overnight) at 2 8 C or centrifuge the blood sample at rpm for 5 minutes. Discard carefully the maximum volume of plasma (less than 3 mm of plasma or buffy coat should be present) Vortex for 5 seconds. IMPORTANT: Remove this excess of plasma before analysis. 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. Samples to avoid: Do not use unsedimented blood samples. Only red cells should be analyzed. Avoid aged (older than 7 days), improperly stored blood samples; the automated hemolysis of samples may be disturbed by viscous aggregates in red blood cells. Then, degradation products (as artifacts) may affect the electrophoretic pattern. Do not use hemolyzed blood samples. 3

4 Capillarys Hemoglobin Assay Principle of the Test 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. The CAPILLARYS System has capillaries functioning in parallel allowing 7 simultaneous analyses for hemoglobin quantification. 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 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. By using alkaline ph buffer, normal and abnormal (or variant) hemoglobins are detected in the following order, from cathode to anode: δa 2 (A2 variant), C, A2/O-Arab, E, S, D, G-Philadelphia, F, A, Hope, Bart, J, N-Baltimore and H. At 415 nm the carbonic anhydrase is not visualized on the hemoglobin electrophoretic patterns; this enables the identification of hemoglobin A2 variants in this migration zone. 4

5 PROCESSING HEMOGLOBIN SAMPLES Loading hemoglobin samples onto a sample rack 1. Place SEVEN (7) opened/non-capped hemoglobin sample tubes (minus the plasma) in positions 1-7 of sample rack. If sample number is less than seven (7) place tubes containing distilled or deionized water in the rack positions that have no sample. 2. Make certain the barcode of each tube is clearly visible in the sample rack opening. 3. Place 4 ml of CAPILLARYS HEMOGLOBIN(E) hemolysing solution in a tube (CAUTION: Do not introduce air bubbles in tube) and place it in position No. 8 of the sample rack. 4. Place a new green Hgb dilution segment on the sample rack. 5. Insert the sample rack(s) into the CAPILLARYS system through the inlet conveyor on the front of the Capillarys. Up to 13 sample racks can be introduced at once. 6. Once testing is complete, Remove analyzed sample racks from the outlet conveyor belt on the left side of the instrument. Discard used dilution segments ***Low volume sample option: Make an offline dilution of 18 ul of the patient s washed red cells with 90 ul hemolysing solution directly into the 8 th well of the green dilution segment 5

6 Warning Flags Yellow Flag: When Hb A and /or Hb A2 fractions are not detected on the electrophoretic pattern within the target range, a yellow flag appears. An automated adjustment is performed within the software using the position of the Hb A fraction on the previous pattern obtained with the same capillary (typical case for homozygote patients). The fractions are not identified and the different variant zones (Z1 to Z15) do not appear on the screen of the system, nor on the printed report. Red Flag: When the automated adjustment is not possible, a red signal appears. (Call Sebia for further assistance.) Note: When a Capillarys unit is initially installed, if the first hemoglobin sample run on the instrument contains no Hb A the same warning will appear. It is then recommended to analyze a blood sample with Hb A on the capillary in question and then reanalyze the sample without Hb A by placing it in a position corresponding to a capillary which has already detected Hb A. 6

7 PROCESSING HEMOGLOBIN CONTROLS Quality control and Migration Control It is necessary to run the Normal Hb A2 control PN 4778 as a migration control after changing the analysis buffer lot number, after changing the technique, after a Capillarys cleaning sequence with CAPICLEAN, or before starting a new analysis sequence. 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 a normal blood sample, the Normal Hb A2 Control, SEBIA, PN 4778 or the Pathological Hb A2 Control, SEBIA, PN All reconstituted controls must be kept refrigerated or frozen. Do not allow controls to remain at room temperature for more than 1 hour. 1) Hb A2 Control (PN 4778) used as the Migration Control Always refer to the package insert for the most up to date information concerning reconstitution of control, storage, and control ranges. Make certain to choose the procedure for Capillarys Hemoglobins when reconstituting the controls. Migration control: The Normal Hb A2 control is designed to be used as a migration control in the following circumstances: (a) When the analysis buffer lot number changes (run x2) (b) When the analysis technique is changed (run x2) (c) After a capillary cleaning sequence with CAPICLEAN or after capillary activations (run x2) (d) After a software upgrade (run x2) (e) Before starting a new analysis sequence i.e. running samples the next day or shift with none of the above mentioned changes. (run x1) 7

8 When using the Hb A2 control for this purpose, the control must be run through the system twice, one run after the other before any other samples or controls are run through the system. Running the Migration Control (1) Pour the reconstituted Normal Hb A2 Control in a micro tube. The micro tube should be placed in the top of a test tube (example 12x75) (2) Cut the cap off of the micro tube or use a micro tube that does not have an attached cap. (3) Place the micro tube into a clean 12x75 mm test tube used as a holder, in position No. 1 on the CAPILLARYS sample rack No. 0 intended for control blood sample and a new green dilution segment. (4) Pipette 4 ml CAPILLARYS HEMOGLOBIN (E) hemolysing solution in a clean 12x75 mm test tube without introducing air bubbles and place it in position No. 8 on the sample rack No. 0. IMPORTANT: Ensure the absence of foam in the tube before placing it on the sample rack. (5) Start the analysis: Slide the sample rack No. 0 into the CAPILLARYS system, select Hb A2 Normal Control in the window which appears on the screen and validate. A drop down menu containing the options Low, Medium, and High QC is located beneath the Hb A2 Normal Control window. Select the appropriate level of control in order to have the values stored in the QC folder and available in the Levy-Jennings chart. (6) After having performed the first series of analyses on the control, slide again immediately the sample rack No. 0 with the same dilution segment containing the Normal Hb A2 Control, previously diluted during the first series. IMPORTANT: Place clean empty tubes in position No. 1 and 8 for this second analysis sequence and for any analysis of a dilution segment containing the previously diluted Normal Hb A2 Control. The results are then automatically considered by the software for the data analysis. 8

9 (7) A warning message appears Warning: SAMPLER- Motor 5 Absence of CDT diluent or hemolysing solution for the Hgb (depending on technique) Select OK to continue. This message is simply a warning to the user that normally the hemolysing solution should be present in order to proceed with Hb testing. In the case of the migration control run for the second time, the solution is not necessary. 2) Hb A2 Normal Control (PN 4778) used as Quality Control Always refer to the package insert for the most up to date information concerning reconstitution of control, storage, and control ranges. Make certain to choose the procedure for Capillarys Hemoglobins when reconstituting the controls. Quality control: After reconstitution, use the Normal Hb A2 Control as a quality control (with the sample rack No. 0). The values obtained must fall within the range provided with each batch of Hb A2 Control. With the sample rack N 0: (1) Pour the reconstituted Normal Hb A2 Control in a micro tube. The micro tube should be placed in the top of a test tube (example 12x75) (2) Cut the cap off of the micro tube or use a micro tube that does not have an attached cap. (3) Place the micro tube into a new hemolysing tube used as a holder, in position No. 1 on the CAPILLARYS sample rack No. 0 intended for control blood sample and a new green dilution segment. 9

10 (4) Pipette 4 ml CAPILLARYS HEMOGLOBIN (E) hemolysing solution in a clean 12x75 mm test tube without introducing air bubbles and place it in position No. 8 on the sample rack No. 0. (5) Start the analysis: Slide the sample rack No. 0 into the CAPILLARYS system, select Hb A2 Normal Control in the window which appears on the screen and validate. A drop down menu containing the options Low, Medium, and High QC is located beneath the Hb A2 Normal Control window. Select the appropriate level of control in order to have the values stored in the QC folder and available in the Levy-Jennings chart. 3) Hb A2 Pathological Control (PN 4779) used as Quality Control Always refer to the package insert for the most up to date information concerning reconstitution of control, storage, and control ranges. Make certain to choose the procedure for Capillarys Hemoglobins when reconstituting the controls. The Pathological Hb A2 Control is designed for the quality control of quantification of human hemoglobin A2 with CAPILLARYS HEMOGLOBIN(E) electrophoresis procedures. The values obtained must fall within the range provided with each batch of Hb A2 Control. With the sample rack N 0 (1) Pour the reconstituted Hb A2 Pathological Control in a micro tube. The micro tube should be placed in the top of a test tube (example 12x75). (2) Cut the cap off of the micro tube or use a micro tube that does not have an attached cap. (3) Place the micro tube into a new hemolysing tube used as a holder, in position No. 1 on the CAPILLARYS sample rack No. 0 intended for control blood sample and a new green dilution segment. (4) Pipette 4 ml CAPILLARYS HEMOGLOBIN (E) hemolysing solution in a a clean 12x75 mm test tube without introducing air bubbles and place it in position No. 8 on the sample rack No

11 (5) Start the analysis: Slide the sample rack No. 0 into the CAPILLARYS system, select Hb A2 Pathological Control in the window which appears on the screen and validate. A drop down menu containing the options Low, Medium, and High QC is located beneath the Hb A2 Pathological Control window. Select the appropriate level of control in order to have the values stored in the QC folder and available in the Levy-Jennings chart. 4) AFSC Control used as an Overlay marker (PN 4791 and PN 4792) Always refer to the package insert for the most up to date information concerning reconstitution of control, storage, and control ranges. Make certain to choose the procedure for Capillarys Hemoglobins when reconstituting the controls. The Hb AFSC Control is designed for the quality control of electrophoretic separations of human hemoglobins with CAPILLARYS HEMOGLOBIN(E) procedures. It should be used as normal human blood. WARNING: The dilution cup well (green segment) containing this control must be position 8. (1) When used as the overlay marker, the AFSC Control can be run in conjunction with patient samples. Make an offline dilution (a) For PN 4791 the dilution is as follows: 36 ul reconstituted Hb AFSC Control with 72 ul hemolysing solution added directly into the 8 th well of the green dilution segment (b) For PN 4792 the dilution is as follows: 18 ul reconstituted Hb AFSC Control with 90 ul hemolysing solution added directly into the 8 th well of the green dilution segment (2) Place SEVEN (7) opened/non-capped hemoglobin sample tubes (minus the plasma) in positions 1-7 of sample rack. If sample number is less than seven (7) place tubes containing distilled or deionized water in the rack positions that have no sample. (3) Make certain the barcode of each tube is clearly visible in the sample rack opening. 11

12 (4) Place 4 ml of CAPILLARYS HEMOGLOBIN(E) hemolysing solution in a tube (CAUTION: Do not introduce air bubbles in tube) and place it in position No. 8 of the sample rack. (5) Place a new green Hb dilution segment on the sample rack. (6) Insert the sample rack(s) into the CAPILLARYS system through the inlet conveyor on the front of the Capillarys. (7) Once the AFSC Control curve image is available for review go to the Edit Curve Screen and select the 4 th icon located beneath the curve image. This is the save current curve as reference pattern icon. (8) The AFSC Control can now be used as the overlay/reference pattern with other patient samples by selecting the 8 th icon located above the curve image. The AFSC image will be overlaid onto the curve image of the patient samples. 12

13 Note for Editing Curves If it is necessary for the user to delete a fraction in order to modify the quantification or to remove the fraction completely, all fractions must be deleted and then manually renamed. When a fraction is deleted and then manually shaded, the order of the fractions may be changed. This change can result in the corresponding names not appear in the correct sequence. INTERPRETING HEMOGLOBIN SAMPLES Result analysis The CAPILLARYS system calculates concentration (%) of each hemoglobin fraction. 1. Normal and abnormal (or variant) hemoglobins are detected in the following order, from cathode to anode: delta A 2(A2 variant), C, A2/O-Arab, E, S, D, G- Philadelphia, F, A, Hope, Bart, J, N-Baltimore and H. 2. Patterns are automatically adjusted with regard to Hb A fraction a. The Hb A fraction is automatically identified and adjusted in the middle of the review window. b. When Hb A is absent (yellow warning signal), the adjustment is performed using the position of the Hb A fraction on the previous electrophoretic pattern obtained with the same capillary. c. When the adjustment is not possible, a red warning signal appears. 3. When Hb C is present, Hb A2 can not always be quantitated precisely. With some hemoglobinopathy disease states, Hb A2 may not be quantified accurately in the presence of Hb C. Such conditions may include homozygous Hb C, Hb SC, and heterozygous Hb C when an A2 variant is present. In such cases, an over/underestimation of Hb A2 may occur. If desired, Hb A2 and C quantification can be reported as one value, as opposed to two separate values. 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; the Hb A fraction is adjusted in the middle of the review window. The resulting electrophoregrams are evaluated visually for pattern abnormalities. 13

14 The potential positions of the different hemoglobin variants (identified in zones called Z1 to Z15) are shown on the screen of the system and zones are indicated on the printed report. The labelling of the zones is software dependant. The table present in this manual is for CAPILLARYS Software Version The table below shows known variants which may be present in each corresponding zone. Hb Fractions and Variants according to Zone: (Table for CAPILLARYS Software Version 5.41) Zone Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z9 Hemoglobins (Hb) Hb δa 2 Hb αa 2 Hasharon Hb A2 variant Winnipeg Hb A2 variant other Hb A2 variants, Hb Constant Spring Hb C Setif Hb A2 variant Hb A2 Hb O-Arab Hb E Hb Köln Hb A2 variants (and degraded Hb C) Hb S Hb Hasharon (and degraded Hb O-Arab) Hb D-Punjab Hb D-Ouled Rabah Hb Lepore Hb G-Philadelphia Hb Korle-Bu Hb Köln Hb Winnipeg Hb Setif (and degraded Hb E) Hb F Hb Porto-Alegre Hb Richmond Hb G-San-Jose (and degraded Hb S) Hb A Hb Okayama Hb Camperdown Hb Toulon Hb Fontainebleau 14

15 Z10 Z11 Hb Hope Hb J-Kaohsiung (and degraded Hb A) Z12 Z13 Z15 Hb Bart s Hb J-Providence Hb J-Mexico Hb J-Baltimore Hb J-Broussais Hb J Hb N-Baltimore Hb H 15

16 Hemoglobin Assay Ranges: Sebia Patient Normal Range: HbA >96.8% HbF <0.5% HbA (slight increase from Normal Range of HbA2 on gel) Analyzer Ranges: Capillarys Quantitative range of HbA2: % Capillarys Quantitative range of HbF: % (Able to detect very small fractions of HbF. On gel 3% HbF difficult to detect) 16

17 Weekly cleaning of capillaries and probe using CAPICLEAN When using the green (100 µl) dilution segments specific for CAPILLARYS HEMOGLOBIN(E) assay, follow the steps below for capillary and probe cleaning using CAPICLEAN: 1. Use rack No. 100 to perform cleaning 2. Add 50 µl of concentrated CAPICLEAN (PN2051) solution to each well of the green dilution segment 3. Add 50 µl of distilled or deionized water to each well of the same dilution segment; avoid formation of foam 4. For sample probe cleaning, place tube containing 200 µl of CAPICLEAN and 200 µl of deionized water in position 1 of rack No. 100; avoid formation of foam 5. Place the dilution segment containing CAPICLEAN and water mix onto the rack No Slide the rack into CAPILLARYS 7. In the MAINTENANCE screen that automatically will appear on your monitor, select LAUNCH THE CAPICLEAN CLEANING and validate 8. Cycle will start automatically and will last approximately 20 minutes.. 17

18 Hb Questions: 1. How many Hb samples can be processed on one Capillarys rack? 2. Under which circumstances is it mandatory to run the migration control? 3. Can whole blood samples be analyzed on the Capillarys? 4. Which zone would Degradated HbA appear? 5. Which rack are each of the controls run in? 6. Where is the most up to date information concerning control material reconstitution, storage, and control ranges found? 18

19 Hb Answers: (a) When the analysis buffer lot number changes (b) When the analysis technique is changed (c) After a capillary cleaning sequence with CAPICLEAN or capillary activations (d) Before starting a new analysis sequence 3. No, only Red Cells 4. Z Rack 0 6. Always refer to the package insert for the most up to date information concerning reconstitution of control, storage, and control ranges. Make certain to choose the procedure for Capillarys Hemoglobins when reconstituting the controls. 19