VI-K. Gel: Preparing Buffered Gel Columns

Transcription

1 Section VI. Alternative Serologic Methods for Antibody Detection Gel: Preparing Buffered Gel Columns Page 1 of 4 VI-K. Gel: Preparing Buffered Gel Columns Purpose To provide instructions for preparing Sephadex gel columns for serologic testing: Antibody detection. Antibody identification. Background Information Sephadex gel is a porous gel composed of alkaline dextran cross-linked with epichlorohydrin and hydrated with a buffer such as PBS or LISS. The gel beads used (Sephadex G100), have a fractionation range from a molecular weight of 4000 to 150,000, which will permit the free passage of single RBCs but not the passage of RBC agglutinates. Lapierre et al described the use of Sephadex gel for conventional saline and enzyme techniques in a simple inert buffer. Also see Appendix VI-3. Operational Policy The centrifuge should be calibrated appropriately to permit even packing of the gel suspension. Limitations Unwanted Positive Reactions: Gel is unevenly packed. Fibrin is present in the test plasma. Reagents are prepared incorrectly. Unwanted Negative Reactions: Gel is unevenly packed. Supernatant buffer has not been decanted sufficiently. Reagent volumes are incorrect. Sample Requirements IgM anti-m and anti-d reagents

2 Gel: Preparing Buffered Gel Columns Page 2 of 4 Equipment/ Materials 6 50-mm clear polystyrene test tubes: Precipitin tubes (# , Elkay Co, Basingstoke, UK) hole precipitin test tube racks. Variable-volume pipettes: 5-50 μl and μl (VWR, Batavia, IL). Benchtop centrifuge with a swing-out rotor. Sephadex G100 superfine powder. 0.1% BSA-LISS. Reagent RBCs diluted to a 1% suspension in LISS. Control All columns must be visually inspected to ensure that the gel is packed evenly before use. Column Preparation Procedure Use the following steps to prepare the columns: Step Action 1. Prepare the gel suspension by mixing 0.5 g of Sephadex G100 superfine powder in 10 ml of 0.1% BSA-LISS. 2. Mix thoroughly to ensure even hydration and suspension of the beads. 3. Pipette 350 μl of the Sephadex gel solution into 6 50-mm test tubes. Centrifuge at 1000 g for 60 seconds to remove air bubbles and pack the gel. 4. Columns may be used immediately, or store columns, covered with Parafilm, upright at 2-8 C for a maximum of 14 days. 5. Evaluate the columns as follows: If the top of the column is level the top of the column is uneven the gel column trails down the side of the tube Then the tubes are ready to use. recalibrate the centrifuge, resuspend the gel column, and recentrifuge. ensure that a swing-out rotor is in use: Recalibrate the centrifuge, resuspend the gel column, and recentrifuge

3 Section VI. Alternative Serologic Methods for Antibody Detection Gel: Preparing Buffered Gel Columns Page 3 of 4 Control Procedure Use the following steps to quality-control the columns: Step Action 1. Take 22 gel columns from the refrigerator and allow them to warm to room temperature. 2. Prepare a doubling dilution of anti-m and anti-d in PBS/1% BSA to a dilution of 1 in Visually inspect the columns to confirm that the gel suspension is level. 4. Decant the supernatant buffer on the columns by a rapid flick into a waste container. 5. Mark two series of 10 tubes: 1, 2, 4, 8, 16, 32, 64, 128, 256, Mark 2 tubes with 1 for the negative controls. 7. Add 30 μl of M+N+ 1% RBCs to each of 10 tubes. 8. Add 30 μl of M RBCs to the negative control tube. 9. Add 30 μl of each dilution to the appropriately marked tube. 10. Repeat 7-9 with D+ and D RBCs and the anti-d dilutions. 11. Centrifuge at 300 g for 10 minutes. 12. Read the tests macroscopically over a light box. Grade tests from 0 (negative) to 4+ (see Appendix VI-4 for representative agglutination patterns). 13. Determine the required dilutions of anti-m and anti-d for positive controls as follows: If there is a dilution that yields a 2+ reaction all dilutions are strongly reactive no dilutions are reactive Then select that dilution for a positive control. prepare and test higher antibody dilutions. select a different example of antibody to titrate. Reference Lapierre Y, Rigal D, Adam J, et al. The gel test: A new way to detect RBC antigen-antibody reactions. Transfusion 1990;30:

4 Gel: Preparing Buffered Gel Columns Page 4 of 4 Effective Date Approved by: Printed Name Signature Date Laboratory Management Medical Director Officer -232-

5 Identification of Single or Multiple Antibodies Page 7 of 8 Table VIII-A-1. Serologic Characteristics of Some Alloantibodies to RBC Antigens System* Anti- Phase Ficin DTT Ig C3 HTR # HDFN** % Comp ABO A RT>37-IAT M G 56 A 1 RT M rare 64 B RT>37-IAT M G 85 CH/RG Ch IAT G 4 Rg IAT G 2 CO Co a IAT G rare Co b IAT G rare 91 CROM Cr a IAT G rare DI Di a IAT G >99 Di b IAT G rare Wr a RT-37-IAT G M >99 DO Do a IAT G 33 Do b IAT G 13 Hy IAT G rare FY Fy a IAT G rare 33 Fy b IAT G rare rare 20 GE Ge 37<IAT G M rare H H RT>37-IAT M G rare I I RT>37-IAT M G rare JK Jk a IAT G M 25 Jk b IAT G M 25 JMH JMH IAT G rare KEL Js a IAT G M >99 Js b IAT G rare K 37-IAT G M rare 91 k 37-IAT G M rare Kp a 37-IAT G 98 Kp b 37-IAT G M rare KNOPS Kn a IAT G 2 McC a IAT G 2 Sl a IAT G 2 Yk a IAT G 8 LE Le a RT>37-IAT M G rare 78 Le b RT>37-IAT M G 28 LU Lu a RT-37-IAT G A rare rare 92 Lu b RT-37-IAT G A rare rare rare LW LW a RT>37-IAT M G 3 LW b RT>37-IAT M G