Blood Bank Testing and Automation PDF

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Summary

Column Agglutination Technology (CAT) Uses acrylamide gel to trap agglutinates. The method eliminated the need for multiple washes with saline and control cells. Provides a very stable endpoint with reproducible results. Also known as the Gel technology test or ID Micro Typing System (MTS). Principl...

Description

MLSC-3200, Transfusion Science Column Agglutination Technology (CAT) -

Uses acrylamide gel to trap agglutinates. The method eliminated the need for multiple washes with saline and control cells. Provides a very stable endpoint with reproducible results. Also known as the Gel technology test or ID Micro Typing System (MTS).

Principle -

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The test is based on the ability of the test to detect Ag-Ab reactions. Based on a controlled centrifugation of RBCs as they pass through dextran-acrylamide microtube containing pre-dispensed reagent. Each microtubule is composed of an upper reaction chamber to allow for the reaction to occur. The column then gradually gets narrower as the RBCs pass through. Measured volume of plasma and RBCs are dispensed into the wider reaction chamber. Ab screening is the only test that needs incubation in this test. Controlled centrifugation causes the cells to pass through the microtube and get trapped by the gel as it gets narrower. Free un-agglutinated cells flow faster and freely passed the gel to form a pellet at the bottom (negative). Agglutinated cells will get trapped in the gel (positive). Large agglutinates are trapped at the top of the tube while small agglutinates get trapped near the bottom. Results can be reviewed for up to 3 days.

Applications -

Used for ABO forward (anti-sera gel card) and reverse grouping (buffered gel cards), Rh typing (cards with Anti-D) and Rh phenotyping (cards with anti-D/C/E/e/c) Microtubules filled with gel containing anti-IgG used for compatibility testing and Ab screening/ID. The FDA and Health Canada approves this technology for ABO/Rh grouping, DAT, Ab screening and ID and compatibility testing.

Types of Gel Cards -

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Neutral Cards, contain no specific reagent. Works the same way as other cards but uses whatever antigen and AB you want to add to it. Used for Ab screening and reverse ABO. Specific Cards, contain specific reagent added into the card. Aids in antigen determination and used for ABO forward grouping using Anti-A, Anti-B and Anti-D in the gel. Antiglobulin Card, used for IAT and DAT testing. AHG is already in the gel and testing requires no washing.

MLSC-3200, Transfusion Science Grading -

Uses the same grading scheme as tube method agglutination reactions. A solid band of agglutinated cells on top of the gel is a 4+ reaction. A solid band of agglutinated cells on the top of the gel with some agglutinates making their way down the tube column is a 3+ reaction. Agglutinates disturbed evenly through the gel column is a 2+ reaction. Agglutinates concentration toward the bottom of the gel is a 1+ reaction. The formation of a pellet at the bottom of the tube is a 0 reaction. Mixed field reactions are seen as a pellet at the bottom with a layer of agglutination at the top of the gel that looks like a 4+/3+.

Pros and Cons of MTS -

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Advantages o Standardization through automation o Stable for 3 days o Well-defined endpoints o Does not require washing o Fewer procedural steps, minimizes the amount of operator error o No need for controls o Consistent reproducible results o Easy to use o Requires little sample volume o Enhanced sensitivity and specificity Disadvantages o Cannot use hemolyzed or icteric samples o Lipemic samples require prior centrifugation to avoid false positives. o Rouleaux can cause false positives. o Requires incubators, centrifuges, pipettes, and RBCs. o Start-up cost

Solid Phase Technology -

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The test system uses one form of test reactant (antigen or antibody) that is bound to a solid support (micro titre wells) before the test starts. The unknown reactant (antigen or antibody) is then added to the well to check for complex formation. Solid-Phase Red Cell Adherence (SPRCA), uses adherence and not agglutination. The first generation of this test uses a target antigen that is added by the user to the well before testing. The second generation has reactants bound to the wells during manufacturing. This technology is approved by the FDA and Health Canada for antibody screening/ID, weak D testing, IgG autologous controls and compatibility testing.

MLSC-3200, Transfusion Science -

Positive results show adherence of indicator RBCs to part or all the well lining, depending on the strength of the reaction.

Pros and Cons of SPRCA -

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Advantages o Stable well-defined endpoints o Reproducible results o No pre-dilution of reagents required o Hemolyzed, lipemic and icteric samples can be used o Detects weak alloantibodies o LISS is used as a quality control Disadvantages o Requires special reagents and equipment o Increased sensitivity may result in weak alloantibody detection.

Capture-R Technology -

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Solid phase testing that involves immobilized reactants already at the bottom of the micro titre wells. IgG specific technology is then used for the detection of clinically significant antibodies. A chemical bonding reagent is placed at the bottom of well. Then, a diluted suspension of reagent RBCs are added to the well. The RBCs will bind to the chemicals and then lysed and dried. This gives us a monolayer of RBC antigens coating the bottom of the well. The layer of antigens will allow antibodies to bind for analysis. Microplates can be stored at RT and have a 90-day shelf life. The cell suspension used to make the antigen layer is very weak, making a single cell layer across the bottom of the well. This makes the serum:cell ratio is much greater making the test very sensitive to weak antibodies. Capture-R Reagents o Capture-R Ready Screen, contains three cells with membranes from the separate group O donors. Also includes a control well. o Capture-R Ready ID Panel, used after Capture-R Ready screen is done. Same method, but instead uses 14 cells for specific antibody ID. o Capture-R LISS, low ionic strength solution that enhances the Ab-Ag binding in the test. o Capture-R Indicator Red Cells, group O RBCs coated with monoclonal Anti-IgG antibodies needed to detect IgG in patient serum. o pHix (Phosphate Buffer), produces a pH of 6.9 – 7.2 in the test well which is optimal for the Capture assay.

MLSC-3200, Transfusion Science -

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Step 1, strips come in a foil pouch. Open up the pouch and ensure that the moisture indicator is blue (valid). Once validated, label the test wells and add two drops of LISS (purple solution) to speed up the reaction and hydrate the antigen monolayer. Step 2, using a transfer pipette add one drop of patient plasma/serum to all wells. Quality assurance is done when wells change from purple to blue upon adding patient sample. Step 3, micro titre wells are incubated at 37 degrees for a minimum of 20 minutes. Allows the Ab-Ag reactions to occur. Step 4, wells are washed in an automated washer for less than 20 seconds per test strip. Removes paraproteins, rouleaux and macro/cryoglobulins. Hemolyzed, lipemic and icteric samples can be used for this test. Step 5, one drop of indicator cells is added to each well. Step 6, centrifuge the wells for 2 minutes. Analyze wells for adherence after centrifugation. Result Interpretation, capture is based on adherence and not agglutination. Grading is similar to the tube method grading system. Negative reactions show tightly defined RBC buttons showing no adherence. Positive reactions show localized (weak) to disperse (strong) covering of antigen layer with Capture cells showing adherence.

Solid-Phase Protein A Technology -

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IgG antibodies are detected using microplate wells coated with protein A. Protein A is a component of the bacterial cell wall of Staph aureus and has a very strong affinity to the Fc region of most Ig classes, especially IgG. Used for compatibility tests, IgG sensitization on RBCs and weak D testing.

Sensitivity and Specificity -

SPRCA is more sensitive than CAT for detecting RBC alloantibodies. However, SPRCA also has a higher rate of non-specific reactions. CAT, SPRCA and Protein A can do all blood bank tests, but only CAT can do DAT and detect IgG/C3d....