Compatibility Testing - Lecture notes 1-2 PDF

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IMMUNO HEMAm o d u l e 12 - l a b n o t e sLESSON 1: Crossmatching _DISCUSSIONThere are wide variety of techniques available for crossmatching. Since some antibodies will react to certain methods, but not all by others, techniques should be chosen to include several incubations and mediums.Those tec...

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m o d u l e 12 - l a b n o t e s LESSON 1: Crossmatching

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DISCUSSION There are wide variety of techniques available for crossmatching. Since some antibodies will react to certain methods, but not all by others, techniques should be chosen to include several incubations and mediums.

7. Hemolysis or agglutination at any stage of the crossmatch indicates an incompatibility. 8. The blood banker should be familiar with the information on incubation, centrifugation, antiglobulin techniques and sources of error, and reading of agglutination. 9. All test tubes should be labeled before use with unit and recipient identification and an indication of the test performed, if appropriate.

Those techniques considered optimal for detection of hemolyzing and agglutinating antibodies include saline or serum at room temperature (18-25°C) for 15 to 30 minutes. Although the detection of agglutinating antibodies would be improved by incubation at 4°C for long period of time, any advantage gained by such a procedure is outweighed by finding ubiquitous cold agglutinins, usually not significant in transfusion therapy; and 4°C incubation is not recommended for routine crossmatching. The optimal method for detecting coating antibodies is the use of antiglobulin serum following incubation at 37°C for 15-30 minutes. This incubation may be carried out in a patenting medium of high dielectric constant, such as albumin, and is customarily read before washing and addition of antihuman globulin serum. Many technical factors must be considered in the performance of a crossmatch. These include: 1. Donor’s red cell for crossmatching should be obtained from a segment of tubing, integral with the container or from a pilot tube attached to a tamper proof fashion at the time of phlebotomy and not removed during processing or storage. 2. The cells used for crossmatching should be saline washed. 3. A 2 to 5% cell suspension is usually recommended. 4. Reaction tubes are generally 10 or 12 x 75mm. 5. Tubes should be examined for hemolysis against a white background before resuspension of centrifuged cells. (gently mix tube to check for hemolysis) 6. An optical aid such as magnifying lens, mirror or microscope is advised for the reading of agglutination.

VIDEO LECTURE NOTES! ● ● ● ● ● ●

Crossmatching is only a part of compatibility testing Crossmatching is the final check for pretransfusion testing Compatibility testing - series of test Donors red cell should be collected from a bloodbag’s segment of tubing (Spx) Job aid - posted on the walls/tables which contains procedures and reference ranges Request form can either be done electronically, written or oral format ○ Written format (hard copy) is recommended

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Two patient identifier required for the recipient (recipients first, middle, last name and ID number) to prevent patient’s misidentification ○ Specific component requested such as RBC, platelets, amount and any special requirement ○ Additional, include patient’s history ( to check for immune antibodies) In pretransfusion testing the ones involved are the patient and recipient Most hemolytic transfusion reactions happen because of labeling errors Plasma is highly recommended since incompletely clotted serum samples may contain small fibrin clots which can trap red cells into aggregates that can cause false (+) results Plasma can be obtained from EDTA Check if spx is hemolyzed or lipemic Pretransfusion samples must be stored at refrigerator temp. For at least 7 days after each transfusion (+) Ab screening then proceed to Ab identification (-) Ab screening, skipped identification then proceed to crossmatching

PRINCIPLE:

3. Incubate 15-30 minutes at room temperature. 4. Centrifuge (2500 rpm); examine for hemolysis and for agglutination with an optical aid; record result - macro and micro (R1) 5. Add 2-3 drops of 22% albumin, mix, centrifuge, record results. (R2) Incubate 15-30 minutes at 37°C. 6. Centrifuge immediately upon removing from the incubator. 7. Examine for hemolysis and agglutination with an optical aid and record result. (R3) 8. Wash 3 times with saline. After last wash, decant completely. Add 2 drops of antihuman globulin serum, mix. 9. Centrifuge, examine for agglutination with an optical aid, record result. (R4) 10. If no agglutination or hemolysis is seen in any phase, the crossmatch is compatible *** One tube crossmatch procedure in major crossmatch *** Uses only 1 test tube .

B) Two tube Crossmatch Procedure (Broad Spectrum Compatibility Test)

Cross matching is based on the principle of serological detection of any clinically significant irregular/unexpected antibodies in either donor or recipient’s blood. MATERIALS: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Test tube 10 or 12 x 75 mm Patient’s serum Magnifying lens/microscope Donor’s red cell Waterbath 22% bovine albumin Serofuge Anti-human globulin serum Saline solution

PROCEDURE A) One tube Crossmatch Procedure 1. Place 2 drops of recipient serum in a labeled tube. 2. Add 1 drop of a 2-5 percent saline suspension of donor cells. (tomato red)

1. Prepare a 2-5% cell suspension of donor’s red cell. 2. Place 2 drops of recipient’s serum in each of two labeled tubes. 3. Add 2 drops of 2-5% saline suspension of donor’s cell to each tube. 4. Place 2 drops of 22% bovine albumin I one tube, then mix. On the other tube, do not add 22% bovine albumin. 5. Centrifuge at 2500 r.p.m. for 1 minute. 6. Examine for agglutination and/or hemolysis with an optical aid; record results. (R1)

7. Incubate the two tubes at 37°C water bath for 15 minutes. 8. Remove the tube with albumin and centrifuge at 2500 r.p.m. for 1 minute. 9. Examine for agglutination and/or hemolysis with an optical aid; record results. (R2) 10. The contents of the second tube are washed 3 times in large volumes at the end of the last washing, decanting the saline completely. 11. Add 2 drops of anti-human serum to the sedimented cells. Mix and centrifuge at 2500 r.p.m. for 1 minute. 12. Examine for agglutination and/or hemolysis with an optical aid. Record result. (R3)

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Detects ABO incompatibility and detect most Ab against donor cells ● Cannot detect all ABO typing error and Rh typing errors ● Detect platelet and leukocyte Ab ● It will not detect immunization and will not ensure normal survival of RBC 2. Minor crossmatch ● involves donor’s plasma + patient’s red cell ● Replaced by Antibody screening

NOTE: 1. Steps 1-6 is the Protein Phase 2. Steps 7-9 is the Thermo Phase 3. Steps 10-12 is the Anti-Human Phase If no agglutination or hemolysis is seen in any phase, the crossmatch is compatible. *** Broad Spectrum Compatibility Testing ● ● ●

Usually it is the one performed All inclusive testing Has 3 phases 1. Immediate spin phase 2. Thermophase 3. AHG phase

1-TUBE VS 2-TUBE ● ● ● ● ●

One tube - w/ 4 reactions Two tube - w/ 3 reactions Two tube is faster and more convenient to use 4 tubes are used in total Autocontrol - detect autoantibodies ○ Patient serum ○ Patient red cell

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Usually* - reacts at 37C except for P since it is an IgM

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1st choice - give the specific blood type (type specific) 2nd choice - only happens when there is shortage or immediate need for transfusion Blood type O RBC - given at any choices (doesn’t contain Ag)

VIDEO LECTURE NOTES: Crossmatching ● ● ●

Final check and last step Determines compatibility of blood unit from donor with the recipient 2 procedures 1. Major crossmatch ● Involves patient’s serum + donor’s RBC

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Blood type AB - doesn’t contain any AB antibodies (Universal recipient)

NOTE: ● ● ●

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Whole blood - must be type specific Red blood cell/Granulocyte - must be compatible w/ recipient’s plasma Fresh frozen plasma - must be compatible w/ recipient’s red cell (to prevent Ag-Ab attachment) Platelets - all are acceptable Cryoprecipitated Hemolytic Factor - all are acceptable

CROSSMATCH INCOMPATIBILITiES 1. Incorrect ABO grouping of patient and donor 2. Alloantibody in patient’s serum reacting w/ donor’s RBC - when tested w/ autocontral (-) no agglutination means presence 3. Autoantibody on patient’s serum - (+) autocontrol w/ agglutination 4. Protein/antibody sensitization of donor’s RBC - positive in AHG phase since there is sensitization 5. Other abnormalities in patient’s serum 6. Contamination ● Inspect blood bag for hemolysis, discolored or there is clot formation ● All antisera must be refrigerated or stored at 2-6C when not in used (room temp. first before using) ● All reagents must be tested daily ● In routine testing, all blood typing should be carried out in TUBE METHOD except for bed side- slide testing is utilized ● Timing of centrifuge must be tested with a stopwatch ● Tachometer - used to check speed every 6 mos ● Waterbaths - are kept at 37C ● Blood bank refrigerator - must be between 1-6C (checked/monitored every 4 hrs) PRETRANSFUSION CASES

TESTING

IN

SPECIAL

● Group O (-) blood 2. Non-blood group specific blood 3. Plasma products - red cell should be compatible w/ the recipient 4. Intrauterine transfusions - often happens in infants w/ HDN (usually blood group O is given since Ab is still not detected) 5. Neonatal transfusions (0-4 mos. old) ● Testing red cells with Anti-A and Anti-B (only test required) ● Reverse typing is not used 6. Massive transfusions (8-10 units in less than 24 hrs or 4-5 units in 1 hr) ● Computer or Immediate spin crossmatch should be carried out in this transfusion DIFFERENT METHODS IN CROSSMATCHING 1. Abbreviate crossmatch ● Blood typing ● Immediate crossmatch ● Antibody screening ● Uses patient’s serum and donor’s red cells 2. Electronic crossmatch ● Uses machines and computers for massive transfusion 3. Emergency crossmatch ● O (-) packed red blood cell is given INTERPRETATION OF RESULTS ● ● ●

(+) Major, (+) minor = incompatible (-) Major, (+) minor = release blood w/ packed RBC only (+) Major, (-) minor = do not release blood

LESSON 2: Gel Test

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GEL TEST PRINCIPLE: Gel testing occurs in small columns filled with a viscous gel (dextran acrylamide gel). The RBCs and plasma being tested are added to the chamber at the top of the column and incubated, followed by centrifugation to try to force the RBCs through the gel to the bottom of the column. Gel matrix acts as a sieve VIDEO LECTURE NOTES

1. Emergency ● release of blood w/out crossmatching ● There should be a signed statement from requesting physician

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Major advantage is standardization RBC’s that are agglutinated will stop earlier in the gel than those that are not agglutinated

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Gel contains anti-IgG which binds to the IgG sensitized coated RBC which inhibits transport to the gel

MATERIALS: 1. 2. 3. 4.

Patient Plasma Donor's Red Cell Gel card Special equipments centrifuge

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incubator

and

In compatibility testing: ●

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NO AGGLUTINATION = NEGATIVE = COMPATIBLE ○ Forms pelet at the bottom of gel AGGLUTINATION = POSITIVE = INCOMPATIBLE ○ Ab is detected MF REACTION - evident

m o d u l e 13 - l a b n o t e s LESSON 1: Antibody Screening_____________ ● ●

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Very critical part of pre-transfusion testing Detect the presence of “unexpected” / “unusual” antibodies (aka Indirect Coomb’s Test) ○ Traditional method of Antibody Screening Unexpected antibodies - result of immunization prior exposure to RBC with such antigens (pregnancy, vaccination). Screen the donor’s plasma After screening, ANTIBODY PANEL is performed to identify the specific antibody present in the sample.

PRE-TRANSFUSION TESTING - Set of tests performed to prevent transfusion reaction in donor and the recipient. OBJECTIVE: To detect the presence of clinically significant antibodies (reactive at 37C / in AHG test) other than anti-A and anti-B in the patient’s/donor’s serum. DISCUSSION The occurrence of isoagglutinins, anti-A and anti-B, in the serum of an individual is a normal finding. However, the presence of antibodies to other blood group antigens is usually the result of immunization and the detection of these antibodies to other blood group antibodies is of obvious importance. This is of concern to the obstetrical patient in whom detection and identification of the antibody prior to delivery allows adequate time for preparations to be made for the possible transfusion of the newborn infant. The second instance in which sera are tested for atypical antibodies is in the case of crossmatch or the investigation of a transfusion reaction. In either case, the detection of the antibody is especially important so that only compatible blood will be selected for the patient. Also, the screening of sera from blood donors for the presence of antibodies is

increasingly utilized instead of a minor crossmatch. This donor-screening can be done at the same time as the other routine tests are performed on the donor blood and, in this way, valuable time required for crossmatch is held at a minimum. Bovine Albumin, 22% solution, is an ideal suspending medium for a variety of serological tests that are performed in the blood bank. The primary use of this medium is in the detection of blood group antibodies, as well as in the compatibility tests. Once antibodies have been detected, 22% Bovine Albumin may be used in the titration of those antibodies. In addition, this solution is used as a control of possible error in Rh typing. Bovine Albumin, 22% solution, is prepared by a unique, patented manufacturing process. The final product is adjusted to a pH of 7.5; the salt and protein concentrations are also adjusted within a narrow range. In addition, a proper ratio of alpha globulin to albumin is maintained to provide optimal avidity. When Bovine Albumin is used in conjunction with the antiglobulin (Coombs) test it will markedly enhance reactions in which antibodies of other blood group systems are involved. Rh antibodies are generally referred to either as (1) saline agglutinins, which agglutinate red cells in saline suspension; and (2) albumin agglutinins, which do not agglutinate saline suspended red cells, but will agglutinate the cells when they are suspended in albumin. Antibody detection tests – either for Rh or other atypical blood group antibodies are conveniently performed by employing SELECTOGEN Reagent Red Blood Cells (Human).

THREE REAGENTS USED FOR ANTIBODY SCREENING 1. RBC Reagent Cells ● Type O blood that contains the most common and clinically significant RBC antigens ● Set of 2-3 red cell suspensions

2. Enhancement Reagents ● 22% Bovine Serum Albumin (BSA) - Reduces repulsive forces between cells and enhances agglutination. - Incubation: 30 mins. ● Low Ionic Strength Solution (LISS) - Decrease zeta potential of RBC. - Incubation: 10-15 mins. ● Polyethylene Glycol (PEG) - Water-soluble linear polymer that potentiates antigen-antibody reaction. 3. AHG Reagent ● Polyspecific AHG is used. (Anti-IgG and Anti-C3b or C3d) MATERIALS 1. Test tube 2. Saline 3. Serofuge 4. 22% Bovine Albumin 5. Waterbath 6. Anti-Human serum 7. SELECTOGEN Reagent (Human) 8. Patient’s serum

Red

Cell

Part II. Thermophase 5. Mix all tubes and incubate for 15 minutes at 37°C. 6. Remove all tubes from the water bath. 7. Centrifuge at 2500 r.p.m. for 1 minute. Examine macroscopically for agglutination. If there is no agglutination or hemolysis in the “Saline” tubes, they may be discarded. Part III. AHG Phase 8. The cell-serum mixtures in the “Albumin” tubes are washed thoroughly with large volumes of saline. Perform this washing for a minimum of 3 times. Decant the saline completely after the last washing. 9. Add 2 drops of anti-human serum to the sedimented cells and mix well. 10. Centrifuge immediately for 1 minute at 2500 r.p.m. Examine for agglutination macroscopically. INTERPRETATION OF RESULTS ● Screen Cell (SC) ● Immediate Spin (IS) ● Autocontrol (AC): Px serum + Px RBC ● (-): No agglutination/hemolysis ● (+): Agglutination/hemolysis CELL

IS

37C

AHG

PROCEDURE

SC I

-

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Part I. Immediate Spin Phase 1. Add two drops of the serum to be tested to each of 4 test tubes. 2. Add 1 drop of unwashed SELECTOGEN I to 2 of the test tubes approximately labeled; add 1 drop of SELECTOGEN II to other 2 tubes. (For example, the test tubes might be labeled – using “S” for saline and “A” for albumin – as I-S, I-A, II-A,II-S). 3. Add 2 drops of Bovine Albumin, 22% solution to those labeled “A” 4. Centrifuge for 1 minute at 2500 r.p.m. Examine all tubes macroscopically for agglutination or hemolysis.

SC II

-

-

2+

AC

-

-

-

POSSIBLE INTERPRETATION: 1. There is a single alloantibody or there are 2 alloantibodies 2. Probably, IgG antibody because it reacts on AHG phase CELL

IS

37C

AHG

SC I

-

1+

3+

SC II

-

-

1+

AC

-

-

-

POSSIBLE INTERPRETATION: 1. There could be multiple antibodies 2. There could be a single antibody but there’s a dosage (Kidd, MN, Duffy) 3. Probably, IgG because both reacts in 37C and AHG phase CELL

IS

37C

AHG

SC I

1+

-

-

SC II

2+

-

-

AC

-

-

-

POSSIBLE INTERPRETATION: 1. There is a single or multiple antibodies since both reacted in Screen cell reagent 2. Probable an IgM antibody because it reacted on Immediate Spin phase CELL

IS

37C

AHG

SC I

+

-

+

SC II

+

+

+

AC

-

-

-

POSSIBLE INTERPRETATION: 1. There could be multiple antibodies both warm and cold antibodies because they both react in 37C, IS phase, and AHG phase. 2. Could be potent cold antibody binding complement in AHG phase (in cases of Cold Agglutinin Disease). CELL

IS

37C

AHG

SC I

-

-

+

SC II

-

-

+

AC

-

-

-

POSSIBLE INTERPRETATION: 1. There could be a single warm antibody 2. Antibody to high prevalence of antigens such as Kidd, Duffy 3. Complement binding by cold antibody that is not detected at IS phase

CELL

IS

37C

AHG

SC I

-

-

+

SC II

-

-

+

AC

-

-

+

POSSIBLE INTERPRETATION: 1. There could be warm antibodies 2. Warm antibodies / Warm autoantibodies 3. There could be transfusion reaction because autocontrol is (+) 4. Probable an IgG antibody because it reacted at AHG phase ANTIBODY PANEL TESTING / IDENTIFICATION ● Done after the antibody screening test ● Identify the specific clinically significant antibody that caused agglutination in the thermo and AHG phase of the screening ● Reagent Red cells: 11-20 RCS ○ Worksheets should never be interchanged with other set/lot STEPS IN ANTIBODY PANEL TESTING / IDENTIFICATION 1. Check the autocontrol. 2. Check the phase with the positive reaction. 3. In the phase with the posi...