COMPATIBILITY TESTING

Compatibility Testing Overview

• The serologic aspect of pre-transfusion testing is called compatibility testing.
• It includes every serologic aspect, beginning with donor blood and ending with the recipient blood sample.
• Key steps include identification of both the patient and donor, as well as collecting appropriate samples for testing.
• Testing encompasses both the donor blood sample and the patient sample.
• Reviewing past blood bank records is also a necessary component.
• Selection of appropriate donor units and crossmatching is performed.
• Re-identification of the patient occurs before the infusion of blood.

Crossmatching Types

• The major crossmatch involves mixing the patient's serum with the donor's red blood cells (RBCs).
• The minor crossmatch is the mixing the donor's plasma with the patient's RBCs-
• Because donor samples undergo screening for common antibodies, it is completely eliminated in most blood banks.

Interpreting Crossmatch Results

• Agglutination and hemolysis indicate an incompatible crossmatch.
• If both major and minor crossmatches are compatible, the blood can be released for transfusion.
• If both major and minor crossmatches are incompatible, the blood should not be released.
• In the instance of a major incompatibility and a minor compatibility, the blood must not be released.
• With a major compatible and a minor incompatibility, the blood is released as packed RBCs.

Sample Collection and Preparation

• Clerical errors resulting in incorrect ABO grouping are the major cause of transfusion-associated fatalities.
• Misidentification of the patient involved in the transfusion is the most common cause of clerical errors, which can lead to transfusion accidents.
• Hemolyzed samples cannot be used to test because hemolysis is caused by complement activation.
• Serum or plasma can be used for pre-transfusion testing, but most blood bank technologists prefer serum because plasma can cause small fibrin clots, which is difficult to distinguish from true agglutination.

Donor Sample Protocols

• Donor testing samples must be drawn when the full donor unit is collected.
• Donor and recipient samples should be stored for a minimum of 7 days after transfusion.
• Samples should be stoppered, refrigerated at 1-6°C, carefully labeled, and of adequate volume to allow for re-evaluation if the patient experiences an adverse reaction to the transfusion.

Selecting Donor Units

• Whenever possible, blood and blood components selected for transfusion should match the patient's own ABO and Rh group.
• If the patient's blood type is unavailable or if some other reason prevents its use, units selected must lack any antigen against which the patient has a significant antibody.

Compatibility Testing Process

• Patient samples require accurate identification and proper sample collection and handling.
• Reviewing past blood bank records and determining ABO and D type, antibody screen, and crossmatch is part of the process.
• Donor units need ABO and D type, antibody screen, and infectious disease testing.
• A double check of the unit's ABO and D typing label is also performed.
• For patient samples, tagging, inspecting, and issuing of blood products is performed.
• Accurate re-identification and monitoring of transfusion is necessary.
• Specimens are collected within 2-3 days of transfusion if a patient has been pregnant or transfused in the preceding months.
• Patient specimens and unit segments are retained at 1-6°C for 7 days after transfusion.

Allogeneic and Autologous Units

• Allogeneic units require ABO typing, Rh typing, antibody screening, and crossmatching.
• Autologous units require only blood typing.

ABO Grouping

• An individual's ABO blood group is determined by red cell grouping and serum grouping procedures.
• For antigen source, Forward Grouping uses patient's RBCs, and Reverse Grouping uses reagent's RBCs.
• As an antibody source, Forward Grouping uses reagent Anti-A and Anti-B and Reverse Grouping uses patient's serum.

Type-Specific vs. Type-Compatible

• Type-specific refers to something that is exact and matches a particular type only.
• For example: A+ blood receiving only A+ blood
• Means something can work with multiple types within a category, as long as they are compatible. Example: type O-negative blood is considered type-compatible because it can be given to people with any other blood type

Blood Components Compatibility

Red Blood Cells

• Blood Type O is the universal donor of red blood cells
• Blood Type AB is the universal recipient for red blood cells

Plasma

• Blood Type AB is the universal donor of plasma
• Blood Type O is the universal recipient of plasma

Rh Typing

• Used to determine the presence or absence of Rh D antigen on red blood cells.

Antihuman Globulin Testing

• It detects IgG and complement components C3b and C3d.

Direct AHG Test

• Detects in vivo antibody or complement coating of RBCs.

Indirect AHG Test

• Detects in vitro antibody coating of RBCs.

Antibody Screening

• This involves a reaction between patient serum and 2-3 reagent cells that have been phenotyped for multiple antigens.
• The purpose is to detect unexpected antibodies, like red blood cell antibodies other than expected anti-A and anti-B antibodies.
• Reagent antibody screening cells as the source of antigens.
• Patient's serum being the source of antibodies.
• Incidence of unexpected antibodies is 0.2-2%.

Mandated Pretransfusion Tests

• Tests must demonstrate clinically significant antibodies through 37°C incubation and an AHG test.
• This refers to the antibodies that are reactive at 37°C and/or in antihuman globulin test.
• Can cause transfusion reaction or unacceptably short survival of the transfused red cells.
• IgG-sensitized cells are used to confirm negative AHG reactions.

Antibody Identification/Panel Testing

• This more definitive test determines if antibodies are in the serum when the antibody screen is positive.
• Reagent antibody panel cells (10-16 cells) are the antigen source.
• The patient's serum is the antibody source.

Terminology for Exclusion Method of Antibody Detection

ANTIGENS

• D: Denotes the presence or absence of the D antigen in the Rh blood group system.
• K: Represents the Kell antigen.
• Fy: Refers to the Duffy (Fya) antigen.
• Jk: Indicates the Kidd (Jka) antigen.
• S: Is the S antigen in the MNS blood group system.
• Lu: Relates to the Lutheran (Lua) antigen.

PHASES

• IS(Immediate Spin): The immediate spin phase, where reactions are observed immediately after centrifuging the sample.
• 37C(37 degrees Celsius): Reactions are assessed after incubation at 37 degrees Celsius.
• AHG (Anti-Human Globulin): The phase where Anti-Human Globulin is added to detect antibodies bound to red blood cells.(Coombs Phase)

Categories for Cell-by-Cell Antibody Detection

These elements are related to the cell-by-cell method for identifying antibodies, which is a process used in blood banking and immunohematology to identify the specific antibodies present in a patient's serum.

• Cell # (Cell Number): Represents individual cells used in the antibody identification panel.
• Auto (Autocontrol): Tests patient's serum with their own red blood cells to detect autoantibodies. Useful for determining if a positive reaction is due to antibodies against self.
• Identified Antibodies are the antibodies that have been specifically identified through the testing process.

Crossmatching Purpose

• It is done to determines the compatibility of donor RBCs with the recipient's blood.
• It uses methods that demonstrate ABO incompatibility and clinically significant antibodies.

Crossmatching Constituents

• A Major crossmatch requires the patient serum to react against donor red blood cell antigens.
• A Minor crossmatch requires donor serum to react against patient red blood cell antigens.

Types of Crossmatch

Immediate Spin

• Done using only an IS recipient serum and donor RBCs only.
• Performed if recipient does not have and never had clinically significant antibody.
• Determines AB incompatibility.
• Antibody screen carried through AHG must be performed and must be negative.

Antiglobulin

• Done with recipient serum and donor RBCs rested through AHG.
• Used if recipient has or had clinically significant antibody.

Computer

• An electronic check of donor ABO and Rh type; and recipient ABO and Rh type

Abbreviated

• A type and screen coupled with immediate spin crossmatch

Broad Spectrum

• A crossmatching method that includes three phases:
  • Immediate spin (IS) phase
  • Thermophase or 37°C phase
  • Antihuman globulin (AHG) phase

Interpretation of Compatibility Tests

• POS indicates a positive antibody screen, while NEG means the test was negative
• The major crossmatch category is for the results of the patient serum reacting against donor RBCs.

Antibodies in the Screening but not RBC Donor Cells

• Possible to transfuse after phenotyping the donor to confirm compatibility

Negligible Antibodies and Low Incidence Antigen

• Do not transfuse

Antibodies in Donor and Screening Receptor Cells

• Do not transfuse

No Antibodies Detected

• Transfusion is possible

Compatibility Tests

• Autocontrol has the recipients serum reacting with their own red blood cell antigens

Phenotype Frequencies of RBC Units

• C Positive = 70%
• K Positive = 9%
• Jka Positive = 57%
• Can be used to perform compatibility testing

Potentiating media

• Used to increase the rate and degree of antigen-antibody complex bonding

LOW IONIC STRENGTH SOLUTION (LISS)

• Made from 0.2% NaCi in glycine
• Increases antibody uptake

BOVINE ALBUMIN (22% OR 30%)

• Makes sensitized cells get closer together to form agglutination lattices

POLYETHYLENE GLYCOL (PEG) ADDITIVE

• Concentrates antibodies and creates solution of low ionic property to increase antibody uptake

POLYBRENE

• A positively charged polymer
• It reduces the zeta potential by getting rid of the reduction of negative charge in the red cell

PROTEOLYTIC ENZYMES

• Commonly used enzymes include papain, ficin, trypsin and bromelin

Special Serologic Techniques

ENZYME TECHNIQUE

• Use of enzyme-treated RBCs to enhance or remove reactivity of some antibody specificities
• Enhanced are: Rh, Lewis, Kidd, Pi, I, ABO
• Destroyed are: M, N, S, Duffy, Xga

ELUTION

• Technique used to dissociate IgG antibodies from sensitized RBCs
• Intact RBC antibody removal uses buffers to remove the antibody from the RBC without destroying it
• Digitonin releases the antibody by destroying the RBCs
• Lui freeze-thaw is used to remove IgM antibodies (usually A or B) present on newborn RBCs

ADSORPTION

• A process of removing antibody from serum by combining a serum sample with appropriate RBCs

COLD AUTOADSORPTION

• Incubate the patient's serum and cells at 4°C for 30-60 minutes

WARM AUTOADSORPTION

• Incubate the patient's serum and cells at 4°C for 30-60 minutes
• Remove serum and use serum for panel to test for alloantibody

AUTOADSORPTION

• Can be performed if patient was NOT transfused within the past 3 months
• If patient was transfused within the past 3 months, use allogeneic cells lacking the same antigens as the patient for adsorption

NEUTRALIZATION

• Uses soluble antigen to inhibit the reactivity of certain antibodies in hemagglutination assays

Anti-P1

• Uses 0.2% Naci in glycine; to increases antibody uptake

Anti-Lewis

• Uses neutralized saliva, serum or plasma

Anti-Chido and anti-Rodgers

• Uses neutralized serum or plasma

Anti-Sda

• Uses neutralized urine

Anti-l

• Uses neutralized human breast milk

CHEMICALS TO ALTER ANTIGEN EXPRESSION

• Proteolytic enzymes such as papain and ficin
• 0.2 M dithiothreitol (DTT)
• ZZAP
• Chloroquine diphosphate (CDP)
• 2-aminoethylisothiouronium bromide (AET or 2-AET)

Technologies in Blood Banking:

TUBE TESTING

• Method uses glass tubes; observe tubes for agglutination or hemolysis

Gel Technology

• Method Uses a plastic microtube containing dextran acrylamide gel that is centrifuged.

• Used for ABO forward and reverse grouping, Rh typing, direct antiglobulin testing, antibody screening, antibody identification and compatibility testing

  -Positive reaction: -Agglutinated RBCs suspended in gel -The position reflects the reaction strength.

  -Negative reaction: -A cluster of unagglutinated RBCs can be seen on the tube’s bottom’

#####ADVANTAGES: - Decreased sample volume needed for testing - Enhanced sensitivity and specificity.

#####DISADVANTAGES: -Need a special incubator and centrifuge -Need specific pipettes

###Agllutination Reaction in the Gel Test ####+

• The red agglutinates can be seen on the lower end of the tube. These reactions are weak

####Mixed Field

• A layer of red aggregate is present at the the top, while agglutinates can be seen at the the bottom the tube.

####Negative

• The red blood is collected at the bottom with no clumping

###Agglutination Reaction in the Gel Test ####4+

• A very apparent dark bands of agglutinates can be seen at the top of the gel

####3+

• The top is heavier, the band is ticker with more agglutinates are slightly lower.

####2+

• The red blood cell clumps are scattered throughout the tube

SOLID PHASE TECHNOLOGY

• Principle: Test reactant (antigen or antibody) is connected to a solid structure (usually a microtiter well)
• Application: To screen antibody or identify antibody or check compatibility

Solid-phase red cell adherence (SPRCA):

Microplate containing RBC membranes bound to wells -Positive indicator is well adherence

• Negative well contains button of RBs at the bottom

AFFINITY COLUMN TECHNOLOGY (GAMMA REACT)

Principle of affinity adherence of IgG sensitized erythrocytes to an immunologically active matrix Used for antibody screening, antibody identification, and compatibility testing

###. LUMINEX-BASED ASSAY Bead-based assay that uses fluorescence and flow cytometry to test for platelet/HA antibodies