Blood Compatibility Testing

Compatibility Testing

• Refers to the serologic aspect of pre-transfusion testing
• Encompasses every serologic aspect from donor blood to recipient blood sample

Key Steps in Compatibility Testing

• Identification of the patient and donor
• Collection of appropriate samples for testing
• Donor blood sample testing
• Patient sample testing and review of past blood bank records
• Selection of appropriate donor units
• Crossmatching
• Re-identification of the patient before blood infusion

Major Crossmatch

• Involves reacting patient's serum with donor's red blood cells (RBCs)

Minor Crossmatch

• Involves reacting donor's plasma with patient's RBCs
• Mostly eliminated due to prior screening of donor samples for prevalent antibodies

Incompatible Crossmatch Indicators

• Agglutination (clumping)
• Hemolysis (destruction of red blood cells)

Crossmatch Compatibility and Blood Release

• Release blood if major and minor crossmatches are compatible
• Do not release blood if major and minor crossmatches are incompatible
• Do not release blood if major crossmatch is incompatible, and the minor is compatible
• Release as packed RBCs, if the major is compatible, but the minor is incompatible

Sample Collection and Preparation

• Incorrect ABO grouping primarily results in transfusion fatalities due to clerical errors
• Patient misidentification is the prevailing cause of clerical errors, resulting in transfusion accidents
• Hemolyzed samples cannot be used for testing due to complement activation.
• Serum or plasma is acceptable for pre-transfusion testing
• Serum is preferred by most blood bank technologists
• Plasma may cause small fibrin clots, making differentiation from true agglutination difficult

Donor Sample Guidelines

• Donor testing samples must be taken when the full donor unit is drawn
• Store donor and recipient samples for at least 7 days post-transfusion
• Stopper and refrigerate samples at 1-6°C
• Ensure samples are carefully labeled and of adequate volume
• Needed for re-evaluation should the patient experience transfusion-related adverse effects

Donor Unit Selection

• As a standard, blood and its components should match the patient's ABO and Rh group for transfusion
• Select units lacking any antigen against which the patient has a significant antibody when the patient's blood type is unavailable

Compatibility Testing Processes

• Accurate patient identification is crucial
• Proper sample collection and handling are required
• Review of past blood bank records is necessary
• ABO and D typing, antibody screen, and crossmatch of patient samples are performed
• ABO and D typing, antibody screen, and infectious disease testing of donor samples are performed
• Double-checking the unit's ABO and D typing label
• Tagging, inspecting, and issuing blood products accurately
• Accurate reidentification and monitoring of transfusion

Specimen Collection Timing

• Collect specimens within 2-3 days of transfusion
• If the patient has been pregnant or transfused in the months prior to the procedure

Post-Transfusion Specimen Retention

• Retain patient specimens and unit segments at 1°-6°C for 7 days following transfusion

Allogeneic Unit Testing

• ABO typing
• Rh typing
• Antibody screening
• Crossmatching

Autologous Unit Testing

• Blood Typing

ABO Grouping Determination

• Determining the ABO group of red cells is done via red cell grouping and serum grouping
• Antigens source is from patient's RBCs (forward grouping) and reagent's RBCs (reverse grouping)
• Antibodies source is from reagent Anti-A and Anti-B (forward grouping) and patient's serum (reverse grouping)

Type-Specific Transfusion

• Using blood that is an exact match to the recipient’s type
• Example: A+ blood receiving only A+ blood

Type-Compatible Transfusion

• Using blood that is compatible with the recipient’s type
• Example: O-negative blood being given to any other blood type

Red Blood Cell Compatibility

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

Plasma Compatibility

• AB blood type is the universal donor of plasma
• O blood type is the universal recipient for plasma

Antihuman Globulin Testing

• Detects IgG and complement components C3b and C3d

Antibody Screening

• Involves reacting patient serum or plasma with 2-3 reagent cells phenotyped for multiple antigens
• Detects unexpected antibodies like red blood cell antibodies that are not anti-A or anti-B
• Incidence of unexpected antibodies is 0.2-2%
• Reagent antibody screening cells are the source of antigens
• Patient's serum is the source of antibodies

Current Requirements for Patient Samples Before Transfusion

• Clinically significant antibodies (tested using 37°C incubation and AHG test)
• Antibodies that react at 37°C and/or in the antihuman globulin test
• IgG-sensitized cells usage validates negative AHG reactions
• Transfusion reactions or unacceptably short survival of transfused red cells

Antibody Identification/Panel Testing

• Used if the antibody screen is positive to determine which antibodies are in the serum
• Reagent antibody panel cells are the antigen source (10-16 cells)
• Patient's serum is the antibody source

Crossmatch Definition

• Compatibility of donor RBCs is determined with the recipient's blood
• Detects ABO incompatibility
• Clinically significant antibodies detection

Crossmatching Components

• Can be computer crossmatched if the antibody screen is negative and antibodies haven't been detected before
• Plasma needs to be ABO compatible with the recipient's RBCs
• With Cryoprecipitate, all ABO groups are acceptable; Rh type is not considered
• With Platelets, all ABO groups are acceptable, recipient RBC compatibility is recommended, ABO-identical blood is preferred, RBC containing apheresis platelets need ABO compatibility and crossmatch

Parts of Crossmatch

Major Crossmatch

• Mixes the patient's serum with the donor's RBCs
• Donor cells are the source of antigens
• Recipient's serum is the source of antibodies

Minor Crossmatch

• Mixes the donor's serum with the patient's RBCs
• Donor cells are the source of antigens
• Recipient's serum is the source of antibodies

Types of Crossmatch

Immediate Spin

• Uses recipient serum and donor RBCs tested in IS only
• Performed if and only if the recipient never had and never had a clinically significant antibody
• It detects AB incompatibility
• Antibody screen carried through AHG must be negative

Antiglobulin

• Recipient serum and donor RBCs are tested through AHG
• Used if recipient has or had clinically significant antibody

Computer Crossmatch

• Comparing donor's ABO and Rh type to the recipient's ABO and Rh type
• Recipient cannot have or have ever had a clinically significant antibody

Abbreviated

• Combines type and screen with immediate spin crossmatch

Broad Spectrum

• Preferred method of crossmatching as it includes 3 phases:
  • Immediate spin phase
  • Thermophase or 37°C phase
  • Antihuman globulin phase

Interpretation of Compatibility Tests

Positive Antibody Screen; Negative Major Crossmatch

• Antibody reacts with an antigen on screening cells but not on donor cells
• A compatible transfusion may be possible, but the donor needs to be phenotyped to confirm compatibility.

Negative Antibody Screen; Positive Major Crossmatch

• Antibody reacts with a low-incidence antigen
• Donor cells test positive for DAT
• Technical error occurred
• NO Transfusion

Positive Antibody Screen; Positive Major Crossmatch

• Antibody reacts with an antigen on the donor cell and screening cell
• NO Transfusion

Negative Antibody Screen; Negative Major Crossmatch

• No antibody detected
• YES Transfusion

Blood Bank Inventory Example

A patient needs blood transfusion of RBC units has anti-C, anti-K and anti-Jka

- C Positive = 70%
- K Positive = 9%
- Jka Positive = 57%

Potentiating Media

• Low Ionic Strength Solution(LISS) has 0.2% NaCi in glycine; increases antibody uptake
• Bovine Albumin(22% or 30%) allows sensitized cells to come closer together to form agglutination lattices
• Polyethylene Glycol (PEG) additive concentrates antibodies and creates low-ionic solution to allow greater antibody uptake
• Polybrene is a positively charged polymer that reduces zeta potential by neutralizing the negative charge in red cell
• Proteolytic Enzymes include papain, ficin, trypsin and bromelin

Special Serologic Techniques

• Enzyme Technique uses enzyme-treated RBCs to enhance or remove reactivity of some antibody specificities
  • Enhanced: Rh, Lewis, Kidd, Pi, I, ABO
  • Destroyed: M, N, S, Duffy, Xga
• Elution technique used to dissociate IgG antibodies from sensitized RBCs
  • Whole RBC antibody removal uses buffers to remove the antibody from the RBC
  • Without destroying the RBC
  • 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 is the process of removing antibody from serum by combining a serum sample with appropriate RBCs - Cold Autoadsorption incubates the patient's serum and cells at 4°C for 30-60 minutes for 30-60 - Warm Autoadsorption incubates the patient's serum and cells at 4°C for 30-60 minutes, then removes serum and uses serum for panel to test for alloantibody - An autoadsorption can be performed if patient was NOT transfused within the past 3 months - If patient was transfused within the past 3months, use allogeneic cells lacking the same antigens as the patient for 'adsorption
• Neutralization uses soluble antigen to inhibit reactivity of certain antibodies in hemagglutination assays
  • Anti-P1: 0.2% NaCl in glycine; increases antibody uptake
  • Anti-Lewis is neutralized by saliva, serum or plasma
  • Anti-Chido & anti-Rodgers are neutralized by serum or plasma
  • Anti-Sda is neutralized by urine
  • Anti-I is neutralized by human breast milk
• Effect of Chemicals on 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)

Blood Banking Technologies

Tube Testing

• Glass test tubes used as reaction containers, observe for agglutination and/or hemolysis

Gel Technology

• Plastic microtube containing dextran acrylamide gel
• Controlled centrifugation of red cells through dextran-acrylamide gel and reagents in a microtube
• Used for:
  • ABO forward and reverse grouping
  • Rh typing
  • Direct antiglobulin testing
  • Antibody screening, identification and compatibility testing
  • Reactions:
  • Positive reaction - Agglutinated RBCs suspended in gel, With stronger reactions having larger agglutinates at the top
  • Negative reaction - Button of unagglutinated RBCs in bottom of microtube

ADVANTAGES

• Standardization
• Stability
• Decreased sample volume needed for testing
• Enhanced sensitivity and specificity

DISADVANTAGES

• Need special incubator and centrifuge
• Specific pipette for 25 microliter of serum or plasma and 50 microliter of 0.8% RCS

Agglutination Reaction in the Gel Test

• 1+ reaction - Red cell agglutinates predominantly observed in the lower half of the gel column with red cells also in the bottom,These reactions may be weak, with few agglutinates remaining in the gel area just above the red cell pellet in the bottom of the microtube
• Mixed Field - Layer of red cell agglutinates at the top of the gel column accompanied by a pellet of unagglutinated cells in the bottom of the microtube - Negative - Red cells forming a well-delineated pellet in the bottom of the microtube, The gel above the red cell pellet is clear and free of agglutinates
• 4+ - Solid band of agglutinated red cells at the top of the gel column, Usually no red cells are visible at the bottom of the microtube
• 3+ - Predominant amount of agglutinated red cells towards the top of the gel column with few agglutinates staggered below the thicker band, The majority of agglutinates are observed in the top half of the gel column
  • 2+ - Red cell agglutinates dispersed throughout the gel column with few agglutinates at the bottom of the microtube,Agglutinates should be distributed through the upper and lower halves of the gel

Solid-Phase Technology

Principle: Test reactant is bound to solid support before test is started Used for antibody screening, identification and compatibility testing Solid-phase red cell adherence (SPRCA) Reaction container: Microplate with RBC membranes bound to the surface of wells Positive Reaction: Indicator RBCs adhere diffusely to the surface of the well Negative Reaction: No adherence of RBCs, button of RBs in bottom of well Solid-phase protein A Solid-phase enzyme-linked immunosorbent assay (ELISA)

Affinity Column Technology (Gamma React)

Main principle: Affinity adherence of IgG sensitized erythrocytes to an immunologically active matrix Used for antibody screening, identification, and compatibility testing

Luminex-Based Assay

Bead-based assay using fluorescence and flow cytometry to test for platelet/HA antibodies