MLS 411

Define antigen and antibody

Antigen: Foreign substance that can trigger an immune response

Antibody: Protein secreted by plasma that specifically bind to an antigen 

What is complement?

A series of 25 serum proteins responsible for

• Cell lysis

• Opsonization

• Mediation of inflammation

When complete: pore forms in RBC, causing intravascular hemolysis

What is zeta potential?

Electric charge difference between RBC surface and surrounding ionic cloud. Keeps RBCs separated and prevents agglutination. Lower zeta potential allows agglutination

What is dosage?

Some antibodies react more strongly when an antigen is present in a double dose (homozygous inheritance) than in a single dose (heterozygous inheritance)

Explain the process of agglutination, as it applies to immunohematology.

When corresponding antibodies bind to antigens on the surface of RBCs, the Fc regions become close in proximity, causing them to clump

Discuss the conditions that must exist to see hemolysis as a positive endpoint in immunohematology tests

• Must be complement-binding antibodies (IgM or close-paired IgG)

• Fresh serum

• Presence of free Ca²⁺ and Mg²⁺ ions

• Corresponding target antigen on RBC surface

Discuss the clinical significance of the Rh system as applied to transfusion medicine

Rh antigens are integral transmembrane proteins only found on the surface of RBCs. They play a role in transfusion medicine because they trigger a strong immune response if there is incompatibility between a recipient and donor.

List the major antigens of the Rh system using Fisher-Race terminology.

D, C, c, E, e

Describe the inheritance of Rh genes.

Discuss the structure and immunogenicity of Rh antigens.

• Rh antigens are integral transmembrane proteins only found on the surface of RBCs

• Highly immunogenic

• Well developed at birth

• Display antigen variability - can be weak/strong

Differentiate the three mechanisms that result in the presence of the weak D antigen.

Discuss situations when weak D testing would be appropriate

1. If testing with anti-D at the IS phase of testing is negative, incubate at 37C and test with AHG reagent to test again

2. Blood, organ, tissue donors

3. Rh-negative infants born to Rh-negative mothers (to see if the baby is positive)

4. Individuals who historically typed as Rh-positive, but currently type as Rh-negative

Compare Rh and ABO antibodies based on:

1. Type of antibody

2. Phase of reactivity

3. Ability to activate complement

4. Ability to cause HTR (hemolytic transfusion reaction)

5. Ability to cause HDFN (hemolytic disease of the fetus and newborn)

6. Exhibits dosage

List acceptable specimens for Rh type determinations.

EDTA or clot tube

Explain the principle of Rh testing, including the weak D test

Rh Testing

• Performed at the IS phase, where patient RBCs have anti-D added to them

• After centrifugation, the presence of agglutination = a positive test

• An inert control (negative result) is always performed (if performing JUST Rh typing)

  • Can use: Rh-hr control, 6% bovine serum albumin (BSA), or DAT

  • Control not needed if ABO/Rh typing is performed together, unless all reactions are positive (AB+)

Weak D Testing

• If the Rh test result is negative at the IS phase, then the sample is incubated at 37C

• Sample is washed multiple times with saline to rid of any unbound antibodies

• AHG reagent is added to the sample and spun

• Agglutination = Weak D positive = Rh positive

List the order of immunogenicity of the Rh antigens from most to least immunogenic

D > c > E > C > e

Discuss the significance of the ABO system as applied to transfusion medicine

The ABO system is the most critical blood classification in transfusion medicine.

Matching donor and recipient ABO types prevents acute hemolytic transfusion reactions, where the recipient's immune system attacks and destroys incompatible donor red blood cells, which can be fatal.

List the most common to least common ABO types

O > A > B > AB

Explain the inheritance of the A, B and H genes

List the concentration of H substance in each ABO phenotype from most to least

O > B > A > AB

What genes are needed to express ABO antigens?

Hh, ABO, Sese (secretor)

Codes for transferases that add immunodominant sugars that form the antigen

Outline the development of A, B and H antigens including the specific transferases and terminal sugars

List acceptable specimens for ABO grouping determinations.

EDTA or clot tube

Explain the principle of ABO testing

ABO Testing involves forward and reverse typing

Reagents used for monoclonal, which are hybridoma-derived

Testing methods: tube, microtiter, gel method

Forward Typing

• Patient RBCs are tested with antisera (Anti-A, Anti-B which contains antibodies) to detect antigens

  • Anti A,B is used only sometimes

• Agglutination = positive test

Reverse Typing

• Patient plasma is tested with known reagent red cells (A₁ and B) to detect antibodies

• Agglutination = positive test

Inert Control: If a patient’s results are AB, then an inert control must be used (6% BSA)

• 6% BSA is a protein base which mimics antisera but doesn’t have the antibody

• MUST yield a negative result for the AB result to be valid

What else can serve as a positive endpoint in immunohematology tests instead of agglutination?

Hemolysis

Conditions:

• Complement activation & complement-fixing antibodies (IgG)

• Fresh serum

• Antigen presence

Describe the phases of testing

Describe the antiglobulin reaction/tests

The antiglobulin reaction (Coombs test) detects non-agglutinating antibodies or complement proteins bound to red blood cells (RBCs).

Compare polyspecific vs monospecific AHG reagent

Polyspecific AHG = contains both anti-IgG + anti-C3

• Used for screening during IAT/DAT

• Created by blending/mixing rabbit polyclonal or mouse monoclonal anti-IgG with mouse monoclonal anti-C3

Monospecific AHG contains either anti-IgG / anti-C3

• Used for identification after a positive IAT/DAT

• Created by isolating or preparing individual antibody specificities (ex: utilizing monoclonal hybridoma cells)

What is used as controls during the antiglobulin test?

Performed when AHG test is negative (no agglutination)

Check cells (IgG or C3-coated RBCs)

• Must yield a positive reaction for a valid result

• If negative, test must be fully repeated

Discuss how the following factors influence antigen-antibody reactions in immunohematology tests:

• Antigen/ Antibody ratio

• Temperature

• Reaction time/Incubation

• Ionic strength of test medium

• Number and position of antigens

• pH

Antigen/ Antibody ratio

• Best when in equal amounts; if not, then lattice can’t be formed

• Prozone: excess antibody

• Postzone: excess antigen

Temperature

• IgM: 4-25C (wide thermal amplitude)

• IgG: 36-38C

Reaction time/Incubation

• IgM: requires little to no incubation

• IgG: requires incubation at 37C

• Too long incubation = antibody dissociation

• Too short incubation = insufficient sensitization

Ionic strength of test medium

• Want low zeta potential, ionic strength, and shielding effect to allow antibodies to sensitize RBC and decrease distance between the RBCs

• Add potentiators to the medium for this (Albumin, LISS, PEG)

Number and position of antigens

• Surface-exposed antigens react more readily

• Homozygous/double-dose antigen expression generally produce stronger reactions than a heterozygous/single-dose

pH

• Physiological pH: 6.5 - 7.5

• Adjusting pH can enhance or decrease some reactivities

Discuss the action of the following potentiators:

• Albumin

• Low Ionic Strength Solution

• Polyethylene Glycol

Albumin

• Reduces zeta potential

• Incubation: 15-30 min

Low Ionic Strength Solution (LISS)

• Lowers zeta potential and shielding effect, promoting antibody uptake onto RBCs

• Incubation: 15-20 mins

Polyethylene Glycol (PEG):

• Same as LISS, removes water from test system, concentrating antibodies

• Incubation: 15-30 mins

Compare IgM and IgG

Hemolysis can only be observed in what type of sample?

Serum (red or gold top)

EDTA tubes bind and remove Ca²⁺ and Mg²⁺, which are needed to activate complement

Antithetical antigens

Antithetical antigens are two different antigens encoded by different alleles of the same gene.

Immunogenicity

Ability of an antigen to stimulate antibody production

• Factors include: non-human antigen sources, host tolerance

The more foreign an antigen, the more likely an antigen-___ individual will make an antibody if exposed

negative

Alloantibody

Antibody that attacks foreign RBC antigens

Autoantibody

Antibody that attacks self RBCs

Who needs an antibody screen?

1. Pregnant (prenatal patients): screens for maternal IgG which could cross the placenta and attach to fetal RBC antigens

2. Pre-Transfusion Patients: if the transfusion recipient has a clinically significant, then the donor unit must lack the corresponding antigen

3. Blood, Organ, and Tissue Donors: donors that have alloantibodies in their plasma could passively transfer that antibody to the recipient

The antibody screen is an application of what method?

Indirect Agglutination Test (IAT)

• Patient plasma is combined with a set of RBCs (screen cells)

  • Positive: if the antibody is present in plasma and the screen cells have the antigen, agglutination/hemolysis occurs

  • Negative: Add check cells

• Performed during AHG phase to bridge sensitized RBCs

What are screening cells (used for antibody screens) composed of?

• 2-4 vials of RBC suspensions in a preservative, which inhibits hemolysis and preserves Ag expression

• Group O (always); Rh negative and Rh positive

• Tube testing: 2-5% suspension; Gel 0.8%

• Homozygous/double-dose of antigens in other major blood group systems

Each set of screen cells comes with an antigram: profile/matrix that lists Ag present on each reagent RBC 

What specimens are used for antibody screens?

• EDTA (specimen of choice)

• Clot tubes

• No gel tubes

• Complement-dependent antibodies

  • Only detected in serum specimens

Describe the phases of testing during an antibody screening

1. IS (Optional)

   1. Detects cold reacting IgM antibodies 

   2. Often omitted to limit detection of clinically insignificant antibodies 

2. 37C (Required) 

   1. 37C required for RBC sensitization 

   2. Enhancement media can be used (Album, LISS, PEG) 

   3. Screen cells may or may not be read for agglutination or hemolysis at this phase 

   4. Strong IgG antibodies can be detected, some cold reacting antibodies (wide temp amplitude) 

3. AHG (Required) 

   1. Detects clinically significant IgG antibodies

   2. Can also detect insignificant IgM antibodies that can activate complement (if using serum/polyAHG)

4. CC (Check Cell Contro - if negative AHG) 

   1. Must see macroscopic agglutination (positive result) 

   2. Proves: AHG reagent was added and adequate, and the wash step was adequate to remove any unbound antibodies

Compare and contrast the following methods for antibody detection and identification:

• Gel (microcolumn agglutination)

• Solid phase adherence

• Gel Technology (Column Agglutination): Uses microtubes pre-filled with a dextran-acrylamide gel matrix. Patient serum/plasma and reagent RBCs are added to the top of the column and centrifuged. If antibodies are present, they bind to the cells and form agglutinates that get trapped in the gel. The denser the agglutinate, the higher up the cell stays in the gel

• Solid Phase (Capture-R) Reagent RBC antigens are bound to the bottom of microwells. Patient plasma and indicator RBCs (coated with anti-IgG) are added. If antibodies are present, they attach to the antigens on the well wall, creating a diffuse "carpet" of RBCs (positive). If no antibodies exist, the cells settle into a distinct, tight button at the bottom of the well (negative)

What are limitations of the antibody screen?

• Will not detect ABO incompatibility 

• Will not detect antibodies to antigens that are not present on the screen cells 

• May not detect antibodies exhibiting dosage 

• May not detect antibodies that are too low in titer

Discuss quality control requirements for reagents used in the antibody screen.

• For prenatal and potential transfusion recipients: Each vial must be tested separately 

• For donors: can use 2 screening cells that have been pooled into 1 vial 

• QC performed every day with plasma or commercial control material that contains antibodies

T/F: Autoantibodies can also be detected in an antibody screen because they can react equally well with all screen cells; can also mask the presence of alloantibodies

True

If an autocontrol is included with an antibody screen

• Patient plasma tested with patient RBCs in all phases alongside the screen

• If AC is reactive = indicative of autoantibodies present

Rosenfield Nomenclature

Assigns a number to each antigen in the system and uses letter combinations to represent the blood group system (Ex: Rh:1)

ISBT Nomenclature

Each antigen is assigned a unique 6-digit number (first 3 digits = blood group system, last 3 = antigen

High frequency antigen

Occurs in 99.9% or more of the population 

Low frequency antigen

Occurs in 10% or less of the population

Outline the antibody identification steps

 

ANTIBODY IDENTIFICATION STEPS
Identification	1. Positive Antibody Screen
	2. Antibody ID Panel	Panel made of:  8-20 Group O RBCs with varied antigen expression  Diverse cell phenotypes to establish a unique pattern of reactivity  Contains heterozygous AND homozygous antigen expression Each lot includes an antigram, which is lot-specific and identifies rare cells (low and high-incidence Ag)
	3. Selected cells or other tools	If panel is inconclusive:  Test additional cells from other panels using the same method  OR retest original panel with a different method
Confirmation	4. Probability	Used to confirm if ID’d correct antibody; ensures reactions are due to the antibody not random chance Follow 95% confidence standards  Confirmation requires:  3 antigen-positive cells - reactive 3 antigen-negative cells - nonreactive
	5. Antigen Typing	Utilize Landsteiner’s Law (must lack the antigen for the antibody identified)  Use commercial antisera with patient RBCs  Want a negative result  If positive Ab ID may be incorrect Patient may have autoantibody  Possible mixed RBC population  Positive and negative controls run once daily  Choosing negative control: select RBCs that lack the antigen  Choosing positive control: select RBC with double dose of antigen
Autocontrol Testing		Not required by regulation; DAT may be used instead  Used at each phase used for panel cell testing (can be run with panel or screen)  Uses 2-5% patient RBCs mixed with patient plasma  Helps detect autoantibodies and newly forming alloantibodies (recent transfusion - sample contains mixed RBC populations of patient and donor; new alloantibody reacts with donor RBCs but not patient’s own)  If DAT is used DAT will be positive with AHG; AC will be positive

Explain the importance of patient history when evaluating antibody screen and panel results.

• History of Transfusion - important!

• History of antibodies

• Transplant

• Pregnancy

• Medications

• Diagnosis

• Ethnicity