Transfusion - Module 1, Part 3

What are the eight methods used to enhance in vitro antigen-antibody reactions?

Protein media, Plasma/Cell ratio, Proteolytic enzymes, Anti Human Globulin, Low ionic strength solutions, Polyethylene Glycol/Polybrene (PEG), Gel test, and Others.

What concentration of bovine albumin is added as a protein media to enhance reactions?

22%.

How does 22% bovine albumin reduce zeta potential?

Albumin molecules are amphoteric (+ and - charges) and react with ions in the suspending medium, decreasing repulsion between RBCs.

Why is protein media considered the least sensitive method of enhancing Ag/Ab reactions?

It may allow agglutination of IgG sensitized RBCs, but it is the least sensitive method.

How does increasing the plasma/cell ratio enhance Ag/Ab reactions?

It may increase the number of Ag/Ab complexes.

What happens if too much antibody is present when increasing the plasma/cell ratio?

It may result in prozone (a false negative).

What are five examples of proteolytic enzymes used to enhance Ag/Ab reactions?

Ficin, Papain, Pepsin, Bromelin, and Trypsin.

How do proteolytic enzymes enhance Ag/Ab reactions?

They release sialic acid residue from the RBC membrane (decreasing zeta potential) and remove surface glycoproteins, making antigens more available to antibodies.

What is the consequence of over-treating red cells with proteolytic enzymes?

False positive reactions.

Which antigens are denatured by proteolytic enzymes, making their antibodies undetectable?

M, N, S, and Fy antigens.

What is the one-stage method for using proteolytic enzymes?

Patient plasma + Test Cell + Enzyme are added together, then read.

What is the two-stage method for using proteolytic enzymes?

Incubate Test cells + Enzyme, remove excess enzyme, then add patient plasma and read.

What are the alternative names for Antihuman Globulin (AHG)?

AHS, antiglobulin, or Coomb's reagent.

What components make up the AHG reagent?

Anti-IgG and anti-C3.

What is the function of AHG reagent?

It bridges the gap between sensitized red cells (IgG or complement attached) to allow lattice formation and visible agglutination.

Low Ionic Strength Solutions (LISS) decrease zeta potential by doing what?

Removing or decreasing positive charges, allowing negatively charged RBCs to more easily combine with positively charged antibodies.

What can happen if the ionic strength of LISS is too low?

False positive reactions may occur.

What is the function of Polyethylene Glycol (PEG) or Polybrene?

Macromolecule additives used with LISS to bring RBCs closer together.

How does the Gel Test (MTS, CAPTURE) work?

Plasma and cells are added to an upper reaction chamber and centrifuged; agglutinates are trapped at different levels in a gel column (larger at the top, negative goes to bottom).

Who discovered the principle of the AHG test and in what year?

Dr. Robin Coombs in 1945.

What is the importance of the AHG test in transfusion services?

It is second in importance only to the ABO blood group.

How is Polyspecific AHG prepared?

Rabbits are injected with human serum containing IgG and complement, producing Anti-IgG and anti-complement, which are purified and pooled.

How is Monospecific AHG prepared?

Anti-IgG or anti-C3d are prepared separately.

What does the Direct Antiglobulin Test (DAT) detect?

In vivo sensitization of red cells (cells coated with IgG and/or complement in the body).

What does the Indirect Antiglobulin Test (IAT) detect?

In vitro sensitization of red cells following incubation of plasma and cells at 37°C.

What is the first application of the DAT regarding pregnancy?

Provides evidence of Hemolytic Disease of the Newborn (HDN).

How does HDN lead to a positive DAT in a newborn?

Mother forms IgG against fetal antigen, crosses placenta, attaches to fetal RBCs causing destruction, resulting in a positive DAT.

What does a positive DAT indicate following a blood transfusion?

Detects a possible transfusion reaction (patient's Ab destroying transfused cells).

What does a positive DAT indicate in Autoimmune Hemolytic Anemia?

The body fails to recognize "self" and produces Abs against its own RBC antigens.

What does a positive DAT indicate in drug sensitization?

A drug coats RBCs, making them appear abnormal, and an antibody attaches to the drug-coated cells.

Why is the Rh control run?

Anti-D reagent and Rh control are high protein reagents; sensitized cells may spontaneously agglutinate in them.

What must be done if a patient's Rh control is positive?

The Rh type is NOT valid and must be repeated using low protein reagents.

Why does a sensitized patient's RBCs invalidate red cell typing by AHG?

If RBCs are already sensitized with IgG/complement, ANY AHG test will be positive and therefore invalid.

What is the best sample type to perform a DAT to prevent non-specific complement uptake?

EDTA red cells.

What is the procedure for the DAT?

Add 1 drop 5% patient RBCs + 2 drops AHG, centrifuge 30s, resuspend, examine microscopically (incubate 5-10m at R.T. if negative and using polyspecific AHG, then re-spin and read).

In the DAT procedure, what is added to all negative tests?

One drop of control cells (CC cells).

What are the three applications of the Indirect Antiglobulin Test (IAT)?

Detection of alloantibodies, compatibility testing, and antigen phenotyping of red blood cells.

What is the purpose of pre-transfusion IAT testing?

To detect clinically significant alloantibodies in patient serum that may react with transfused red cell antigens.

What is the purpose of prenatal IAT testing?

To detect clinically significant alloantibodies in Mom's serum that may react with fetal antigens (HDN).

Can you perform an antigen phenotype (e.g., K phenotyping) if the patient has a positive DAT?

No, the phenotyping would be invalid.

What is the procedure for an Antibody Screen (IAT)?

Add 1 drop reagent cells (SCI/SCII) + 6 drops patient plasma, incubate 37°C for 15-60 min, wash 3-4x, add 2 drops AHG, centrifuge 30s, resuspend, examine.

What are the five reasons red cells are washed in AHG testing?

To remove trace plasma proteins, fragile cells, soluble blood group substances, anticoagulant, and commercial reagent solutions.

Why must unbound plasma proteins be washed away in AHG testing?

They may neutralize the AHG reagent, resulting in a false negative reaction.

Why must fragile cells and stroma be washed away?

They might hemolyze and compete for antibody.

Why must soluble blood group substances be washed away?

They may neutralize the AHG reagent.

Why must anticoagulant be washed away?

It is anti-complementary.

What is the purpose of the control cells (CC cells) added to negative AHG tests?

To validate the test by proving AHG was added, is active, and cells were washed adequately.

What agglutination grade must be observed with CC cells for the test to be valid?

1+ to 2+ visible agglutination.

What three things does a positive CC cell control tell you?

AHG reagent was added, AHG reagent was active, and cells were washed adequately.

What four things does a positive CC cell control NOT tell you?

That incubation was long enough, that temperature was correct, that the correct sample was used, or that the original result was truly negative.

What is a common cause of a False Positive AHG reaction related to the test cells?

Use of test cells with a positive DAT in an IAT procedure.

What is a common cause of a False Positive AHG reaction related to storage?

Non-specific absorption of complement components from serum onto red cells when stored together at 4°C.

What is a common cause of a False Positive AHG reaction related to centrifugation?

Over centrifugation.

What is a common cause of a False Positive AHG reaction related to the AHG reagent?

Incomplete absorption of anti-species antibody, return of antibody activity, or presence of anti-transferrin contaminant.

What is a common cause of a False Negative AHG reaction related to washing?

Inadequate washing or prolonged washing/interruptions in the wash cycle (causing elution).

What is a common cause of a False Negative AHG reaction related to the AHG reagent?

Failure to add AHG or AHG contaminated.

What is a common cause of a False Negative AHG reaction related to the sample?

Old sample used with loss of complement activity.

What is a common cause of a False Negative AHG reaction related to centrifugation?

Under centrifugation (not enough time for Ag/Ab reaction) or over centrifugation (weak agglutination shaken away).

What are IgG Sensitized Red Cells (CC cells)?

Reagent red cells (usually group O Rh positive) weakly sensitized with IgG antibody (usually anti-D) used as a positive control.

What do IgG Sensitized Red Cells indicate?

The AHG reagent is capable of detecting weak IgG antibodies attached to red cells.

What are Complement Sensitized Red Cells used for?

To indicate the AHG reagent is capable of detecting complement components attached to red cells.

What is the negative control for AHG testing?

Unsensitized Red Cells.