Lec4: Antibody Screen and D

AHG

• Anti-Human Globulin

• AHG is a prefix and describes the test

  • AHG Crossmatch

IAT

• Indirect Antiglobulin Test

• Patient Plasma + Donor Cells + AHG

• Indirect Coombs Test

DAT

• Direct Antiglobulin Test

  • Patient Cells + AHG

  • Direct Coombs Test

Antibody Screen

An IAT that is meant to look patient antibodies to common RBC antigens

AHG is made by injecting

rabbits with human IgG

Unexpected antibodies

Antibodies that is development ONLY

If you been exposed to a blood product

(Ex: transfusion, unclean IV drugs)

Clinically significant antibodies

Antibodies that can cause hemolysis 

These are the expected ones 

What are sources of alloummunization

• Transfusion

• Pregnancy

• Drugs

• Parasites

• Disease

IgM is stronger than IgG. So we use anti-

IgM for agglutination. IgG need two steps

What are the two stages of agglutination

• Sensitization

• Lattice formation

Sensitization

Antibody and Antigen are mixing and binding

Lattice Formation

Antibodies start cross-linking and binding to multiple red cells

Dectecting IgG antibodies steps (indirect)

1. Recipient’s serum is obtained containing antibodies (Ig’s)

2. Donor’s blood sample is added to the tube with serum

3. Recipient’s Ig’s that target the donor’s RBC form antibody-antigen complexes

4. Anti-human Ig’s (coombs antibodies) are added to the solution

5. Agglutination of RBC occurs, because human Ig’s are attached to RBC

IgM

IgG

Anti-IgG

Describe AHG antisera

• Monoclonal or polyclonal

• Directed at Ig’s, complement, whatever

• Originally from animals

• Now commercially made

• Depending on manufactuer, can be IgG or IgM

What are some potentiators (ubstance or drug that increases the strength, effectiveness, or duration of action of another substance)

• Temperature (at body temp 37C)

• Time

• Medium

• Concentration

• pH

• Enzymes (ish)

What is Prozone

Occurs when there are far too many antibodies relative to antigens. stopping proper cross-linking

• falsely low or false-negative test results

Equivalence

optimum proportions of antigen and antibody

Postzone

Antigen excess. A similar false-negative issue caused by an overwhelming excess of antigen rather than antibody.

Zeta potential

net negative electrical charge at the slipping plane of the cell membrane that causes erythrocytes to repel each other and stay separated in the bloodstream.

• What need to be overcome for agglutination

What are ways to enhance the agglutination (based on the lecture)

• Standard saline incubation 37C – 30-60 min

• LISS - Low Ionic Strength Saline

• 22% Albumin

• PEG - Polyethylene Glycol

• Combination of the above

  • Searching for the perfect combination of specificity and sensitivity

Describe LISS – Low Ionic Strength Saline

• 0.2% saline

• Decreases negative repulsion (Zeta Potential)

• Decreases incubation time to 5-15 min

Describe 22% Albumin

• Also decreases zeta potential

• Incubation time 15-60 min

Describe PEG – Polyethylene Glycol

• Decreases water between RBCs

• Decreased hydrophobic repulsive force

• Brings cells closer together

• Incubation time at 37C is 10-30 min

What are the Indirect Antiglobulin Test Principle

• Central question for IATs: does this patient have antibodies that could bind to RBCs?

• Patient sample (plasma) is the unknown while the screening cells are the known

• If antibodies are present, cells will be sensitized

• This test is in vitro and does not reflect patient status

Describe the tube method for Indirect Antiglobulin Test

• Patient plasma is incubated with screening cells in an enhancement media

• Cells are than washed to removed excess IgG

• AHG is added and sample is centrifuged

• Test is positive if cells agglutinate

• If no agglutination is seen, check cells are added and should agglutinate upon centrifugation

  • Check cells are sensitized RBCs

Was this a good wash?

This picture is a bad wash because it has free floating antibodies lead to a false negative. These are neutralize and are binding to free floating antibodies instead of the RBCs.

What is a another name for check cells

as control cells, Coombs cells

What are check cells

• RBCs coated with IgG antibody

  • Usually Rh pos RBC coated with anti-D

  • Also available in RBCs coated with C3

• They are added to every AHG reaction tube with a negative result

  • They are a guard against false negatives

    • Bad washing, no AHG added

What is the appearance for Check cells

• Mixed field appearance

  • Checks will agglutinate, screening cells will not

  • Two different cell populations

Check cells test are vaild if

they agglutinate

If CC do not agglutinate, then it can be cause by

• Bad washing

• No AHG added

• Test is invalid and must be repeated

What are the pros for tube method?

gold standard, inexpensive, flexible

What are the cons for tube method?

Labor intensive, not stable

Describe the GEL

• Patient plasma, LISS and screening cells are incubated in a microtube

• Microtube is filled with gel which is laced with AHG

• If cells are sensitized, red cells will be held in gel, otherwise will form pellet at bottom

What are the pros of GEL?

• Smaller sample size

• No cell washing step

• Clear, stable endpoint

• Sensitivity comparable

• Can be automated 

What are the cons of GEL?

• Expensive

• More false positive

What method is this?

• Patient plasma, LISS and screening cells added to microtiter well lined with Protein A which has a high affinity for Fc portion of AHG and incubated.

• Wash

• AHG added

• Centrifugation

• Graded via computer system.  

• Positive will show as cells lining entire well

Solidphase

What is solidphase?

Red blood cell membranes or specific antigens/antibodies are bound (immobilized) to the bottom surface of small plastic microplate wells (typically 96-well plates). - all labs are different

What does this mean in solidphase?

Neg. (solid pellet); tube was 0

What does this mean in solidphase?

tube was 1+

What does this mean in solidphase?

tube was 2+

What does this mean in solidphase?

tube was 3+

What does this mean in solidphase?

Pos. diffuse; tube was 4+

Pros of solidphase?

• Reduced volume needed

• Increased sensitivity

• Less technique needed

cons of solidphase

• Learning curve

• Expensive

• Reagent expiration

Check cells only check for if the

AHG was added or if there was a improper wash

Direct Antiglobulin Test Principle

• Central question for DATs : does this patient have antibodies or complement already stuck to their red cells in vivo?

• Test Procedure: Washed patient red blood cells + AHG

Is this direct or indirect antiglobulin test

Direct Antiglobulin Test

Why use DATS?

• Ordered less frequently

• Transfusion reactions

• Suspected hemolysis

• Can tell you if IgG and/or complement is bound to RBC

• Neonates:

  • Do NOT have functional complement

  • Only test for IgG

  • Looking for maternal IgG causing hemolysis (HDFN)

Complete redrawing of the thing we did in class (on ipad)

complete