BB reagents

Reagent regulations

• how is blood regulated

• what certifies reagents

• regulation criterias

Blood regulated like drug by FDA

• CBER certifies reagents → establish minimum criteria

  • Package insert (instructions)

    • Intention + how it is made + testing info

  • Facility policies

  • Reagent QC done on day of use

Polyclonal vs monoclonal Abs vs blended

• Polyclonal: mixture of IgM and IgG Abs made by B cell clones to detect multiple epitopes on Ag

• Monoclonal: hybridomas derived from myeloma cells to target specific epitope w/ IgM OR IgG

• Blended: diversity (either monoclonal or poly + mono)

  • overcomes disadvantages of over specificity of monoclonal

ABO antigen typing

• forward grouping

• use of monoclonal IgM

D antigen typing

• high vs low protein

Reagent control

• when to use

ABO antigen typing

• forward grouping: looking for Ag

  • determine ABO phenotype of pt cells (use anti-A and anti-B + agglutination)

• monoclonal IgM helps detect weak A and B Ag expression

D antigen typing

• High protein = historical- not used anymore

• Low protein = monoclonal or mono/poly blends

  • IgM Abs = immediate spin

  • IgG Abs = weak D testing

Reagent control

• high-protein reagents

• A, B, and D testing all positive

Reagent RBCs

• what is it?

• types and use

• Known Ag for detecting unknown Ab

• A1 and B cells:

  • used for reverse grouping: looking for Ab → confirms forward testing

• Screening cells:

  • detects non-ABO Abs

• Panel cells

  • wider range of cells to specify Ab detected

Antihuman globulin

• purpose

• examples

• DAT vs IAT

• AHG control cells types

• bridges gap bc IgG too tiny for agglutination

• anti-IgG, anti-C3d, polyspecific AHG (both), monospecific AHG (seperate)

• DAT: in vivo; 1 step (cells alr sensitized)

• IAT: in vitro; 2 steps (incubate for sensitization)

• AHG control cells:

  • IgG coated or C3 coated group O cells

  • confirm neg rxns

Potentiators

• what is it

• what does it do

• what types + what they do + effect

• “Enhancement media”

• looks for IgG in IAT

• low-ionic strength solution (LISS)

  • reduces electrostatic charge around RBC = better Ab uptake + agglutination

• Polyethylene glycol (PEG)

  • removes water molecules → RBC get closer + concentrates Ab enhancing uptake

• Bodine albumin

  • allow Ab sensitized cells to come together = better agglutination

Enzymes

• proteolytic?

• destroys what

• enhances what

• common enzymes

• Proteolytic: removes neg charges and glycoprotein fragments from RBC

• Destroyed by enzymes: duffy, MNS

• “Enhanced” by enzymes: ABO, Rh, Lewis, Kidd, H, I, Lutheran

• Common enzymes:

  • Ficin- from figs

  • Papain- from papauas

  • Bromelain- from pineapples

Lectins

• what?

• types?

• Alternate to antisera → cause agglutination 

• Types

  • Dolicho biflorus: anti-A1 lectin 

  • Ulex europaeus: anti-H lectin

Sources of errors in agglutination

• false pos

• false neg

False positive	False negative
Dirty glassware	Delayed testing → not adding AHG immediately
agglutinated RBC present before washing then added AGH	Failed to wash before adding AHG
Over centrifugation	Failed to ID weak pos rxn
	No adding AHG
	Loss of reagent activity
	Under centrifugation
	Inappropriate RBC concentration → RBC suspension fall outside optimal 2-5%

Are the following IAT or DAT?

1. Drug-related mechanisms (IgG–drug complex on RBC)

2. Antibody screening (detect atypical non-ABO antibodies before transfusion)

3. IgG and/or C3 bound to patient RBCs

4. Abs produced by passenger lymphocytes (donor B cells attack recipient RBCs)

5. Weak D test (detects presence of D antigen)

6. Passively acquired alloantibodies (transfused or infused antibodies)

7. Nonspecifically absorbed proteins (globulins coating RBCs)

8. Crossmatch (tests serologic compatibility)

9. Donor or fetal cells sensitized with IgG antibodies

10. Antigen typing

11. Activation of complement (C3 binds RBCs and is detected by AHG reagent)

12. Antibody identification (determines antibody specificity in patient or donor)

1. DAT

2. IAT

3. DAT

4. DAT

5. IAT

6. DAT

7. DAT

8. IAT

9. DAT

10. IAT

11. DAT

12. IAT