W6 -ABID, Cross, Phenotype

What is the dosage?

The quantitative difference in strength

What are the proteolytic enzyme?

Ficin, Papain, Bromelein

Which antigens are destroyed/ sensitive to enzyme?

MNS and Duffy

Which antigens are enhanced by enzymes?

Rh, Kidd, Cold-reacting (I, H, Lewis, P)

What are some types of chemical modifications?

• DTT

• Chloroquine

• 2ME

• Acid glycine/EDT

What is ZZAP and what is it’s purpose?

A combination of enzyme + DTT; reduce the disulfide bonds on IgM molecules, decrease their ability to agglutinate red cells

What are the characteristics of IgG?

• Incomplete antibody

• Most stimulated via transfusion or pregnancy

• crosses the placenta

• reacts best at 37C

• not affected by DTT

What are the characteristics of IgM?

• Complete agglutinating antibody

• Fixes complement

• Inactivated by DTT

• Does NOT cross the placenta

• reacts best at room temp or lower

What is the word for too much antibodies?

Prozone

What is the word for not enough antibodies/ antigen excess?

Postzone

What are some factors that can affect agglutination?

• Antigen concentration

• # of antigen sites

• Class of antibodies

• Ionic strength of suspending medium

• Time

• Temperature

• pH

• Centrifugation

What are some things that can enhance agglutination?

• Reduce concentration of antigen

• Increase concentration of antibodies

• Use enhancement media

• Use of enzyme

What is the first step in antibody identification?

ABO/Rh and RS

What is the second step in antibody identification?

Investigate patient history (aka did they get transfusion?)

What is the 3rd step in antibody identification?

• Evaluate initial panel and autocontrol

• Auto control pos?

• adsorption required?

• allogeneic adsorption

• autologous adsorption

What is the purpose of rule out?

• Evaluate panel cells that are not reactive at all phases tested

• Rule out w/ one homozygous cells first and 2(or 3) heterozygous

What is the first step in ruling out?

Examine the non-reactive cells, cross-out all antigens that are + (Homo or hetero on the panel cell)

What is the second step in ruling out?

Examine the pattern of reactivity of those that not crossed out

What is the 3rd step in ruling out?

If a pattern fits a specific antigen, select panel cells of certain phenotype to exclude or include the presence of other antibodies

Which antigens are common causes of intravascular RBC destruction? What is the main antibody structure that causes this?

ABO, some Kidd, Kell and Duffy antibodies; IgM

Which antigens are common cause of extravascular RBC destruction? What is the main antibody structure that causes this?

Rh, Kell, Duffy, Kidd, Ss antibodies; IgG

What is the first step in ABID?

Run initial panel: 8 cells + autocontrol

What is the 2nd step in ABID?

Evaluate results of autocontrol (a/c)

• +a/c = do DAT

• + DAT IgG = do elution

What is the 3rd step in ABID?

Evaluate reaction patterns:

• Strength of rxn

• Phase of testing : IS, RT, 37C AHG

• Specificity match antigen expression on the panel

What is the 4th step in ABID?

Antibody exclusion (Rule out)

• Evaluate all non-reactive cells on the initial panel and rule out homozygous cells then heterozygous cells

What is the 5th step in ABID?

Figure out which antibodies are not ruled out

What is the 6th step in ABID?

Identify suspected antibody or antibodies

What is the 7th step in ABID?

Select cell panel

• select cells that are antigen negative for the suspected antibody

• all rxns must be accounted for

What is the 8th step in ABID?

Rule in antibody confirmation

• Must demonstrate P value: 3 and 3 rule for each antibody

• all other antibodies must be ruled out

What is the 9th step in ABID?

Phenotype the pt for the antigen that corresponds to the antibody identified

• If DAT is + —> use monoclonal antisera

What would you do if DAT is positive on step 9 of ABID?

Treat pt cells with acid/glycine/EDTA or chloroquine to remove the antibodies coating the RBC

What do you expect the antisera reagent when you QC them?

Positive —> must be heterozygous

Negative —> must lack the antigen

What are some special techniques that can be used when identifying antibodies?

1) Enzyme treatment

2) Chemical treatement

3) Neutralization

4) Elution

5) Adsorption

6) Cold panel

7) Pre-warm testing

What is the significant of enzyme treatement?

• Reduces zeta potential

• remove hydrophilic glycoproteins

• separate antibodies

What is the significance of chemical treatement?

• Removes IgM reactivity

• destroy antigens in the K system and some high-prevalent antigen

• Allows us to see any IgG reactions

What is significance of neutralization?

Neutralize the antibodies

What is the significance of elution?

To free the antibodies that are coating the RBC

What is the significance of adsorption?

Testing to see if the antibodies in the plasma was picked up by the antigens

What is the significance of cold panel?

Evaluates specificity of cold reacting antibodies

What is the significance of pre-warming?

To inhibit the activity of cold reacting antibodies

What are the phases of testing of ABID?

IS, RT, 37C, AHG, IgG / IgM

What is used to neutralize Anti-P1?

Hydatid cyst fluids, turtledove eggwhite, pigeon dropping

What is used to neutralize Lewis antibodies?

Plasma, serum, saliva

What is used to neutralize Anti-Chido/ anti-Rodgers?

Serum with complements

What is used to neutralize anti-Sda?

Urine

What is used to neutralize Anti-I?

Human breast milk

What are some techniques of elution?

• Acid Elution

• heat Elution

• Lui Freeze-Thaw

What is eluate?

Fluid that contains antibody after we shake them off the RBC

What is adsorbed plasma?

The plasma after antibodies in the plasma is removed by corresponding antigens

What is autoadsorption?

Autoantibodiess absorbed by patient’s own cells —> thus we treat their cells with ZZAP

What is allogeneic adsorption?

Phenotypically matched cells but differential adsorption

What are some examples of cold reacting antibodies?

• Anti-M

• anti-N

• anti-LeA

• Anti-Leb

• anti-P1

• Anti-IH

• Anti-I

What is the difference between WAA and pan agglutinin?

WAA is when pt does NOT have a hx of transfusion when PAN has a hx of transfusion

What is WAA?

Warm autoantibody where pt own antibodies attack itself at optimal body temp

What is panagglutinin?

A pan-reactive antibody of undetermined specificity in the plasma and /or eluate of a patient transfused in the past 3 months

What are some steps to take when you have ABO/Rh discrepancies?

• Washed RBC w/ warm saline

  • Prewarm Reverse typing