Detection and ID of RBC antibodies

Sample requirements

Less than 72 hours, (expires in 3 days) kept for additional 7 days = 10 days total

Free of hemolysis

Full name, ID#, Date, Time, collectors initials

Plasma EDTA (Pink top)

Pt history

Age, sex, race

Diagnosis, Medication, IV solutions,

Transfusion/pregnancy history

Historical antibodies

Screen cells

Antibody detection

Pt plasma tested against screen cells that represent commonly inherited, clinically important blood group antigens

Pooled

Single vial from 2 donors

Donor units

Not for transfusion

Unpooled cells

Single donor

2-3 individual pts

Contains homozygous expression of Rh antigens

Homozygous

expression of an Antigen

2 genes code for same antigen

Jk (a+b-) usually from Jka Jka donor

Displays dosage

Heterozygous

expression of an antigen

Donor has one copy of the gene

Expression 2 different antigens

JkaJkb (a+b+)

Antibody panel

Positive screen

11-16 vials of group O cells

Ensure lot #s match - antigram

Auto control

Auto control

Pts plasma and cells

If +ve probable autoantibody

Potentiators

LISS

PeG

Enzymes

Solid phase gel

LISS

Low ionic strength solution

Lowers zeta potential

Increases speed of reaction

Enhances sensitization

PeG

Polyethylene glycol

Modified LISS

Enhances sensitaztion

Enzymes

Remove sialic acid/proteins

Decrease zeta potential

Enhance sensitization

Denature antigens

Denatured by enzymes

MN and Duffy

Enhanced by enzymes

Rh lewis, P, I, and Kidd

Solid phase

Pooled O cells bound and dried to surface of microtiter wells

Plasma antibody bind to RBC in well

Addition of indicator cells (anti-human IgG coated RBCs)

-ve = bottom of well

Gel column

Micro column contain dextran acrylamide gel particles and antiglobulin reagent

Does not require washing

Gel particles have sieving effect that reflects the size of the RBC agglutinates

AHG

Cross link RBC already sensitized

Polyseptic

Monospecific

IgG sensitized Coombs control cells

AHG polyspecific

Complement and IgG

Check cells

IgG sensitized Coombs control cells

Rh positive RBCs coated with human IgG antibody

Used to ensure AHG was added

Required to add to each AHG test that is negative

Rule out

Eliminate antibodies to antigens present on non-reactive cells

Rule out Rh MNSs, Kidd and Duffy using homozygous expression Rh

Rule of 3 probability value <0.05 statistically valid

Presence of antibody

ABO discrepancy

Positive antibody screen

Positive auto-control or DAT

Incompatible crossmatch

Grading reactions

Consistent grading

Hemolysis = +ve Ab/Ag RXN

RBCs must be resuspended

Plasma cell ratio

2:1

Add plasma first, then cells, then potentiator

+ve = agglutination / hemolysis

-ve = no agglutination / hemolysis

Variations in antigen expression

Dosage = Duffy, Kidd, Rh, MNS

Variation unrelated to zygosity = I, P1, Lewis, Sda

Antigen deterioration = P1, M, Duffy

Cord cells = I, Lewis, Sda

Variation Dosage

Duffy, Kidd, Rh, MNS

Variation unrelated to zygosity

I, P1, Lewis, Sda

Variation antigen deterioration

P1, M, Duffy

Variation Cord Cells

I, Lewis, Sda

Antibodies to low incidence antigens

<1%

Panel screen may be negative, crossmatch may be incompatible

Transfusion therapy should not be delayed

Kell = exception, more prevalent

Antibodies to high incidence antigens

>98%

Uncommon

Uniform RXN with all panel and donor cells tested

Transfusion therapy more challenging - test siblings

Anomalous reactions

Antibodies to drugs and additives

Antibodies in plasma + dye/drug - antibody complex

Spontaneous agglutination or aggregation

Anomalous RXNs positive results

DAT negative

Acriflavin = Yellow dye

Caprylate = bovine albumin/LISS

Thimersol = LISS

Rouleaux

Plasma protein seen in reverse

Appearance of agglutination

Elevated plasma proteins, fibrinogen, Wharton’s jelly, plasma extenders

Wash w/ saline = -ve disperse RBCs

Agglutination w/ saline = other issue

Enhancement

Plasma:cell ratio

Enhancement media

Change temp of incubation

Decrease pH

Sulfhydryl reagents

DTT, 2-ME, ZZAP

Sulfhydryl reagents DTT

Dithiothreitol

Use when IgM mask IgG

Dissolves IgM disulfide bonds

DTT destroys some RBC antigens (Kell)

Must give kell neg blood if working w/ DTT

Sulfhydryl 2-ME

2-mercaptoethanol

Cleave IgM into monomers

Diminish or destroy IgM antibody activity

Sulfhydryl ZZAP

A combination of DTT and proteolytic enzyme

Used in adsorption

enhance warm antibodies to pts RBCs

Neutralization

Soluble blood group substances

Saliva, urine, plasma

Inhibition can be used to neutralize antibody that masks another antibody

Lewis = saliva

P1 = hydatid cyst fluid; pigeon egg

Neutralize P1

Hydatid cyst fluid; pigeon egg

Adsorption

Sponge soak up / take out alloantibodies

Remove autoantibody activity

Separate multiple antibodies

Elution

Frees antibody molecules from sensitized RBCs; recover bound antibody in usable form

Investigate +ve DAT

Prepare free RBCs for phenotyping

Acid elution

Titration

Titer is determined by testing serial two fold dilutions of plasma against select RBC

Reciprocal of the highest dilution that gives 1+ result

Amount of antibody present / strength

Prenatal studies, antibody identification, separating multiple Abs, HLA