Week 9 Part 2 - Resolving Complex Antibody Mixtures Flashcards
Examples of Complex Antibody Mixtures
Unexplained reactions
Antibodies to high and low frequency antigens
Multiple antibodies
High titre, low avidity “like” antibodies
Positive auto control
Examples of Unexplained Reactions
Reactions that don’t match a pattern on panel sheet
Technique/reagent problem
Antibody against reagent
Antibody against a low/high frequency antigen not listed on panel sheet
Antibody demonstrating dosage
- anti-Jka and anti-M as examples
Low Frequency Antigens
Occurs in <10% of population
Kpa, Cw, Lua, Jsa, Dia, etc
Antibodies against these are rare
Not identified on screen
Identified in combination with another antibody, or after an incompatible XM with a neg screen
Easy to find blood for transfusion
High Frequency Antigens
Present in > 98% of the population
k, U, Kpb, Jsb, Lub, Coa
Antibodies against these are rare
All panel cells pos w/ uniform rxn strength, neg auto control
How to identify High Frequency Antigens?
Want to test cells that are negative for high incidence antigens
Enzyme-treated/chemically modified cells
- eg. DTT destroys Kell and Lutheran ag’s
- rare Rhnull and K0 cells
Phenotyping
Diego Blood Group
Contains 22 antigens
Dia/Dib; Wra/Wrb; Wu/DISK - antithetical pairs
16 low-incidence
Carried on band 3 - RBC integral membrane protein
The Di(a-b+) is the most prevalent phenotype
Anti-Dia
- IgG
- causes HTRs and HDNB
Results seen with Multiple Antibodies
Positive reactions against many panel cells
A single antibody doesn’t account for the pattern of reactions
Varying strength of reactions
Negative autocontrol
Anti-G
G antigen
- an antigen in the Rh blood group
- present on C and D antigens, and therefore C+ and/or D+ RBCs
Individuals who are G- can produce an anti-G antibody
- most Rh(D) neg individuals are rr, and therefore are also G-
- sensitised to G antigen after exposure to r’r cells
Anti-C+D vs Anti-G
Anti-G will react to C+ D+ cells on ID
Is the antibody present anti-G, or are two aby’s present (i.e. anti-C+D)?
- anti-C+D anti-D component is usually stronger than anti-C
- anti-G - apparent anti-C component (i.e. reactivity against D-C+ cells) is stronger than apparent anti-D component (i.e. reactivity against D+C- cells)
Why is it Important to Differentiate between Anti-C+D and Anti-G
Because someone producing anti-G (or anti-C+G) is eligible for Rh(D)Ig, whereas someone producing anti-D+C is not
Phenotyping
React patient RBCs with known antibody to determine which antigens they express
Can’t phenotype if recently transfused (< 3 months)
Controls used in Phenotyping
Need to ensure that the system can detect the weakest examples of antigen expression
Positive Control
- RBCs which have heterozygous expression of the target antigen
- i.e. M+N+
Negative Control
- RBCs which do not express the target antigen
- i.e. M-N+
Adsorption to Separate Multiple Antibodies
Plasma is mixed with cells expressing only one of the antigens to which the suspected antibodies are directed
- i.e. D+, Fy(b-) and D-, Fy(b+)
Antibody binds to antigen
Single antibody remaining in adsorbed plasma
Antigen Separation to Separate Multiple Antibodies
Chemical treatment of cells to enhance or degrade antigen
Antigen Modification - Enzyme Treatment of Cells
E.g. ficin, papain, bromelin, trypsin
Inactivate Duffy, MNS, Ch, Rg, antigens (others too)
Enhance Rh, Kidd, Lewis, P, I antigens
Can only cross-out antigens which are not destroyed
Antigen Modification - DTT or AET Treatment of Cells
Reducing agents
Destroy Kell antigens
Neutralisation to Separate Multiple Antibodies
Some antigens are soluble and are found in various fluids/substances
Incubated with plasma before antibody ID to neutralise the antibody present
Examples of Soluble Antigens that Require Neutralisation
Lewis antigens - plasma or saliva of Le(a+b-) and Le(a-b+) individuals
P1 substance - hydatid cyst fluid, pigeon droppings, turtledove egg whites
Sda - urine of Sd(a+) individuals
Chido and Rodgers antigens - plasma of most people
High Titre, Low Avidity ‘Like’ Antibodies (HTLA)
Formerly known as HTLA antibodies
React against most panel cells w/ neg auto control
Tubes - weak-1+ agglutination that easily break up
Cards - 1-2+ agglutination
HTLA Titre
Classically have high titres
Titre
- doubling dilutions of patient plasma containing Aby
- react against cells carrying corresponding Ag
- reciprocal of the highest dilution giving a 1+ reaction
HTLA-‘Like’ Antibodies - Chido/Rodgers (Ch/Rg)
Components of C4 complement protein
Rg - C4a, Ch - C4b
Adsorbed onto RBC surface from plasma
Ch/Rg neg individuals can make anti-Ch/Rg antibodies
Antigens are destroyed by enzymes, resistant to reducing agents
Testing Setup for HTLA-‘Like’ Antibodies Ch/Rg
Identify by neutralising antibodies by plasma containing Ch/Rg
Test sample - patient plasma + normal plasma (1:1 ratio)
Control sample - patient plasma + albumin (1:1 ratio)
Test both using same method that detected suspected aby
If aby is anti-Ch/Rg, reactivity will become negative
HTLA-‘Like’ Antibodies - John Milton Hagen (JMH)
Antigen is present on CD108, a glycosylphosphatidylinositol (GPI) linked protein
Destroyed by enzymes and reducing reagents
HTLA-‘Like’ Antibodies - Knops
Antigens are present on CR1, the RBC C3b/C4b receptor
- Kna, McCa, Sla, Yka
Weakened by enzymes, destroyed by reducing agents
Patient has anti-D and anti-Fyb antibodies. Requires two units of blood. Have 56 units of ABO group-specific blood in the fridge. Are 2 compatible units likely to be present? How many units do you need to screen to find 2 compatible units?
19.7% of the population are Fy(b-)
17.9% of the population are D-
Found in frequency table
Convert numbers to decimals then multiply together
- i.e. 0.197 x 0.179 = 0.035, which = 3.5%
So 3.5% of the population should be D-, Fy(b-)
Number of compatible units in fridge
- total no of units x % of popn that is compatible
- 56 x 3.5%
- 1.96 units
How many units do I need to screen to find 2 compatible units?
- no. of units reqd / % of popn that is compatible
- i.e. 2/0.035, = 57.14 units
