Week 9 Part 2 - Resolving Complex Antibody Mixtures

Question 1

Examples of Complex Antibody Mixtures

Answer 1

Unexplained reactions
Antibodies to high and low frequency antigens
Multiple antibodies
High titre, low avidity “like” antibodies
Positive auto control

Question 2

Examples of Unexplained Reactions

Answer 2

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

Question 3

Low Frequency Antigens

Answer 3

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

Question 4

High Frequency Antigens

Answer 4

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

Question 5

How to identify High Frequency Antigens?

Answer 5

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

Question 6

Diego Blood Group

Answer 6

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

Question 7

Results seen with Multiple Antibodies

Answer 7

Positive reactions against many panel cells
A single antibody doesn’t account for the pattern of reactions
Varying strength of reactions
Negative autocontrol

Question 8

Anti-G

Answer 8

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

Question 9

Anti-C+D vs Anti-G

Answer 9

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)

Question 10

Why is it Important to Differentiate between Anti-C+D and Anti-G

Answer 10

Because someone producing anti-G (or anti-C+G) is eligible for Rh(D)Ig, whereas someone producing anti-D+C is not

Question 11

Phenotyping

Answer 11

React patient RBCs with known antibody to determine which antigens they express
Can’t phenotype if recently transfused (< 3 months)

Question 12

Controls used in Phenotyping

Answer 12

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+

Question 13

Adsorption to Separate Multiple Antibodies

Answer 13

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

Question 14

Antigen Separation to Separate Multiple Antibodies

Answer 14

Chemical treatment of cells to enhance or degrade antigen

Question 15

Antigen Modification - Enzyme Treatment of Cells

Answer 15

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

Question 16

Antigen Modification - DTT or AET Treatment of Cells

Answer 16

Reducing agents
Destroy Kell antigens

Question 17

Neutralisation to Separate Multiple Antibodies

Answer 17

Some antigens are soluble and are found in various fluids/substances
Incubated with plasma before antibody ID to neutralise the antibody present

Question 18

Examples of Soluble Antigens that Require Neutralisation

Answer 18

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

Question 19

High Titre, Low Avidity ‘Like’ Antibodies (HTLA)

Answer 19

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

Question 20

HTLA Titre

Answer 20

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

Question 21

HTLA-‘Like’ Antibodies - Chido/Rodgers (Ch/Rg)

Answer 21

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

Question 22

Testing Setup for HTLA-‘Like’ Antibodies Ch/Rg

Answer 22

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

Question 23

HTLA-‘Like’ Antibodies - John Milton Hagen (JMH)

Answer 23

Antigen is present on CD108, a glycosylphosphatidylinositol (GPI) linked protein
Destroyed by enzymes and reducing reagents

Question 24

HTLA-‘Like’ Antibodies - Knops

Answer 24

Antigens are present on CR1, the RBC C3b/C4b receptor
- Kna, McCa, Sla, Yka
Weakened by enzymes, destroyed by reducing agents

Question 25

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?

Answer 25

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