COMPARE IGM & IGG

COMPARE PRIMARY AND SECONDARY IMMUNE RESPONSE

STAGES OF AGGLUTINATION
 incubation No visible agglutination
Antibody binds to antigen by
immunologic recognition

Serum to cell ratio Antibody antigen concentration

ionic environment =

 shielding effect
between antigen
antibody
Between cells
Collision between sensitized
RBC to develop cross linkage
or agglutination
MONOCLONAL VS POLYCLONAL ANTIBODY

CAUSES OF POSITIVE DAT

FALSE POSITIVE ERROR IN AHG TEST

 FALSE NEGATIVE ERROR IN AHG TEST

POLYETHYLENE GLYCOL

MENDEL LAW OF INDEPENDENT SEGREGATION

 • removes water and concentrates the antibodies in the sample
• Create low ionic environment
• Can only be used for ICT
• No need to centrifuge
• Non specific agglutination with polyspecific AHG

Passing of one gene from each parent to the offspring

 MENDEL LAW OF INDEPENDENT ASSORTMENT

ABO ANTIGEN IN NEWBORN

COMMON CARBOHYDRATE STRUCTURE FOR ABO

Alleles of different genes are inherited independently of one another.

• Chromosome 9
• Detectable at 5-6 weeks in utero
• Newborn carry fewer antigen than adult
• Antigen structure not fully develop
• Full expression of adult level 2-4y/o

• H antigen
• I/i antigen
• P antigen
• Lewis antigen
Composition of type 1 and 2 chain

H antigen

A & B antigen
 (Mainly glycoprotein)

• Chromosome 19
• h allele is amorph
• H gene codes for L-fucosyltransferase (FUT-1)
• Transfer L-fucose to type 2 oligosaccharide chain in RBC and secretion
• Clinical significant antibody
• High thermal activity and complement activation
• igM type
• Also present in A1 and A1B but not potent

• Chromosome 9
Variation of H antigen concentration

A1 & A2 subgroup

A subgroups
• Quantitative and qualitative difference in Ag expression
• 80% of group A is A1
• Ag assembled on branched and linear chain
• Dolichos Biflorus agglutinate A1 cells
• 20% of group A is A2
• Ag assembled on the linear chain
• Anti A1
• 1-8% A2 individuals
• 22-35% A2B individuals
• Reactive at RT or lower
• Not clinically significant
• Problem at
• IS
• ABO phenotyping

Serological classification of rare subgroup of A

• Anti-AB can detect weaker subgroup of A • Test with anti-A and anti-AB
• A3 – mixed field agglutination • Test with anti-A1 lectin
• Resolution • Test for presence of anti-A1 in serum
• Saliva study to detect soluble A and H antigen • Test with anti-H lectin
• Anti H lectin – (3+ to 4+ reaction) • Test for A and H antigen in saliva
• Ael subgroup – adsorption and elution for detection • Adsorption and elution study
 ABO discrepancy

Group A with acquired B antigen

B(A) phenotype
 Test with A1 lectin, then A1 &
A2 cells
Cold - Le, M, N, P1

Malignancy – leukemia
- Hodgkin

Tn polyagglutination Genetic defect - incomplete synthesis of normal residues on glycophorins A and B -

exposure of a normally hidden Tn antigen (cryptantigen)

• Caused by bacterial deacetylating enzyme

• Obtain more information on patient
• Lower GI disease
• Colon/rectum ca
• Gram negative septicemia
• GI obstruction
• Test against autologous cell, it wont react
• Test with additional monoclonal anti B reagent
• incubating the acquired B RBCs with acetic anhydride will lead to “re-acetylation”
• acidifying the reaction mixture of the patient’s RBCs with anti-B (non-self) can eliminate the incompatibility with that source of anti-
B

• Trace amount detected due to potent monoclonal antibody

• B gene transfer B sugar and also trace amount of A sugar.
• Resolution
• Obtain diagnosis and history
• Use Anti-A from other manufacturer
• Polyagglutination if used human derived ABO sera (contains other antibodies)
• Abnormal concentration of serum protein (causes nonspecific aggregation)
 Weak A resolution

Mixed field resolution

Extra antibody resolution

• Obtain diagnosis and history
• Repeat with extended incubation (enhance antigen-antibody reaction)
• Include human polyclonal anti-AB, or monoclonal blend anti-AB

Serological classification of rare subgroup of A

• Test with anti-A and anti-AB
• Test with anti-A1 lectin
• Test for presence of anti-A1 in serum
• Test with anti-H lectin
• Test for A and H antigen in saliva
• Adsorption and elution study

• Diagnosis and history

• Determine
• Recent hematopoietic progenitor cell recipient
• Transfusion with group O

For A3 subgroup and Aend

Serological classification of rare subgroup of A

• Test with anti-A and anti-AB
• Test with anti-A1 lectin
• Test for presence of anti-A1 in serum
• Test with anti-H lectin
• Test for A and H antigen in saliva
• Adsorption and elution study

• Diagnosis and history

• Resolution
• Test with screening cells and AC at RT
• If AC pos – cold autoantibody - anti-I
• If only screening positive - cold alloantibody - Le, M, N, P1

• If A1 cell weak positive

• Test with A1 lectin, then A1 & A2 cells – to look for A2 subgroup
 Rouleaux resolution

Missing antibody resolution

Extra antibody resolution

• Disease associated
• Multiple Myeloma
• Waldenstrom macroglobulinemia

• Obtain diagnosis and history

• Wash red cell suspension and repeat the phenotyping
• Perform saline replacement technique
• Incubate serum and RBC and centrifuge
• Remove serum and replace with saline
• Centrifuge
• No agglutination – Rouleaux
• Agglutination – true agglutination

• Diagnosis and history

• Age
• Immunoglobulin level
• Serum protein electrophoresis
• Resolution
• Incubate serum testing for 15 minutes at RT, then centrifuge, if negative
• Place serum testing at 4 Celsius for 30 mins with AC
• If A1&B cells pos + AC neg – group O
• If A1&B cells pos + AC pos – cold autoantibody

• Diagnosis and history

• Resolution
• Test with screening cells and AC at RT
• If AC pos – cold autoantibody
• If only screening positive - cold alloantibody - Le, M, N, P1

• If A1 cell weak positive

• Test with A1 lectin, then A1 & A2 cells – to look for A2 subgroup
Secretor status

Para-Bombay

A2 resolution
• Se allele produces
• FUT-2 – responsible for soluble A, B, H in secretion
• L-fucosyltransferase
• Add fucose to type 1 oligosaccharide chain only in secretory gland
• Non secretor
• sese
• Don’t have A,B, H antigen in secretion, but RBC has A,B, H if H gene intact

• The genetic basis

• mutated FUT1 (H gene) with or without an active FUT2 gene (Se gene)
• silenced FUT1 gene with an active FUT2 gene
• Antibody present
• Ah serum - +anti-B, no anti-A, although anti-A1 is usually present
• Bh serum - +anti-A, and anti-B may be detected

silenced FUT1 gene with an active FUT2 gene

• FUT2 produces H, A, B, on type 1 antigens in secretions, including plasma.
• These type 1 antigens in plasma may adsorb onto the RBC membrane,
• yielding very weakly expressed H, A, and B antigens

• First step:
• determine if group A1 or group A2. (If group A1, the discrepancy with the A1 cells is NOT due to anti-A1).
• type RBC with the anti-A1 lectin which is Dolichos biflorus . If the red cells agglutinate, the person is group
A1. If the red cells do not agglutinate, the person is not group A1, and probably is group A2 assuming the red
cells reacted strongly (3+ or 4+ with anti-A).

• Second step:
• If RBC group A2, prove the extra antibody is anti-A1, and not some other IgM irregular antibody.
• test serum against a panel of 3 A1 cells and 3 A2 cells. If anti-A1 is present, only the A1 cells should
agglutinate.