An introduction to Blood Group Serology

Question 1

Blood Group Antigens are?

Answer 1

• Glycoproteins and glycolipids present on the surface of red cells
• Some (eg ABO) may be present more widely on endothelial surfaces
• Genetically determined
  
  • Generally autosomal and co-dominant
  • Xg system sex linked
• Limited understanding of biological function of the antigens

Question 2

Describe the Genetic control of Blood groups

Answer 2

1. Protein Determinants
   
   • Gene codes for the antigenic determinant itself
   • Rh, Kell, Duffy and Kidd systems
2. Glycolipid Determinants
   
   • Gene codes for production of enzymes that add or remove carbohydrate or lipids, through this you get definition of an antigen!
   • ABO, Lewis group systems

Question 3

Duffy Blood Group System and Malaria?

Answer 3

• The Duffy antigen on the RBCacts as the entry point to the red cell for the malarial parasite
  
  • in caucasian populations the Fy^a-Fy^{b negative}phenotype is rare
  • In individuals with a black african ethnic backgroup up to 40% are Fy^a-Fy^b- negative this represents the impact of natural selection for malarial resistance

Question 4

The McLeod Phenotype

Answer 4

• Kx null phenotype associated with Chronic Granulomatous Disease and acanthocytosis!

Question 5

Blood Groups Variation with Populations

Answer 5

• Varies significantly in different population types
• Most likely due to Genetic drift
• Usually no biological advantage apparent
• May have a clinical impact on the provision of compatible blood!!

Question 6

Blood group antigens/systems are clinically important because they have the ability to

Answer 6

generate Blood Group Antibodies

Antibodies rcognise ‘foreign’ antigens

• May be IgM, IgG or occasionally IgA
• May be naturally occurring or immune stimulated

Question 7

Difference between Naturally occuring and Immune stimulated Antibodies

Answer 7

Naturally Occurring:

• No exposure to foreign red cells but exposure to bacteria containing, for example, ‘A-like Antigens’.
• At birth baby has no ABs againsts ABO antigens, but by 6 months almost all babies will have formed some form of anti-A or Anti-B. This is because A or B antigens are very close to the antigens formed on the surface of bacteria
• Can Activate the Complement Cascade > intravascular destruction
• Anti-A produced

Immune Stimulated:

• exposure to foreign red cells (for example Rh+) by transfusion or pregnancy (Rh+ fetus)
• Cannot fully activate Complement Cascade > extravascular destruction
  
  • can activate early phases, and coat early proteins
• Anti-Rh produced

Question 8

Naturally Occurring Red Cell Antibodies

Answer 8

• Antigens that develop in the absence of exposure to the red cell antigen
• Most likely stimulated by cross reacing antigens derived from bacteria (as they’re very similar)
• Not present at birth but develop during 1^st year of life
• Usually related to lipid antigens
• Significant IgM component to antibody, but IgG may also be present
• ABO and Lewis antigens fall in this category

Question 9

Immune Stimulated Red Cell Antibodies

Answer 9

• Develop only following exposure to specific antigen
• May be produced following
  
  • transfusion
  • pregnancy
  • injection (eg IVDU)
• Normally IgG in nature

Question 10

A brief overview of the history of Transfusion.

Just for extra info

Answer 10

• Earliest record of transfusion in the early 19th century, for women bleeding heavily following childbirth. Would use husbands blood via cannuler. “Blundell Transfusion”
• Some died immediately, and some survived.
• Later on Dr Karl Landsteiner discovered the ABO blood group system
• the ABO blood group system is the most likely to kill you, and the best to know about

Question 11

Where are ABO antigens found?

Answer 11

Present on the surface of

1. Blood cells
2. Epithelial cells
3. Body fluids

Question 12

What determines the ABO phenotype?

Answer 12

The phenotype is determined by a series of glycosyltransferase enzymes. These are resposible for addition of CHO molecules to the basic membrane structure.

The H antigen is neccessary for the ABO phenotype to be expressed
this is present in (almost) everyone

Question 13

The ABO groups all have an H group attached, but whats the molecular difference in this!

Answer 13

H:** base structure with a D-galactose and fucose sugar. **This alone is known as the O blood group!!

A: N-acetylgalactosamine added to the surface of the red cell

B: Galactose added to the surface of the red

Therefore your blood group is determined by the presence of a sugar molecule

Question 14

What does it mean by the ABO blood group being ‘co-dominant’

Answer 14

You inherited one gene from your mum and one from dad, and this COLLECTIVELY determines phenotype.

By 6 months of life you’ve developed antibodies against the A or/and B antigens which you lack

O is the most common phenotype!

Question 15

Clinical Relevance of ABO systems

Answer 15

• ABO antibodies: naturally occuring and appear ~3-6months old
• Most important blood group as → to fatal transfusion errors
• ABO incompatible transfusion results in complement activation leading to…
  
  • ​intravascular haemorrage
  • renal failure
  • DIC

Question 16

Therefore for a tranfusion we select antigens that don’t have ___________

Answer 16

Therefore for a tranfusion we select antigens that don’t have corresponding antibodies present in the recepiants plasma

eg; blood group O (has therefore anti-A and Anti-B present) cannot be transfused with A or B groups. Can only be transfused with O

O is therefore the universal Donor but as a small population they can’t sustain everyone!

Question 17

The Rh Blood Group System

Answer 17

1. Second most important blood group system
2. Protein antigen
3. Expressed only on RBC
4. antibodies only produced post exposure to the D+ red cells ‘immune stimulation’
5. Rh(D) is highly immunogenic,
   
   • if you expose a group to D+ red cells 80-90% will have a reaction

Question 18

What is Rh(D)?

Answer 18

• Most important antigen of the Rh system
• Everyone is either Rh(D) positive or Rh(D) negaticed antigen is amorph

Rh(D) negative: dd

Rh(D) positive: DD or Dd

Question 19

population frequency of Rh(D) in NZ?

Answer 19

Question 20

Rh(D) and transfusion

Answer 20

• 90% of Rh(D) negative individuals transfused with a unit of Rh(D) positive red cells will produce anti-D strong immune reaction
• Anti-D is an IgG antibody that is unable to bind complement, Red cell destruction is extravascular
• Anti-D is the most common cause of Haemolytic Disease of the newborn: fetus RBC cross into mum, if fetus is D positive from Dad and mum is negative > anti-D formation > problems for mum in subsequent pregnancies
• Normally transfuse red cells of the same Rh(D) type as the recipient
• Never transfuse Rh(D) positive red cells to an Rh(D) negative female of child bearing age; male would only require D negative cells, not a biggie

Question 21

Describe what the Rh antigen is??

Describe the Expanded Rh system

Answer 21

• THe Rh system is the most complex of known blood group systems
• Rh antigen: product of 3 genetically closely linked alleles; which make allelic pairs
  
  • C and c
  • D and d (d is an amorph)
  • E and e
• 3 allelic antigens behave as a single entitiy
• a number of common alleles can therefore be defined

Question 22

Minor Blood Group Systems

Answer 22

• Over 400 blood group systems have been defined
• Majority not of clinical interest, but some relevance to
  
  • frequency of antigen in pop
  • frequency of antibody production following transfusion
  • ability of antibody to destroy transfused red cells
• Relevance is higher if a small amount of the population will react

Question 23

What are the 3 blood group systems of common clinical interest?

Answer 23

1. Kell: K (Kell) and k (cellano)
2. Kidd: Jka and Jkb
3. Duffy: Fya and Fyb

Don’t need to know gene frequencies

Question 24

Laboratory Techniques for detecting antigens and antibodies

Answer 24

• Often involves an agglutination technique; when the antibody + antigen put together they CLUMP
• Antigen testing usually uses commercially sourced monoclonal reagents
  
  • these have high specificity and sensitivity
  • are IgM in nature
  • produce direct agglutination
• Molecular typing methods increasingly available

Question 25

Laboratory Techniques; Red cells and *zeta potential*

Answer 25

* Red cells are negatively charged, so called ***zeta potential*** * **IgM** molecule is **larger** then the **ZP** so can produce cross-linking → agglutination * **IgG** molecule is too **small** to produce cross-linking; therefore a **potentiator (enables red cells to get closer)** is need to have agglutination * usually **Anti-human Globulin** * also enzymes, albumin

Question 26

Immunoglobulin Structural differences of the IgG and IgM?

Answer 26

IgG: dimer with 2 antigen-binding sites that are very close together, soif a red cell is bound to one site the other is blocked IgM: Pentamer, with 5 binding site, therefore you can get several RBCs around to form an agglutination via crossinglinking (see pic)

Question 27

Describe what would happen via IgG and RBC, and what you need to do in the lab to fix this

Answer 27

As the IgG is too small, you get it coating the cell, but no cross link reaction occuring, and the IgG can't overcome the pushing apart of the RBC (zeta potential) and you can't see anything. 1. Wash the solution 4x to remove any free antibody present 2. Take those free cells and expose them to anti-human globulin; antibody directed at human IgG 3. IgG of the AHG will react with the IgG bound to the RBC, and you will get a reaction ***This is partiularly important in detecting IgG antibodies***

Question 28

How is Anti-human Globulin produced?

Answer 28

* Used to be in rabbits, inject with serum containing IgG as well as other proteins * Rabbit reacts against IgG and forms ***Antihuman Globulin*** against this (also forms anticomplement antibodies)) * Select the AHG elements you require. Nowadays mixtures of monoclonal reagants more comonly used; as easier to produce and standerdise

Question 29

How do we form/detect Antibodys?

Answer 29

**_Tile Groups_** 1. Have a line of Antibodies (eg; anti-A and Anti-B) and a line of blood cell types (A, B O cells) 2. Note if you get agglutination **_Test Tubes:_** Postitive: Agglutination in bottom of testtube Negative: Dispersed cells **_Micro Tetre Plate_** No reaction: cells sink to bottom Reaction: red cells will coat the surface of the cell;carpet like phenomenon **_Chromotographic Gel Cards_** Reaction: red cells can't pass through so float on top Can also do this testing Antibodies

Question 30

What causes **Haemolytic Disease of the Newborn**?

Answer 30

* When **maternal antibody crosses placenta** → ***destruction of fetal RBCs*** * Always involves **IgG** antibody (IgM too large to cross) * Most frequently from *Anti-D* * Followed by *Anti-c* * And Anti-Kell * Frequency reduced by **immunoprophylaxis**

Question 31

Describe the course of events of Haemoytic Disease of the Newborn

Answer 31

*maternal antibody crosses the plancenta → destroys fetal red cells → baby becomes progressively anaemic → develops cardiac failure → develops **Hydrops Fetalis*** ***Hydrops Fetalis*** : severe conjestive cardiac failure. If untreated can cause death in utero. Usually in will survive.

Question 32

Usually babies will haemolytic disease will survive to birth, but what happens to them then?

Answer 32

They will have the maternal antibody present in their system for a few weeks. (T_1/2 of IgG is 3 weeks) , baby continues to produce red cells that continue to be destroyed by IgG and the baby becomes jaundiced. *This doesn't happen in utero as the bilirubin is sucked out into the maternal circulation.* The infants *blood-brain-barrier* is poorly developed → bilirubin crosses to brain surface → CSF→ irritant to the neuro system → causes a range of neurological issues/abnormalities. Can cause **deafness** or **Kernicterus** (spasm), which can cause cerebal palsy

Question 33

What is happening here?

Answer 33

1. D positive infant 2. Cells cross into maternal circulation * *usually at childbirth, but can be due to a variety of other reasons during the pregnancys* 3. The D positive red cells lead to the formation of anti-D in the mother 4. This Anti-D will then travel back into the fetus and destroy its RBCs ## Footnote *This occurs less where there's an ABO incompatibility between mum and baby, because the ABO antibodies would remove the D + red cells before anti-D formation could occur.*

Question 34

What has cause the decline of Anti-D sensitisation in births, and how does in work?

Answer 34

Immunoprophylaxis. (combined with improved obstrectric antenatal care) * The basis of it is to break the cycle; and stop mum forming anti-D when the babies cells enter the circulation * As this occurs at childbirth (And you have a D - mum and D+ baby) we give the mum an injection of Anti-D * This clears the D+ red cells and prevents the mums immune response from occuring *"passive immunisation"*

Question 35

Prevention of Rh (D) HDN?

Answer 35

* Anti-D immunoglobulin given routinely following birth of an Rh (D) + baby to a Rh (D) - mother * Standard postnatal dose in NZ is **625 IU destroys 5ml fetal blood** * **Kliehauer test:** used to detect women with larger foetomaternal bleeds (as they'll need a larger dose)

Question 36

When is Anti-D immunoglobulin given during pregancy?

Answer 36

Following a *potentially sensitising event* * *Abortion* * *Termination* * *Amniocentesis* *As this is a point where theres more bleeds, give in the 3rd trimester as bleeds occur most here!*

Question 37

ABO Haemolytic Disease of the Newborn?

Answer 37

* We all have ABO antibodies * ABO incompatibility between mum and baby is common * but Haemolytic ABO disease is rare * AB antigens weakly expressed in fetus/newborn * AB antigens widely distributed in placental tissue and absorb antibody before it reaches fetus * When ABO HDN occurs it's usually **mild** and transfusion rarely required