Please enable JavaScript.
Coggle requires JavaScript to display documents.
Transfusion (Principles of compatibility (ABO Blood groups (Most…
Transfusion
Principles of compatibility
Haemolytic Transfusion Reactions (HTR)
Immediate or Acute HTR
Intravascular haemolysis often results in severe morbidity or mortality
Potent complement fixing alloantibodies haemolyse the transfused donor cells
IgM anti-A and anti-B - ABO incompatibility
Delayed Haemolytic Transfusion Reaction
Extravascular haemolysis causes delayed destruction of transfused red cells
Clinically moderate morbidity but may be severe
Normally caused by a secondary immune response to infused red cells resulting in the production of a clinically significant IgG antibody
Hypersensitivity type II immune reaction
Donated blood split into three components for therapeutic use
Red cell concentrates
To correct anaemia
Mostly has a limited volume of donor plasma
Apply rules for red cell compatibility to avoid transfusion of incompatible A and B antigens
HT status can be ignored if red cells stored in additive solution
Platelet Concentrates
Treatment of thrombocytopenia
Avoid transfusion of high titre anti-A and/or anti-B to non ABO-identical recipients
ABO identical platelet concentrate is the first choice
Fresh Frozen Plasma
Prevent microvascular bleeding due to abnormal coagulation
Treatment of coagulation factor deficiencies
Avoid transfusion of high titre anti-A and/or anti-B to non ABO-identical recipients
ABO identical FFP is normally available
Aims in selecting blood components
To avoid immediate-type HTR
select blood components for transfusion which are ABO identical or compatible
To limit production of clinically significant alloantibodies
match for RhD antigen to avoid recipient immunisation
select K negative red cell units for pre-menopausal females
To avoid 'other' HTRS
detect clinically significant 'irregular' alloantibodies
select and transfuse with antigen negative (phenotyped) red cells
ABO Blood groups
Most clinically significant blood group system
Incompatible ABO red cell transfusions are the cause of the majority of immediate-type HTR (can be fatal)
A and B antigens widely distributed in body on cell membranes
Landsteiner's Law - Individuals lacking either the A or B antigens on their red cells will have the corresponding antibody in their plasma
Anti-A and Anti-B are very potent 'natural' IgM antibodies with a wide thermal range
Selecting ABO type of red cells for transfusion
First choice - ABO group identical Second choice - ABO group compatible
Group O is the universal donor
A or B antigens are not present on the red cells
Group AB is the universal recipient
anti-A or anti-B are not present in the plasma of the patient
Dangerous Universal Donors (DUDs)
The high titre test (HT test)
Detects group O donations with a high titre of anti-A and/or anti-B
These could otherwise cause clinically significant haemolysis if transfused to a non- group O recipient
Group O HT neg (HT-) red cell concentrates are the ideal choice for emergency situations
Rh Blood group system
Second most important blood group system in terms of transfusion practice
RhD antigen is highly immunogenic
UK population: 85% are RhD Pos; 15% are RhD Neg
RhD positive: the D antigen is present on the red cell surface
RhD negative: the D antigen is missing from the red cell surface
Transfusion of an RhD Neg patient with RhD Pos blood may cause an immune response resulting in the production of IgG anti-D
anti-D can cause significant HTR and severe/fatal HDFN
Selecting suitable RhD type of red cells
RhD Negative patients (or unknown group)
Should be given RhD Neg red cells
If females of child-bear potential - must be given RhD neg red cells or platelets
RhD Positive patients
Should be given RhD positive components (RhD negative components are still suitable)
If cannot match RhD negative status
a medical officer/ lab policy may authorise use of RhD Pos red cells to RhD Neg recipient in particular circumstances
Best emergency blood = group O RhD Neg HT-
Selecting antigen negative (phenotyped) red cells
To avoid 'other' haemolytic transfusion reactions
Patient's samples are screened to detect any 'irregular' red cell antibodies
Patients with a clinically significant antibody must be transfused with antigen negative donor red cells (e.g. patient with anti-Fya will require antigen neg blood (Fya-))
Red cell donations are 'phenotyped' to determine the presence/ absence of specific red cell antigens on the membrane
This involves use of antibody reagents of known specificity (e.g. anti-Fya reagent is used to determine whether selected donations are Fya positive or Fya negative
Status displayed on blood group label
Determining the ABO and RhD type
a variety of methods used
test tube
microplate
gel cards
a saline (direct) technique is used
centrifugation speeds up reaction
Guidlines
'Guidlines for pre-transfusion compatibility procedures in blood transfusion laboratories (2012)'
Procedure for basic ABO typing
Two parts
Cell group
determines presence of A and/or B antigens on red cells
react with known anti-A and anti-B reagents
sometimes called the forward group
Serum/ Plasma group
determine presence of anti-A and/or anti-B
react with A1 and B reagent red cells
also called back or reverse group
Include positive and negative test controls with each batch
must give expected results
Both halves must agree before overall group can be allocated
Procedure for basic RhD typing
test patients specimen with one or two monoclonal anti-D reagents
Include positive and negative controls with each batch - must give expected results
test results designated as RhD Pos or RhD Neg
patients blood type result should be verified by matching with previous historical records
Red cell antibodies and their role in immune haemolysis
Antibodies are immunogens
Immunogens are capable of eliciting an immune response and reacting specifically with the product of that response
Immunogenicity is a measure of the ability of an antigen to provoke an immune response
Degree of polymorphism
Molecular shape, charge, size and complexity
Accessibility
Number of copies
Homozygous vs. heterozygous
Humoral immune response
Primary antigen challenge - IgM predominantly then IgG
4 phases - Lag, Log, Plateau and Decline
Lag phase below threshold of detectability
Immunoglobulins class switch from IgM to IgG (normally T cell dependent)
Occurs during B cell maturation
Secondary antigen challenge - IgG produced rapidly in much higher quantities. Low amounts of IgM produced.
Lag time significantly shortened
Memory B cells respond in a T cell independent way resulting in an immediate IgG response
Antibody has improved affinity and avidity
Affinity = strength of bond between individual antibody and antigen interaction
Avidity = overall strength of bond between multi-epitope antigen and polyclonal antibody
Red cell membrane antigenic structures
Proteins (Very strongly immunogenic)
Rh antigens = T-cell dependent response (mainly IgG Abs)
Carboydrates (not always immunogenic)
ABO antigens = mostly T-cell independent response (mainly IgM Abs)
Glycoproteins (Often strongly immunogenic)
K, Fy antigens = mostly T-cell dependent response (mainly IgG Abs)
Lipids (poorly immunogenic)
Glycolipids (moderately immunogenic)
P1 antigens = mostly T-cell independent response (mainly IgM Abs)
Red cell antibodies
Five classes (IgG, IgM, IgA, IgE and IgD)
IgG and IgM function as blood group antibodies
IgM - carbohydrate/ glycoliipid - mostly T-cell independent
MW approx 900,000 Da
Optimal temp. 4oC
Clinically significant IgM reactive at/near 37oC --> cause intravascular haemolysis
Readily activates complement
aggultination caused by IgM detected in vitro by direct saline methods
IgG - protein/glycoprotein - mostly T-cell dependent response
MW approx 160,000 Da
IgG1 and IgG3 are most significant blood group Abs
Optimal temp 37oC - most are clinical signficant
All subclasses cross the placenta
IgG1 and IgG3 best, IgG2 and IgG4 worse
small size means enhancing techniques are required to cause agglutination in vitro
extravascular haemolysis
IgA - major Ig involved in muscosal immunity
MW approx.160,000
Also found in plasma - transfusions can cause 'anaphylactoid' reactions in IgA deficient recipients
Clinical significance
Haemolytic transfusion reactions (HTR)
IgM or IgG alloantibodies may cause significant levels of red cell destruction
Haemolytic disease of the foetus and newborn (HDFN)
IgG alloantibodies may cause foetal/ newborn morbidity and mortality
Autoimmune haemolytic anaemias (AIHA)
IgM or IgG auto-antibodies mediate removal of self red cells
Often chronic condition but patient may eventually succumb
Role in immune haemolysis
Antibody mediated RBC destruction
Action of phagocytic cells
Carry FcyR on membrane
Macrophages with receptor bind to cells coated with IgG antibody
Results in extravascular haemolysis
Activation of complement
Destruction of pathogens
Intravascular haemolysis (mostly caused by IgM antibodies still active at 37oC
Extravascular haemolysis - removal of antibody coated red cells by macrophages outside the vascular circulation (mostly caused by IgG antibodies)
Cells coated with C3b and large amounts of IgG aree destroyed by phagocytic macrophages with FcyR and C3b receptors in liver
Cells coated with only small amounts of IgG are removed by phagocytic cells with FcyR in spleen
Generation of inflammatory mediators
C3a, C5a (&C4a) are anaphylatoxins
Induce smooth muscle contraction
Cause release of cytokines from macrophages
Bind to basophils and mast cells
Cause release of histamine which acts as a vasodilator
Attracts phagocytes to site
C5a acts as a chemotactic factor
Accumulation of neutrophils at site of infection
Classical and alternative pathways
Classical
Destruction by haemolysis (intravascular) or opsonisation (extravascular)
Alternative
evolved as part of innate immunity - not dependent on antibody acitvation
activated by biological factors
Involves plasma proteins (Factor B,D and P)
Role in transfusion science
In vivo
Haemolytic transfusion reactions
Autoimmune haemolytic anaemias (AIHA)
Causes some adverse effects of transfusion due to complement activation by white cell antibodies
In vitro
Aids detection of clinically significant complement binding red cell antibodies (serum samples only)
causes haemolysis in a test = a positive reaction
Sensitises red cell surface with complement components
Haemagglutination reactions and antibody detection techniques
Ab-Ag reaction
Specificity is dependent on structure and charge
Must have complementary shape and opposite electrical charge (creates force of attraction)
Held together by weak non-covalent forces
Electrostatic forces (Ionic bonding)
Hydrogen bonds
Hydrophobic bonds
van der Waals forces
'Goodness of fit' affects antibody affinity
Immune complex formation follows the law of mass action
Rate of a chemical reaction is proportional to the concentration of the reacting substances
Ka (association constant) = [Ab-Ag]/[Ab][Ag]
Measures the affinity of the interaction between antigen and antibody
Detection in immunological assays
Agglutination
End-point for most techniques
antibody cross links antigen of adjacent red cells - results in clumps of red cells
2 interrelated stages
Primary stage - sensitistion (coating)
Factors affecting=
Ag:Ab ratio
increased serum/plasma: red cell ratio increases amount of Ag-Ab complexes formed
Low concentrations of red cell suspensions used - decrease in Ag concentration causes increased uptake of Ab per cell and therefore increased sensitivity
pH
Most blood group Ab optimal at pH 6.5-7.5. Optimum pH used is 7.0 +/- 0.2
Ionic strenth
Charged groups on Ag and Ab molecules attract ionic cloud which hinders the association of Ab with Ag and reduces rate of reaction
1 more item...
Temperature
IgM Abs react best at low temp. (Carb Ags associated with 'cold' Abs)
IgG Abs react best at 37oC (Protein Ags associated with 'warm; Abs)
Time
Allow optimal time of incubation
Steric Hinderance
Abs physically blocked from reaching Ag (use of enzyme techniques reduce this effect)
Secondary stage - haemagglutination (forms 3D latticework of visible red cell aggregates)
Factors affecting=
Forces of Repulsion
Ab size and electrostatic repulsion
2 more items...
Hydration of water
1 more item...
Frequency of collisions
Ab binding sites - more binding sites of IgM molecules increases the chances of random Ab-Ag collisions
Location and number of antigen sites
*Effect of centrifugation/ gravity
Used to detect Ab present in patient or donor serum/plasma
Techniques used to detect Ag present on patient or donor red cells (phenotyping)
Precipitation
Haemolysis
May also indicate presence of Ag:Ab reaction
Radioimmunoassay (RIA)
Immunofluorescence
Enzyme linked immunosorbant assay (ELISA)
Solid phase adherence
Formation of visible Ab:Ag complexes
Follows a bell-shaped curve: three zones
Ag excess = no visible reaction
Equivalence zone = Ab:Ag ratio is optimum for maximum visible agglutination reaction
Ab excess (Prozone) = no visible reation
Detection techniques
Direct 'saline' technique
Will detect IgM antibodies (will not detect IgG antibodies)
IgG are incomplete antibodies
Enhancing techniques are required for their detection
Antiglobulin test
Formation of a bridge between antigen-associated IgG molecules on adjacent cells
Detects antibody already coating the cells in the patients sample
Use of enzymes
To overcome the zeta potential and allow cells to come into closer contact
Proteolytic enzymes break glycoprotein molecule peptide bonds and remove varying amounts of protein and sialic acid
This reduces net negative charge on the red cells, improves antigen accessbility by removal of charged groups, release bound water, may cause antigen site clustering which will improve cross-linking
reagent cells pre-treated with papain then resuspended in 3% PBS
test serum/plasma against enzyme treated cells by direct method --> read for agglutination and haemolysis
Advantages - some Abs give enhanced reactions by enzyme technique
Disadvantages - loss of antigen sites/ unwanted reactions/ tests difficult to read
1 vol serum/plasma: 1 vol reagent cells --> incubate at room temp --> read for agglutination
Centrifuge at low speed allows reduction of incubation time
Can be performed by tube, gel and microplate methods
Indirect saline technique
Antiglobulin technique
Agglutination is achieved by the addition of anti-human IgG (AHG) reafent which cross-links IgG molecules on sensitised red cells.
AHG binds to the Fc portion of human IgG of any specificity
Detects antibody in patient serum/ plasma by first incubating with reagent screening cells - any antibody will attach to its corresponding antigen and thus coat the cells
Polyspecific AHG contains anti-IgG and anti-C3d
Monospecific AHGs may be used
Incubate serum and reagent cells together at 37oC --> wash with Phosphate Buffered Saline (PBS) --> Add AHG --> Read for agglutination and record result --> validate all negatives
Benchmark standard for detection of clinically significant red cell antibodies
Aims to detect any irregular red cell antibodies present in the patient's serum/plasma
NISS IAT (Normal Ionic strength solution indirect antiglobulin test)
PBS at 0.150M NaCl
Add 2-4 vols serum/plasma: 1 vol of 2-3% cell suspension --> Incubate at 37oC for 45-60 mins
LISS IAT (Low ionic strength saline indirect antiglobulin test)
PBS at 0.03M NaCl
Add equal vols of serum and cells of 1-2% cell suspension --> incubate for 15-20 mins 37oC
Controls for IAT technique
Ideally include a weak antibody with each batch of tests
Affinity column/ column agglutination technology (CAT)
Incubate serum/cell mixture in reaction chamber at 37oC
Centrifugation to push sensitised cell/ agglutinates through the individual columns of the cassette
Diamed-ID Micro Typing System
Principle is to trap agglutinates in a Sephadex gel matrix
Free cells will pass through the matrix during centrifugation phase
In the LISS IAT cassette, cells sensitised with antibodies will form agglutinates as they pass through the AHG impregnated gel matrix
BioVue System
Uses a slurry of glass beads in the microtube to trap agglutinates
Solid-phase technology
IgG antibodies present in sample onto a solid surface
Normally used to detect antibody by Antiglobulin techniques
Red cell antigens are bound to surfaces of microtitre wells --> test serum/ plasma is incubated in microplate --> excess serum/plasma is washed off --> IgG coated indicator cells plus anti-IgG AHG are added
Positive test - indicator cells will adhere to the well sides (seen as mat in the well)
Negative test - if no antibody bound, the indicator cells fall to bottom of well - seen as a button in the well
Reading and grading agglutination reactions
For standard tube technique a grading system is used
(5 - 0) 5=complete reaction, all red cells agglutinated in one clump 0=negative - no agglutination - very red cloudy mixture
Tip and roll - be gentle, read over white light