Week 1/ ABO and Rh blood group systems
H and ABO blood group system
[Hh system]
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H gene is dominant, h gene is recessive
H gene 100% in all population
hh gene approx. 1 in 106 caucasian
abnormal phenotypes e.g. Bombay and para-bombay group due to point mutation in H gene
H antigen is the precursor for production of A and B antigens
H antigen
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Type 1 found in secretion, plasma, adsorbed to RBCs
Type 2 found in RBC membranes
Type 3 found in mucin, repetitive structure, RBCs
Type 4 found in glycolipids on kidney and RBCs
Membrane associated form of antigen is a glycosphingolipid
Low expression at birth and increase as branched forms later in life
Amount of H antigen expressed is determined by A and B gene and efficiency of resultant glycosyl transferase
O > A2 > B > A2B > A1 > A1B > Oh
Type 1 H antigen (product of secretor gene) in secretions is a glycoprotein
Membrane and soluble forms if Se gene inherited
Found on most cells in body
Present in gram negative bacteria as membrane associated and soluble lipopolysaccharide
Found on cells of other animal species
Histo-blood group (associated with cell interaction and tissue development)
Infections
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O blood group protection against P. falciparum, pancreatic Ca but susceptible to Y pestis (plague)
A blood group more susceptible to smallpox, high level of coagulation proteins and thromboembolism
B blood group more susceptible to gonorrhoea, S. pneumoniae, E.coli
H abnormal phenotypes
Para-bombay groups
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Single point mutation in H gene lead to fructosyl transferase defect
Results in reduced H antigen production leading to reduced A and/or B antigen expression
Ah, Bh or ABh, OH
Antibodies and serology
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Alloantibody is very rare, only found in Bombay and some para-bombay individuals
Autoantibodies is common, most often found in compound anti-IH in A1 pregnant women
Usually IgM cold reactive antibody
Occasionally IgG antibody that fixes complement
Clinically significant in Bombay individuals
Test for H antigen using lectin Ulex europaues (gorse) 凝集素
Genetics
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Gene locus in chromosome 9 q34.1 – q34.2
A and B gene are co-dominant
O gene is amorph (deletion at codon 87 produces frameshift resulting in premature stop codon can lead to nonfunctional protein)
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Point mutations results in enzymes with different substrate specificities and glycosylation efficiencies – leads to
variant antigens including O variants
Blood group
A
B
AB
O
Antibodies in
plasma
Antigen in RBCs
Anti-B
Anti-A
None
A antigen
B antigen
A and B antigen
Anti-A and AntiB
None
Phenotypes patterns
Antigen
A1
A2
B
O
A1B
A2B
Oh
Genotype
A1A1, A0, A1A2
A2A2, A2O
BB, BO
OO
A1B
A2B
hh, any ABO gene
Antibodies
Anti-B
Anti-B (2-8% anti-A1)
Anti-A, anti-A1
Anti-A, A1, B, A,B
None
None
Anti-A, A1, B, H
Importance of ABO blood group
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Performed IgM antibodies in the circulation
Ag/Ab interaction will activate classical pathway of complement quickly
Induces intravascular haemolysis through incompatible ABO transfusion
ABO blood group system has high density and distribution in body
Soluble forms of ABH in the body
Highly immunogenic
In HDFN IgG anti-A, B can cross the placenta
ABO discrepancies
1) Variant antigens
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A3 and B3: characterised by mixed field agglutination (many small agglutinate in agglutinated cells)
Ax: no reaction with anti-A, weak reaction with anti-A,B, may have anti-A1, soluble A-antigen if secretor
Am: no reaction with anti-A, weak/no reaction with anti-A, B, no anti-A, no soluble A-antigen
Cis-AB: appears as group AB but have allo-anti-B in reverse group due to abnormal A gene that produces enzyme
capable of adding both A and B carbohydrates
B(A): detecte with monoclonal antisera which anti-B detects weak expression of A antigen on B cells
Ael, Abantu, Alae
2) Mixed filed reactions: many small agglutinates in a sea of unagglutinated cells
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Often missed with tube techniques
Best seen with tile or CAT
Accidental/deliberate transfusion of ABO mismatched blood
Feto-maternal bleed
Maternal contamination of cord specimen
Twin-twin transfusion in utero
Chimeras
Bone marrow transplant
Subgroups of A and B (A3, B3 and some other will not be seen with monoclonal reagents)
Polyagglutination (will not be seen with monoclonal typing reagents)
3) Antigen alteration
Unexpected antigens
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Whartons jelly in cord samples
Autoagglutinable cells
Polyagglutination
Acquire A or B antigen
Preservative/dye dependent reactions
Weakened antigens
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Subgroups
Cord cells
Disease related (leukaemia and hodgkins disease)
Variant antigens
e.g. A2
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A1 = A+A1, A2 = A, therefore A2 can produce anti-A1
A1 =Aa, Ab, Ac, Ad, A2 = Aa and Ab, therefore anti-A1 = anti-Acd
A2 allele leads to substitution of leucine for proline at position 156 in the N-acetylgalactosamyl transferase leading
to elongation of protein, results in less efficient enzyme leading to qualitative and quantitative difference between
A1 and A2
e.g. cis-AB (ABO genotype)
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Genotype AB + OO offspring with unequal cross-over (rare) to have genotype ABO (cis-AB) offspring
Cis-AB expression in a number of cases is due to structural mutation in either A or B gene producing single
abnormal enzyme with bifunctional activity
Bombay
Testing Oh
1. Anti-A1 + Oh = no reaction
2. Anti-B + Oh = no reaction
3. H-lectin (act as anti-H) + Oh = no reaction (because Oh group do not have any H-antigen therefore will not
react with anti-H)
ABH antigen and disease
1. Reduced expression maybe found in
1) Translocation involving chromosome 9
2) Leukaemia
3) Haemopoietic stress conditions
2. Acquired antigens
Rh blood group system
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Gene location: 1p36.11, locus linked to elliptocytosis gene locus
Number of genes = 2, RH(D) and RH(CE) polypeptides
Genes are inherited as haplotypes (50:50)
Rh protein
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Multi-pass membrane protein
Cross RBC membrane 12 times
No sugars attached
D negative Caucasians lack the RH(D) gene
D negative blacks and Japanese have abnormal RH(D) gene
Weak D type – do not make anti-D
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Missense mutation in RH(D) encoding transmembrane/cytoplasmic portion of D
Less protein inserted into RBC membrane can type as Rh(+) or Rh(-) by direct agglutination with monoclonal IgM
anti-D regents
Type 1 and 2 (3 and 4) account for 90% of weak D, do not produce anti-D
Type 4.2, 5, 11, 15, 19, 20 known to form anti-D when exposed to D+ RBCs
IS: 0, D IAT: 3, Ct. IAT: 0
Partial D type - Weak expression of D able to make anti-D
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Partial D: hybrid RH(D) allels (DVI, DIIIa, DIVa/B)
Del: detection by adsorption/elution
D epitopes on RH(CE) gene
IS: 0/3, D IAT: 3, Ct. IAT: 0
E.g. partial DVI gene results from 3 exons of RH(CE) gene inserted into RH(D) gene
Del
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Type as D-nega (IS&IAT) only adsorb and elute anti-D
Severely reduced protein
Individuals may make anti-D after receiving D+ blood
Cause by exon 9 deletion in Asians (10-30%)
Reasons to resolve weak expression
1) Conserve Rh-neg blood for D-neg recipients (high risk of making anti-D)
2) Avoid giving RhIG to women who do not need it (Rh status is confirmed for historical discrepancies)
3) Resolve early in pregnancy to eliminate false-positive rosette tests
Weak Rh(D) antigen expression
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Partial D results from lack of D epitopes (these people may type as D-pos but can make anti-D to the epitopes they
lack)
Numerous categories based on epitope profile: DII, DIII, DVI, DDBT
D weak due to error in pre-mRNA processing or due to C in trans relationship to D (C and D genes are on opposite
chromosomes)
Summary
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3-5% RhIG goes to women with weak D types
Switch of Rh status is needed when: molecular test is permanent solution, weak D types 1-4 are RH+ as a recipient
and donor
Informed consent for administration of RhIG: avoid blood product where it is not needed, RhIG shortage, new
infectious disease
Rh variants
Mechanisms
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Rearrangement of RH(CE) and RH(D)
D - - : lack of expression of C and E antigens
Increased expression of D (as IgG anti-D may directly agglutinate these cells)
Point mutation
Non-sense mutation
Deletion of nucleotides with resultant frameshift
Compound antigens
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C and E antigen combinations when the genes are in cis relationship (on the same chromosome)
ce (f) present in R0 and r
Ce present in R1 and r’
cE present in R2 and r’’
CE present in Rz and ry
G antigens
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Antigen present on both RhD and RhC proteins
Due to common residues in second extracellular loop encoded by exon 2
Not a compound antigen
Antibody often seen as anti-CD mixture, actually could be anti-D+G or anti-C+G or anti-D+C+G
Very rare to get a pure anti-G
2 stage adsoption to prove/disprove the presence of anti-G
Rh null
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Lack expression of ALL normal Rh antigens
Haemolytic anaemia of varying severity
Increase osmotic fragility (24 hrs)
Stomatocytic red cell morphology
Abnormal cation flux of RBC membrane
Inheritance: amorph type DcE/--- + DCe/--- = DcE/--- or - - -/- - Regulator type: (due to mutation affecting RHAG gene that affects RhAG needed for normal Rh antigen
expression) DCe/Dce, X1r/X0r + DCe/dce, X1r/X0r = Rh null, X0r/X0r or DCe/DCe, X1r/X1r
Rh and disease
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Rh and HDFN in Caucasian patients
Reduced expression of RhD has been seen in patients with myelodsplastic syndromes (patient change from D-pos
to D-neg)
Rh linked with one form of HE
Reduced expression of Rh antigens in SE Asian Ovalocytosis
Rh null and compensated haemolytic anaemia
Rh and warm AIHA
Landsteiner-Weiner system (LW)
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LW(a+b-) 97%, LW(a+b+)3%
More LW on D-pos than D-neg cells
More LW on cord cells than adult cells
No LW expression on Rh null cells
Accessory proteins and cytoskeletal interaction
Function of Rh and RhAg proteins
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Evidence suggests RhAG is involved in ammonia transport
Non-erythroid homologues presence in kidney, liver, skin, testis and brain
RhAG homologs identified in earthworms
Structural role in maintaining red cell shape
RhD and RhCE have possible role in CO2 transport
Rh phenotyping
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Reagents: polyclonal (rarely used)
1. mixture of high avidity anti-D sera,
2. potentiaed to produced direct agglutination,
3. MUST run a reagent control.
Reagents: monoclonal (commonly used)
1. Human cell lines
2. IgM and IgM/IgG blends
3. Not potentiated
Donors: true RhD-neg (i.e. rr), tested for weak D (all variants, especially DVI)
Patients: test for PhD using reagent that doesn’t detect DVI (maternity cases are the exception), if negative
transfuse with RhD-neg donors
Rh alloantibodies
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Generally immune (IgG1 and IgG3)
Involved in HTR and HDFN
Naturally occurring antibodies do exist: Anti-E most common but anti-Cw and anti-D exit as well
Mixtures of antibodies common e.g. Anti-D+C, anti-E+c in R1R1 person
React best by enzyme (low avidity antibodies) and antiglobilin techniques
DO NOT fix complement (Rh antibodies do not activate complement due to the distance between antigens but is
probably due to a lack of mobility)
Reactivity of Rh antibodies is enhanced by enzyme treatment of the test RBCs and most react optimally at 37C
Week 2/ Haemagglutination and serological techniques – compatibility testing
Haemagglutination and serological techniques
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Affinity: related to the closeness of fit between the hapten/epitope and the antibody binding site
Avidity: related to the closeness of fit between individual epitope/binding site unions and the number of different
epitope/binding site reaction
Blood group antigens are heterogenous with respect to biochemistry, epitope configuration, distribution w/n
body, # and cellular density
Antigen epitope configurations: AB antigens: at least 4 different configurations, Rh antigens: D antigen known to
have 30 different epitopes
Antigen distribution: AB antigens: on virtually all cells in body (soluble antigen in Se positive individuals ~80%)
Kell and Rh antigens only found on red cells
Primary antigen-antibody interaction (Ag + Ab  AgAb)
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Specific, fast, invisible, energy changes occurs
Factor influencing primary interaction: ags/abs concentration, proteolysis of red cell membrane, ionic strength,
pH, temp
Ags/abs concentration: promote formation of ag/ab complex: increase conc. of abs relative to ags by adding larger
volume of serum/plasma, decrease ag conc relative to abs by using weaker cell suspensions
[if abs conc is low, use weak cell suspension (tube or CAT tech), if ags expression is weak, use strong cell suspension
(tile technique)]
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Proteolysis of RBC membrane: treating RBC with proteolytic enzymes (papain, ficin) can remove surface structures
including some blood group antigen and additional antigen sites and decrease negative charge of RBC. Proteolytic
digestion of RBC membrane can expose 30% more Rh(D) antigen sites
Ionic strength: reduction the ionic strength of a red cell suspension can enhance the rate of primary union of
antigen and antibody by up o 1000-fold, this is achieved by increasing the ionisation of cell surface antigens and
promoting the formation of ionic bonds
pH (H+ conc) generally ag-ab reactions work best at neutral pH, some reactions work best at slight acidic pH
(pH6.5) e.g. reactions favouring acid conditions include some anti-M and anti-P1 abs
temp: influence of temp is determined by the thermal characteristics of ag-ab reaction, the thermal
characteristics of reaction are influenced by the predominating non-covalent bonds, predominating non-covalent
bonds are largely influenced by the biochemistry of the antigen
non-covalent bonds (e.g. ionic, H, hydrophobic, dispersion forces): relatively weak bonds which contribute to the
reversible nature of ag-ab reaction, bond strength is dependent on the proximity of the epitope and ab-binding
site
Secondary antigen-antibody interaction (Ag + Ab + AgAb  AgnAbn)
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Specific, slow, visible, little energy change
Factor influencing secondary interaction: size relationship b/w ab and RBC surface, zeta potential, water of
hydration
RBC interspace 25nm, best fit for IgM but not best for IgG
Zeta potential: cloud of positive cations (Na+) surrounded negative charged RBC surface, resulting in the repulsion
from other negatively charged red cells.
1) Inversely proportional to dielectric constant and ionic strength of medium
2) Dielectric constant is a measure of the charge dissipating ability of the suspending medium
3) Zeta potential can be altered by changing RBC surface charge and/or altering the suspending medium
4) Dielectric constant can be increased by the addition of material such as albumin
5) Increase in ionic strength will decrease zeta potential but at the same time decrease the rate of primary ag-ab
reactions
Water of hydration: electro-negative charge RBC surface binds water through H bond, abs with stronger H-bond
ability displace the H-bonded water when they combine with their epitope, the more water released the closer
the cells can become leading to cross-linking by IgM and IgG abs
Reducing the electro-negative charge of cell (e.g. proteolysis, decreases the amount of H bonded water) where
antigen conc is high (e.g. A and B antigens, large amounts of specifically bound IgG antibody can displace sufficient
H bond water allowing cells to come close enough for IgG ab to crosslink them
Haemagglutination
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Ag determines: the Ig class of the ab, how much and where ab can attach to the RBC surface, the thermodynamics
of ag-ab reaction
Both zeta potential and wate of hydration operate to keep RBC apart and required modification if IgG ab are to
produce direct haemagglutination
Titre: measure of ab conc, significant difference requires at least 2 tube difference in titre
Scors: measure of ab concentration and avidity, significant difference requires a score which differs by 10 or mote
Reaction vessels:
1. Test tube (easty to hanle, can be centrifuged, good for weak abs);
2. Microtiter: plates U and V bottom (convenient for batch testing, economical with reagents, good for weak ab,
require special microplate carriers for centrifugation)
3. Tiles: glass or plastic: possible to perform multiple tests, good for weak ag expression and cell mixtures, tiles
retained for repeat observation
4. Capillary tubes (chown): very economical on regents, difficult to handle and label capillary tubes, reading difficult
5. Solid-phase red cell adherence: reading based on monolayer or pellet of indicator cells, economical on reagents,
solid-phase coated plates can fixed/stored, good for batch testing and weak abs, requires microplate carriers and
accurate centrifugation condition, can be read using image analysis, can’t discriminate cell mixtures
6. Lateral flow: immobilised ab captures ag positive cells and unattached cells are washed away leaving bands of red
cells to indicate phenotypes
7. Magnetised red cell separation: red cells are coated with magnetised beads that allow them to be separated
based on a magnetic field and carefully controlled shaking of a microtitre plate
Serology techniques – Saline
Direct haemagglutination (ABO, H, I, P, Lweis, MNS, IgM warm reacting abs can be detected)
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Primary or secondary AgAbs reactions occur
Mainly IgM abs but can be IgG
Usually react best at RT and below, can react up to 37C
Usually performed as IS and following 15min incubation at 37C
Serology techniques – anti-human globulin
Techniques to detect IgG and/or C3b/C3d’ sensitised red cells (primary ag/ab reaction only)
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Direct: sensitisation has occurred in vivo
Indirect: sensitisation has occurred in vitro
Variation in technique relate to optimisation of primary ag/ab reaction (LISS, PEG)
37C saline test performed before washing can indicate direct agglutination which could be carried through to the
AGH phase
Low avidity abs can be eluted form the cells during the washing steps
CAT DO NOT require washing step
Serology techniques – manual anti-human globulin
False negative
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Inadequate washing of cells
AHG regent neutralised or deteriorated
AHG not added
Serum/plasma not added in indirect test
Serum non-reactive (C’ inactive)
Inadequate incubation conditions
Cell suspension too weak/heavy
Poor reading techniques
False positive
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Auto-agglutinable cells
DAT positive cells used in indirect test
Bacterial contamination of cells’saline
Poor reading techniques
Preservative or LISS dependent abs
CAT: diamed only abs, warm/weak reactive no appaerent specificity (WRNAS)
Serology techniques – anti-human globulin
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Detects: Rh, Kell, Kidd, Duffy, S s U, Diego, Vel and others
Manual technique detects 200-300 IgG molecules/RBC
CAT more sensitive than manual technique
Manual technique may miss low avidity abs
Serology techniques – enzyme (proteases: papin, ficin, bromelin)
Detects Rh, Jk and some Kell abs, cold, auto and enzyme only abs
Destroys Fy(a) Fy(b), M, N, S antigens (In, Kn/McC, Ch/Rg, JMH)
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Can be performed as single (mixed red cells, enzyme and serum, two-stage: mix red cells and enzyme, wash and
add serum/plasma, techniques)
Tube two stage most sensitive of the enzyme techniques
Considered to be overly sensitive to non-clinically significant abs hence not used routinely
False positive if red cells are over-treated with enzyme
Can be used with CAT
Manual technique can be adapted to an enzyme-AHG technique
Summary
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Different abs react best by different techniques therefore resolving ab problems requires use of a range of
serological techniques
CAT has introduced standardisation, automation and simplification to red cell serology
CAT has limitations in its flexibility to resolve ab investigations and sometime is too sensitive
Clinical significance of blood group antibodies
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Clinical significance of blood group antibodies are those that results in 1) shortened survival of transfused cells, 2)
results in products from an antigen antibody reaction that produce adverse clinical effects in the recipient
Generally react at 32C+ temp and results in complement activation and/or phagocytosis and/or ADCC (can be
sequestration of agglutinated RBCs, apoptosis, membrane perturbation/eryptosis)
Complement activation pathways: activation > amplification > Membrane attack complex MAC formation
1. Classical pathway: ag-ab interaction (C1qrs, Ca2+) > C2, C4 amplification > C3 amplification > C5,6,7 > C8,9 > MAC
2. Alternate pathway: C3 hydrolysis > factor D/B, properdin,C3 amplification > C3 amplification> C5,6,7 > C8,9 > MAC
3. MBL pathwaylectin binding to bacteria mannose > C2, C4 amplification > C3 amplification> C5, 6, 7 > C8, C9 > MAC
Classical pathway activation sequence
1. Recognition phase: ab (IgM, IgG3, IgG1, IgG2) + C1q (ca2+), C1r and C1s
2. Amplification phase:
a) C1s acts on C4 > produce C4a, C4b; act on C2 > produce C2a, C2b
b) C4b and C2a combine to produce C4b2a (C3 convertase) which is stabilised by binding to CR1 sites on RBC
c) C4b2a acts on C3 > produce C3a, C3b.
d) C3b combines C4b2a > produce C4b2a3b (C5 convertase), some C3b binds to other CR1 sites for further
activation
e) C4b2a3b act on C5 > produce C5a, C5b
f) C5b combine C5 convertase (C4b2a3b) or attach weakly to other sites on RBCs
3. MAC formation
a) C5b attract C6, C7 > produce C5b67 on RBC surface
b) Lesion stat to appear on surface
c) C8 combine C5b67 > produce pores in RBC membrane
d) C9 combine C5b678 > act as catalyst to enhance pore formation
e) Cell is unable to maintain osmotic balance, water flows in and the cell eventually bursts
Complement activation products
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C2b: kinin like activity
C3b opsonisation
C3a and C5a: chemotaxis and anaphylaxis
C5b67: reactive lysis
C8, C9: membrane lysis
Phagocytosis
1. Splenic sequestration: IgG3, IgG1 [EVH]
2. Hepatic sequestration: C3b (IgG3, IgG1) [IVH]
ADCC
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Perforin and granzymes
IgG3 and IgG1, macrophages, NK cells, neutrophils
Mediators of clinical effects
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4.
Anti-inflammatory: IL-1ra
Proinflammatory: IL1, TNFa, IL6 > fever, shock, coagulation
Chemokines: IL8, MCP > infiltration degranulation
C3a, C5a activated monocyte/macrophage
Factors influencing clinical significance
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Antigen: immunogenicity, biochemistry, density and distribution in the body
Antibody: Ig class and subclass, conc, avidity
Complement: activation and regulation
Monocyte/macrophage system: activity
Immunogenicity of blood group antigen: D > K > c > E > k > e > Fy(a) > C > Jk(a) > S
Summary
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Antigens are the primary factors in determining clinical significance in blood transfusion
Antibody characteristics and the reaction with antigens can caused red cell destruction by number of mechanisms
which are not mutually exclusive
The interaction between antigen and antibody can produce a range of biologically active mediators that cause
clinical changes in a transfusion recipient
Week 3/ Clinical significance of blood group antibodies
Antibody detection
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ABO reverse groups
Screening cells
Purpose to detect abs to the most common clinically significant blood group ags
Consist of: 2-3 group O cells containing all of the ags to the most commonly occurring abs, with homozygous
expression of ags known to demonstrate dosage e.g. Rh, Fy, Jk, MNS
Dosage Effect is a phenomenon in which red cells from homozygous people possess more antigens per red cell
than do cells from heterozygous people. The effect is exhibited by the corresponding antibodies reacting more
strongly with cells from homozygous people
With increasing reliance on abs screening to release blood, screening cells should express important low frequent
antigens
Zygosity and dosage
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4.
Jka/Jka (homozygous): dosage 4+/12 > antibody production: anti-Jkb
Jka/Jkb (heterozygous): dosage 2+/8 > antibody production: none
Jkb/Jkb (homozygous): dosage 0/0 > antibody production: anti-Jka
Jk/Jk: dosage 0/0 > antibody production: anti-Jk3
Antibody detection – techniques
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Those capable of detecting clinically significant red cell abs reactive by haemagglutination and/or haemolysis at
37C: IS, 37C, IAT
CAT indirect antiglobulin technique (IAT)
IS: detecting MNS, P1, Lewis (I, H)
IAT: detecting Rh, Kell, Fy, Jk and others
Enzyme: detecting Rh, [Kell], Jk, P1, Lewis, (I, H)
Antibody identification Panel cells
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Purpose to confidently identify the single abs or simple ab mixtures
Consist of 11 to 16 group O cells of known phenotype that provide sufficient ags positive and negative cells to
allow 95%confidence of abs identification
Should express ags which may be unique to the racial groups (e.g. V, VS, Mia, Mur, Gerbich)
Indication of high and low frequency ags and additional ag typings
May include a pool of group O cord cells which are negative for the following ags: I, lewis, Sid
Collected into preservative colution giving expiry of at least 8-10 weeks from collection
Basic rules
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Include original reactive cells where possible: if performing investigation following initial ID in crossmatch
Include patients own cells (autocontrol)
Use a variety of techniques
Record results quantitatively
Include A1 and A2 cells as appropriate: if querying anti-A1 or anti-H
Panel interpretation
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Autocontrol: allo- or autoantibody or both
Single o multiple abs: reaction in different tech, different strength of reaction in one tech (dosage or multiple abs)
Consider negative reactions first: abs can not be directed against antigens on cells which are non-reactive, start
with the techniques that has most negative reactions
Match pattern of reactions
Interpretation of reaction
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In which techniques are the reaction occurring? IgG or IgM (warm or cold abs)? Is haemolysis present in 37 saline?
Do reactions occur with different cells in different techniques? Multiple abs reacting with different ags in their
preferred techniques e.g. Rh enhanced in enzyme, Fy negative in enzyme
Do reaction strength differ with different cells in one technique (multipe abs, single abs showing dosage)
All cells reacting: autoabs (note auto-control), abs to high frequency ags or HTLA, oreservative dependent (panel
cells positive, auto-control negative, DAT negative), reagent dependent abs (all cells positive including auto,
patient DAT negative)
Confirmation of identification
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Consider reaction characteristics of possible abs
What is/are the most probable abs and what can’t be excluded
Extend the panel to eliminate abs that can not be excluded
Must have at least two cells with only one of the antigens for the most probable abs reactimg positively and/or
negatively
Phenotype the patient and donor units if part of a cross match
Calculate probability
Phenotype patient and donor units
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Ethnic background of patient may provide clue to likely abs specificity
If allo-abs, patient should lack ags to most probable abs
Compatible donor units must lack ags to most probable abs
Incompatible donor units must express one or more of the antigens which have not been excluded
Calculate probability
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95% probability requires 3 ags positive and 3 ags negative cells reacting accordingly (fisher race calculation and
AABB guidelines)
BCSH and AABB guidelines indicate 2 ags positive and 2 ags negative cells are all that is required to confirmed abs
specificity
Extent the panel when mixtures existed
Should be used in conjunction with serological information e.g. anti-Fya reactivity by IAT and NOT by enzyme is
strong evidence of presence, irrespective of probability calculation
Unexplained reaction
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Poor technique
Antibody only reacts with homozygous cells
Abs reacting with ags not indicated on panel i.e. low frequency/private ags
Preservative or reagent dependent abs e.g. neomycin, thiomersal, inosine, glucose, EDTA etc
Problem - Complex mixture/abs to high frequency ags
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Autocontrol negative, no negative reactions in panel
Reactions are wek and confusing (maybe HTLA or HLA related)
Problem - Autocontrol is positive
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Autoabs present in sample and may mask presence of alloabs
Resolving complex mixture:
1. Separate antibodies
a) Dilution of sample: low titre abs don’t react
b) Change incubation time and/or temperature: limits reactivity of some abs in mixture
c) Differential absorption and elution: absorb some specificities from mixture, use cells that are giving strong
reactions to absorb multiple abs or those at highest titre, or weakest reaction to absorb fewer abs and those
at lower titre
2. Extend the panel by selecting appropriate antigen positive and negative cells, use null phenotype cells
3. Modify antigens on cekks
a) Enzyme destroy MNS, P, Fya, Fyb, Xg1, In, Kn/McC, Ch/Rg and JMH
b) Aminoethylisothiouronium bromide AET and dithiothreitol DTT destroy Kell system antigens, destroy/weaken
LW, In, Kn/McC, Gya, Hy, Joa, Yt
4. Neutralise antibodies
a) ABH, Lewis using saliva
b) P1 using hydratid cyst fluid, pigeon egg ovalbumin
c) I using colostrum
d) Sid using urine
e) Ch/Rg and HLA using fresh inert serum
5. Adsorb the antibodies e.g. Bg/HLA abs can be absorbed using expired pooled platelets
6. Use special techniques: combine enzyme/AHG (where abs masked by Fy, weak Jk abs)
7. Use rare and different cells (Reference labs)
a) Rabbit red cell stoma: high in I ags
b) Monkey, horse cell cells: high in i ags
c) Dog red cells: use in differentiating Pr abs
d) Pig red cells: express A(p) ags
Compatibility testing
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Purpose to prevent transfusion reaction
Blood product will be of maximum benefit to the patient
Clinical decision on need for blood and type of product
Request for blood/sample collection
Grouping, antibody screening and crossmatching
Release of blood and setting up transfusion
Monitoring the patient
Crossmatching
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Check for major discrepancies in ABO grouping
Check for presence of pre-formed abs which may lead to premature destruction of transfused red cells
Cannot detect all ABO mismatches and mistake RhD typing
Do not guarantee transfused RBCs will have normal survival
Do not guarantee recipient will not have a reaction or produce abs to transfused blood
Require: patient ID (full name, dob, UR, ward), full history (diagnosis, therapy, pregnancy, known abs), type and
volume of produce required, date and time required, request signed
Acute blood loss (injury/surgery)
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Type of product and rate of transfusion dependent on situation
Massive blood loss (volume equivalent to patient blood volume transfused in 24hrs or 5 units in 4 hrs)
Massive transfusion protocol/pack (MTP)
a) 4 units RBC (ABO matched but uncrossmatched blood or O RhD neg)
b) 4 units fresh frozen plasma (FFP) (group AB if group unknown)
c) One platelet pool (4 individuals) or one apheresis pack
Laboratory protocol for need to crossmatch in place (e.g. don’t crossmatch after 10 units given)
Elective surgery or chronic blood loss
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Maximum (surgical) blood order schedule (MSBOS)
Indication of likely need for blood associated with different surgical procedures determined by hospital
transfusion committee, regularly reviewed and updated
Crossmatch transfusion ratio
≤2:1 considered to be desirable (>2:1 unnecessary testing being performed)
Patient blood samples collected
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Within 72hrs of transfusion if previously transfused
Positive patient ID (direct questioning for full name and dob), policy in place for ID of unconscious, irrational,
foreign speaking ir patient w/o wrist band
Volume and anticoagulant appropriate (EDTA plasma or serum)
Labelling: patient name written on tube immediately after collection, date and time of collection noted on tube
and request slip, request slip signed
Transport immediately to laboratory
Laboratory check on ID, match sample to request form, consult laboratory records for previous testing
ABO and Rh(D) type sample
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First time testing: test twice, two people independently confirm results, use twoo different anti-D reagents
Repeat testing: confirm result with laboratory records
Antibody screen
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Three cell panel to cover ag system showing dosage
Techniques: manual procedure IS, 37C saline, IAT or CAT IAT
Antibody screen negative and no previous history of antibody detection:
1. Issue ABO and Rh(D) matched units
2. IS to check ABO mismatch
3. Check computer crossmatch/electronic release
Antibody screen positive or history of previous antibodies
1. Identify antibodies
2. Crossmatch ABO, RhD matched and antigen negative donor unit
a) Negative for identified antibodies and any antigen likely to stimulate more antibodies e.g. K for pregnant
women, R1R1 with anti-c
3. Record results which should include:
a) Full patient ID
b) Sample collection date/time and expiry
c) ABO and Rh(D) type, antibody screen and antibody ID
d) Blood product type and donation/batch numbers
e) ABO, Rh(D) type and extended phenotype as appropriate
f) Compatibility results, time performed and by whom
g) Date and time of issue
Blood of same group not available
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AB use group A red cell concentrate first (note expirt of available unites)
A or B use group O red cell concentrate change to patients true group once any transfused isohaemagglutinins no
longer detected by IAT
RhD positive blood only to RhD negative males or post-menopausal females
BMT patient with different ABO donor group
Laboratory protocol for group of products to be used at different times post-transplant
Change patient record for blood group
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Check ABO and RhD type of donor units before release for transfusion
Label units for patient and hold in appropriate section of the blood bank refrigerator for agreed time
Print compatibility or issue report (including patient ID, ABO Rh(D) group, product detail, testing results,
appropriate interpretation and clinical comments, details of any special requirements e.g. pre-warm to
transfusion
Autologous blood
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Autologous blood collected by the red cross must meet all of the normal donor requirements
Autologous blood collected by other agencies may not meet the red cross requirements but the individual must
be healthy enough to have two-three units collected in the two to three weeks prior to surgrty
Individual should sign each of the packs collected and they should be stored in the blood bank fridge in a special
section for autologous donations
Prior to transfusion the normal crossmatching protocols should be adopted before the blood is released for
transfusion e.g. check blood groups, antibody screen
If conscious, the person should identify their signature on the blood pack prior to transfusion commencing
If blood not transfused during or post-surgery, it should be discarded
Releasing product for transfusion
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Visually check products for evidence of gross contamination prior to release to ward or theatre
Release units only with positive patient ID being provided
In ward or theatre two people positively ID patient and donor units
Laboratory must be able to trace every blood component/product received through to its final destination e.g.
patient, clinical area (ambulance), another facility or disposal
See remote release
Computer crossmatch/electronic release can be used when:
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ABO and Rh(D) group confirmed
No antibodies in screening test
No history of clinically significant antibodies
Comprehensive validated software system exists
Computer crossmatch/electronic release software features
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Only release group specific blood if records on current G and S exist
Group specific blood not released on historic record
O Rh(D) pos or neg blood only released in emergency where no current G and S exists
Warning for special blood products provided e.g. CMV negative, irradiated, autologous
Mechanism to ensure that blood component is labelled for specific patient
Unique label generated at release of unit and checks that group of blood component and patient match
Issue report produced with release of blood component to allow documentation of transfusion episode
Use of software
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Must be validated before use
a) Cross tabulation of donor and recipient groups for all crossmatched blood products
b) Validation recorded and checked by senior staff member
Must be revalidated if any software modification undertaken
Software modification only by authorised staff
All transaction logged and traceable to individual staff e.g. audit trail
Random access to database prohibited
Remote release
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Used where blood stocks are held at local centre but all testing is performed centrally
a) Extension of computer crossmatch
b) Local centre can interrogate the patient records for ABO, Rh(D) group, G and S results and history
c) If all conditions are met, check through stocks for appropriate unit and generate label and transfusion
documentation.
Summary
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Proper patient and donor ID essential throughout all stages of compatibility process
Serological crossmatch is only required on patient with identified antibody/ies or history of antibodies
Validated software can be used to safely release blood products for transfusion