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AP0509 Cell Transfusion > NHS BT > Flashcards

NHS BT Flashcards

1
Q

Blood group antibodies

A

In Transfusion there are two classes of immunoglobulin which are of interest as far as blood group antibodies are concerned
IgM
IgG

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2
Q

IgM

A

Pentameric structure (MW c. 900,000)
10 antigen binding sites
Red cells repel one another and do not come closer than within 12 nm of one another
A single IgM antibody can bridge the gap and cause agglutination of red cells

Work best at temperatures below 37oC
Are generally of minor clinical significance
May bind complement
Do not cross the placenta
ABO blood group antibodies are mainly IgM (yet are highly clinically significant)

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3
Q

IgG

A

Monomeric structure (MW c. 180,000)
2 antigen binding sites
Binding site can only reach 10 nm apart
A single IgG antibody cannot cause agglutination of red cells

Work best at 37oC
Generally of clinical significance
Do not usually bind complement
Are able to cross the placenta
Primarily cause the removal of red cells by extravascular haemolysis (Fc regions of red cell bound IgG interacts with Fc Receptors on macrophages of reticuloendothelial system)
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4
Q

ABO groups in UK

A

Group O 47%
Group A 42%
Group B 8%
Group AB 3%

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5
Q

ABO Genes

A

The presence of the A and B blood groups is
governed by three genes which encode for
glycosyltransferase enzymes

H gene Located on Chromosome 19
A gene Located on Chromosome 9
B gene Located on Chromosome 9

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6
Q

Blood Group A Genotype

A

AA AO

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7
Q

Blood Group B Genotype

A

BO BB

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8
Q

Blood Group AB Genotype

A

AB

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9
Q

Blood Group O Genotype

A

OO

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10
Q

ABO subgroups

A

A1 – 80%
A2 – 20%

Is quantitative difference, but may be qualitative in some cases where anti-A1 is produced.

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11
Q

Blood Group A1 Genotype

A

A1A1, A1A2, A1O

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12
Q

Blood Group A2 Genotype

A

2A2, A2O

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13
Q

Blood Group A2B

A

A2B

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14
Q

ABO Antibodies

A

Present in the general population
“Naturally” occurring
Not present at birth
Develop over the first four months after birth

Some immunoglobulin just happens to ‘fit’ (c.f. Lectins)
Via maternal milk
From environmental factors (Experiments with chickens)
Genetic basis

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15
Q

Landsteiners Law

A

ABO antibodies are found in the plasma against the ABO antigens an individual lacks

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16
Q

Landsteiners Law Blood Groups

A 
Blood Group	    Antigen on		  Antibody in
				    red cells		  plasma
		O		      None		    Anti-A + B
		A		         A		    Anti-B
		B		         B		    Anti-A
		AB		      A and B	        	    None
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17
Q

Development of ABO Ag and Abs

A 
Antibodies
Not present at birth
Development during first 4 months
Reach maximum strength at 5 yrs old 
Antigens
Not fully developed at birth
Development over first 1 - 2 yrs of life
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18
Q

Clinical significance of ABO

A

IgM antibodies
Work best at low temperatures

But…….
Highly clinically significant
Cause intravascular haemolysis (complement activation)
May cause fatal reaction in cases of incompatible transfusion

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19
Q

Antibody Identification ISBT View

A

No antibody investigation technique or combination of techniques can be guaranteed to detect all red cell antibodies.

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20
Q

Red Cell Ab Panel

A 
Identify a single antibody
Separate common mixtures
If possible in a single pass
Not give the same pattern of results with two different antibodies
Give “confidence” in the findings
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21
Q

Panel Cells

A

Panel cells
usually cell samples from 10 selected donors
stored liquid in preservative solution

Reactivity in vitro is a surrogate marker for red cell destruction in vivo

All group O  
Include R1R1 (Cde) and R1wR1
Include R2R2 (cDE), r’r (Cde) and r”r (cdE)
3 rr (cde) homozygous expression of k, Fya, Fyb, Jka, Jkb, S, s and also express K
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22
Q



BCSH* Guidelines for Compatibility Procedures.

A

“When an alloantibody is detected in the screening procedure its specificity should be determined and its clinical significance assessed”

Transfusion Medicine 14 59-73 2004

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23
Q




Antibody risk assessment
for transfusion SIGNIFICANT

A 
Significant
ABO
Rh (all of them!)
Kell system
Kidd system
Duffy system
anti-M 37oC active
Anti-S, -s
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24
Q




Antibody risk assessment
for transfusion Insignificant

A 
anti-Lea
anti-Leb
anti-P1
anti-H(I) (Not anti-H in “Bombay” phenotype)
anti-N
anti-M (20oC only)
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25
Q

Indirect Antiglobulin Technique

A

detects 37oC active antibodies
detects IgG antibodies
detects complement fixing antibodies
Detects all Rh antibodies, anti-K, k, Fya, Fyb, Jka, Jkb, M (reactive at 37 deg C), S, s, Kpa, Kpb, Lua, Lub

“The gold standard”

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26
Q

Enzyme treated cell techniques (Papain)

A

good for all Rh antibodies and anti-Jka, Jkb, Lea, Leb, P1
does not detect anti-Fya, -Fyb, -M, -N, -S because these blood groups are destroyed by papain treatment
excellent in resolving mixtures

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27
Q

Saline 20oC

A

detects IgM antibodies, e.g anti-M, N, P1, Lea, Leb, H, I and ABO
detects antibodies which are usually clinically insignificant (ABO antibodies excepted)
detects antibodies which may give unclear IAT
good for resolving mixtures

28
Q


Tools of the trade: other techniques

A

PEG IAT, 2-stage IAT, enzyme IAT,
Capture R, albumin displacement, polybrene,
37oC saline, 31oC saline 4oC saline,
NISS, LISS, BLISS, adsorptions, elutions, neutralisations………….

29
Q


BCSH Guidelines for Compatibility Procedures.

A

“The specificity of the antibody should only be assigned when it is reactive with at least two examples of reagent cells carrying the antigen and non reactive with two examples lacking the antigen”

30
Q

Identification criteria

A

To be certain of the identification of an antibody the panel used must give
Positive results against two cells which contain the antigen
Negative results against two cells which lack the antigen
Exclude the presence of any other antibodies

31
Q

Antibody ID tips

A

Enzyme sensitive (don’t work with papain-treated cells) helps to resolve mixtures: anti-M, -N, -S, -s, -Fya, -Fyb

Dosage: stronger reactions with homozygous cells (~twice as much antigen on cells)
anti-Fy, -Jk (especially), MNSs,
anti-c, -D (anti-D may react stronger with R2R2 cells)

IgM antibodies (20oC active): anti -M,-N,-P1, -Lea,-Leb

Masked antibodies
Expect the expected!

32
Q


Antibody mixtures:
How do you know it’s a mix?

A

No fit for a single specificity, auto negative
? Different strengths of reactions
? Different patterns with different techniques?
Additional reactions to those in previous sample

33
Q


How to identify elements of a mixture

A 
Getting some negative results?
Try to identify at least one component
(there’s usually some Rh in there)
Use negatives to eliminate something!
Try to find more negatives (more panels)

Performing a patient phenotype can help
(ABO, D, C, E, c, e, Cw, MNSs, P1,Lea, Leb, K, (k if K+), Kpa(Kpb if Kpa+), Fya, Fyb ,Jka, Jkb

How does the phenotype help?
Tells you what alloantibodies patient can form
Helps you to find more panel cells
May help in providing safe transfusion

BUT BEWARE
The transfused patient
The “untransfused” patient
The Positive Direct Antiglobulin Test

34
Q

Indirect Antiglobulin Test

A

IAT
Antigen – Antibody complex forms

AHG added
(Anti-Human Globulin)

Agglutination seen

35
Q

Molecular RBC typing

A

Can provide a full genetic type for highly prevalent antigens
Simple 3 step procedure with minimal ‘hands-on’ time, giving results in under 3 hours
Most technology (PCR cyclers) already present in H&I laboratories
Specialised Reader

36
Q

Clinical Value of Molecular RBC typing

A 
DAT positive patients
Multi-transfused patients
Patients with pan-reactive autoantibody that resists absorption procedures
Patients with weak expression of antigen
Where liquid reagents are unavailable
37
Q

RBC genotyping

A

Extract DNA (30 mins)
Prepare tests - microplate (10-30 mins)
Amplify DNA – thermocyclers (c2 hours)
Analysis (10 mins)

38
Q

Taqman Probe PCR SSP

A

….

39
Q

Taqman Workflow

A

Samples processed in a single day
3 Samples per run, up to 8 runs a day
Results available in approx 2.5 hours
Good reliability with low failure rate

40
Q

RBC FluoGene Validation Results

A 
Newcastle and Sheffield 1056 donor samples
6 fails (0.6%) – All resolved on repeat
7 ‘indeterminate’ single types – 4 resolved on repeat

Similar findings in routine processing of samples

41
Q

Patient Evaluation for Genotyping

A

AIHA
DAT positive
Antibody combinations/SNDT
Ethnic representation

42
Q

RBC Genotyping Advantages

A

Most useful in multi-transfused and AIHA cases
Results can be obtained the same day
Reproducible, Low fail rate
Good concordance with serological typing results
Simple to use and not labour intensive
No significant wastage
Direct download of results

43
Q

RBC Genotyping Disadvantages

A

Not suitable for urgent cases
Not as fast as serological typing (see urgent cases above)
Not suitable for rare genotypes – require sequencing

ABO – simple serologically but complex genetically (not performed on this assay).

44
Q

RBC Genotyping COnclusiona

A

Genotyping improves ability to provide patients with suitable blood.
Costs currently prohibit routine use for all patients.
Likely greater use in future for both donors and patients

CE marking
Direct download
Reporting (Hematos / SP-ICE)
Price
What we do with the info;
Recommendations
Selection of blood
45
Q

Rh blood group system History

A

Landsteiner and Wiener (1941) found that
an immune serum produced in guinea pigs immunised with blood from Rhesus monkeys reacted with red cells from 85% of the New York population.
They named this new finding the ‘Rhesus factor’
85% were ‘Rhesus’ pos, 15% were ‘Rhesus’ neg

It was discovered that
all Levine & Stetson’s women were ‘Rhesus’ negative and
all their husbands were ‘Rhesus’ positive
Following this discovery (and the subsequent confirmatory work) it was proposed that all ‘Rhesus’ negative people should be transfused ‘Rhesus’ negative blood.

46
Q

Rh D Antigen

A

The Rh system comprises five commonly occurring antigens. The most important one is called D

The RhD gene is found on chromosome no 1
Defines Rh status in donors and patients
The unlinked RhAG gene on chromosome 6 incorporates the antigen into the cell membrane
D positive individuals have the RhD gene
Most D negative individuals have an RhD gene deletion

47
Q

RhD Uk Frequencies

A

85% RhD positive

15% RhD negative

48
Q

Rhesus Weak D

A

Weak D

Formerly known as Du
Possesses all normal D antigens, but has quantitatively less
Is RhD positive as a donor, patient and an antenatal patient
Cannot produce anti-D

49
Q

Rh Partial D

A

Lacks part of a normal D antigen

Can produce anti-D (as is missing some of the D antigen)
Can cause the production of anti-D in a D negative recipient (as possess some of the D antigen)

Regarded as RhD Pos as a donor
RhD Neg as a recipient
Antenatal patients require anti-D prophylaxis

50
Q

Expanded Rh System

A 
Composed of 5 commonly occurring antigens
D
C
c
E
e

These 5 Rh antigens are inherited as a group of 3 antigens which are inherited together

C and c antigens are alleles on the RHCE gene
E and e antigens are alleles on the RHCE gene

There is no d antigen, most RhD negative individuals lack the D antigen (have no RHD gene)

51
Q

Rh Combinations

A

Antigens Short
CDe R1 1st order
cDE R2 1st order
cde r 1st order

Cde r’ 2nd order
cdE r’’ 2nd order

Antigens Short
cDe Ro 1st order in blacks
rare in whites

CDE RZ Rare
CdE ry Extremely rare

52
Q

Rh Antibodies Anti D

A

Anti-D

Anti-D is the most potent and remains a major cause of Haemolytic Disease of the Fetus and Newborn (HDFN)
Produced in women as a result of sensitisation by red cells from D positive fetus
May be produced in males as a result of transfusion with D positive blood.

53
Q

Rh Antibodies Anti c

A

Clinically significant
Relatively common
May cause HDFN or Haemolytic Transfusion Reaction (HTR)
Often found together with anti-E

54
Q

Rh Antibodies Anti E

A

Anti-E

Clinically significant
Relatively common
May cause HDFN or HTR
Less clinically significant than anti-c
Often found together with anti-c in individuals who lack c and E antigens
55
Q

Rh Antibodies Anti C

A 
Anti-C
Clinically significant
Relatively common, often seen with anti-D
May cause HTR
HDFN rare when on its own
56
Q

Rh Antibodies Anti e

A

Anti-e
Clinically significant
May cause HTR

57
Q


Prevention of Rh antibody production

A

Type patients for full Rh group and match the donations
This is important for:
All female patients under 60 yrs of age
Patients who may need regular transfusions (e.g. Sickle cell disease)
Patients who have already produced other antibodies (‘responders’)

58
Q


Haemolytic Disease of the Fetus and Newborn (HDFN)

A

A condition affecting fetuses and newborn infants caused by a blood group antibody in the mother attacking red cells in the infant which carry the corresponding antigen inherited from the father.
The most potent form of the disease results when the mother is RhD negative and the fetus is RhD positive

59
Q

First pregnancy post delivery

A

Mother is sensitised against foreign red cell antigens on fetus
Antigens which are more fully developed on fetal cells are more likely to cause sensitisation (e.g. Rh, but not ABO)

60
Q

Main causes of HDFN

A 
Antibodies involved include
Anti-D (or D + C)
Anti-Kell
Anti-c
Anti-E
Anti-Fya
Other IgG antibodies e.g –S, -Jka, other anti-Rh
ABO
61
Q

Relationship between antibody 
strength and HDFN outcome

A 
Theoretically: More IgG antibody in the maternal circulation = more crosses the placenta = more severe HDFN
But…
Moderate anti-D and anti-c 
Poor anti-K and anti-E
Variable other specificities
Useless ABO
62
Q

Diagnosis and treatment of HDFN

A

Direct antiglobulin test (DAT) on cord blood.
This detects IgG antibody bound to the surface of the infant’s red cells.

A positive result is diagnostic of HDFN.
Mild – Phototherapy under UV light
Moderate – Top-up or exchange transfusion
Severe – Exchange transfusion at birth or Intra-Uterine Transfusion (IUT) during pregancy

63
Q



Treatment - Requirements for IUT / exchange transfusion red cells Intrauterine transfusion

A 
5 days old or less, CPD red cells
CMV negative
Sickle cell negative
Negative for any red cell antibodies
Low level of anti-A and anti-B
Gamma irradiated
Hct 0.5 – 0.6 for ET up to 0.7 for IUT
64
Q

Prevention of HDFN – History

A

1960’s – Sir Cyril Clarke at Liverpool
Known that ABO antibodies offered some protection
Prevention began in 1970 (post-natal prophylaxis)
Standard dose of prophylactic anti-D (polyclonal, human derived)
500 IU up to 4mL bleed
1500 IU up to 12mL bleed
FMH calculated by Kleihauer test or Flow cytometry

Antenatal prophylaxis
Monoclonal anti-D (not as effective)

65
Q

Significant antibody risk for HDFN

A 
Significant
Anti-D
Anti-c
Anti-K
Other Rh
Kidd system
Duffy system
anti-M (37OC), -S -s
66
Q

Insignificant antibody risk for HDFN

A 
Not significant
•anti-Lea
•anti-Leb
•anti-P1
•anti-H(I)  
•anti-N
•anti-M (20oC only)

AP0509 Cell Transfusion (6 decks)

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