Auto-Immune Haemolytic Anaemia Flashcards
What are the two types of AIHA?
Cold and warm
In general what is the main difference between cold and warm AIHA
Warm is more frequent then cold
However warm is rarely seen in the lab while cold is often seen
Warm doesnt really affect the patient
What are the four different types of AIHA that we will look at, classify AIHA?
Warm
Cold/Cold Agglutinin Disease (CAD/S)
PCH -> paroxysmal cold haemoglobinuria
Drug Induced haemolytic anaemia
Give a general overview of AIHA, incidence, what is it, causes
(7)
Incidence of 1/80,000 people
Occurs at all ages but more common in people over 40 with a peak in the 70s
Caused by autoantibodies direct against self antigens
Often due to a loss of T suppressor cells
Can be either primary/idiopathic or secondary
Red cell loss is either extravascular or intravascular
What is the incidence of AIHA?
1/80,000 people
Who is most likely to be affected by AIHA
Rarely seen in anyone under 40
Mostly in those over 40 with a peak in 70s
More common in females vs males
Higher frequency in males with CLL (10% develop AIHA)
Compare idiopathic vs secondary AIHA, compare the frequency
Primary or idiopathic AIHA means we dont know where the AIHA has arisen from
In secondary we know the cause and can treat it by treating the cause
Its 50/50 primar/secondary in warm AIHA
Its 90% secondary in cold
Compare the antibodies of warm vs cold AIHA
Cold = IgM
Warm = igG
How frequent are the different classifications of AIHA?
Warm is seen in 80% of cases
Cold is not as common as warm but has characteristic features in lab
Drug induced is very rare
Talk about anaemia in those with AIHA
Anaemia may not always occur due to compensatory mechanisms but there will always be a reduced red cell survival
AIHA requires patient to be anaemic but this only happens when compensation mechanisms fail
Drug induced AIHA tends to give the most severe anaemia, other types tend to be insidious and not as severe
Talk about characteristic agglutination in cold AIHA
Sample will come to the lab and look clotted - entire sample will look like a big blood clot
When sample is warmed up bood will look anticoagulated as normal
Very obvious in lab
Talk about the antibodies in warm AIHA
IgG autoantibodies which preferentially react at 37 dgrees
Causes increased red cell removal usually through extravascular haemolysis (liver and spleen)
Fix complement only to a level of C3, if at all
50% of cases are secondary/associated with other conditions such as CLL, SLE, transpants etc
what are the main sources of extravascular haemolysis in WAIHA?
Liver
Spleen
Talk about complement fixation in WAIHA
Not always complement fixing, if any it is only to the C3 level
Fc(IgG)/C3b complex on red cells are recognised by receptors on macrophages -> this is what causes the extravascular haemolysis
Give some examples of conditions which cause WAIHA
lymphoproliferative disorders e.g. CLL ((10%=AIHA)
autoimmune disordered e.g. SLE
Post transplant
50% of WAIHA are caused by these conditions
How might we detect a WAIHA in the lab?
Screen might be negative or weakly positive due to low levels of antibody in plasma
DAT will be positive
Why is the screen usually negative in AIHA?
Most of the autoantibody will be bound to red cells leavin very little left free in plasma
What will a AIHA blood film look like?
Spherocytes
Reticulocytes/polychromasia
RBC fragmentation
NRBCs
Will look like hereditary spherocytosis
Evidence of extravascular haemolysis but might see signs of intravascular haemolysis in severe disease
Film is typical of any kind of extravascular haemolysis e.g. transfusion reactions
How do we distinguish between AIHA and HS?
AIHA will be DAT+
HS will be DAT-
Why does WAIHA result in spherocytes?
Its from the extravascular haemolysis system attempting to destroy the red cells
This compresses the red cells into spherocytes
What does the degree of haemolysis in WAIHA depend on?
The level of haemolysis increases with the titre of the autoantibody
WAIHA only fixes complement to C3 level, why is this significant?
This is what haemolysis is only extravascular
Not enough complement fixing to warrent intravascular haemolysis
At what temperature does WAIHA antibodies bind?
Igs will bind at all temperature but 37 degrees is preffered
What kind of antibodies are seen in WAIHA primary/idiopathic type
IgG antibodies directed against a single RBC membrane protein
They will appear as non specific i.e. will react with most rbc samples but wont react against Rhnull cells -> Rh associated glycoprotein antibodies
What kind of blood do we give for WAIHA patients?
Blood is consequently difficult to find as antibodies are often against Rh glycopeptide antigens
-> only Rhnull blood in IBTS will come up crossmatch negative but we dont have this available to give to paients
We usually ABO, Rh and K type the patient and give back their groups
Very important to ask if patient has been transfused or pregnant though as these could possibly have other antigens such as Kidd antibodies or duffy
Why do we use the DAT for WAIHA?
As the DAT indicates in vivo red cell coating
How do we treat WAIHA?
Treat the primary condition
Steroids, splenectomy, transfusions, typical courses of treatment for primary conditions etc
Steroids = gold standard for WAIHA - very successful - antibodies usually disappear after a few weeks
Talk about transfusions in AIHA
WAIHA rarely warrents need for a transfusion
In WAIHA patients Hb usually drops really slowly, over a long period of time, eventually patient will present to GP with tiredness and maybe jaudice if really anaemic
Patient Hb can be as low as 5 but since it is a gradual drop in Hb steroids are still the gold standard for treatment and not transfusion
Difficult to get blood for these patients and steroid tretment will begin to see increase in Hb immediately
How do patients present with WAIHA
Anaemia
Severe pallor
Weakness
Dyspnoea
Fever
Sometimes jaundice
How does WAIHA affect ABO and Rh typing?
No affect on ABO typing usually
Used to cause problems with our Rh control
- used to come up positive due to high levels of potentiators used in RhD antisera
- improvements in antisera (now use monoclonal) in recent years has meant we no longer see this positive Rh control (potentiator - Rhantisera)
- cleaner reagent requiring less enhancement
Talk about the use of DAT in WAIHA
DAT is usually positive
20% are IgG only DAT positive
67% are IgG and C3 positive
13% are C3 only positive
How does the type of antibody affect complement fixation in WAIHA?
Some types of antibodies are better at complemen fixing then others:
IgG1+IgG3 are better than IgG2 or IgG4
Its based on the affinity for FcR and increased complement activation
IgG1 and 3 are more haemolytic then 2 and 4
What would you do with a warm AIHA in the lab?
Antibody screen - will be positive if enough free antibody
DAT - will be positive
Adsorptions - adsorb autoantibodies out of patient plasma so that you can look for alloantibodies
Elutions of limited value as eluate will react against everything
Why is it important to do adsorptions for WAIHA?
There are allo antibodies in 40% of cases
-> increased if patient has been transfused or pregnant
Why is it important to do adsorptions for WAIHA?
There are allo antibodies in 40% of cases
-> increased if patient has been transfused or pregnant
Why is it important to do adsorptions for WAIHA?
There are allo antibodies in 40% of cases
-> increased if patient has been transfused or pregnant
How do you carry out an adsorption
adsorb autoantibody from patients plasma
React patient red cells with their own plasma -> red cells will be coated in the autoantibody
Separate out plasma again
Now react plasma - autoantibody againist antibody panel to look for other alloantibodies
WAIHA antibodies are produced against what?
Rh related glycoprotein
Band-3
Glycophorin A
Talk about steroid treatment for WAIHA
Corticosteroids/glucocorticoids e.g. prednisone
Decreased FcR on macrophages
Mainstay treatment???
20% will go into complete remission - no evidence of disease
10% will have no response to treatment at all i.e. Hb will continue to drop
List the treatment methods for WAIHA
Steroids
Splenectomy
MABS e.g. Rituximab
Immunosuppressive therapy
Plasmapheresis
IVIG
Talk about splenectomy for WAIHA
Splenectomy only considered if patient is young
Patient will require lifelong antibioti treatment
Corticosteroid treatment is preferred
Decreases destruction and autoantibody production
Will need 6x the antibody titre to produce the same amount of haemolysis without a spleen
Talk about rituximab for AIHA
Monoclonal antibody treatment against CD20
Affects B cells - decreases B cells
Thus decreasing amount of monoclonal antibody production
Talk about Immunosuppressive therapy for AIHA
Only used if all other drugs have failed
Azothioprine, cyclophosphamide etc -> cytotoxic immunosuppressants
If all else fails then chemo drugs such as methatrexate are considered
Talk about IVIG for WAIHA
Were not 100% sure how it works
Think it works by flooding macrophage Fc receptors
- macrophages unable to detect that rbcs have been coated with antibodies
Known as liquid gold - very expensive and rarely used
When is transfusion given for WAIHA
In the short term where anaemia is sudden and severe
Fabian has never seen this been done
Usually cant get crossmatch neg blood -> just give ABO and Rh matched
Clinican is responsible for transfusion, they will take accountability
Nuissance to investigation, lot of work for one patient, follow ups must be done every week
Talk about CAIHA/CHAD antibodies
Environmentally stimulated IgM cold agglutinins
They have a low thermal range
They do not cause clinical problems usually
Abnormal cold aggs can have an extended thermal range, i.e. they may be active at 30-32 degrees which can cause clinical symptoms in periphary (where body temp is lower)
Autoantibodies prefer low temperatures but some IgM can bind up to 30 degrees -> called high titre cold agglutinins
Can cause intravascular haemolysys by complement activtion
What are abnormal IgM cold aggs
CAIHA antibodies with an extended thermal range
These function at 30-32 degrees
Thus cause symptoms when peripheral blood reaches these temperatures e.g. when patient’s hands or feet get cold etc
What are high tire cold aggs in CAIHA?
Auto antibodies which prefer low temperatures e.g. 4 degrees but can also bind to rbcs at 30 degrees
What is the preferred temperature for cold aggs?
4 degrees
Talk about complement activation in CAIHA
Can cause intravascular haemolysis by complement activation known as cold haemolysins
But this is much less common
Typically only complement is found on rbcs
What percentage of CAIHA is secondary?
90% of CAIHA is secondary to other conditions
Talk about cold aggs
these are only an affect of our testing - only reactive at room temperature not at body temperature
Dont actually affect the patient
These antibodies will disappear at 37 degrees
Not true Cold agglutinin disease
CAIHA antiobdies are specific to what?
They are often anti-i or anti-I
(Ii are antigens found on rbcs structures bearing ABH antigens)
Talk a little abobut Ii blood group, when would you see this
i has no branches, I has many branches
A fucose is added on to end of branch to become a H antigen
Cannot tell if an antibody is anti-H or anti-i
- same specificity really
All cord blood is little-i, i.e. I-
i is converted to I as you get older
What are the two kinds of CAIHA
Chronic or transient
What is transient CAIHA
This is a phenomenom that occurs in children infected ith Mycobacterium pnneumonia or infectious mononucleosis
It is a secondary type of CAIHA
The antibodies produced are acute but transient i.e. high titre but will disapear when infection is cleared
What antibody specificity is the transient CAIHA antibody in infectious mono vs M. pneumonia?
IM = anti-i
M. pneumo = anti-I
How do we demonstrate high titre cold agglutinins, i.e. cold aggs that react at 30 degrees?
ABO and typing must be carried out at 37 degrees, sample must be transported to lab in flask etc etc
Specimen will demonstrate rough agglutination at room temperature
ABO group carried out with washed cells (prewarmed saline) at 37 degrees
Rh and DAT carried out
DAT + with C3d
Can do a serial dilution afterwards to quantify titre of antibody but usually this isnt done, patient is just treated
A cold autoadsorption could also be possible if query any alloantibodies
How do you know if your 37 degrees investigation has worked/failed?
Your reagent controls are vital in this scenario
Everything will remain positive if youve faileed i.e. if not everything at 37 degrees
These are really hard to read though and clinicians dont like transfusing off these results due to the interpretive nature etc
How are cold auto adsorptions carried out now?
You could use patients own cells at low 30 degrees or below or use REST
- Rabbit erythrocyte stroma ?
- will adsorb out any anti H or I present
How is CAIHA treated?
Keep warm - no affects if body temp doesnt reach 30 degrees etc
Corticosteroids - not as effective as in WAIHA
Transfusion - must prewarm blood - not often required
- crossmatch at 37 degrees and use anti-IgG
- tend to give scruffy results - tend to not transfuse
Treat underlying disorder e.g. pneumoniae
Plasmapheresis if acute
What exactl is drug-induced immune haemolytic anaemia?
Rare anaemia as a consequence of therapy with a particular drug
What drugs are associated with Drug induced immune haemolyic anaemia?
There are over 125 drugs implicated in DAIHA
Historically:
- 67% = Methyldopa
- 23% = IV penicillin
Nowadays:
- 2nd and 3rd generation cephalosporins (80-90%) especially cefotetant (72%) and ceftriaxone (10%)
What cephalosporins are most indicated in DAIHA nowadays?
72% cefotetan
10% ceftriaxone
What is the mortality of DAIHA?
40% of reported DAIHA are fatal
What antibodies are causative of DAIHA?
Mostly IgG
-> simply antiobodies produced against the drug
Compare DIHA to AIHA in terms of eluate reactivity and seum
Eluate
- Drug dependent antibody (DDA) negative or weak
- Warm auto antibody (WAA) will be strongly positive
Serum
- DDA disappears within days if drug is discontinued e.g. with penicillin or quinolone whereby DDA disappears immediately after stoping treatment
- WAA persists -> body will continue to produce it
Why would the eluate be negative in DIIAHA?
Red cells coated in DDA
DAA washed off red cells
Antibodies are directed only against drugs such as penicllin which is obviously not found on our reagent red blood cells - hence why panel will be negative
Describe how we would carry out an acid elution
Wash x4 to remove unbound protein
Add acid to lower pH to dissociate bound antibodies
Centrifuge
Transfer supernatant to new tube
Buffer to neutral pH
Test to determine specificity of antibody
How exactly does Drug induced anaemia occur?
Drugs provoke antibody production through one of three methods
Antibodies bind to red cells
Coated red cells are removed by spleen or liver resulting in anaemia
How do we treat DAIHA
Anaemia is corrected by removal of drug from patient
What are the three mechanisms of Drug-induced immune haemolytic anaemia?
Drug adsorption mechanism
Immune complex mechanism
True autoimmune mechanism
What is the drug adsorption mechanism of DIIHA, give examples of drugs which cause this?
Drug binds firmly to rbc surface and stimulates production of an antibody which targets the drug when bound to rbcs
Examples:
- penicillins
- cephalosporins
What is the immune complex mechanism of DIIHA, give examples of drugs which cause this?
Drug binds transiently to red cell membrane and together they form an epitope
The antibody binds to this forming an immune complex
This is what also happens in HIT
Examples:
- Quinidine
- Quinine
- Isoniazid
What is HIT
Heparin Induced thrombocytopenia
A disorder caused by antibodies that recognise complexes of platelet factor 4 and heparin
When on heparin therapy patients experience thrombocytopenia
What is the autoimmune mechanism of DIIHA, give examples of drugs which cause this?
This is where drugs cause production of autoantibodies
These autoantibodies react with normal red cells
Cross reactivity of antibody causing an autoimmune reaction
They are usually indistinguishable from WAIHA
Eluate will be positive
Examples:
- methyldopa
- Ibuprofen
How exactlydoes immune complex or drug dependent antibody mechanism cause anaemia
Drug binds to and reacts with red cell surface proteins creating a neo-epitope
Antibodies recognise altered protein/drug complex as foreign
Antibodies bind and initiate process leading to red cell destruction
Red cells removed as “innocent bystander” of destrucion of immune complexes
Only occurs when drug is in the system
C3 bound mainly -> hence serious anaemia
In short what are the three types of DIIAHA antibodies
Hapten-specific -> antibody specific to hapten (drug)
Neonatigen -> antibody specific to hapten and membrane components together
Crossreactive autoantiody produced to hapten-modified RBC membrane -> antibody mainly produced against membrane components (autoantibody)
In short what are the three types of DIIAHA antibodies
Hapten-specific -> antibody specific to hapten (drug)
Neonatigen -> antibody specific to hapten and membrane components together
Crossreactive autoantiody produced to hapten-modified RBC membrane -> antibody mainly produced against membrane components (autoantibody)
What is the fourth, non-immune, form of haemolytic anaemia?
Drugs alter red cell surface charge
Nonimmunologic adsorption of proteins onto RBCs
Drugs might change the rbc membrane so that many proteins attach to the membrane
Results in red cell removal
Leading to a positive DAT and possibly DIIHA without the production of any antibodies
How might we detect a DIHA in the lab?
Can be hard to spot, wouldnt expect them, overall rare
DAT positive with IgG
Hb as low as 2-4g/dl -> hence 40% mortality
Drug treatment and transfusion history
Clinically significant drug induced antibody titre>100
Eluate might be reactive against untreated cells in 15% of cases
What is DIIAHA often mistaken as?
Can appear like an autoantibody with anti-e specificity
-> anto-immune anti-e is actually quite common particularl in Black individuals so it could easily be mistaken for this
The eluate will be positive in what percentage of DAIHA?
15% of cases are eluate positive
What is Paroxysmal cold haemoglobinuria, what kind of antibodys involved, when seen, effects etc
A transient acute AIHA
Seen usually in children following viral infection
Caused by IgG with anti-P specificity
Causes complement fixation
Reactive at 30 degrees i.e. low peripheral body temperature
Causes mild HA, fabian has never seen one requiring transfusion
What antibody causes PCH, what is it often called?
IgG anti-P
Also called Donath-Landsteiner antibody
What are the signs of PCH in the lab?
DAT positive
- positive with C3b/d
patient details history
- child with viral infection
- historically assocaited with syphilis
- anti-P associated with living with pigeons
How is PCH treated
Corticosteroids
Warm climate
Transfusion - fabian never seen one required
How frequent is PCH
Is causes aout 30% of immune haemolytic anaemia
- as it is associated with viral infection
- actually quite commonly seen
What disease was PNH associated with?
Syphilis
