-3 Ensherah Mokheemer Rama Nada Tareq Aladily...Note: the first type the antigenic drugs is not a true autoimmune disease because the antibodies were not produced against self-antigens

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- Ensherah Mokheemer

- Rama Nada

- Tareq Aladily

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In this lecture we will continue talking about autoimmune hemolytic

anemia.

Autoimmune hemolytic anemia

- There are several types that shares the same phenomena which is

the presence of an immunoglobulin attached to their RBCs’

surface which causes destruction later on by macrophages in the

spleen. “A group of anaemias in which an abnormal immunoglobulin is

attached to RBC membrane”.

- Coombs Test it is a test done in the hospital to detect the

presence of the antibodies coating the RBCs, we have two tests

the direct and indirect, The direct Coombs test is used to test for

autoimmune hemolytic anemia, we give antibodies that binds to

the immunoglobulin (autoantibody) on the RBC membrane, this

binding leads to agglutination** of the RBC, we can also give RBCS

which will bind the autoantibodies -if they are present- and cause

agglutination. “Coombs test: the patient's blood is mixed with serum

containing antibodies that are specific for human immunoglobulin. If the

autoantibody is present, agglutination of RBC occurs”. ** Agglutination means large aggregates of RBCs that becomes

clump to each other and the become viscous and turbid.

- We have two types of autoimmune hemolytic anemia “we classify

them based on their site of activity in the blood”:

1) Warm Antibody Type: usually occurs in the trunk of our bodies

where the temperature is 37.

2) Cold antibody type: which occurs in the periphery of the body

(Fingers, nose, ears) where the temperature is below 37.

Now let’s talk about them in detail:

1) Warm antibody Type: - Most common 70% of the cases.

- 50% are idiopathic (primary) there is no obvious cause; the others

are related to either exposure to a drug (mostly penicillin and

cephalosporins) which is more common, or they have other

autoimmune diseases such as systemic lupus erythematosus.

- Most causative antibodies are of the IgG class; less commonly, IgA

antibodies

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- A common target is the Rh antigen on RBCs.

- These abnormal Antibodies (autoantibodies) that coat the RBCs,

have receptors on the macrophages so when they go to the spleen

they will bind to the fc receptor on the phagocytes, the

phagocytes will remove the part of the RBC membrane which

antibodies are bound to ( it pinches that part of the membrane) ,

this process is called partial phagocytosis because we lose small

fragments of the RBC membrane not the whole RBC is lost. With

the continuous loss of fragments, the RBCs will become smaller

and rounder (spherocytes/ ball like). In the secondary circulation

when they go back to the spleen, they will be destroyed due to

their abnormal shape.” The red cell haemolysis is mostly extravascular.

IgG-coated red cells bind to Fc receptors on phagocytes, which remove red

cell membrane during "partial" phagocytosis. As in hereditary

spherocytosis, the loss of membrane converts the red cells to spherocytes,

which are removed in the spleen”.

- Moderate splenomegaly and extravascular haemolytic anaemia.

Drug induced hemolytic anemia:

A) Antigenic drugs: when certain drugs (especially antibiotics and

antimalarial drugs) are given at high dose (intravenous) these

drugs bind to the cell membrane of the RBC they act as a hapten “small molecule that binds to another molecule attracting antibodies”

eventually the body will recognise them as antigens and produce

IgG antibodies against them.

- The haemolytic anaemia occurs in 1 to 2 weeks after therapy is

initiated that is because the synthesis of antibodies IgG takes

time.

- Example of these drugs are: Penicillin, cephalosporin, anti-malaria

drugs.

- Antigenic drugs mean that the drug works as an antigen.

B) Tolerance-breaking drugs

-Unknown mechanism but there is no binding of the drug to the

RBC

-Activates production of autoantibodies against Rh group

-α-methyldopa: anti-hypertensive drug commonly used in

pregnancy

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Note: the first type the antigenic drugs is not a true autoimmune

disease because the antibodies were not produced against self-

antigens directly, they were produced against drugs (not self-

antigens) but they attacked the self-antigen RH that’s why we put

them along with autoimmune diseases (but they are not true

autoimmune diseases). As for the second type tolerance breaking

drugs it is a true autoimmune disease because the antibodies

were directly produced against self-antigens (Rh antigen).

2) Cold Agglutinin Type: - IgM antibodies (IgM is the largest antibody is pentamer (5 binding sites))

- IgM bind red cells at low temperatures, but the binding is weak

when it reaches the trunk of the body where the temperature is

high it dissociates. But when it is bound to the RBC in the

periphery it attracts small amounts of the complement system

(C3b), which is recognized by macrophages in the spleen and

removed “the same mechanism as the warm type first we have

spherocytes then they are removed by the spleen.”.

- IgM is functioning the best at low temperatures (0-4 c) and it is

seen in the nose, fingers, ears.

- less common than warm immunohymolytic anaemia:

• Acute: Self-limited, following infection by Mycoplasma

pneumonia (Atypical anemia), Epstein-Barr virus,

cytomegalovirus, influenza virus, and HIV.

• Chronic: associated with B- cell lymphoma, severe.

Why does it occur in lymphoma? In B-cell lymphoma there are

large numbers of B lymphocytes and they are not normal which

increases the risk of formation of auto-antibodies.

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- Under the microscope we see RBC agglutination in autoimmune

haemolytic anaemia only in cold AIHA. Because IgM is a

pentamer an it can bind to many RBCs. In IgG in the warm AIHA

we don’t see agglutination we only see spherocytes.

Haemolytic Anaemia Resulting from Trauma to Red Cells - This type of anaemia is secondary to mechanical damage to t RBCs

- Physical damage to RBCs:

• (1) Cardiac valve prosthesis (we replace the soft tissue with

metal which will injure the RBCs).

• (2) Vigorous exercise, but it’s mostly not significant (that’s mean

if you see schistocytes in the blood film for an athlete it doesn’t

mean medically significant anemia), its rarely seen.

• (3) Microangiopathic diseases (diseases in the small vessels) the

most important category:

(a)disseminated intravascular coagulation (DIC)

(b)thrombotic thrombocytopenic purpura (TTP)

(c)hemolytic-uremic syndrome (HUS), aggregates of fibrin and

platelets causes damage to RBCs

**These three diseases share wide spread thrombosis in the small

blood vessels.

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- RBCs appear as fragments (schistocytes). “torn RBCs”.

- Now we will star talking about hemoglobinopathies, disorders

which are related to abnormal haemoglobin synthesis.

Thalassemia - Heterogeneous group (not a single disease) of disorders caused by

inherited mutations that decrease the synthesis of adult

hemoglobin, HgA (α2β2).

- Autosomal Recessive.

- We classify it according to the severity and the type of the chain

that is deficient (α or β).

- Endemic (always persistent in large number) in Middle East,

tropical Africa, India, Asia (not common in Europe).

- β-Thalassemia is caused by deficient synthesis of β chains,

whereas α-thalassemia is caused by deficient synthesis of α

chains.

- The consequence of the diminished synthesis of one globin chain

leads to the relative excess in the other globin chain and this

excess causes harm to the RBC, leading to their haemolysis.

- Hence that in thalassemia the bone marrow is producing RBCs but

they are deficient (empty) and this is by definition called anaemia

because it is a decrease in the RBC mass not in their number.

- The hematologic consequences of diminished synthesis of one

globin chain stem not only from hemoglobin deficiency but also

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from a relative excess of the other globin chain, particularly in β-

thalassemia.

β-Thalassemia: - caused by mutations that diminish the synthesis of β-globin chains

(chromosome 11).

- There are two types of mutations which differ in their severity:

1- β0 mutations, associated with absent β-globin synthesis.

Remember that we have two alleles one is maternal, and the

other is paternal so of there is a β0 mutation in one of them

and the other is normal the patient survives.

2- β+ mutations, characterized by reduced (but detectable) β-

globin synthesis. In this case it is either a decrease in the globin

synthesis or slower rate of production.

- 100 different causative mutations, mostly consisting of point

mutations, the severity of anemia vary according to the mutation.

- Pathogenesis:

Lower production of HgA so the RBCs will appear

hypochromic, microcytic and the patient will suffer from

hypoxia.

β-Thalassemia does not appear early in life, because in new-

borns we have a higher percentage of foetal haemoglobin,

so it masks the deficient of beta chain (remember that HbF

is composed of two alpha chains and two gamma chains). At

the ag of 6 month the foetal haemoglobin level becomes

low and not sufficient to mask the deficiency eventually

thalassemia symptoms appear.

Unpaired α chains precipitate (insoluble) in erythroid

precursors and RBCs, causing membrane damage which

leads to haemolysis in BM, blood and spleen. So, it is a

haemolytic anaemia.

In bone marrow: Due to hypoxia, erythropoietin is released

from the kidney activating the bone marrow to produce

erythroid cell there is a marked erythroid hyperplasia

eroding bone and shifting oxygen and nutrients leading

skeletal deformity and growth retardation (this occurs in

thalassemia major).

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Ineffective erythropoiesis which means we have large

number of erythroid precursors but still the anaemia is not

corrected because we have a genetic disease in order to

compensate the ineffective erythropoiesis the body will

activate other sites to produce RBCs and this is called

extramedullary haematopoiesis (Haematopoiesis outside

bone marrow results from persistent stimulation of

erythropoietin, such as in spleen liver and sometime lymph

nodes) and this will lead to enlargement of these organs.

We treat them by Repeated blood transfusion + Hepcidin

will be suppressed as a result of increased erythropoietin

these two factors will cause hemosiderosis.

In blood transfusion the patients receive RBCs and RBCs

have iron inside them, so this will increase the iron in the

body. As for hepcidin it is a protein that reduces iron uptake

from the intestinal cells so when it is supressed iron uptake

will increase leading to increase in the iron level in the

body. These two factors contribute to the occurrence of

hemosiderosis in patients with thalassemia major.

Hemosiderosis means excess amounts of iron, iron will

accumulate in the tissues of the body causing physical

damage especially to the heart, kidney and endocrine

system.

- Clinical classification:

1- β -thal minor: loss of one β alleles, asymptomatic it could be

β+/β or βo/β. In these patients, RBCs colour and size is below

normal, so in the blood film of these patients you will see

microcytic, hypochromic anaemia. this is the requested test

before marriage.

2- β – thal intermedia: mutations in two β alleles. Patients have

moderate anaemia, but stable and it could be β+/β+ or βo/β+.

These patients do not receive blood transfusion.

3- β – thal major: loss of two β alleles. Severe symptoms (βo/βo).

α- Thalassemia

- It is usually deletion mutations

- We have 4 alpha genes.

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- In case of α thalassemia there is excess of β chains and γ chains.

- Since free β and γ chains are more soluble than free α chains,

haemolysis and ineffective erythropoiesis are less severe than in

β-thalassemia.

- As in β-thalassemia, the anaemia occurs both from inadequate

haemoglobin synthesis and the effects of excess unpaired non-α

chains (β, γ, and δ).

- Tetra gamma haemoglobin it is called haemoglobin Bart, tetra β is

called haemoglobin H. we use these two in diagnosis of α

thalassemia.” In newborns with α-thalassemia, excess unpaired

γ-globin chains form γ4 tetramers known as hemoglobin Barts,

whereas in older children and adults excess β-globin chains form

β4 tetramers known as HgH, resembles β- intermedia”.

- Clinical classification: remember we have 4 α genes:

Silent carrier: a single gene deletion, patients have slight

microcytosis but no anemia, asymptomatic.

α-Thalassemia Trait: deletion of two genes, but still we have

two functional genes. Clinically identical to β-thalassemia

minor: microcytosis, minimal or no anemia, aymptomatic

Hemoglobin H Disease: deletion of 3 alpha genes, common

in Asia, clinically resembles β-thalassemia intermedia. In

this type we have excess beta and gamma chains.

Hydrops fetalis: deletion of 4 alpha genes. Patients die in

utero, that is because alpha chains are present in foetal

haemoglobin in addition to adult haemoglobin.

The babies are incompatible for life unless they are

transfused, and this is not accessible in most patients.

- Diagnosis of thalassemia:

1- Blood film: hypochromic microcytic anaemia, target cells**,

basophilic stippling*.

**Target cells: the RBC appear dark in periphery then pale in the

centre and then dark again this appearance is not specific for

thalassemia, it is for all the diseases in which there is abnormal

haemoglobin synthesis (Iron deficiency and sickle cell anemia), so

it’s not specific for thalassemia.

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Target cells Basophilic stippling

*Basophilic stippling: small basophilic dots everywhere in RBCs,

they are aggregates of ribosomes, appear as fine blue inclusions in

RBCs.

2- Hg electrophoresis: different globin chains have different

electrical charges. Hg is separated on gel and an electrical current

is applied. Each type of Hg migrates a specific distance and hence

can be recognized, the globin with low concentration will appear

as small band (in case of beta thalassemia, HbA will give small

band and HbA2 will give band larger than normal). This test gives

us the definite diagnosis.

3-In β-thalassemia, HgA2 in increased because it does not have

beta chains only alpha and delta and they both increase in beta

thalassemia.

4- Genetic test: this is the most accurate and definite test as it

tests the type of mutation.

Sickle cell anaemia. - autosomal recessive.

- Common in Africa, Middle East.

- Point mutation (substitution mutation) on β gene,

glutamate(hydrophilic) residue is replaced with

valine(hydrophobic) (HgS). So, the end result is change in the

characteristic of beta chain.

- Mutation in one gene: sickle cell trait (silent carrier), HgS =40%,

HgA=40% and the remaining 10% is HgA2 and HbF.

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- Mutation in 2 genes: sickle cell disease, HgS =90%, we don’t have

HgA.

- Both conditions protect against Malaria falciparum infection.

- Pathogenesis:

HgS is less soluble so it tends to polymerize longitudinally

upon hypoxia, acidity, dehydration these factors lead to

decrease in the affinity of haemoglobin toward oxygen, so

oxygen is released, and we have more deoxygenated Hb

which is polymerised.

Needle-like shape

Membrane damage

Intravascular haemolysis occurs due to membrane damage

and extravascular haemolysis occurs when the sickle

shaped RBCs are homolysed due to their abnormal shape.

Sickled RBCs can bind each other and form a thrombus

which will cause Capillary occlusion and ischemic infarction.

HgA, HgF prevent sickling, that’s why carries do not develop

symptoms because they still have HbA which prevents

polymerization. Also, symptoms do not appear until the age

of 6 months because of HbF prevents polymerization in the

first six months.

Remember that in thalassemia and sickle cell anaemia

symptoms appear at the age of six months and not

immediately after birth.

With chronic haemolysis: BM, bone changes &

extramedullary haematopoiesis similar to thalassemia

major.

Splenic changes:

1- Early disease: splenomegaly due to extravascular

haemolysis.

2- Sequestration crisis: massive entrapment of RBC in

spleen, leading to hypovolemia and shock, the spleen

becomes engorged with blood until it affects the whole

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circulation. The patients will show hypovolemic

symptoms and extravascular bleeding.

3- Advanced disease: splenic infarction (autosplenectomy).

Because sickle cells form thrombi leading to splenic

infarction and atrophy.

Vaso-occlusive crisis:

Thrombosis, tissue infarction, can be developed in any

organ.

Bone and joint pain due to multiple bone infarction.

Skin ulcer (leg) if it was severe it will lead to infection.

Myocardial infarction it is lethal, and it occurs at early age.

Acute chest syndrome thrombosis in lungs or bones of the

rib cage, which will cause severe pain in the chest.

Penis (priapism), continuous erection, the drainage of the

penis is blocked by thrombi so eventually it will lead to

penis infarction.

Aplastic crisis:

• Parvovirus B19 it affects the erythroid progenitors.

• Destroys erythroid cells.

When normal people get infected their body can cope with the

virus, so no symptoms appears. But in patients with

thalassemia and sickle cell anaemia, the virus becomes

stronger and they suffer from long standing chronic anaemia

and aplastic crisis (there is no production of erythroid cells in

the bone marrow).

• Patients present with sudden, severe, worsening of the

anaemia.

• we can diagnose it using serology “antibodies against the

virus” or we can take a biopsy from the bone marrow we can

see inclusions in the Nuclei of erythroid precursors (viral

particles).

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• Also complicates thalassemia.

- Diagnosis:

1- Blood film:

Sickle cells, Howell Jolly bodies (after splenectomy or the

atrophy of the spleen), target cells (due to abnormal

haemoglobin synthesis and distribution in the RBC).

2- Haemoglobin electrophoresis.

3- Crew-cut appearance of skull of X ray: shows the shadow of

erythroid cells in the BM, secondary to marked erythropoiesis

in sickle cell anaemia and B-thalassemia and they have specific

facial features (prominent bones)

called sickle face.

This is due to very active bone marrow

but ineffective erythropoiesis, so they

cannot compensate anaemia.

Also, they have extramedullary

haematopoiesis but still can’t

compensate anaemia.

The End, Sorry for any mistake

Good luck 😊

Aplastic crisis:

pronormoblast shows

nuclear inclusions