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Autoimmune haematological disorders
Dr. Demeter Judit
demjud@bel1.sote.hu
Semmelweis University, Ist Department of Medicine
Thrombocytopenia
Examination of the thrombocytopenic patient
1. Exclusion of pseudothrombocytopenia
EDTA dependent platelet clumping - in vitro phenomenon
Repeat sample in citrate-containing tube
Clinical examination of the thrombocytopenic patient
Clinical
manifestation
Bleeding disorder
Platelelet defect Coagulation factor
deficiency
Site of bleeding
Skin, mucous
membranes (oral
mucosa, GI tract)
Deep in soft tissues
(joints, muscles) or
preformed cavities
Bleeding after
minor trauma+ Generally not
petechiae + –
ecchymoses Small, superficial Big, palpable
hemarthros,
muscle hematomarare frequent
Bleeding after
surgical
intervention
Immediate, mild Prolonged, severe
Examination of the thrombocytopenic patient
• Normal platelet count: between 150.000 – 450.000/ml
• thrc < 150.000/ml thrombocytopenia
• thrc < 50.000/ml bleeding tendency following
trauma, intervention
• thrc 10 – 20.000/ml !! – needs hospitalisation
• thrc < 10.000/ml dangerous
Young, big platelets are haemostatically more active –
severe bleeding in ITP is rare
Platelet count required for specific interventions
Intervention Platelet count recommended
Major surgery,
Surgical procedures involving brain
or eye
>100 G/L
Minor surgical procedure, vaginal
delivery, cesarean section, lumbal
puncture
>50 G/L
Tooth extraction >30 G/L
Minor dental procedure >10 G/L
Examination of patient with severe thrombopenia
• exact (drug) history
• physical examination
Basic evaluation
Patient/family history
Physical examination
Complete blood count and reticulocyte count
Peripheral blood film
Quantitative immunoglobulin level measurement*
Bone marrow examination (in selected patients)
Blood group (Rh)
Direct antiglobulin test
H. Pylori**
HIV**
HCV**
ITP - Diagnosis
*should be considered in children with ITP
**for adult patients regardless of geographic location
Glycoprotein-specific antibody
Antiphospholipid antibodies (including anticardiolipin and lupus anticoagulant)
Antithyroid antibodies and thyroid function
Pregnancy test in women of childbearing potential
Antinuclear antibodies
Viral PCR for parvovirus and CMV
Tests of potential utility
ITP - Diagnosis
• There is no ‘gold standard’ test that can reliably
establish the diagnosis
• Diagnosis based on the exclusion of other causes of
isolated thrombocytopenia (see next slide)
• Bone marrow examination is appropriate in
patients >60 years old, in those relapsing after
remission and in patients not responding to first-line
therapy options
• The detection of H. pylori infection should be
included in the initial work-up
• Quantitative Ig level testing is indicated to exclude an
immune deficiency syndrome or when treatment with
intravenous Ig is considered
ITP – Basic considerations
Differential diagnosis of thrombocytopenia
Exclude pseudo-
thrombocytopenia
EDTA-dependent in vitro clumping –
repeat sample in citrate-containing tube
Preceding conditions causing
secondary
immunethrombopenia
HIV, HCV, SLE, autoimmune disease,
malignancy, recent vaccination
Liver diseaseAlcoholic liver disease, hepatitis virus
infections, toxic liver damage
Chemical compoundsAlcohol abuse, quinines (eg. tonic
consumption), enviromental toxins
Bone marrow diseasesMDS, leukemias, malignancy , fibrosis,
aplastic anemia, megaloblastic anemia
Recent transfusion Posttransfusion purpura
Heparin induced
thrombocytopenia
Unfractionated heparin, or LMWH
administration within previous 100 days
Hereditary causes
TAR syndrome, radioulnar synostosis,
congenital amegakaryocytic
thrombocytopenia, MYH-9 associated
diseases, Bernard-Soulier sy. etc.
Physical examination of the
thrombocytopenic patient
• Skin, mucous membranes (lower legs, oral mucosa)
• hepatosplenomegaly ?
• lymphadenopathy ?
• Retina by fundus examination (central nervous system
bleeding is the most frequent cause of death)
Petechiae
• Small capillary bleedings, in groups, especially where the
venous pressure is incresed. Lower legs, ankle! Not on the
soles (there the vessels are protected by strong connective
tissue)
Ecchymosis
• Multiple superficial cutaneous bleedings
Purpura
• Purple discoloration of the skin, due to multiple petechiae
• A: dry purpura (skin only)
• B: wet purpura (bleeding on the mucous membranes)
Wet purpura is more dangerous, might predict life-
threatening bleeding
BM, normal megakaryocytes
Mature multinucleated megakaryocyte
• Viral infection?
• drugs
• Family history (bleeding? – thrombopenia?)
• Known oncohaematological disorder? (leukemia,
lymphoma, MDS)
• non-hematological cause (e.g. sepsis)
• Alkohol abuse and/or B12 folic acid deficiency
Medical history of the thrombocytopenic patient
Easy and important to ask!
• Products of plant origin
• drinks containing chinin
• aspirin
• non-steroid anti-inflammatory drugs
Thrombocytopenic patient drug history
• heparin (central vein catheters!) (HITT)
• gold containing drugs
• chinin, chinidin
• sulfonamids
• interferons
• Inhibitors of glycoprotein IIb / IIIa (e.g. abciximab)
Maing drug causing thrombocytopenia
Recommendations – overview of management options1
Clinical situation Therapy option
First line (initial treatment for newly
diagnosed ITP)
Anti-D
Corticosteroids: dexamethasone, methylprednisolone, prednis(ol)one
Intravenous immunoglobulin
Second line* Azathioprine
Cyclosporin A
Cyclophosphamide
Danazol
Dapsone
Mycophenolate mofetil
Rituximab
Splenectomy
Thrombopoietin-receptor agonists
Vinca alkaloids
Treatment for refractory ITP patients
(patients failing first- and second-line
therapies)
Category A: treatment options with sufficient data
Thrombopoietin-receptor agonists
Category B: treatment options with minimal data and considered to have
potential for considerable toxicity
Campath-1H
Combination of first- and second-line therapies
Combination chemotherapy
Hemopoietic stem cell transplantation
*All treatment options are listed alphabetically and thus do not imply a preferred treatment option. Treatment should be individualized to meet patient needs
1. This research was originally published in Blood online. Provan D et al. International consensus report on the investigation and
management of primary immune thrombocytopenia. Blood. Prepublished October 21 2009; DOI 10.1182/blood-2009-06-225565
ITP – Summary of diagnostics
• no gold standard
• Exclusion of thrombocytopenia from other causes
• Medical history, examination of perpheral blood smear
+ HIV serology
+ bone marrow aspiration, exlusion of MDS above 60 years
Splenomegaly is NOT characteristic
ITP case presentation
Past medical history:
• Female patient. born: 1981, age 18 at dg
• Jan 1999.: menorrhagia, gynecological iron
deficiency anaemia, iv. iron treatment
• June 1999.: thrombopenia (Thr 30 G/l), temporary
hormonal inhibition of bleeding
First hospitalization in the clinic
Aug 1999: presenting at haematology outpatient clinic,
because of irregular antibodies on serology exam
• Status: 168 cm, 70 kg, physically normal.
• Laboratory: Hb 84, Htc 0.27, Ret 2.7%, Thr 17, LDH
422
• Peripheral smear: anisopoikiocytosis, polychromasia,
microspherocytes, very few platelets
• Bone marrow smear: increased erythro- and
thrombopoesis, megakaryocytic thrombocytopenia
• Serology: two types of irregular antibodies in the
serum
PLT RBC HB Ret ANC MCV
1999.12.28 6 1,85 48 0,070 5,6 83
Direct Coombs slightly pos, ANA: slightly pos; Anti-DNS: norm;
LDH: 1800 (norm: 230-460); test for occult blood in stool ++++
2000.01.11.
High dose steroid, Solu-Medrol, dexamethason followedby i.v. immunoglobulin.
Evans syndrome: ITP + AIHA
Splenectomy
AIHA ceased, ITP persisted
Steroids were ineffective in ITP relapse, cyclosporin A was effective
2007.01.31. 140 4,8 128 0,006 4,54 84
ITP case – hospital admissions
Thrombopoietin (TPO)
eTPO is the endogenous
cytokine for megakaryocyte
growth and platelet formation
eTPO binds specifically to
the TPO receptor (TPO-R)
eTPO also plays a central
role in the survival and
proliferation of hematopoietic
progenitor cells
TPO structure adapted from: Feese et al. Proc Natl Acad Sci USA. 2004;101:1816–1821.Deutsch & Tomer. Br J Haematol. 2006;134:453–466.
Thrombopoietin Production Is Constant
Liver
TPO
TPO structure adapted from: Feese et al. Proc Natl Acad Sci USA. 2004;101:1816–1821.
Thrombopoietin Levels Drive Platelet Production
Maintenance of normal
platelet number
General
thrombocytopenia
Blue arrow represents amount of free or unbound TPO in the system.Adapted from: Kuter et al. Thrombopoiesis and Thrombopoietins; 1997.
Liver
Normal Physiology of Platelet Turnover
Aging platelet
Macrophage
Spleen
TPO-
bound
platelets
Immune Thrombocytopenia
Pathophysiology of ITP
Traditional Concept of Thrombocytopenia
Impaired production:
CIT and aplastic anemia
Destruction:
ITP
Spleen
Antiplatelet
antibody
TPO
CIT, chemotherapy-induced thrombocytopenia.
Pathophysiology of Immune Thrombocytopenia: Accelerated Platelet Destruction
TPO-
bound
platelets
Macrophage
SpleenAutoantibodies bind to
healthy platelet
Gamma receptor
B cell
Pathophysiology of Immune Thrombocytopenia:A Disease of Accelerated Platelet Destruction and Suboptimal Platelet Production
Blue arrow represents amount of free or unbound TPO in the system.
Spleen
Liver
Thrombopoietin: Thrombopoietin Regulation in Immune Thrombocytopenia
General
thrombocytopenia
ITP
Blue arrow represents amount of free or unbound TPO in the system.
Spleen
Liver
Normal(n = 96)
Aplasticanemia(n = 23)
ITP(n = 170)
0
100
200
300
400
0
250
500
750
1,000
1,250
1,500
1,750Platelet count (× 109/L)
TPO level (pg/mL)
Pla
tele
t co
un
t (×
10
9/L
)T
PO
level (p
g/m
L)
Adapted from: Nichol. Stem Cells. 1998;16(Suppl 2):165–175.
Relative Thrombopoietin Deficiency in Immune Thrombocytopenia
ITP: A Disease of Suboptimal Platelet Production and Increased Platelet Destruction
Autoantibodies result in accelerated platelet destruction1
Platelet production may not be maximal in patients with ITP
– Autoantibodies inhibit megakaryocyte growth in vitro and
promote apoptosis resulting in impaired thrombopoiesis2,3
– Platelet production is reduced or normal in two thirds of
patients4–6
– TPO levels are normal, rather than increased, in 75% of
thrombocytopenic (< 30 × 109/L) patients with ITP
Latest treatment options aim at increasing platelet production
and no longer focus on decreasing platelet destruction
1. Cines & Blanchette. N Engl J Med. 2002;346:995–1008.2. McMillan et al. Blood. 2004;103:1364–1369. 3. Chang et al. Blood. 2003;102:887–895. 4. Stoll et al. Blood. 1985;65:584–588.5. Heyns Adu et al. Blood. 1986;67:86–92.6. Ballem et al. J Clin Invest. 1987;80:33–40.
Immune Thrombocytopenia
Mechanisms of ITP therapies
X
Immune
suppressionT cell
TPO-
bound
platelets
Macrophage
Spleen
B cell
Treatment Options for ITP:Immune Suppression
T cell
TPO-
bound
platelets
Macrophage
Spleen
B cell
Immune globulin
Treatment Options for ITP:Immune Globulin
X
ChemotherapyT cell
TPO-
bound
platelets
Macrophage
Spleen
B cell X
Treatment Options for ITP:Targeted Chemotherapy
T cell
TPO-
bound
platelets
B cell
Spleen removed
Treatment Options for ITP:Splenectomy
Platelet production
eTPO production
TPO-R agonistPlatelet pool
Antibody-mediated splenic platelet
destruction
Increased platelet production
Normal splenic destruction
Treatment Options for ITP:TPO-Receptor agonists
Immune Thrombocytopenia
Current treatment recommendations
Current treatment recommendations:an overview
Clinical situation Management options
First-line therapy • Anti-D*
• Corticosteroids
• IVIg
Second-line therapies • Azathioprine
• Cyclosporin A
• Cyclophosphamide
• Danazol
• Dapsone
• Mycophenolate mofetil
• Rituximab
• Splenectomy
• TPO-receptor agonists
• Vinca alkaloids
Treatment of refractory ITP Category A: treatment options with sufficient data
• TPO-receptor agonists
Category B: options with limited data and high potential toxicity
• Campath-1H
• Combination chemotherapy
• Combination of first- and second-line therapies
• Hemopoietic stem cell transplantationManagement options are listed alphabetically and do not imply a preferred treatment option. Treatment should be individualized.
* Anti-D was withdrawn from the EU market in June 2009
Provan et al. Blood. 2010;115:168–186.
Summary
In ITP, antiplatelet antibodies result in increased platelet destruction and suboptimal platelet production
eTPO is an important regulator of platelet production
– ITP is a disease state of functional eTPO deficiency
Traditionally, therapies for ITP acted to reduce platelet destruction
TPO-R agonists are the first therapeutic agents aimed at increasing platelet production
TPO-R agonists are now recommended for 2nd line therapy
Heparin-induced thrombocytopenia
(HIT)
Heparin-induced thrombocytopenia
(HIT)
• HIT type 1: slight drop in platelet counts after
heparin administration
– Frequent, but no real consequence
– Nonimmune, heparin effect on platelet activation
• HIT type 2 (HIT & thrombosis = HITT):– Immune- mediated
– Severe dip in platelet counts (>50%↓)
– Platelet-rich arterial and venous thrombi
– Late onset (4-10 days), may be immediate if heparin
exposure in previous 100 days.
HITT - presentation
• Tendencies:
– More frequent following UFH (2,6%) than in LMWH
treatment (0,2%)
– More frequent in surgical than in medical patients
– More frequent in females than in males
• Monitoring:
– If administering unfractionated heparin (UFH), platelet
count monitoring is required every other day through
days 0-14.
– No routine monitoring required for LMWH therapy
HITT - management
• Immediate cessation of heparin!!!
• Begin non-heparin anticoagulants:
– Heparinoids (eg. danaparoid)
– Direct thrombin inhibitors (eg. bivalirudin)
– Direct fX inhibitors (eg. rivaroxaban, argatroban)
• Oral anticoagulation should be started after platelet
count returns to normal:
– For 2-3 months without evidence of thrombosis
– For 3-6 months following manifest HITT thrombosis
Autoimmune haemolytic anaemia
(AIHA)
Recognizing hemolysis
- new onset of pallor and anemia
- jaundice with increased indirect bilirubin concentration
- gallstones
- splenomegaly
- presence of circulating spherocytic red cells
- increased serum lactate dehydrogenase (LDH)
- reduced (or absent) level of serum haptoglobin
- a positive direct antiglobulin test (Coombs test)
- increased reticulocyte percentage or absolute reticulocyte
number, indicating the bone marrow's response to the anemia
Confirming hemolysis
The combination of an increased serum LDH and a
reduced haptoglobin is 90 percent specific for the
presence of hemolyis
while the combination of normal serum LDH and a serum haptoglobin >25
mg/dL is 92 percent sensitive for ruling out hemolysis.
Peripheral blood smear abnormalities suggesting
extravascular hemolysis:
spherocytes,
microspherocytes,
elliptocytes, "bite" or blister cells,
acanthocytes,
teardrop red cells.
Abnormalities that suggest that the hemolysis is
intravascular:
presence of free hemoglobin in plasma or urine,
a urine sediment positive for iron (hemosiderinuria)
in rare cases, the presence of circulating red cell
"ghosts."
High power view of a normal peripheral blood smear. Several
platelets (black arrows) and a normal lymphocyte (blue arrow)
can also be seen. The red cells are of relatively uniform size and
shape. The diameter of the normal red cell should approximate
that of the nucleus of the small lymphocyte; central pallor (red
arrow) should equal one-third of its diameter.
a microangiopathic hemolytic anemia with marked red cell
fragmentation.
spherocytes
microspherocytes and elliptocytes
SITES OF RBC DESTRUCTION
The severity and type of red cell alteration determine the cell's site
of destruction. In most cases (eg, oxidant attack, metabolic insult,
hemoglobinopathy) this leads to an alteration of the RBC
membrane.
If the damage is severe enough, immediate lysis occurs within
the circulation (ie, intravascular hemolysis).
If the damage is less severe, the cell is destroyed within the
monocyte-macrophage system in the spleen, liver, bone marrow,
and lymph nodes (ie, extravascular hemolysis).
Intravascular hemolysis
Destruction of RBC within the intravascular space requires a
considerable amount of structural damage to the RBC
membrane.
• Direct trauma (bongo drummers and march hemoglobinuria)
• Shear stress (defective mechanical heart valves)
• Heat damage (thermal burns)
• Complement-induced lysis (paroxysmal cold hemoglobinuria)
• Osmotic lysis (following infusion of hypotonic solutions)
• Lysis from bacterial phospholipase toxins
(eg, clostridial sepsis)
Incompletely hemolyzed RBCs, which have lost some of their
membrane, can deform and become spherocytes or
microspherocytes, while others are destroyed outright.
The released hemoglobin appears in the plasma, either as red-
colored oxyhemoglobin or the brownish-colored oxidized
form, methemoglobin.
Free hemoglobin binds to haptoglobin; the resulting
hemoglobin-haptoglobin complex is rapidly removed by the
liver, leading to a reduction in plasma haptoglobin, often to
undetectable levels
Course of intravascular hemolysis
•severe intravascular hemolysis due to sepsis with Clostridium perfringens.
Neutrophils show toxic changes: toxic granulation and vacuoles.
• spherocytes (blue arrows)
• polychromatophilic red cells (ie, reticulocytes, red arrow).
•large number of red blood cell ghosts (black arrows), due to the intravascular lysis
of red cells from the phospholipase and other lytic enzymes elaborated by the
Clostridial organisms.
Extravascular hemolysis
Severely damaged RBCs, especially those coated with
complement, are primarily destroyed in the liver, since
this organ receives a larger proportion of the cardiac
output than the spleen.
However, the spleen represents more of a challenge to
poorly deformable RBCs (eg, spherocytes) due to the
cords of Billroth. These unique vascular channels end
blindly, unlike other vascular channels in the body. The
only way for a RBC, which has a diameter of
approximately 7 to 8 microns, to escape from these cords
and return to the general circulation is to deform
sufficiently to pass through 2 to 3 micron slits in the walls
of the cords.
Scanning electron microphotograph of normal murine red blood cell
passing from a splenic cord (below) through the sinusoidal barrier and
into the splenic sinusoid (above). Note the deformation necessary to
squeeze through the slit in the sinusoidal wall and how a surface area
depleted spherocyte would be incapable of transversing the barrier.
Senescent or damaged RBCs remain in the cords and are phagocytosed
by elements of the macrophage-monocyte system.
AIHA can be a medical emergency !
In case of AIHA transfusion of matched RBC-s only in
the case of life threatening anaemia
Underlying malignancy e.g. lymphoma should be
excluded (BM biopsy)
AIHA – General considerations
First line: corticosteroids
Other agents:• Immunosuppressive agents (cyclophosphamide,
azathioprine, CyA)
• Rituximab: alone or in combination with
glucocorticoids
• Splenectomy
• (Alemzutumab: anti-CD52 monoclonal antibody –
limited evidence).
AIHA – Treatment
Paroxysmal cold haemoglobinuria
(PCH)
Paroxysmal cold haemoglobinuria (PCH)
• AIHA subtype characteristically presenting in children, triggered by
viral infections
• Caused by the Donath-Landsteiner antibody: direct specificity
against the surface P-proteins of RBCs
• The antibody has unique characteristics:
• Only binds to RBC surface on low temperatures (ca. 4°C)
• When temperature returns to normal (37°C), complement
mediated lysis takes place
• This leads to intravascular haemolysis and haemoglobinuria
• Diagnosis:
• Verify presence of Donath-Landsteiner antibody
• Rule out other causes of haemoglobinuria
• Treatment: usually self-limited
Cold agglutinin disease (CAD)
Cold agglutinin disease (CAD)
• Typically presents in elderly patients
• Caused by IgM antibodies, that have an anti-I specificity (RBC
surface protein)
• The antibody reacts poorly at 37°C, but binds strongly in lower
temperatures
• Prevention of exposure to cold may amelliorate haemolytic
attacks
• In some cases antibody concentration is high enough to present
as a monoclonal spike in plasma protein electrophoresis IgM
characteristics CAD is a subtype of Waldenström
macroglobolinaemia (WM)
• Treatment
• Minimize cold exposure
• Immunosuppression, steroids and splenectomy have limited to
no effect on the disease
• Rituximab: promising results (as in Waldenström disease)
Paroxysmal nocturnal
haemoglobiuria (PNH)
• Nocturnal haemoglobinuria:– Intravascular hemolysis (LDH ↑, haptoglobin↓)
– Urine HB-content and color changes by the hour
• Cytopenia of multiple cell lines:– Partly due to complement-mediated lysis, but
mainly as a result of T-cell dependent aplasticchanges
• Development of visceral thrombosis:– Mesenterial thrombosis
– Budd-Chiari syndrome
– Portal vein thrombosis
– Thrombosis of cerebral, cutaneous vessels
PNH - symptoms
PNH - haemoglobinuria
Toronto Medical Hospital
PNH - pathomechanism
• Prevalence: 1-5 / 1 million
• Acquired defect of the PIG-A gene
• The product of the gene enables the expression of
glycosil-phosphatidilinositol (GPI) on cell surface,
wich serves as an anchor for various surface proteins.
• CD59 és CD55 are examples of GPI-anchored
molecules
– The role of these proteins is to prevent complement
activation. The lack of their expression leads to profound
activation of the serum complement system.
PNH - diagnostics
• TODAY: flow cytometry:
– Analysis of surface CD55 and CD59 expression on RBCs
– Analysis of involvement of granulocytecell line.
• Previously:– Sucrose test: addition of isotonic sucrose potentiates the effect
of complement haemolysis?
– HAM-test (acidified serum test): activation of complement bylowering sample pH to 6,4.
PNH - treatment• Corticosteroids: efficiacy is controversial.
Mainly used to treat acute haemolyticexacerbations.
• (Androgen therapy): to limit anemia
• Iron replacement therapy
• Folic acid replacement: necessary becauseof the ongoing haemolysis
• Propylaxis of thrombosis: controversial, role may depend on PNH clone size
• ANTI-COMPLEMENT TREATMENT:
Eculizumab: anti-C5 monoclonal antibody.
Effect of eculizumab on transfusion need
P Hillmen et al: N Engl J Med 2006; 355:1233-1243.
Effect of eculizumab on long term
survival
Kelly R J et al. Blood 2011;117:6786-6792.
PNH – speculations about
underlying causes
• Limited number of GPI-deficient cells is also present in normal
subjects
• There is evidence for T-cell mediated immunological reactions
playing a role in the aplastic changes of the bone marrow observed
in PNH
A possible explanation for the development of PNH:
PNH may be also consided a special subtype of aplastic anemia,
where the GPI myeloid stem cell clone evades the immunological
changes attacking the bone marrow
the GPI deficient clone may expand as a result of this survival
advantage
Thank you for your
attention!