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!