THROMBOCYTOPENIA

Malvika Tripathi Department of Pathology

Platelets were described by Addison in 1841 as “extremely minute — granules” in clotting blood.

They were termed platelets by Bizzozero, who also observed their adhesive qualities as “increased stickiness — when a vascular wall is damaged”.

The same elements were identified by microscopic examination of blood smears by Osler and Schaefer and by Hayem in the late 19th century.

Megakaryocytes have been recognized as rare marrow cells for nearly two centuries, but it was the elegant camera lucida studies of Howell in 1890 and his coining of the term megakaryocyte that led to their broader appreciation as distinct entities.

In 1906, James Homer Wright put forth the hypothesis that blood platelets are derived from the cytoplasm of megakaryocytes and the basic elements of thrombopoiesis were established.

Platelet production begins in the yolk sac and, like the remainder of hematopoiesis, shifts to the fetal liver and then to the marrow at the time of gestation.

Based on the adult blood volume (5 L), the number of platelets per micro liter of blood ( 2 x 10∼ 5), and their circulatory half-life (4-10 days), it can be calculated that each day an adult human produces 1 x 1011 platelets.

The platelet count varies among the healthy population (1.5 to 4.5 x 109 lakhs / cumm) but remains within a fairly narrow range in any given individual.

In times of increased demand, platelet production can rise 10-fold or more.

MegakaryopoesisTotipotent stem cell Pluripotent stem cell Hematopoietic stem cell

Common myeloid progenitor IL-3,SCF,TPO

Megakaryocyte erythroid progenitor TPO,IL6,IL11

CFU-Meg

Megakaryoblast (stage1)

Promegakaryocyte(stage2)

Granular megakaryocyte(stage3) Mature megakaryocyte(stage4) PLATELETS

Structure of platelets :-

Sol gel zone

Peripheral zone

Organelle zone

Platelet organelles & their contents :-

1. Alpha granules :-

Platelet specific protein – PF4, PDGF, thrombospondin, β thromboglobulin.

Coagulation specific protein- Fibrinogen, Factor V, vWF, High molecular weight kinogen.

Fibrinolytic system protein – a 2 antiplasmin , plasminogen , Platelet aggregation inhibitor-1.

Others – fibronectin, albumin.

2. Dense granules :-

Anions –ATP, ADP, GTP, GDP.

Cations - Serotonin, Calcium .

Role of platelets in hemostasis :-

1.Adhesion :- • Via GPIb on the surface of platelets.

• Congenital absence of GPIb results in Bernard’s

Soulier syndrome

• Congenital absence of vWF in plasma results in

VWD .

2. Release reaction ( secretion) :-• Begins immediately after adhesion.

• ADP from dense granules promotes aggregation of

platelets.

• PF4 neutralises anticoagulant activity of Heparin .

• PDGF stimulates proliferation of vascular smooth muscles,

skin fibroblasts.

• TxA2 induces aggregation of other platelets & local

vasoconstriction.

3. Aggregation :-• Binding of platelets to each other.

• Gp IIb & III a (fibrinogen receptors) exposed to

surface

• Binding of fibrinogen molecule causes aggregation of

platelets.

What is Thrombocytopenia

Thrombocytopenia refers to decrease in the number of platelets in peripheral blood below normal (<1.5 lacs/cumm).

Although the normal platelet count in humans (150–400 x 109/L) far exceeds the minimal level required to avoid pathologic hemorrhage (<50 x 109/L), a number of medical conditions cause either increased destruction or reduced production of platelets, increasing the risk of pathologic bleeding.

It may result from one of the following causes –

1. Inadequate platelet production2. Increased destruction of platelets3. Platelet trapping4. Abnormal platelet distribution/ pooling

Pathogenesis of Thrombocytopenia

Classification Pseudo

thrombocytopenia

1. Platelet agglutination2. Platelet Satellitism3. APLA4. GpIIa-IIIa antagonists5. Giant platelets6. Miscellaneous

associations

Accelerated platelet destruction

1. Immune (Idiopathic) thrombocytopenic purpura(ITP)

2. TTP/HUS3. DIC 4. SLE5. Neonatal alloimmune

Thrombocytopenia6. Post transfusion purpura7. Drug induced8. Infections

Impaired platelet production

1. MYH9 related thrombocytopenia syndromes

2. Mediterranean macro thrombocytopenia

3. Paris trousseau syndrome

4. Wiskott-Aldrich syndrome5. Fanconi anemia6. Aplastic anemia7. Megaloblastic anemia8. Marrow infiltration9. Drugs10. Viral infections

Platelet Trapping

1. Kasabach Merrit syndrome

Abnormal distribution/Pooling

1. Spleenomegaly2. Hyperspleenism3. Massive transfusion

Idiopathic Thrombocytopenic purpura/Autoimmune Thrombocytopenic purpura It is a common acquired autoimmune disorder

defined by a low platelet count secondary to accelerated platelet destruction or impaired thrombopoiesis by antiplatelet antibodies.

It occurs in 2 forms – Acute and Chronic.

Acute ITP occurs in children following viral infection or vaccination. Sudden in onset.

In acute ITP, Immune complexes bind to Fc receptor on platelets that leads to immune destruction of platelets by macrophages in spleen.

Chronic ITP occurs predominantly in adult women (20-40 years) and is not preceded by infection or any underlying disease, Insidious onset, Self limiting.

In chronic ITP, antibodies directed against specific glycoproteins IIb/IIIa or Ib/IX (Mainly IgG type). These antibodies are also directed against megakaryocytes.

These antibody coated platelets are recognized by Fc receptors on macrophages and destroyed

mainly in spleen.

GPIIb/IIIa are sites for fibrinogen binding during platelet aggregation.

These antibodies also block GPIIb/IIIa and cause platelet dysfunction in addition to platelet destruction.

Parameters Acute ITP Chronic ITPAge Childhood (2-4 years) Adults (15-40 years)

Sex No sex preference F>M

History of preceding viral infections or vaccination

Common No history

Onset of bleeding Sudden Insidious

Type of bleeding Purpuric spots and ecchymosis

Superficial

Site of bleeding Cutaneous and mucous membranes (Gums,

Nose, GIT and hematuria)

Skin and mucous membrane

(Menorrahegia)

Degree of thrombocytopenia

Severe Moderate

Spleen Just palpable Non palpable

Spontaneous remission Usual Need therapy

Recurrence Uncommon Common

Duration 1-6 months Months to year

Prognosis Very good Fair

Blood loss may lead to anemia.

Lymphocytes and Eosinophils are frequently increased in acute ITP.

Platelets are markedly reduced (<20,000/cumm) to moderate and Macrothrombocytes are found.

Number of large platelet is proportional to megakaryocytes in marrow.

Megakaryocytes are normal or increased in number in bone marrow and frequently show morphological changes such as hypo granularity of cytoplasm, vacuolization and dense nuclear chromatin.

If clinical features, complete blood counts and blood smear are indicative of ITP then bone marrow examination is not necessary for diagnosis of ITP.

Levels of platelet associated immunoglobulins are raised in majority of patients with ITP.

Drug induced thrombocytopenia Development of thrombocytopenia after quinine was

first described by Vipan in 1865, and since then a large number of drugs have been found to cause thrombocytopenia.

Drug-induced thrombocytopenia generally affects only a small percentage of patients taking a particular drug, and is usually not severe, although it can be fatal.

Genetic or environmental factors both influence susceptibility to drugs.

Drugs may cause thrombocytopenia by different mechanisms.

Dose-dependent myelosuppression and immune destruction of the platelets are two well-known causes.

One of the most severe and life-threatening immune thrombocytopenias is heparin-induced thrombocytopenia (HIT), an immune-mediated disorder caused by antibodies that recognize a neoepitope in platelet factor 4 that is exposed when platelet factor 4 binds heparin.

The result is activation of platelets and the coagulation cascade and, ultimately, thrombosis.

HIT affects up to 5 percent of patients exposed to heparin.

Other drugs causing drug induced thrombocytopenia –

1. Sulfamethoxazole2. Penicillin3. Gold salts4. Quinidine5. Quinine6. Diazepam7. Lithium8. Amiodarone9. Acetazolamide10. Amphotericin B

Neonatal Thrombocytopenia

When the fetal platelets possessing paternally derived antigens lacking in the mother enter maternal circulation during gestation or delivery, formation of alloantibody is stimulated.

These maternal antibodies cross the placenta and

cause destruction of fetal platelets.

The most common antigen against which antibodies are formed is HPA-1a.

The condition is usually resolved by 3 weeks (maximum 3 months) after delivery.

In severe cases purpura and hemorrhages are evident at birth or manifest within few hours.

Other causes of neonatal thrombocytopenia – 1. Thrombocytopenia with absent radius syndrome2. Infections3. Drug induced4. Congenital megakaryocytic hyperplasia5. Congenital leukemia

Post transfusion purpura Rare

Sudden onset

Bleeding occurs about 7-10 days after blood transfusion.

Donor platelet (HPA-1a antigen) + Transfusion = Destruction of patient’s platelets (already sensitized) = Thrombocytopenia (severe).

IV gamma globulins and plasmapheresis - treatment modalities.

Disseminated intravascular coagulation An acute, sub acute, or chronic thrombohemorrhagic

disorder, disseminated intravascular coagulation (DIC) occurs as a secondary complication in a variety of diseases.

It is caused by the systemic activation of the coagulation pathways, leading to the formation of thrombi throughout the microcirculation.

As a consequence of the widespread thromboses, there is consumption of platelets and coagulation factors and, secondarily, activation of fibrinolysis.

DIC is characterized by –

Intravascular activation of extrinsic pathway of coagulation with generation of thrombin and fibrin.

Reduction in level of endogenous anticoagulants (antithrombin, protein C).

Suppression of fibrinolytic system which causes delayed and inadequate removal of fibrin.

These 3 factors in combination leads to generalized deposition of fibrin in circulation and form micro thrombi.

2 types – Acute and chronic.

Acute/ decompensated DIC – Rapid and extensive activation of coagulation

leading to significant bleeding from consumption of coagulation factors and widespread micrvascular thrombosis with consequent end organ damage.

Ex – DIC due to sepsis or trauma.

Sudden onset of spontaneous bleeding from multiple sites like skin (petechie and ecchymosis), GIT, urinary system, epistaxis, and oozing from venepuncture sites.

Chronic / Compensated DIC – Slow activation of coagulation in small amount

with slow consumption of coagulation factors ; Coagulation factors are normal or increased, Clinical features are minimal or absent. Laboratory abnormalities are the only evidence of DIC.

Ex – IUD, Liver diseases, giant hemangioma, eclampsia, malignancy.

Mild and protracted disease, manifests only with venous thrombosis.

Laboratory features –

Acute DIC – Low platelet or falling platelets on repeat testing, prolonged PT and aPTT, Low fibrinogen or falling levels on repeat testing, Low plasma level of coagulation inhibitors i.e. ATIII or protein c, schistocytes on blood smear.

Chronic DIC – Platelet count normal or slightly reduced, PT and aPTT are normal.

Thrombotic thrombocytopenic purpura 2 types – Idiopathic and familial.

Idiopathic – Auto antibodies against ADAMTS13 lead to deficiency of

ADAMTS13 and accumulation of ultra large vWF multimers that bind large number of platelets.

Familial – ADAMTS13 deficiency results from mutation in ADAMTS

13 gene.

ADAMTS13 – A disintegrin and metalloprotease with thrombospondin type 1 motif 13.

Affects mainly young adults.

More common in females.

Pentad of manifestations include -

1. Microangiopathic hemolytic anemia2. Bleeding manifestations secondary to severe

thrombocytopenia3. Fluctuating neurological dysfunctions4. Renal abnormalities5. Fever

These 5 features may not be present in all patients.

Hemolytic uraemic syndrome

Characterized by triad of features :- 1) Acute renal failure 2) Thrombocytopenia 3) Microangiopathic hemolytic anaemia.

Two types :- Typical and Atypical.

Typical HUS –occur predominantly in children<5 yr and is associated with Shiga toxin –producing E.coli o157:H7.

It is characterized by a prodrome of diarrhoeal illness followed by Microangiopathic hemolytic anaemia, Thrombocytopenia and renal failure.

Atypical HUS- has similar clinical features but is not preceded by diarrhoeal prodrome.

Pseudo thrombocytopenia/Spurious thrombocytopenia

Uncommon phenomenon caused by ex vivo agglutination of platelets.

This leads to platelet clumping.

Due to this, platelet counts are reduced on automated cell counters because they cannot differentiate platelet clumps from individual cells.

Causes – 1. Use of EDTA anticoagulant2. With platelet cold agglutinins3. Multiple myeloma.

Antibody induced platelet agglutination Caused ex vivo either by anti platelet antibodies

or by activation of platelets during collection.

Antibodies which cause platelet agglutination do not appear in any pathologic process as they are present in normal individual.

These antibodies recognize the platelet membrane glycoproteins which are modified or exposed when calcium is chelated.

These antibodies are typically of IgG type ; but IgM and IgA are also described.

This phenomenon is most common in presence of EDTA as anticoagulant.

But other anticoagulants can also cause antibody induced agglutination of platelets such as –

1. Sodium citrate2. Sodium oxalate3. Acid citrate dextrose4. Heparin

Antibodies cause agglutination at room temperature therefore the reaction can be prevented if blood sample is kept at 37 Celsius.

Clumping is usually evident in 60 minutes after blood is drawn.

In most cases antibodies are directed against GPIIb/IIIa.

Platelet Satellitism Antibodies directed against GPIIb/IIIa react

simultaneously with Fc receptor III (FcRIII) of leukocytes and attach platelets to neutrophils and monocytes.

Platelets form a rosette around periphery of leukocytes.

Neutrophils are most commonly involved.

Monocytes can also be involved.

These antibodies are naturally occurring and their presence does not clearly correlate any clinical situation, disease or drug.

These antibodies fail to produce Satellitism in – Platelets of patients with Glanzmann

Thrombasthenia (Absence of GPIIb/IIIa) or Patients with congenital absence of FCIII

receptor .

Antiphospholipid antibodies(APLA) Some anti platelet antibodies from patients with

pseudo thrombocytopenia cross react with negatively charged phospholipids and exhibit anticardiolipin activity.

Sera of these patients lose their ability to clump platelets.

Therefore antibodies against phospholipids can bind to antigens modified by EDTA on platelet membrane and cause platelet clumping.

Antibodies from patients with thrombocytopenia can induce platelet agglutination with donor platelets in presence of EDTA.

This agglutination can be prevented by – 1. Warming donor platelets to 37 celsius.2. Pretreating donor platelets with aspirin,

Prostaglandin E1.3. Monoclonal antibodies against GPIIb/IIIa.

MYH9 related thrombocytopenia syndromes May-Hegglin anomaly, Fechtner syndrome,

Sebastian syndrome, and Epstein syndrome are autosomal dominant macrothrombocytopenias with mutations in the MYH9 gene.

This gene is located on chromosome 22q12–13.

This gene encodes NMMHC IIA , which is expressed in platelets, kidney, leukocytes, and the cochlea.

In all cells in which the gene product is expressed, except platelets and leukocytes, other NMMHC isoforms (IIB and IIC) are also expressed, and these can compensate functionally for the defective IIA isoform, restricting the most profound manifestations of NMMHC-IIA deficiency to platelets and leukocytes.

The NMMHC-IIA protein appears to be an important cytoskeletal contractile protein in hematopoietic cells.

One mutation in the MYH9 gene produced a highly unstable protein with abnormal organization of the megakaryocyte cytoskeleton.

The defect in platelet number is likely a defect in platelet maturation from proplatelets, as when MYH9-deficient stem cells were differentiated to megakaryocytes they produced proplatelets normally.

These syndromes include a triad of Thrombocytopenia, Macrothrombocytes and Dohle body like inclusions and other features.

One hallmark feature of MYH9-related disorders is revealed on the blood film, where neutrophilic inclusions that appear blue with Wright- Giemsa stain are noted.

The inclusions correspond to cytoplasmic aggregates of NMMHC-IIA, which are readily detected by immunocytochemistry.

Mediterranean macro thrombocytopenia Mild congenital thrombocytopenia with an

autosomal dominant pattern of inheritance.

Many of the patients share clinical and molecular features with the heterozygous Bernard-Soulier syndrome phenotype.

Linkage analyses reveal a heterozygous Ala156Val missense substitution in the GPIb gene (also known as the Bolzano mutation), which is also present in patients with Bernard-Soulier syndrome.

The clinical manifestations of Mediterranean macro thrombocytopenia are variable, with the severity of bleeding related to both platelet number and function.

A related syndrome with concomitant stomatocytosis and hemolysis (Mediterranean stomatocytosis/ macrothrombocytopenia) and autosomal recessive transmission is caused by mutations in the genes ABCG5 and ABCG8.

Paris Trousseau syndrome/Jacobsen Thrombopenia Congenital dysmorphology syndrome in which

affected individuals manifest trigonocephaly, facial dysmorphism, heart defects, and mental retardation.

Result from deletion of the long arm of chromosome 11 at 11q23, a region that includes the FLI1 gene, the product of which is a transcription factor involved in megakaryopoiesis.

All affected patients have mild to moderate thrombocytopenia and dysfunctional platelets.

The blood film shows a subpopulation of platelets containing giant granules.

Marrow examination reveals two distinct subpopulations of megakaryocytes with expansion of immature megakaryocytic progenitors, dysmegakaryopoiesis, and many micromegakaryocytes.

Pathologic bleeding usually is mild.

Kasabach-Merritt Syndrome Profound thrombocytopenia related to platelet

trapping within a vascular tumor, either a Kaposi-like hemangioendothelioma or a tufted angioma.

The syndrome presents predominantly during infancy, but several adult cases have been reported.

These vascular tumors should be differentiated from vascular malformations such as classic benign hemangiomas.

Thrombocytopenia in KMS usually is severe and associated with DIC.

Contributing factors include "platelet trapping" by abnormally proliferating endothelium within the hemangioma and platelet consumption associated with DIC.

Platelet trapping has been demonstrated by immunohistochemical staining of the tumors with anti-CD61 antibodies (a marker of platelets and megakaryocytes)141 and by nuclear studies using 51Cr-labeled platelets and 111 In.

The mainstay of treatment is eradication of the tumor.

Thrombocytopenia Associated with HIV Infection Thrombocytopenia is common in patients infected

with HIV, with the prevalence of thrombocytopenia, depending on the subpopulation of patients studied.

Among HIV-infected drug users, the prevalence is approximately 36.9 percent, compared to 8.7 percent in drug users without HIV infection.

In homosexual men, the prevalence is approximately 16 percent in the HIV-infected group and 3 percent in the HIV-negative group.

The high prevalence of thrombocytopenia in homosexuals and intravenous drug users without HIV probably results from the high frequency of hepatitis in these populations.

Causes –

1. Accelerated platelet destruction primarily related to immune complexes.

2. Decreased platelet production specially in advanced disease.

3. Splenic sequestration.

4. Platelet consumption associated with thrombotic thrombocytopenic purpura(TTP).

5. Medications, Concurrent infections such as hepatitis C, Hemorrhagic malignancies may contribute to development of thrombocytopenia.

Platelet kinetic studies performed in HIV-positive patients with thrombocytopenia demonstrate that the mean platelet life span usually is very short, an indication of accelerated platelet destruction.

HIV appears capable of triggering a large repertoire of immune complexes that participate in the destruction of platelets.

Immune complexes containing anti-F(ab')2 antibodies are found in homosexual individuals with HIV and thrombocytopenia.

Antibodies against CD4 and the CD4 receptor gp120 have been found in HIV-positive patients with and without thrombocytopenia.

These antibodies are capable of forming complexes through their specificity-determining regions.

These immune complexes can bind platelets and have been postulated to play a role in the thrombocytopenia associated with HIV.

In addition to increased platelet destruction, reduced platelet production appears to play a role in the thrombocytopenia observed in HIV patients.

A direct effect of viral infection on platelet production is suggested by the observation that platelet production increases when patients are treated with zidovudine.

Infected patients with thrombocytopenia also have increased levels of TPO, again supporting the notion of ineffective platelet production in the origin of HIV-associated thrombocytopenia.

The defect appears to lie at the level of megakaryopoiesis, as decreased levels of megakaryocyte progenitors in marrow have been observed.

The response to antiretroviral therapy suggests that the virus infects megakaryocytes or their precursors in the marrow.

HIV-1 entry into cells requires sequential interaction of the viral envelope gp120 with CD4 and a co receptor on the host cell plasma membrane, either C-C chemokine receptor-5 (CCR5) or CXC chemokine receptor-4 (CXCR4).

All of these receptors are expressed by megakaryocytes.

Patients with HIV are at higher risk for developing thrombotic microangiopathies.

Thrombocytopenic patients with HIV rarely experience clinically important bleeding (except, of course, those with hemophilia).

The platelet counts rarely dip below 50 x 109/L, and the thrombocytopenia often spontaneously resolves.

Nutritional deficiencies and alcohol induced thrombocytopenia

Thrombocytopenia may be seen in association with vitamin B12 deficiency when the latter results from auto antibodies against parietal cells or intrinsic factor and is associated with immune thrombocytopenia.

Various other autoimmune disorders can coexist with pernicious anemia, including autoimmune vitiligo and autoimmune thyroiditis.

Thrombocytopenia in alcoholic patients almost always results from liver cirrhosis with relative TPO deficiency (the liver is the primary origin of circulating TPO levels), congestive Spleenomegaly, and/or from folic acid deficiency.

Suppression of platelet production sufficient to produce thrombocytopenia requires consumption of large quantities of ethanol over several days.

Thrombocytopenia usually resolves in 5 to 21 days with cessation of ethanol ingestion, sometimes with a transient rebound thrombocytosis.

Myelodysplastic syndrome(MDS) Myelodysplastic syndromes are clonal myeloid disorders

characterized by blood cytopenias in combination with a hyper cellular marrow that often exhibit dysplastic changes in any of the three hematopoietic lineages.

Thrombocytopenia is present in approximately 50 percent of patients and usually occurs in conjunction with other cytopenias.

Moreover, platelets from patients with MDS often display functional abnormalities and, when present, usually augment the thrombocytopenia-related bleeding disorder.

The presence of micromegakaryocytes or micro mononuclear megakaryocytes in marrow from MDS patients indicates altered megakaryopoiesis.

Maturation of megakaryocytes is arrested in MDS, as suggested by immunohistochemistry studies showing an increased number of megakaryocytic precursors in the marrow of affected patients.

Besides maturation arrest, an increased rate of apoptosis is seen in all subtypes of MDS.

Abnormal megakaryopoiesis is due to dysfunctional TPO receptor or an abnormality in the downstream signaling pathway rather than from diminished expression of receptor itself.

Supportive therapy in addition to chemotherapy is the treatment of choice.

Aplastic anemia Aplastic anemia is a pancytopenia that results

from failure of marrow hematopoiesis.

However, some patients with MDS or amegakaryocytic thrombocytopenia present with low platelet counts and then progress to pancytopenia and aplastic anemia.

In aplastic anemia, blood testing reveals markedly decreased cell counts, reticulocytopenia, and lack of circulating blasts.

A considerable amount of data support the hypothesis that aplastic anemia results from an autoimmune attack directed against HSC.

Activated T-helper type 1 T cells that produce interferon-, tumor necrosis factor, and IL-2 are responsible for suppression of the hematopoietic cell compartment.

This cytotoxic activation leads to a Fas-mediated cell-cycle arrest and death of CD4+T cells.

ReferencesI. Williams hematology 8th editionII. De Grutchy’s clinical hematology 6th

editionIII. Robbins basic pathology 8th edition

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