Posts by Avinash Deo

I am a haematologist and medical oncologist

78 year old Alcoholic With Anaemia


An 78 year old man presented with fatigue and breathlessness. Examination showed pallor. There was no icretus, clubbing, lymphadenopathy or hepatomegaly. There was mild splenomegaly. There was no history of haemoptysis, malena or haematochesia.

An haemogram was performed that showed a haemoglobin was 8g/dL with an MCV of 101fl. The leucocyte count was 4.5 x 109/L and the platelet count was 265 x 109/L. The differential leucocyte count was neutrophils 58% lymphocytes 32%, monocytes 6% and eosinophils 4%. The Reticulocyte count was normal. 


Anaemia in alcoholics is multifactorial. The causes of Anaemia include

  1. Direct toxic effect of alcohol: Most alcoholics have macrocytosis in the range of 100-110fl. Macrocytosis is seen before anaemia develops. Anaemia reverses after a few months of abstaining from alcohol.
  2. Iron deficiency from gastrointestinal blood loss from varices and alcoholic gastritis may presents with hypochromic microcytic anaemeia. The transferrin saturation and serum ferritin are low. Alcohol and alcohol associated disease affect MCV, transferrin saturation and serum ferritin making interpretation of test difficult.
    • Microcytosis may not be seen in alcoholics with iron deficiency. A study showed 48% of the patients with chronic liver disease and iron deficiency diagnosed by absence of bone marrow iron had an MCV more than 100fl (J Intern Med. 1998 Mar;243(3):233-41).
    • Alcohol effects transferrin saturation. Transferrin levels may be decrease because of infection or inflammation. In a study of transferrin saturation in cirrhotics a transferrin saturation of <12% was of value in diagnosing iron deficiency. But transferrin saturation did not add to the value of ferritin in diagnosis.
    • Ferritin is an acute phase reactant and can be increase in chronic liver disease. A study has suggested different cutoffs be used in patients with chronic liver disease. Iron deficiency should be diagnosed with a ferritin ≤50 μg/L. Values >400 μg/L were associated with a low probability of iron deficiency (Annals of Gastroenterology (2017) 30, 1-9).
    • sTrasnferrin receptors levels is a iron deficiency (sensitivity 91.6%, specificity 84.6%) in patients with chronic liver disease provided that hemolysis and recent blood loss have been excluded (Clin Lab Haematol. 1999 Apr;21(2):93-7)
  3. Folate and Cobalamine deficiency: Folate and cobalamin deficiency causes macrocytosis and should be suspected in patients with MCV greater than 110fl, those with decreased platelet and/or leucocyte counts and when peripheral smear shows hypersegmantation of neutrophils.
  4. Hypersplenism: Hypersplenism causes anaemia and pancytopenia. The spleen is enlarged but there is no correlation between the degree of anaemia and size of the spleen. Unlike patients with folate or cobalamin deficiency the MCV patient with hypersplenism do not have an markedly (>110fl) increased MCV.
  5. Anaemia of chronic disease: Anaemia of chronic disease may be seen in alcoholics because concurrent infection or inflammation.
  6. Haemolytic anaemia with spur cells: Patients with severe hepatocellular disease develop spur cells that result in haemolytic anaemia. This may be associated with increased jaundice and/or deteriorating liver function. Appearance of spur cells may be related to continued alcohol abuse in the presence of severe chronic liver disease. Transfused blood cells also become acanthocytic and are haemolysed. Abstinence from alcohol can eliminate/reduce spur cells. Spur cell anaemia has been cured by liver transplant.

 

The patient has a serum iron of 24 µg/dL the total iron binding capacity of 202µg/dL and a  transferrin saturation of 11.9%. The serum ferritin was 30 µg/L. The B 12 and folic acid levels were normal. The serum albumin was 3.2g/dL. The AST, ALT, prothrombin time and bilirubin were normal.

The patients was diagnosed to have iron deficiency. An upper gastrointestinal endoscopy showed gastro-oesophageal varices. Colonoscopy did nor show any cause of bleeding. A diagnosis of iron deficiency anaemia was made, iron supplementation given and varices banded.

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Nodular Lymphocytic Predominant Hodgkin’s Lymphoma


Hodgkin lymphoma (HL) is of two types. Classical (cHL) and nodular lymphocyte predominant (NLPHL). NLPHL is rarer and runs a more indolent clinical course.

Epidemiology

NLPHL accounts for about 5% of all HL.

Age: The disease is characterised by two peaks. The first one in childhood and the second between the ages of 30-40.

Gender: NLPHL shows a male predominance. About three-fourth of the patients are males. Male preponderance is less marked in blacks.

Racial Differences: Black patients are younger, more often female and more often present with axillary involvement. Little is known of NLPHL in other races (Cancer 2015; 121:3472-80).

Familial Susceptibility: Family members of patients with NLPHL at increased risk NLPHL. The standardised incidence ratio in one study was reported to be 19 (J Clin Oncol 2013; 31;938-43).

Histology

The normal architecture of the node is effaced and replaced by large nodules. Occasionally there may be large nodules with diffuse areas. Sometimes uninvolved nodal tissue may be seen. This is usually located peripheral in a sub-capsular area.

Microscopically NLPHL shows the malignant cell, LP cell, in a background mainly made up of small lymphocytes and with a prominent follicular dendritic cell (FDC) network. The follicular dendritic cell meshwork is absent from the diffuse areas. Unlike most other malignancies (and like cHL and T cell/Histolytic rich large B cell lymphoma) the normal reactive cells form the bulk of the enlarged node.

The LP cell has a nucleus that shows complex lobulation. It resembles a exploded kernel of corn and hence the cell is also referred to as the popcorn cell. The nucleolus is smaller than that of the RS cell and lies peripherally and is basophilic. There is a thin rim of cytoplasm.

The infiltrate in a nodule mainly consists of small lymphocytes. Unlike cHL, Eosinophils and plasma cells are occasional or may be absent. Most of the small lymphocytes making up the nodule are CD20+, CD79+ small B lymphocytes. The LP cells is however immediately surrounded by CD20, CD3+ T helper cells that express PD-1 and CD57. Diffuse area have CD4+ T cells and areas between nodes have CD3+ parafollicular T cells.

Varient histological patterns are known, associated with adverse prognosis and should be reported (Am J Surg Pathol 2003;27:1346-56).

Immunophenotype helps in diagnosis and has given clues to the origin of LP cells. The LP cells show a B cell phenotype and express CD20, CD79, CD22, PAX-5 and CD45. They express BCL-6 indicating the germinal centre origin. They do not express BCL-2. They strongly express the B cell transcription factor OCT-2 and its cofactor BOB.1. This distinguishes then from the Reed-Sternberg (RS) cells of cHL. RS cells show a weak expression or do not express these factors. RS cells express CD15, CD30 and fascin that are not expressed by the LP cells. About a fifth of the patients express IgD. These patients tend to be male, present with cervical adenopathy and have a greater risk of having a variant histology.

The normal counterpart of the LP cell appears to be the germinal centre B cell at the cenrtoblastic stage of differentiation.

NLPHL as well as cHL are diseases characterised by malignant cells surrounded by an infiltrate of normal cells. Unlike other cancers, the normal cells form the bulk of the tumour mass in both the cases. The malignant cells affect and are affected by the normal cells surrounding them. LP cells, like normal germinal centre cells, appear to depend on normal immunoglobulin receptor signalling. RS cells depends on other signalling receptors e.g. CD30 and CD40. The growth of normal germinal centre cells depends on The FDC and follicular T cells. These cells also support the growth of LP cells. The LP cell do not produce cytokines at levels seen in the RS cell. B symptoms are less common NLPHL less common than cHL.

 

 

Clinical Presentation

The most common presentation of NLPHL is isolated lymphadenopathy, most often in the cervical, axillary or the inguinal region. The swelling is usually present for a long time and has been growing slowly. About 80% of the patients present with localised disease and less than 20% with stage III/IV (Ann Hematol. 2016; 95: 417–423). B symptoms are uncommon (about 5%). Extranodal disease is very uncommon.

NLPHL runs a more indolent course that cHL. It is characterised by a relapses and transformation to high grade lymphoma diffuse large B cell lymphoma (including T cell/ histiocyte rich large B cell lymphoma). Relapses usually respond to treatment.

Staging

NLPHL, like cHL is classified by the Ann Arbor staging system with Cotswolds modifications. The stages are summarised below. A more detailed staging can be found here.

  1. Stage I: Involvement of one nodal region, lymphoid structure or one extra-nodal site
  2. Stage II: More than one region involved but disease limited to one side of the diaphragm.
  3. Stage III: Disease on both sides of the diaphragm but limited to the lymphoid system.
  4. Stage IV: Disease disseminated to one or more extra nodal organs.

Patients with fever with hight sweats and significant (>10% in the preceding 6 months) are said to have B symptoms.

The staging workup should include clinical examination, haemogram, ESR and biochemistry. NLPHL is PET avid. PET-CT is better than CT for staging. It is of value in to exclude diseases dissemination in patients where observation or local treatments are being considered. The value is interim PET-CT is NLPHL is uncertain. The bone marrow is very uncommonly involved (about 1-2%). Only patients with advanced disease should be subjected to bone marrow examination.

 

Differential Diagnosis

  1. Lymphocyte Rich Classical Hodgkin lymphoma
  2. T cell/ Histiocyte Rich Large B Cell Lymphoma
  3. Progressively Trasnformed germinal centres
  4. Follicular Lymphoma
  5. Mantle cell Lymphoma

 

Treatment

Early disease (Stage I/IIA)

Patients who have undergone excision biopsy that has resulted in a complete removal of all disease may be observed. Despite a lower progression free survival the patients who are observed do not show an inferior overall survival. This indicates that delaying treatment (radiation, chemotherapy or both as may be appropriate) does not hamper it’s efficacy.

Advanced Disease (Stage IIB, III, IV)

These patients need chemotherapy with the anti-CD20 antibody, rituximab. Three approaches are possible

  1. Classical Hodgkin lymphoma like therapy with Rituximab with ABVD: R-ABVD (Rituximab, doxorubicin, bleomycin, vinblastine and dacarbazine) should be administered to patients needing chemotherapy.
  2. B cell non-Hodgkin Lymphoma like therapy: R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) is the standard treatment for high grade B cell non-hodgkin lymphoma. R-CHOP has been shown to effective in disease control and reducing the risk of transformation. It may be preferred in patients at a high risk of transformation, though there is not comparative trial with R-ABVD. Males and those with variant histology are at a higher risk of transformation. Models for predicting transformation are available.
  3. Single agent Rituximab: Single agent rituximab is indicated in patients with co-morbidities. The risk of relapse remains high.

Treatment of Relapse

Relapses must be rebiopsied to confirm NLPHL and to exclude transformation to a high grade lymphoma. Localized relapses may be treated with radiation. Chemotherapy should be used for other patients. Patients who have a chemosensitive relapse may be considered for allogenic stem cell transplant (Am J Haematol 2017 Oct 3. doi: 10.1002/ajh.24927).

Treatment of Transformation

Patients who undergo transformation are treated with regimen for regimens for high grade B cell lymphoma. The limited data suggests that the outcome is no different from that of de novo large B cell lymphoma.

 

Prognosis

The prognosis of NLHPL is better than conventional HL partially because of a more favourable disease profile – early stage, no B symptoms, no Bulky disease. One study showed a 94% overall survival at 10years (Ann Hematol. 2016; 95: 417–423). The progression free survival was 75% indicating relapses are common but are curable. Progression to diffuse large B cell lymphoma is seen in 5-10% of the patients. Atypical histology increases the risk of relapse (Blood. 2013 Dec 19;122(26):4246-52).

 

 

Lymphocytosis in Adults


Normal Peripheral Blood Lymphocytes

Lymphocytes are small leucocytes with a round nucleus and a thin rim of cytoplasm. About 10% of the peripheral lymphocytes are large, have a more abundant cytoplasm that has granules. These are called large granular lymphocytes (LGL). About 85% of the peripheral blood lymphocytes are T lymphocytes. The remaining are B lymphocytes and NK cells with B lymphocytes dominating.

Lymphocytosis is increase in the lymphocyte counts to what is normal for age. Lymphocyte count is highest at birth and falls with age. Normal lymphocyte counts in adults is 1000 to 4800 cells/mm3.

Causes of Lymphocytosis

Lymphocytosis may be reactive (see table below) or due to a malignancy. Reactive lymphocytosis is polyclonal. Lymphocytosis due to malignancy is monoclonal. The most common cause of reactive lymphocytosis is infectious mononucleosis due to Epstein-Barr virus infection. Reactive lymphocytosis may be seen in other viral infections, drug hypersensitivity, thymoma and after splenectomy. Persistent polyclonal B cell lymphocytosis is a condition found in middle aged women who smoke.

Monoclonal B cell lymphocytosis and chronic lymphocytic leukaemia are the commonest causes of primary lymphocytosis.

Causes of Lymphocytosis
Reactive lymphocytosis (reactive lymphocytes unless mentioned otherwise)

  1. Infections
    1. Viral: Infectious mononucleosis (EBV), Adenovirus, CMV, Coxsackie virus, Hepatitis, Acute HIV infection, Human T-lymphotropic virus type I, Influenza, Measles, Mumps, Poliovirus, Rubella
    2. Bacterial: Pertussis, Cat scratch disease and other chronic bacterial infections
    3. Parasitic: Toxoplasmosis
  2. Drug hypersensitivity reactions
  3. Stress (normal looking lymphocytes)
  4. Persistent polyclonal B cell lymphocytosis (lymphocytes have a distinct nuclear cleft)
  5. Post-splenectomy  (normal looking lymphocytes)
  6. Thymoma  (normal looking lymphocytes)
  7. Hyperreactive malarial splenomegaly  (normal looking lymphocytes)

Primary Lymphocytosis

  1. B Lymphocytic
    1. Monoclonal B cell lymphocytosis
    2. Chronic lymphocytic leukemia
    3. B-Prolympocytic leukaemia
  2. Leukaemia Phase of non-Hodgkin lymphoma (common
    1. B cell Follicular lymphoma
    2. Mantle cell lymphoma
    3. Splenic marginal zone lymphoma
    4. Lymphoplasmacytic lymphoma
  3. T Cell Lymphoma
    1. Prolymphocytic leukaemia
    2. Sezary cell leukaemia
    3. Adult T-cell leukaemia/lymphoma
    4. Large Granular cell leukaemia
  4. Acute lymphoblastic leukemia

Clinical Profile of Patients with Lymphocytosis

Infectious Mononucleosis: Infectious mononucleosis (IM) is a self limiting disease of adolescents that presents with prodrome of fatigue, myalgia lasting 1-2 followed by fever pharyngitis and lymphadenopathy, splenomegaly, hepatomegaly and rash. The peripheral smear shows lymphocytosis with atypical lymphocytes. These are irregular, have slightly larger nuclei, a more open chromatin and abundant cytoplasm. Some cells may appear blastoid. Atypical lymphocytes are predominantly CD8+. Differentiation from lymphocytosis of malignancy is made by

  1. Clinical Picture: Patients with IM have fever, malaise, pharyngitis and other features of IM.
  2. Morphology of Lymphocytes: Reactive lymphocytes of IM show variations in size and morphology. Cells from patients with malignant lymphocytoisis are more uniform.

Other infection: Atypical lymphocytosis is a feature of other infections listed in the table above. Typical features of the causative infection may or may not be present. CMV associated mononucleosis syndrome is indistinguishable from IM. Lobulated lymphocytes are a feature of human T-lymphotropic virus type I (HTLV-I ) infection.

Lympocytosis with B pertussis: Pertusis caused by Bordetella pertussis with upper respiratory symptoms that evolves into a paroxysmal cough in about 1-2 weeks. Pertusis is an exception amongst acute respiratory infections in that it causes lymphocytosis rather than neutrophilic leucocytosis. Unlike other viral infections lymphocytes of pertussis are small and have cleaved nucleus.

Stress Lymphocytosis: Stress has been associated with an increased lymphocyte count. Counts may range 4,000 to 10,400/mm3. All subsets of lymphocytes increase. The counts normalises after the stressful event passes (Am J Clin Pathol. 2002 May;117(5):819-25).

Persistant Polyclonal B cell lymphocytosis: Persistent polyclonal B cell lymphocytosis is seen in young to middle-aged women who are smokers. The patients show the presence of large binucleate lymphocytes. There is polyclonal increase in IgM in the serum. The patients do not have lymphadenopathy and splenomegaly.

Splenectomy: Lymphocytoisis that persists has been reported in patients who have undergone splenectomy (Clin Lab Haematol. 1995 Dec;17(4):335-7).

Thymoma: Paraneoplastic T cell lymphocytoss may rarely be seen in patients with thymoma. The patients present with a mediastinal mass and lymphjocytiosis. They need to be differentiated from T Lymphoblastic leukaemia who can present with mediastinal mass with lymphoblasts (Ann Oncol 2007; 18:603-604).

Hyperreactive malarial splenomegaly: Hyperreactive malarial splenomegaly is seen in residents of malarious areas. It presents with left upper quadrant pain, fatigue and dyspnoea. The patients have a massive spleen. The haemogram shows anaemia, leucopenia and thrombocytopenia. Some patients may have lymphocytosis.

Monoclonal B cell lymphnocytosis: Monoclonal B cell lymphocytosis is a premalignant condition that has a risk of progressing to chronic lymphocytic leukaemia. It it is diagnosed in a patients with lymphocyte count <5000/mm3 without any other evidence of lymphproliferative disease. The morphology and phenotype of the cells is identical to that of chronic lymphocytic leukaemia. Patients are asymptomatic and the disorder is detected incidentally on a haemogram performed for another reason.

Chronic lymphocytic Leukarmia (CLL)/small lymphatic leukaemia: Chronic lymphocytic leukaemia is the leukaemia phase of small lymphocytic lymphoma. Patients with CLL have lymphocytosis with normal looking lymphocytes. A few prolymphocytes may bee seen. If the percentage of prolympocytes is greater then 55% a diagnosis of prolymphocytic leukaemia should be made. The cells express CD19, CD20(usually weak), CD23 and the T cell marker CD5. Patients may have lymphadenopathy, splenomegaly and hepatomegaly. Anaemia and/or thrombocytopenia may co-exist, some of which may be due to autoimmunity.

Prolymphocytic Leukaemia: Prolymphocytic leukaemia is a misnomer. The malignant cell is actually a activated mature lymphocytes. Prolympnocytic leukaemia may be of B cell or T cell type. The cells are twice the size of a normal lymphocyte. The nucleus is round with a moderately condensed cytoplasm. A prominent central nucleolus is present. The cytoplasm is faintly basophilic. Morphiology of T Prolymphocytic leukaemia is similar. Patients present with a high count (usually > 100X109/L), massive splenomegaly in the absence of lymphadenopathy. T PLL may show skin infiltration in 20% of the cases. They may also show serous effusions.

Peripheral blood involvement with non-Hodgkin Lymphoma: Peripheral blood involvement with non-Hodgkin Lymphoma presents with mononucleated cells. Morphological features may  suggest the type of lymphoma. These include villous lymphocytes of splenic marginal zone lymphoma and atypical hairy cell leukaemia, cells with cribriform nuclei in Sézary syndrome plasmacytoiod lymphocytes of lymphplasmacytic lymphoma and lobulated lymphocytes in adult T-cell leukaemia/lymphoma. 

Large Granular Cell Leukaemia: LGL leukaemia is characterised by a count of 2-10X109/L. The cell are large granular lymphocytes with abundant cytoplasm and fine and coarse azurophilic granules. 

 

Myeloproliferative Neoplasm


Myeloproliferative neoplasm are a group of disorders characterized by bone marrow proliferation with increase in the number of blood cells. The proliferation my be limited to one cell line or may involve more than on cell line. They are distinct from acute leukaemia but carry the risk into evolving into leukaemia as a terminal event. This article discusses general aspects of myeloproliferative neoplasm. The disease entities will be discussed elsewhere.

Progressive myelofibrosis was the first myelroliferative neoplasm to be described. It was described by Gustav Heuck in 1879. This was followed by chronic myeloid leukaemia (CML) by John Hughes Bennett in 1845, polycythaemia vera (PV) described by Louis Henri Vaquez in 1892 and essential thrombocytosis (ET) described by Emil Epstein in 1934. The term myeloproferative disease was coined by Dameshek in 1951 to describe seven conditions that he thought were manifestations of “proliferation of bone marrow to a hitherto undiscovered stimulus” (Blood 1951; 6:372-75). The seven conditions were chronic granulocytic leukaemia (now chronic myeloid leukaemia), polychthaemia vera, agnigeic myeloid metaplasia (now primary myelofibrosis), thrombocytosis (essential thrombocytosis), megakaryocytic leukaemia and erythroleukaemia (including DeGuglielmo’s syndrome). As the malignant nature of these disease was not apparent till recently they were referred to as myeloproliferative disorder. The recognistion of the neoplastic in nature of the diseases has resulted in the change in the name from myelproliferative disorder to myeloprlferative neoplasm.

The WHO 2016 classification of myeloproliferative neoplasm includes the following disease

  1. Chronic myeloid leukaemia (CML)
  2. Chronic neutrophilic leukaemia (CNL)
  3. Polycythaemia vera (PV)
  4. Progressive myelofibrosis (PMF)
  5. Essential thrombocytosis (ET)
  6. Chronic myeloproliferative neoplasm unclassified
  7. Chronic eosinophilia leukaemia NOS
Myeloproliferative disease

Pathogenesis of myeloproliferative diseases

Pathogenesis of Myeloproliferative Neoplasm

The bone marrow proliferates in response to an initiating event (see figure above). This event remains to be discovered. Bone marrow proliferation results in increased blood counts. Increase in erythrocytes results in polycythaemia and features of hyperviscosity. Increased leukocyte counts result in leukostasis. The leucocytes counts that can cause leukostasis are only seen in CML. Thrombocytosis increases the risk of thrombosis. With time a MPN disease progresses. Progression either results in blast transformation or myelofibrosis as the terminal event. CML is more likely to progress to blast crisis and non-CML MPNs to myelofibrosis. Splenomegaly is an early feature of CML. Spleen in enlarged because of infiltration of the spleen by neoplastic haemopoietic cells despite there being no myelofibrosis. In non-CML splenomegaly occurs late and is a result of extra-medullary haematopoiesis due to myelofibrosis.

Clinical Aspects og Myeloproliferative Neoplasm

The myeloproliferative disease have the following common features

  1. Clonal proliferation of bone marrow: The Philadelphia chromosome associated with CML was the first chromosomal abnormality associated with a malignancy. The Philadelphia chromosome is a t(9;22) translocation that results in the formation of the BCR-ABL1 fusion gene. It proved the clonal nature of CML. Mutations proving the clonality of non-CML myeloproliferative neoplasm have been discovered. These include the JAK2, MPL and CALR mutations found in PV, PMF and ET. CNL is characterised by mutations in CSF3R. These mutations are the “undiscovered stimulus” Dameshek wrote about.
  2. Increased Blood Counts: Myeloprolifarative neoplasm have increase leucocytes, erythrocytes and thrombocytes alone or in combinations.
  3. Splenomegaly: Splenomagaly is a future of all myeloproliferative neoplasm. It is most pronounced in CML and PMF. The underlying mechanisms of splenomegaly in the conditions are different. Patients with CML have infiltration. Some develop myelofibrosis. Massive splenomegaly in myeloproliferative neoplasm than CML indicated bone marrow fibrosis.
  4. Overlapping clinical features: The clinical features of myeloproliferative neoplasm overlap. Polycythaemia is only seen in PV. Leucocytosis is a feature of all conditions other than ET. The type of leucocytosis can distinguish entities. Exteme leucocytosis with premature myeloid forms points to CML. But all patients with CML do not have extreme leucocytosis. Neutrophilic leucocytosis is a feature of CNL. Thrombocytosis is a featured shared by myeloproliferative neoplasm. As discussed below myelofibrosis develops in all myeloproliferative diseases. All myeloproliferative neoplasia are at risk of transforming into acute leukaemia. Despite the use of BCR-ABL1 tyrosine kinase inhibitors the risk is of acute leukaemia transformation is highest for CML.
  5. Development of bone marrow fibrosis: As myeloproliferative neoplasia progress bone marrow fibrosis increases. Myelofibrosis develops fastest in progressive myelofibosis. CML often does not develop clinically evident fibrosis but transforms to acute leukaemia as a terminal event. Development of fibrosis results in pancytopenia. Once fibrosis sets in it is not possible to tell the nature of the pre-fibrotic myeloproliferative neoplasm.
  6. Transforamation to acute leukaemia: All myeloproliferative neoplasia have a risk of evolving to acute leukaemia. This is known as blast crisis. The risk is greatest in CML.
  7. A tendency for thrombosis: Myeloproliferative neoplasm increase the risk of thrombosis. The risk is highest in ET and PV.

Treatment of Myerloproliferative Neoplasm

The goals of therapy of myeloproliferative neoplasia are

  1. Prevention and treatment of complications of increased cell counts
  2. Prevention of progression of MPN
  3. Erradicating the malignant clone

Prevention and treatment of complications of increased cell counts

Acute complication of myeloproliferative neoplasia include Leukostasis hyperviscosity and thrombosis. Polycythaemia is addressed by phlebotomy. Leukostasis is treated by measures to reduce counts (see Hyperleukoytosis and leukocytosis). Thrombocytosis of ET is treated low dose aspirin with or without hydroxyurea. The exact combination depends on the diagnosis and the risk stratification. Low dose aspirin is administered to patients with PV and PMF as they are at risk of thrombosis.

Prevention of progression of MPN

The only MPN where progression can be retarded is CML. The use of BCR-ABL1 tyrosine kinase inhibitors (BCR-ABL1 TKI) like imatinib, nilotinib or dasatinib prevent progression. The longest results are available for imatinib. Eithty five percent of the patients are alive at 10 years compared to a median survival of 45.4 with busulifan and58.2 months with hydroxyurea (Blood. 1993 Jul 15;82(2):398-407).

Eradicating the malignant clone

Supression of the BCR-ABL1 positive clone does not result in eradication of the malignant clone. More than half the patients in deep remission have a relapse of CML in one year off stopping BCR-ABL1 TKI therapy. Allogenic stem cell transplant offers the only possibility of cure in CML. The safety and efficacy of BCR-ABL1 TKIs has reduced the role of allogenic stem cell transplant in CML. It is now used in patients who are poorly controlled with upfront BCR-ABL1 TKI therapy. Allogenic stem cell transplant may be used in selected patients with PV, ET and PMF but does not form a front line therapy.

Anaemia of Renal Insufficiency


Richard Bright discovered the relationship of renal failure and anaemia in 1836. Anaemia of renal insufficiency is mainly results from decreased erythropoietin production and impaired supply of iron to erythroid precursors. Anaemia results in fatigue, cognitive dysfunction and cardiovascular consequences and increased cardiovascular mortality. Correction of anaemia decreases LVH, LVH related hospitalizations and mortality of cardiovascular disease. Without erythropoietin supplementation only 2% (Int J Artif Organs 1981; 4:277-9) needing haemodialysis have a hematocrit of >0.4. Availability of erythropoietin and the use of parental iron has reduced the prevalence of anaemia in patients with renal failure.

Pathogenesis of Anaemia of Renal Insufficiency

Anaemia associated with renal insufficiency results from decreased erythropoietin and iron availability.

Erythropoietin, produced by the kidney, is essential for erythropoiesis (see Erythropoietin and Erythropoietin Receptor Signalling). Renal insufficiency results in decreased renal mass and consequently in decreased erythropoietin production. Erythropoietin levels in patients with renal failure are normal or slightly elevated, but they are 10-100 times lower than a normal individuals with similar levels of anaemia. Decreased erythropoietin hampers erythropoiesis contributing to anaemia (no 1 in figure 1). Erythropoietin supplementation corrects anaemia in most but not all patients.

Three other causes of anaemia have been identified in patients with renal insufficiency. These are

  1. Impaired supply of iron
  2. Folate deficiency due to losses in dialysis
  3. Aluminium intoxication resulting in microcytic hypochromic anaemia

Folate deficiency and aluminium intoxication are of historical importance. Current management of dialysis patients includes folate supplementation and measures to prevent aluminium intoxication. Impaired iron supply to erythroid precursors remains the main cause of anaemia that does not respond to erythropoietin.

Iron is a component of heme (see heme synthesis). Patients with renal insufficiency have decreased iron availability from blood loss, impaired iron absorption and inability to use body iron stores (functional iron deficiency)

Causes of blood loss in patients with renal failure include (2 in figure 1)

  1. Gastrointestinal blood loss due to uraemia induced platelet dysfunction
  2. Blood loss due to dialysis
  3. Iatrogenic blood loss due to investigations (Transfus Med 2009; 19;309-14)

Iron supply to the erythroid precursors is also limited by impaired absorption (3 in figure 1) and a functional iron deficiency (4 in figure 1). Hepcidin plays a central role disrupting iron supply in patients of renal insufficiency. It reduces iron absorption in the gut and hampers the release of macrophage iron. The latter results in a phenomena called as functional iron deficiency i.e. diminished iron availability despite adequate iron stores. Hepcidin levels are increased from increased production because of inflammation and decreased elimination as a result of falling GFR. Infections, exposure of leucocytosis to foreign surfaces during dialysis and from the underlying cause of renal insufficiency are the sources of inflammation.

Clinical Features

The degree of anaemia co-relates with the degree of impairment of renal failure. Anaemia is rarely observed in patients with creatine clearance of greater than 45ml/1.72m2. This corresponds to a serum creatinine of 2-2.5mg/dL.  The anaemia is mild to moderate with the haematoctit stabilizing between 15-30%. Anaemia is less severe in polycystic kidney disease and to a lesser extent in hypertension. It may be more severe in diabetics. The presence of severe anaemia is an indication for evaluation for another cause of anaemia.

Signs and symptoms are those of anaemia and the underlying disease causing renal failure.

Diagnosis

A complete haemogram should be performed in all patients with chronic renal insufficiency at diagnosis. The diagnosis is made by the presence of a mild to moderate normochromic normocytic anaemia, presence of renal insufficiency and the absence of other causes of anaemia. Macrocytosis may be seen. The investigations that are usually performed in a patients with suspected anaemia of renal insufficiency include

  1. Complete haemogram with erythrocytes indices to determine severity of anaemia and to exclude co-existing diseases like iron deficiency and aluminium intoxication (microcytosis) or Folic acid/vitamin B12 deficiency (macrocytosis).
  2. The reticulocyte counts are normal but may rise with azotaemia
  3. Serum iron, total iron-binding capacity, percent transferrin saturation and serum ferritin need to be performed to asses the iron stores and iron delivery

Despite the fact that there is a relatively deficiency of erythropoietin, erythropoietin levels are not useful in the diagnosis of anaemia of renal insufficiency. The bone marrow tends to be moderately hypercellular with a slight erythroid hyperplasia.  A bone marrow aspiration is not necessary for diagnosis.

Treatment

The treatment of anaemia of renal insufficiency in transplant ineligible patients consists of erythropoiesis stimulating agent (ESA), iron and blood transfusion. Transplant when indicated cures anaemia in 80% of the patients. Most authorities define anaemia as a haemoglobin of 13-13.5g/dL in men and less than 12g/dL in women.

Iron therapy

All patients with chronic renal insufficiency need iron supplementation. The route of supplementation may be oral or intravenous. Severity of anaemia, patients preference and adherence and previous experience with that route iron therapy are few of the factors determining the choice. In a meta-analysis of oral iron vs. parental iron, parental iron raised the concentration of haemoglobin by >1g/dL more often than oral iron. The risk of hypotension was higher and gastrointestinal side effects lower with parental iron (Am J Kidney Dis. 2016 Nov;68(5):677-690). As the safety of intravenous iron preparation has increased so has their use. Many trials comparing oral with intravenous iron therapy have shown a better response with intravenous iron. However intravenous iron therapy has not been show to be superior in some trials (Clin J Am Soc Nephrol. 2016 Jul 7; 11(7)).

Erythropoiesis Stimulating Agenst (ESA)

ESA are indicated in patients who have adequate iron reserves. These are patients with transferrin saturationis >25 percent and ferritin >200 ng/mL. ESA should be used in patients with iron deficiency (actual or functional) after iron replenishment. Two ESAs available are erythropoietin and darbepoietin. Treatment with ESA results in improved quality of life, improved cognitive function and reduces left ventricular hypertrophy. The target of therapy is to increase the haemoglobin to 11-12g/dL. Higher targets are associated with more side effects. The rise is typically achieved in 6-8 weeks of therapy. Erythropoietin is administered in a dose of 100-150 units every week. Hypertession is seen in about 35% of the patients. Darbepoietin as administered in a dose 0.45 μg/kg/week for patients on dialysis.

Erythropoietin resistance is failure to respond to erythropoietin. The causes of erythropoietin resistance include inadequate iron availability (decreased reserves and/or functional iron deficiency) inadequate dialysis, marrow fibrosis associated with secondary hyperparathyroidism, folate deficiency, aluminium intoxication and co-existing inflammation. Dialysis should reduce blood urea to less than 35% to allow for optimal erythropoietin action.

 

Renal replacement therapy

Renal Allograft: Renal allograft is effective correction of anaemia in 80% of the patients. The rise is seen in 8-10 weeks. About 20% of the patients may get post-transplant erythrocytosis. The causes of failure of haemoglobin to rise after transplant include hemorrhage, intense immunosuppression or graft rejection

Dialysis: Dialysis has a small effect on the anaemia presumably by clearance of inhibitors. Peritoneal dialysis (PD) is more effective than haemodialysis. Reason a higher efficacy of PD is more effective in unknown but more effective removal of middle molecules (500-1500kD) and lesser of inflammation compared to HD may explain the higher haemoglobin in patients with on PD.

 

 

Anaemia with Thrombocytosis


A 49 year old woman presented with weakness and fatigability. On examination, other than pallor of the skin and mucosa there was no other finding.  A haemogram was done that showed a haemoglobin: 6.7g/dL, leucocyte count: 9.1 X 109/L and a platelet: 664 X 109/L.

Anaemia and thrombocytopenia is a feature of

  1. Myeloproloferative neoplasm
  2. Chronic inflammation
  3. Underlying malignancy (Paraneoplastic)
  4. Iron deficiency

A haemogram from a automated haematological counter hides a lot of information. Before more investigations are done it is important to assimilate all the information in the haemogram. The haemogram in the above listed conditions shows:

  1. Myeloproliferative Neoplasm: The myeloproliferative neoplasm are diseases characterised by proliferation of bone marrow. They are distinct from acute leukaemia. According to the 2016 WHO classification myeloproliferative neoplasm include chronic myeloid leukaemia (CML), chronic neutrophilic leukaemia (CNL), polycythaemia vera (PV), progressive myelofibrosis (PMF), essential thrombocytosis (ET), chronic myeloproliferative neoplasm unclassified (CMPN-U) and chronic eosinophilic leukaemia (CEL).  Myeloproliferative diseases associated with anaemia and thrombocytosis are CML, prefibrotic phase of PMF and CMPN-U. Haemogram of patients with myeloproliferative diseases shows leucocytosis with presence of immature leucocyte forms. This is most pronounced in patients with CML. In fact leucocytosis with the presence of immature leucocyte forms is the dominant feature of the haemogram of patients with CML. Anaemia of myloproliferative is normocytic and normochromic.
  2. Chronic Inflammation and Paraneoplastic Diseases: Anaemia and thrombocytosis can also be seen in patients with chronic inflammation and as a paraneoplastic finding. An occasional immature leucocyte form may also be seen in these conditions. This picture may be indistinguishable from that myeloproliferative diseases other than CML. The anaemia is normocytic and normochromic. When the chronic disease or neoplasm is associated with blood loss as may be the case in cancers of the gastrointestinal tract or inflammatory bowel disease, microcytosis due to a coexisting iron deficiency may be seen.
  3. Iron Deficiency: Iron deficiency is associated with microcytic hypochromic anaemia. The degree of microcytosis co-relates with the degree of iron deficiency. The leucocyte counts depends on the cause of iron deficiency. Commonly the leucocyte is normal or slightly decreased. Patients who have iron deficiency because of blood loss due to an inflammatory condition may have leucocytosis. Iron deficiency from blood loss due to helmethiasis may cause eosinophilia.

The differential leucocyte count showed 67% polymorphs, 27% lymphocytes, 2% monocytes and 4% basophils. The erythrocyte indices were MCV 61fl, MCH 15.3pg and MCHC 24.9g/dL. The red cell distribution width was 28.5%. The peripheral smear showed hypochromia, microcytosis, anisocytosis and poikilocytosis.

Of the causes of anaemia and thrombocytosis listed above only iron deficiency is characterised by hypochromic microcytic anaemia. Iron deficiency is also characterised by anisocytosis and poikilocytosis. This manifests as increased red cell distribution on the haemogram.

The serum iron was 27.9 µg/dl, the total iron binding capacity 488 µg/dl with a transferrin saturation 5.7%. The serum ferritin was 8.53 ng/ml. The haemoglobin electrophoresis showed a haemoglobin A2 of 2.8%, the HbA 96% and haemoglobin F 1.2%.

Iron deficiency is diagnosed by documenting low body iron stores and/or impaired iron delivery of iron to the erythroid precursors. The gold standard for depletion of iron stores is absence of stainable iron in the bone marrow. Serum ferritin accurately reflects body iron stores. It has become the preferred method to demonstrate depletion of body iron stores because of the invasive nature of bone marrow aspiration. Levels less than 15ng/ml strongly suggest iron deficiency. Serum ferritin is specific but not sensitive for iron deficiency. Its has a sensitivity of 59% if the cutoff is 15ng/mL and 75% if the cutoff is less than 16ng/ml. The low sensitivity makes the test of limited value to exclude iron deficiency. Ferritin in an acute phase reactant. It has a limited value in the presence of inflammation.

Unlike low serum ferritin, low serum iron is of limited value in diagnosis of iron deficiency. Iron delivery to the haemoglobinizing erythroid precursors is a function of transferrin saturation rather than the serum iron levels. One can have a low serum iron and a low total iron binding capacity as may be seen in anaemia of chronic disease and yet have a normal transferrin saturation. Such patients do not benefit from iron supplementation. Patients with iron deficiency have a low transferrin saturation indicated impaired iron delivery to the developing erythroid cells. Lower the iron saturation higher the probability of iron deficiency being present. Patients are considered to be iron deficient if the transferrin saturation is less than 16%. This patient had a transferrin saturation of 5.7% and a serum ferritin of 8.63ng/ml along with microcytic hypochromic anaemia. A diagnosis of iron deficiency anaemia was made.

The diagnosis of iron deficiency is incomplete without diagnosing the cause of anaemia. Iron deficiency in a 49 year old woman frequently is a result of blood loss that is often menstrual.  This woman had attained menopause and is being evaluated for a gastrointestinal blood loss.

Heme Synthesis


Heme, a porphyrin, is a co-factor in haemoglobin, myoglobin, cytochrome, catalase, heme peroxidase, and endothelial nitric oxide synthase. It has a complex structure with four pyrrole rings with a ferrous iron in the centre that allows it to carry oxygen. The synthesis of heme takes place from glycine and succinyl CoA in eight steps and is extensively studied. Mutations in genes encoding for enzymes involved in heme synthesis result in porphyrias.

Steps in Heme synthesis

About 85% of the heme is synthesised in the developing erythroid cells and almost all the remaining is synthesised in the liver. The control of synthesis differs in erythroid and non-erythroid cells reflecting the exceedingly high heme requirement of the former for haemoglobin synthesis. Heme synthesis takes place in the mitochondria as well as cytosol. The first step, formation of δ-aminolevulenic acid, takes place in the mitochondrial matrix. The next few steps take place in the cytosol. The heme precursor, corpoprophyrinogen III, returns to the mitochondria, is converted to protoporphyrin IX and iron incorporated. The steps in heme synthesis are as follows

  1. Synthesis of δ-aminoleuvelinic acid: Synthesis of δ-aminoleuvelinic acid (ALA) from glycine and succnyl CoA catalysed by ALA synthase (ALAS) is the first step in the synthesis of heme. This is a rate limiting step. ALA synthase is encoded by two genes ALAS1 (OMIM 125290) and ALAS2 (OMIM 301300). ALAS2 codes for the erythroid ALAS and ALAS1 for the non-erythroid (housekeeping) ALAS. The gene ALAS1 is located on chromosome 3p21.1. The product has 12 exons and undergoes is alternate splicing to yield two distinct forms, isoform 1 (640 amino acids) and isoform 2 (657 amino acids). The erythroid specific gene (ALAS2) on X chromosome at Xp11.21. It has 12 exons and also undergo alternate splicing to yield two forms, isoform b (587 amino acids), isoform c (574 amino acids). ALAS is synthesised in the cytosol and transported to the mitochondria. It has a short half life. Heme synthesis is consoled by regulating levels and activity of ALAS (discussed below).
  2. Synthesis of prophobilinogen: ALA moves to the cytosol and is dimerised to prophobilinogen by the action of prophobilinogen synthase (ALA dehydratase). The enzyme is a homo-octomer (made of eight similar units) and needs zinc. The gene (gene ALAD, OMIM 125270) encoding the enzyme is located at 9q32. It has 15 exons. Four isoforms from alternate splicing 361 amino acid, 344 amino acid, 321 amino acid and 304 amino acid are known.
  3. Synthesis of hydroxymethylbilane: Prophobilinogen is converted to hydroxymethylbilane by the action of hydroxymethylbilane synthase. This enzyme is also known as propohbilinogen deaminase. The gene (HMBS OMIM 609806) is located at 11q23.3, has 15 exons. Four alternately spliced forms with 361, 344, 321 and 304 amino acids are known.
  4. Synthesis of uroporphyrinogen: Hydroxymethylbilane is converted to enzymatically to uroporphyrinogen III as well as non-enzymatically to uroporphyrinogen I. The enzymatic conversion is catalysed by the enzyme uroporphyrinogen III synthase. Uroporphyrinogen III synthatase is encoded by a gene (UROS, OMIM, 606938) on 10q25.2-q26 that has 16 exons and encodes for a 265 amino acid protein.
  5. Synthesis of corpoporphyrinogen III: Uroporphyrinogen III is decrboxylated to corpoporphyrinogen III by uroporphyrinogen decarboxylase. The gene (UROD, OMIM 613521) for thes enzyme is at 1p34. It has 10 exons and encodes for a protein 367 amino acid long. This is the last step in the cytosol.
  6. Synthesis of protoporphrinogern IX: Coproporphyrinogen III is converted to propoporphyrinogen IX by a reaction catalysed by corpoporphyrinogen oxidase  in the mitochondria in an oxygen dependent reaction. The gene for corpoporphyrinogen oxidase (COPX, OMIM 612732) is at 3q11.2-q12.1 8 exons. The product has 454 amino acids.
  7. Synthesis of protoporphyrin IX: Propoporphyrin is the final product of the pathway into which iron is incorporated. Protoporphyrin IX is synthesised by the action of protoporphyrinogen oxidase. The gene (PPOX, OMIM 600923) for this enzyme is located at 1q22  and has14 exon. It encodes for a 477 amino acid enzyme.
  8. Synthesis of heme: Ferrochelatse (protoporphyrin ferrochelatase) catalysed the incorporation of iron into protoporphyrin IX. The gene (FECH, OMIM 612386) for ferrochelatse is located at 18q21.31 and has 11 exons. It encodes for a 477 amino acid enzyme.

Control of heme sythesis

The rate limiting enzyme of heme synthesis is the synthesis of ALA. ALA synthase has a short half life. Heme synthesis is regulated  by controlling the levels and activity of ALA synthase.

  1. Inhibition of ALA synthase: ALA synthase is subject to feedback inhibition by heme and and it’s oxidation product hemin. ALA synthase is synthesised in the cytosol and transported to the mitochondrial matrix. In addition to being an inhibitor of ALA synthase hemin also inhibits the metochondrial transport of the enzyme.
  2. Promotion of ALA synthase activity: Cellular iron and factors promoting erythroid differentiation increase the synthesis of ALAS-2, the enzyme responsible ALA synthesis in erythroid cells. Erythroid specific factors like GATA-1 promote the transcription of the ALAS-2 gene. Untranslated portions of the ALAS-2 mRNA have iron responsive elements (IRE) that promote translation. The activity of ASLS in iron deficient cells is low.

Porphyrias

Porphyrias are inherited diseases resulting from a mutation of genes involved in heme synthesis. With one exception, X-linked porphyria that results from a gain of function mutation of ALAS synthase 2, porphyrias result from a partial deficiency of the enzymes involved in heme synthesis. Enzyme deficiency results in accumulation of substrates for the reaction catalysed by the enzyme encoded by the gene. Symptoms of porphyrias may be intermittant and/or chronic. The symptoms are diverse and include skin changes, photosensitivity, abdominal pain, muscle weakness, CNS disturbance, seizures, hyponatremia, discolouration of urine. Enzyme deficiencies associated with porphyrias as as follows:

  1. ALA synthatase 2: Gains of function mutation in X linked protoporphyria
  2. ALA dehydratase: ALA dehydrate deficient porphyria (ADP). Lead displaces zinc from binding sites inhibiting the function of the  with enzyme. In patients with tyrosinaemia type 1 Succinylacetone (4,6-dioxoheptanoic acid) accumulates in tyrosinaemia type I. It is structurally similar to ALA and a potent inhibitor of ALA dehydratase.
  3. PBG Deaminase deficiency results in acute intermittent porphyria
  4. Uroporphyrin III synthatase deficiency results in congenital erythrocytic porphyria
  5. Uroporphyrin decarboxylase deficiency results in porphyria cutanea tarde. All patients with porphyria cutanea trade do not have a mutation. Only type II has gene mutations. Types I and III are due to mulifactorial effects on the gene.
  6. Coproporphyrin III oxidase deficiency results in hereditary coproporphyria
  7. Protoporphyrin oxidase results in varigate porphyria
  8. Ferrochalase results in erythropoietic porphyria

Further Reading

Porphyrin and Heme Metabolism
Erythroid Heme Biosynthesis and Its Disorders (doi:  10.1101/cshperspect.a011676)