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)

 

Granulocyte Colony Stimulating Factor (G-CSF)


Neutropenia is a dose limiting toxicity of chemotherapy. It results in delay and dose reduction both of which adversely affect outcomes of treatment. Myeloid growth factors are biological agents that stimulate the production go granulocytes and offset the myelosupressive effect of chemotherapy. Two myeloid growth factors are available Granulocytic colony stimulating factor (G-CSF) and granulocytic monocytic colony simulating factor (GM-CSF). This article will discuss G-CSF as it is used more often than GM-CSF. Commertially available G-CSF is made by recombinant DNA technology and may be produced in E. coli (Filgrastim) or chinese hamster ovary cell lines (lenograstim). The half life of filgrastim can be increased by covalently linking it to polyethylene glycol (PEG) and converting it to pegfilgrastim.

Mechanism of Action of G-CSF

G-CSF is a 174 amino acid peptide the gene for which is on chromosome 17. It has a molecular weight of 18kDa. It is produced by monocytes, macrophages, fibroblasts, endothelial cells and keratinocytes in response to inflammatory cytokines and bacterial endotoxin.

G-CSF acts via the G-CSF receptor. G-CSF receptor is a transmembrane receptor that form a homodimer on binding G-CSF. Activation of G-CSF receptor results in activations of  JAK/STAT, SRC family of kinases, PI3/AKT and Ras/ERK 1/2. The details of the pathway are not completely understood.

G-CSF AAB.002

Activations of G-CSF has the following effects that lead to increased production of neutrophils

  1. Increased Proliferation of Neutrophilic precursors
  2. Shortened neutrophilic precursor bone marrow transit time
  3. Functions maturation of neutrophils – increased chemotaxis, phagocytosis and antibody dependent cytotoxicity

G-CSFs Available for Clinical Use

G-CSFs for clinical use is manufactured by recombinant DNA technology. Two molecules are available for clinical use. Filgrastim is produced using E. coli and lenograstim is obtained from Chinese hamster ovarian cells. Lenograstim is glycosylated (4% glycosylation).

Filgrastim on subcutaneous administration filgrastim has a half life of 2.5-5.8 hours. The drug is eliminated by uptake be G-CSF receptors on neutrophils and glomerular filtration. Pegylation, that involves attaching a 20kDa polyethylene glycol (PEG) molecule to the N terminal eliminates renal elimination prolonging the half life to 27-47 hours. The product, pegfilgrastim, is only eliminated by binding to neutrophil G-CSF receptors, patients with low neutrophil counts have a lower clearance. Prevention of glomerular filtration allows administration of pegfilgrastim only once in a chemotherapy cycle.

Indications for G-CSF

The discussion that follows applies to filgrastim and perfilgrastim as these drugs are used more commonly than lenograstim. The general principle apply to lenograstim but readers are advised to refer to information on lenograstim for details of use and adverse effects.

  1. Primary prevention of febrile neutropenia (FN) in patients with non-Myeloid malignancy on chemotherapy: Patients where on chemotherapy protocols that have a risk of febrile neutropenia equal to or greater than 20% should be administered G-CSF.
  2. Prevention of recurrence of febrile neutropenia: G-CFS may be used to prevent recurrence of febrile neutropenia in patients who have had an episode of infection in a previous chemotherapy cycle.
  3. Treatment of patients with febrile neutropenia: Initiating therapy with G-CSF after febrile neutropenia has set in has not been shown to decrease mortality of antibiotic use. It may however be used in patients who are at high risk of mortality.
  4. Mobilisation of stem cells for stem cell transplant
  5. Use in patients with myeloid malignancies: There is an apprehension that G-CSF may stimulate leukaemia cells and G-CSF is not used in induction. It may however be used after induction to reduct the duration of neutropenia.

Filgrastim is administered in a dose of 5μg/kg/day subcutaneously, by a short iv infusion or prolonged intravenous infection. therapy should be initiated at least 24 hours after the  chemotherapy. The adult dose of pegfilgrastim is 6mg. The paediatric dose depends on the weight of the child. Children less than 10 kg: 0.1 mg/kg, those between 10 to 20 kg be administered 1.5 mg, between 21 to 30 kg be administered 2.5 mg and between 31 to 44 kg administered 4 mg. Children weighing 45kg or more should be administered the adult dose of 6 mg. Pegfilgrastim should not be administered less than 14 days after a cycle of chemotherapy. It should be administered more than 24 hours after a cycle of chemotherapy.

Adverse Effects

  1. Bone Pain: Bone pain is the commonest side effect with about 20-30% of the patients suffering the side effect.
  2. Rare but serous side effects include splenic rupture, acute respiratory distress syndrome,  precipitation of sickle cell crisis and capillary leak syndrome

Clinical Features of Megaloblastic Anaemia


Megaloblastic anaemia is a macrocytic anaemia resulting from the deficiency of vitamin B12 or folic acid characterised by the presence of megaloblasts in the bone marrow. It has haematological and neurological manifestations. The haematological manifestations are seen with folate as well as vitamin B12 deficiency. Folate deficiency in adults does not affect the nervous system.

Cobalamin deficiency is slow and “pure”. Folate deficiency is rapid and “impure”. Deficiecy of vitamin B12 occurs because of loss of intrinsic factor resulting in an isolated defect of B12 absorption. No other nutrients are affected. The body stores of B12 can last months. This results in B12 deficiency being a slow and “pure” deficiency. Symptoms come on slowly, over months. Folate deficiency evolves relatively quickly and is most commonly because of alcoholism or malabsorption. It is associated with other deficiencies and is rapid and “not pure”.

 

Manifestationf o megaloblastic anaemia

Figure 1. Clinical Manifestations of Megaloblastic Anaemia

Haematological Manifestations

Haematological changes resulting from vitamin B12 deficiency and folate deficiency are indistinguishable. Megaloblastic anaemias are macrocytic anaemia but macrocytosis is not specific to megaloblastic anaemia. It is however exceptional for other diseases characterised by macrocytosis to have an mean capsular volume (MCV) > 110fl.  This value can considered the threshold above which an anaemia is unlikely to be anything other than megaloblastic anaemia.

The earliest change in a megaloblastic anaemia is macrocytosis. This precedes changes in erythrocyte indices. Changes in mean capsular haemoglobin (MCH) follow and then the MCV rises. Haemoglobin usually falls after the MCV increases to >97 fl. As the severity of anaemia increases the peripheral smear shows aniscytosis and poikilocytosis, nucleated cells, Howell-Jolly bodies and Cabot’s ring. Microcytes and erythrocyte fragments that represent dyserythropoiesis may be seen. Polychromasia is absent and this distinguishes megaloblastic anaemia from haemolytic anaemia.

The term megaloblatic anaemia is a misnomer. The disease is actually a panmyelosis.  Erythroid, myeloid and megakaryocytic series are affected. Thrombocytopenia and leucopenia (neutropenia and to a lesser extent lymphopenia) usually occur late in the course. It is uncommon for patients with mild anaemia to have platelets and neutrophils but occasionally changes in leucocytes and/or platelets may dominate.

Iron deficiency or β-thalassaemia trait result in microcytosis and hypochromia and may incidentally co-exist with megaloblastic anaemia. Co-existence of either of these diseases with megaloblastic anaemia may mask macrocytosis of megaloblastic anaemia. Presence of hypersegmented neutrophils in a patients with normocytic normochromic anaemia should raise the suspicion of a megaloblastic anaemia co-existing with Iron deficiency or β-thalassaemia trait.

Neurological Manifestations

Cobalamine deficiceny causes neurological dysfunction. Folate deficiency causes symptoms only in children. Children with inborn errors of folate metabolism may have myelopathy, brain dysfunction and seizures.

The neurological manifestations of B12 deficiency are a result of a combination of upper motor neuron manifestations from subacute combined degeneration of the spinal cord, sensory and lower motor neuron manifestations from peripheral neuropathy and neurophychiatratic manifestations. Subacute combined degeneration of the spinal cord (SACD) is a degerative disease of the spinal cord involving the posterior and lateral column (corticospinal and spinoceribellar tracts) that starts in the cervical and the thoracic region.

The earliest neurological manifestations are impaired sense of vibration and position and symmetric dysesthasia that involve the lower limb. This is frequently associated with sensory ataxia. With progression spastic paraparesis develops. The patients have brisk knee reflexes, reflecting an upper motor neuron involvement and depressed ankle reflex, reflecting a peripheral neuropathy. Bladder involvement is unusual. Some patients may have optic atrophy.

Neuropsychiatric manifestation include memory loss, depression, hypomania, paranoid psychosis with auditory and visual hallucinations.

Other manifestations

Skin and nails can show pigmentations. Mucosa of the villi undergoes megalobkastic change resulting in temporary malabsorption.

Response to therapy

Haematological Recovery

  • Day 1: Feeling better
  • Day2-3: Reticulocytosis appears
  • Day 7-10: Peak retuculocytosis
  • Day 15 onwards: Neutrophilic hypersegmentation disappears
  • Day 56 (8 weeks): Blood counts become fully .normal

Neurological Recovery

Neurologic improvement begins within the first week also and is typically complete in 6 weeks to 3 months. Its course is not as predictable as hematologic response and may not be complete.

 

 

The M-Band


Monoclonal Gammopathy-02

Figure 1. Each plasma cell produces a different type of antibody. Normal γ globin band is depicted in the left column. The plasma cell numbers are normal and each produces an antibody with a different amino acid structure and electrophoretic mobility. Patients with monoclonal gammopathy have expansion (increase number) of a plasma cell clone (red in the diagram) resulting in the production of a disproportionate large amount of immunoglobulin from one type of plasma cell. This results in the M Band (see below). Patients with polyclonal gammopathy have an expansion (increased number) of plasma cells. This is usually occurs in response to infection/inflammation that result in production of a diversity of antibodies. The diversity is reflected in increase in the γ but as no one clone dominates the sharp M band is not seen.

What is an M-Band?

Immunoglobulins are antigen binding molecules secreted by plasma cells. Immunoglobulins bind antigens and play a role acquired immunity. Plasma cells develop from antigen exposed B-lymphocytes. The process of maturation of lymphocytes involves inducing mutations in region of the immunoglobulin gene that encodes for antigen binding regions, the hypervariable regions. This makes the DNA and consequently the amino acid sequence of the immunoglobulin secereted by a plasma cell unique. This is true even when two plasma cells make antibody against the same antigen or antigenic epitope (see figure 1).

Monoclonal Gammopathy-01

Figure 2. The serum protein separate into many bands on electrophoresis. The albumin is a dark band closest to the anode. This is followed by the α1, α2, β and γ bands. The immunoglobulin are mainly found in the γ globulin band but some may be found in the β globin band. The electrophoretic mobility of a molecule depends on the charge it carries which in turn depends on the amino acid sequence. Amino acid sequence determines the antigen specificity and differs between antibodies resulting in a slight variation in electrophoretic mobility of immunoglobulins and resulting in the γ region being a broad band.


The amino acid sequence determines the charge on the immunoglobulin. The electrophoretic mobility is determined by the charge. Majority of the immunoglobulins move to the γ-globulin fraction of serum proteins, some move with β-globulin. The γ-globulin band is a wide electrophoretic band reflecting the diversity in electrophoretic mobility of immunoglobulins arising from the diversity in amino acid sequences (figure 2).

Monoclonal Gammopathy-03

Figure 3. Patinets of monoclonal gammopathies have an expansion of one clone of plasma cells. This reflects in production of a disproportionally large amount of immunoglobulin with identical electrophoretic mobility resulting in a dense band with in γ globin region


Patients of monoclonal gammopathies have clonal expansion of plasma cells. The cells of a clone have identical DNA and produce identical immunoglobulin molecules. When the clone grows to level that it forms a significant proportion of the plasma cell pool the immunoglobulin it produces forms a significant proportion of the total serum immunoglobulins. The identical electrophoretic mobility of molecules produced by the clone results in a disproportionately large number of immunoglobulin concentrating to a point on electrophoresis forming a band.  This is known as the M band.  Lymphoma cells, notably those of lymphoplasmacytic lymphoma, can secrete immunoglobulin and are associated with an M band for similar reasons.

Diseases associated with an M-Band

The M-Band is a serum marker for plasma cell dycrasias and Waldenström macroglobulinemia. IgM and non-IgM (mainly IgG and IgA) monoclonal bands have differing clinical implications. The former is more commonly associated with lymphoproliferative disease and the latter with plasma cell dycrasias. The presence of an M band only indicates a clonal expansion of immunoglobulin producing cells. It does not indicate malignancy. The diagnosis of malignancy is made by features that suggest end organ damage. The absence of end organ damage indicates a premalignant disease including monoclonal gammopathy of uncertain significance (MGUS), soldering multiple myeloma or smoldering Waldenström macroglobulinemia.  The evidence of end-organ damage includes

  1. non-IgM Monoclonal Gammoathies: CRAB (elevated calcium, renal involvement, anaemia and osteolytic (bone) lesions) creatinine,
  2. IgM Monoclonal Gammapathies: Anemia, constitutional symptoms, hyperviscosity, lymphadenopathy, or hepatosplenomegaly that can be attributed to the underlying lymphoproliferative disorder if diagnosis is Waldenström macroglobulinemia or CRAB (elevated calcium, renal involvement, anaemia and osteolytic (bone) lesions) creatinine if the diagnosis of IgM myeloma

False positive M-Band

The presence of M band indicates presence of a clonal expansion of plasma cells. When end organ damage co-exists with M band a diagnosis of a malignancy (multiple myeloma or Waldenström macroglobulinemia) is made. In the absence of end organ damage the diagnosis of a premalignant disease is made. Proliferation a of plasma cells are seen in infections/inflammation. These are polyclonal and result in s polyclonal gammopath. They do not result in the presence of an M-band.

 

 

Evolution and Spread of HbS


The gene for β globin (OMIM  is present on chromosome 11 (11p15.4) along with other globin genes (ε, γ, γ and δ). This is known as the β-globin cluster . Individuals carrying identical genes on the β-globin gene cluster may not have identical DNA sequences in non-codeing regions of the DNA of the cluster. The non-coding regions include segments of DNA between genes and introns within genes. . Differences in DNA exist between individuals every 1000-2000 bases in the form of single nucleotide polymorphisms (SNPs). Single nucleotide polymorphisms are variations in a single nucleotide that occurs at a specific position in the genome. Many of these differences have no consequences on gene expression because either they do not result in change in amino acid sequence or they occur in regions of DNA that neither code for the gene nor regulate the gene. SNPs evolve by spontaneous mutations over time. The lesser the number of such differences between two individuals closer the individuals are the each other genetically (and in terms of evolution). Fewer differences in SNPs between individuals mean a more recent common ancestor.

One of the meanings of the word haplotype is a pattern of SNPs. A haplotype may be considered as a DNA “environment” in which the gene(s) occurs. This “environment” is created by the sequence of single nucleotide polymorphisms in which the gene(s) exists. As mentioned above differences in SNPs (and hence the “environment” the gene(s) exist in) evolve by spontaneous mutations over period of time. Fewer the differences between the “environments” the genes occurs in the more the likelihood that they come from related individuals.

HbS results from a single base substitution in the codon 6 of the β-globin gene. GAG becomes GTA resulting in substitution of valine for glutamate. This change results in a haemoglobin that crystallizes in hypoxic conditions resulting in a haemolytic anaemia. HbS occurs in diverse population groups including African, Mediterranean, Middle-Eastern and Indian. Is the haplotype of the HbS gene in these regions similar?

The HbS mutation occurs on five different haplotypes four African and one Arab-Indian. The mutation is the same (GAG to GTA on codon 6) but the SNPs are different. The haplotypes are

  1. Senegal: The Senegal HbS haplotype is found in Atlantic West Africa and Portugal
  2. Benin: The Benin HbS haplotype is found Central West Africa, Northern Africa and Mediterranean Europe (Greece, Sicily)
  3. Central African Republic or Bantu: The Central African Republic or Bantu is found in South Central and Eastern Africa
  4. Cameroon: The Cameroon haplotype is found in the Eton ethnic group of eastern Cameroon
  5. Arab-Indian: The Arab-Indian haplotype is the only non-African phenotype of HbS found in the eastern oasis of Saudi Arabia and India.

Origin of Haplotypes

There are two theories about the origin of haplotypes. The first, and the more accepted one, states that the five haplotypes arose from five independent mutations. An alternative hypothesis states that HbS mutation occurred only once and spread to other haplotypes by gene conversion.

 

Haplotypes and Severity of Symptoms

Symptoms of sickle cell anaemia are a consequence of crystallisation of haemoglobin under hypoxic conditions. HbF inhibits sickling. Patients with high HbF have fewer symptoms. The Arab-Indian and the Senegal haplotype are associated with higher HbF levels (17% and 12.4% respectively). In general patients carrying these haplotypes have milder symptoms than the Bantu or Benin haplotypes (Blood 2014; 123: 481)

 

Haplotypes and Human Migrations

Trade, conquests and human migrations (voluntary and slave trade) have disseminated the African haplotypes beyond Africa.

  1. The Mediterranean: Most of the Mediterranean (Greece and Scilly) has the Benin haplotype. This reflects pre-historic migrations from Central West Africa along the then fertile Sahara to North Africa. From here it spread to the Mediterranean via the interactions (Trade and wars) between the two regions. The only exception is Portugal. Portugal has the Senegal haplotype which reflects the trading contacts between Portugal and Atlantic West Africa (Angola and Mozambique).
  2. Americas: Neither the native americans nor the original European settlers to the Americas carried the HbS gene. HbS was imported to the Americas with the slaves from Africa. Jamaica was an important slave import hub and records for where tthe slaves arrived from are available. Jamaica has 73% Benin haplotype, 17% Bantu and 10% Senegal haplotypes. These numbers are close to the actual number of slaves who arrived in Jamaica from regions of Africa where these haplotypes are prevalent. Similarly the distribution of haplotype correspond to the origins of slaves in Baltimore and South Carolina (Mariam Bloom. Understanding Sickle Cell Disease, Page 34).
  3. Arab or Indian: It is not clear if the Arab-Indian haplotype originated in India or Saudi Arabia. But considering that all of tribal India has only one haplotype but the East and West Arabian Peninsula have different haplotypes it is possible that the haplotype originated in India.
  4. Spread to Other Parts: As opposed to the era of slave trade modern migration of people in the recent past have been voluntary. These populations have spread across the world as have those form mediterranean but to a lesser extent. These migrations have introduced the HbS gene in areas where it was not indigenous.

 

Anaemia with Hyperbilirubinaemia


A 49-year-old female presented with dyspnoea on exertion of 1 month duration. Examination reviled pallor and icterus. There was no lymphadenopathy, clubbing, koilonychia, platonychia, petechiae or purpura. There was no oedema of feet. The pulse was 90/min and the blood pressure 130/70 mm of Hg. Examination of the respiratory, cardiac and nervous systems did not show any abnormality. There was no organomegaly.

The haemoglobin was 4.9 g/dL with an erythrocyte count 1.37 x 1012/L, haematocrit of 16%, MCV of 116.78 fL, MCH of 35.77 pg and MCHC 30.63 of g/L.  The leucocytes count was 2800 with 35% neutrophils and 65% lymphocytes. The platelet count was 90 x 109/L. The peripheral smear showed macrocytosis and anisocytosis. Hypersegmented neutrophils were seen. The reticulocyte count was 3%.

The bilirubin was 2.1 mg/dL with a direct bilirubin of 1.8mg/dL and an indirect bilirubin of 0.3mg/dL. The Lactate dehydrogenase was 1417IU (normal 105 – 333 IU/L).

Anaemia and unconjugated hyperbilirubinaemia are characteristic of haemolysis. Does this patient have haemolytic anaemia?

Haemolysis shortens erythrocyte lifespan and results in increases haemoglobin breakdown. Haemoglobin is made of heme and globin. Heme consists of porphyrin ring at the centre of which is iron in the ferrous state. Iron released from catabolism of heme is reused. The porphyrin ring is catabolised to bilirubin. The bilirubin is transported to the liver for conjugation and excretion (see haemoglobin catabolism). Patients of haemolytic anaemia have unconjugated hyperbilirubinaemia because the increased bilirubin production overwhelms the hepatic bilirubin conjugation capacity.

One of the characteristics of megaloblastic anaemia is ineffective erythropoiesis. Ineffective erythropoiesis is defined as a sub-optimal (fewer) production of mature erythrocytes from a proliferating pool of immature erythroblasts. Each immature erythroblast produces less than the optimal number of erythrocytes because of premature death of erythroid precursors including haemoglobinized precursors. The haemoglobin released from haemoglobinized erythroid precursors is catabolised in the same manner as haemoglobin released from lysed erythrocytes (see haemoglobin catabolism). Megaloblastic anaemias are associated with unconjugated hyperbilirubinaemia because of death of haemoglobinized erythroid precursors.

The treatment of haemolytic anaemia and megaloblastic anaemia are different? How does one differentiate megaloblastic anaemia from that because of haemolytic anaemia? Does this patients have a haemolytic anaemia or megaloblastic anaemia?

Haemolytic anaemia is characterised by shortened erythrocyte survival. Erythrocytes survival is estimated by the use of radionucleotides something that is not possible at most centres. In clinical practice, a shortened erythrocyte survival is inferred from a high reticulocyte count. Reticulocytes are erythrocytes that have been produced in the preceding 24 hours. The erythrocytes survival is about 120 days and about 1% of erythrocytes are produced every day. Consistent with this the normal reticulocyte count is 0.5-1.5%.In patients of haemolytic anaemia, ddestruction of erythrocytes is matched by an increased production by the bone marrow. This manifests as reticulocytosis (see reticulocyte count). Megaloblastic anaemia occurs because of decreased production of erythrocytes and this manifests as reticulocytopenia. The difference between haemolytic anaemia and megaloblastic anaemia is the reticulocytosis in the former reticulocytopenia in the latter. This patient had a high reticulcoyte count but after correction both the reticulocyte production index [0.43] and corrected reticulocyte count [1.07%] were low excluding haemolysis. This patient was evaluated for megaloblastic anaemia.

The haemogram has clues to differentiate between haemolytic anaemia and megaloblastic anaemia. These include

  1. A very high MCV: The MCV is very high. Patients with haemolytic anaemia have a mild elevation in MCV. An MCV value >110fL is almost exclusively found in megaloblastic anaemias because of folate and/or B12 deficiency.
  2. Pancytopenia: B12 and folate deficiency impair DNA synthesis impairing erythrpoieis, myelopoiesis and megakaryopoiesis. Nutritional megaloblastic anaemias because of vitamin B12 and/or folate deficiency may show pancytopenia.
  3. Hypersegmented neutrophils (>5% neutrophils with >5lobes) is a feature of megaloblastic anaemia

Other features of megaloblastic anaemia include rise serum transferrin receptor, increased serum iron, serum ferritin and methemalbumin levels. Like haemolytic anaemia the serum haptoglobin is low and the LDH high. LDH levels in megaloblastic anaemia can ve very high.

This patients had a low serum B12 and was treated with parental B12 (1mg alternate day for 5 doses) and was evaluated for cause of vitamin B12 deficiency. As Schilling’s test was not available a diagnosis of pernicious anaemia was made by documenting gastric atrophy and anti-parietal cell antibodies.