Erythropoiesis

Normal marrow erythropoiesis is said to be NORMOBLASTIC.

When the essential factors vit. B₁₂ or folic acid are deficient, the red cell precursors show clear morphological changes – erythropoiesis is said to be MEGALOBLASTIC (p.386).

Hypoplastic and Aplastic Anaemias

These are rare conditions and, as the names imply, are due to marrow failure with diminished numbers or absence of haemopoietic cells. Usually all three marrow cell lines are affected, resulting in pancytopenia in the peripheral blood.

Marrow failure of this type is dealt with in detail on page 407.

Marrow failure due to extensive tumour infiltration or fibrosis also occurs.

The anaemias associated with miscellaneous chronic diseases (‘Secondary’ anaemias) are dealt with on page 408.

Dyshaemopoietic Anaemias

The usual cause of these anaemias is deficiency of an essential factor required for proper haemoglobin synthesis or erythroblast maturation and development. They are associated with a hypercellular marrow and are divided into two main groups:

(1) normoblastic and (2) megaloblastic, depending on the type of erythroblastic maturation in the marrow.

Deficiency of Essential Factor

Iron Metabolism

Iron is absorbed mainly in the duodenum and upper jejunum. Only small amounts are normally required to replace iron losses. Since the average diet contains more iron than is required, its absorption is controlled by the mucosal apoferritin mechanism.

The iron (X) in the diet enters the mucosal cell through an apical uptake transporter (DMT-1) and having combined with apoferritin (O) is retained in the cell as ferritin (⊗).

Iron unbound by apoferritin passes through the cell through a basolateral transporter (ferroportin) and is transported in the blood to join the iron recycling system.

Note: New haemoglobin formed in the bone marrow contains 95% iron from recycling system, 5% from diet.

Note: This bound iron is subsequently shed along with the cell into the lumen.

The state of the iron stores controls the apoferritin (a form of intracellular transferrin) content of the intestinal mucosal cell by a feed-back mechanism.

Acute Iron Overload

If a large dose of medicinal iron preparations is taken (particularly by children in error) the absorption and transport mechanisms are overwhelmed and free iron radicals exert very toxic effects.

The iron balance may be summarised as follows.

INPUT (Adult male)	BODY IRON Total 3–6g	OUTPUT
Average: 1 mg/day derived from foods (a) animal muscle (b) vegetables.	(a) Functional iron in haemoglobin, myoglobin, enzyme systems, transferrin 70% at least	Average: 1 mg/day Skin desquamation and miscellaneous secretions
The average diet contains 10–20 mg iron, of which about 10% is absorbed. In the adult female, the average daily input is about 2 mg.	(b) Storage iron in liver, spleen, bone marrow as ferritin, haemosiderin 30% or less	Menstruation This extra loss of about 0.5 to 1 mg requires extra input in the female

Anaemia results when this balance is upset in:

Usually anaemia develops slowly (except in cases of serious haemorrhage).

Folic Acid (Pteroyl-Glutamic Acid) Deficiency

Absorption	Malabsorption
As mono-glutamate in jejunum	Coeliac disease Surgical by-pass

Note:

Haemolytic Anaemias

In all haemolytic anaemias, there is a reduction in the life span of the red cells; due to an increased rate of red cell destruction – haemolysis.

Red cells may be destroyed

Functional reserve can compensate for a certain level of haemolysis but this fails when the degree of red cell loss is extreme or when the marrow function is compromised by other factors.

Effects of the Increased Degradation of Haemoglobin

In most haemolytic conditions, the red cells are removed and the haemoglobin degraded in the usual way, i.e. by phagocytosis by the macrophage system.

Note:

1. The bilirubin is unconjugated and is attached to protein so that renal excretion does not occur – acholuric jaundice.

2. Unconjugated bilirubin is toxic to the central nervous system of neonates; kernicterus (see p.394) is a serious complication of haemolytic disease of the newborn.

3. If haptoglobin reserves are saturated, Hb remains free in plasma causing kidney damage and haemoglobinuria.

Reactive Bone Marrow Changes

There is a marked hyperplasia ——— Extension of RED MARROW into long bones

Many young red cells are released into the blood. The reticulocyte count may be 20–30%.

(1A) Autoimmune Haemolytic Anaemia

Auto-immune haemolytic anaemias are classified according to the temperature at which the reaction occurs and the presence of an underlying cause.

warm (37 °C – usually IgG)	cold (< 30 °C – usually IgM)
Primary – so-called idiopathic haemolytic anaemia: usually in adults.	Idiopathic cold agglutination disease (i) lymphomas. (ii) Mycoplasma pneumoniae. (iii) viral infections e.g. measles.
Secondary – associated with (i) lymphomas e.g. chronic lymphocytic leukaemia, Hodgkin’s disease. (ii) other cancers. (iii) connective tissue diseases e.g. systemic lupus erythematosus, rheumatoid arthritis. (iv) drugs e.g. methyl dopa.	Mechanism
Mechanism: Coating of RBC with IgG antibodies often against Rhesus ‘e’ antigens. Destruction in spleen which is often enlarged.

(1B) Destruction of RBCS is due to Iso-Antibodies

In these, the antibodies act against antigens which are derived from another individual of the same species.

Incompatible ABO blood transfusion is a classic example.

(1C) Haemolytic Disease of the Newborn (HDN)

This occurs in Rhesus positive fetuses conceived by Rh-negative mothers. The usual mechanism is as follows:

But during this pregnancy, iso-immunisation of the mother may occur.

Towards term and particularly during labour the placental barrier is breached and fetal RBCs enter the maternal circulation.

Intrinsic Defects – Usually Hereditary

In

(iii) molecular structure of haemoglobin (haemoglobinopathies).

1. Cell membrane defects

(a) Hereditary spherocytosis: the majority of cases are familial (autosomal dominant).

The primary defect is in the proteins which support the plasma membrane – SPECTRIN and ANKYRIN.

The cells assume a spherical shape due to instability of the plasma membrane.

Premature sequestration and destruction in splenic pulp. Splenomegaly is common.

Note: Splenectomy is usually ‘curative’ but the red cell defect remains.

The severity of the haemolysis is variable; many cases are compensated and not anaemic, but crises are common, e.g. if folate deficiency supervenes. Intercurrent infections may precipitate increased red cell destruction with jaundice or temporary bone marrow hypoplasia, e.g. parvovirus infection, with severe anaemia.

The laboratory diagnosis depends on:

(1) typical blood film with spherocytes,

(2) high reticulocyte count and

(3) increased osmotic fragility of RBCs.

(b) Hereditary elliptocytosis is similar to spherocytosis in many respects, but it is usually not severe enough to cause anaemia or jaundice. Haemolysis is usually mild.

(c) Hereditary abetalipoproteinaemia: a rare disease in which the red cell membranes are abnormal and the cells become spiky (acanthocytes).

(d) Paroxysmal nocturnal haemoglobinuria – a rare, acquired condition in which anchorage of proteins to the red cell membrane is abnormal. The cells are particularly susceptible to the action of complement (usually activated by the alternative pathway) at low pH. Intravascular haemolysis results.

2. Enzyme defects

Usually hereditary. Glucose is the source of energy with which the red cell metabolism is maintained. Glycolysis is effected by two classic enzyme pathways.

The following simplified diagram outlines the pathways and indicates only the main enzymes and substrates.

Over 400 different subtypes of G-6PD are described with a spectrum of activity. In G-6PD deficiency, a common defect especially in Africa, spontaneous anaemia is not common, but crises with anaemia and Heinz body formation occur under oxidant stress, e.g. chemicals and drugs; favism (bean products). The presence of the defect may seriously aggravate haemolysis due to other causes, e.g. haemolytic disease of the newborn.

G-6PD deficiency shows X-linked inheritance so that in the male the defect is fully expressed, while in the female heterozygote there are two populations of red cells in the blood (1) normal cells and (2) defective cells.

In both these defects and in the rare defects of other enzymes in the pathways which have been described the expression of the abnormal gene is very variable from case to case.

Polycythaemia

Polycythaemia (erythrocytosis) is defined by a haemoglobin level above the normal range: in true polycythaemia the red cell mass is increased and the haematocrit is always elevated.

Note: Relative Polycythaemia is the result of fluid loss with a decrease in plasma volume and a normal red cell mass.

Secondary Polycythaemia

The red cell increase is the result of increased stimulation of marrow by erythropoietin. Two main groups of conditions are associated with this:

Primary Polycythaemia (Polycythaemia Rubra Vera)

This is a myeloproliferative disorder with increased red cell production. It may be associated with an increase in other marrow elements. Splenomegaly is usually present. A minority of patients develop acute leukaemia.

Neutrophil Neutrophil Granulocyte (Polymorphonuclear Leucocyte)

The essential function of these cells is protection against microbial infection by phagocytosis and killing. They are produced alongside red blood cells, platelets and monocytes from a common stem cell in the marrow.

Marrow Production - Stained by a Romanowski Method

Note the nuclear changes progressing from large open nucleus and several nucleoli to the mature condensed multi-lobed state. The cytoplasm matures with the acquisition of specific granules.

An increase in the number of circulating neutrophils reflects an increased activity in the tissues at the site of an acute inflammatory reaction

Neutrophil Activation

Chronic Granulomatous Disease (CGD)

It is a rare hereditary disorder in which the neutrophils have normal phagocytic function but are unable to kill certain bacteria. The disease is fully expressed only in males. It presents as multiple chronic abscesses and granulomas affecting skin, lungs, bones, spleen and liver.

The rapid production of H₂O₂ is defective (in the neutrophils and also in macrophages due to deficiency of NADPH-oxidase).

About 2/3 of cases are X-linked (fully expressed in males): the remainder are recessive. The neutrophils are unable to reduce nitroblue tetrazolium (used as a diagnostic slide test).

Abnormalities of Coagulation

Thrombocytopenia

Although the normal platelet count ranges from 150–400 × 10⁹/l, a risk of dangerous spontaneous haemorrhage is unusual unless the count falls below 30 × 10⁹/l.

The causes of thrombocytopenia fall into two main groups:

PURPURA is the term used for disorders involving bleeding from capillaries, with the production of small petechial spots. Mild to moderate thrombocytopenia or defective platelet formation are the usual causes. Two examples are illustrative:

2. Defective platelet function has many forms – von Willebrand’s disease (vWD) is the commonest. The gene encoding vWF is located on chromosome 12.

vWF is a large protein which potentiates the binding of platelets to subendothelial collagen and also acts as a carrier for coagulation factor VIII.

THROMBOCYTOSIS (increased platelet numbers) may occur in a mild form (with no clinical significance) reactive to a variety of disorders of chronic inflammation and cancer. Larger counts are seen in myeloproliferative disorders (e.g. essential thrombocytosis, chronic myeloid leukaemia) and are of serious clinical significance due to thrombosis.

Haemophilia A

Haemophilia is due to deficiency of Factor VIII – an essential cofactor in the activation of Factor X in the intrinsic system.

Factor VIII is encoded by a gene on the long arm of the X chromosome – haemophilia is inherited as an X-linked recessive, i.e. almost all patients are male. Their mothers are usually carriers, but about 30% of cases are due to new mutations. Around 1 in 10 000 is affected.

Clinical Features

The severity depends on the levels of Factor VIII.

		Level of Factor VIII
Severe:	Frequent spontaneous bleeding.
		<2% of normal
Moderate:	Bleeding after minor trauma – rarely spontaneous.	2–10%
Mild:	Bleeding severe only after major trauma or surgery.	10–50%

Important sites of bleeding are:

Note: In the past, the use of virus infected Factor VIII in treatment has caused HIV (AIDS) and Hepatitis C infection. Similar concerns apply to variant CJD (p.555).

Haemophilia B (Christmas Disease)

This is due to deficiency of Factor IX – also carried by a gene on the X chromosome. The clinical features are similar, but the prevalence is lower (1 in 30 000 males).

Rarely deficiencies of other coagulation factors are seen, e.g. fibrinogen, Factor XI and Factor V.

They are treated by infusion of the appropriate factors.

Vitamin K Deficiency

Vitamin K, a fat soluble vitamin, is essential for synthesis of Factors II, VII, IX and X in the liver. Deficiency leads to spontaneous bleeding, e.g. into skin and mucous membranes, and failure of blood clotting.

In adults, deficiency may be due to

(c) INHIBITION – by COUMARIN anticoagulants, e.g. WARFARIN.

In neonates, vitamin K deficiency leads to bleeding – from GI tract and bruising. Breast milk contains little vitamin K – the disorder is uncommon in those receiving bottled milk. A severe deficiency may be seen in infants whose mothers took anticoagulants or anticonvulsants.

Liver Disease

The bleeding tendency in these patients is multifactorial.

This complicates bleeding, e.g. from OESOPHAGEAL varices.

‘Acquired Haemophilia’

Rarely, antibodies develop which block the effects of serum coagulation factors, especially Factor VIII. This may be idiopathic or follow auto-immune disease or drug treatment.

Disseminated Intravascular Coagulation (DIC) (see next page).

Lymph Nodes

The basic structure reflects the main functions.

Reactive Hyperplasia

Several patterns are seen.

Acute Bacterial Lymphadenitis

This is seen draining an infected area.

Cat Scratch Disease

This infection by a bacterium Bartonella henselae follows scratching by an infected cat. Similar histology is seen in lymphogranuloma venereum, caused by Klebsiella granulomatis. A small transient painless genital ulcer is followed by inguinal lymphadenopathy (figure). The diagnosis can be confirmed by the Frei test (injection of purified chlamydial antigen stimulates a delayed hypersensitivity reaction).

Foreign Material

Lymph nodes to which foreign particulate material drains often show marked enlargement due to accumulation of macrophages which have ingested the foreign material.

For example:

CHRONIC SKIN DISEASE (so-called dermatopathic lymphadenopathy) e.g. psoriasis, eczema and mycosis fungoides (p.435).

Sarcoidosis

In this disease of unknown aetiology non-caseating granulomas are seen.

The appearances clearly resemble tuberculosis, but the granulomas remain discrete and there is no caseation.

Sarcoid type granulomas are also seen in the lymph nodes of patients with other granulomatous diseases, e.g. Crohn’s disease and in lymph nodes draining tumours.

Hyposplenism

Hyposplenism is not usually a cause of major disability. It occurs following splenectomy and in cases of splenic atrophy.

The effects are considered under two main headings:

1. On the cells of the blood

2. On resistance to infection

Infarction

(i) Embolism causing infarction is not uncommon but usually clinically unimportant.

(ii) Enlarged spleens are particularly susceptible to infarction without embolism.

Secondary Tumour Invasion

Carcinoma of the breast e.g.

Note:

(i) The nodal enlargement is not necessarily due to secondary tumour.

Reactive hyperplasia is also common in nodes draining tumours especially if there is ulceration. The distinction between these 2 causes of node enlargement can only be made with certainty by histological examination.

(ii) The invasion of a group of nodes occurs in a step-wise fashion: a single node (the sentinel node) is invaded initially and spread to adjacent nodes follows.

Lymphomas

Lymphomas are malignant tumours derived from lymphoid cells. They may arise within lymph nodes or at other sites (extra-nodal lymphomas).

Classically, they are divided into 2 main groups.

In recent years, modern immunocytochemical, molecular biological and cytogenetic techniques have been applied to the study of lymphomas. The traditional division between Hodgkin’s and non-Hodgkin’s lymphoma has been challenged but remains of major clinical importance.

Degree of Malignancy

The natural history tends to separate lymphomas into two main groups which have links with the morphological appearances.

Pathological Complications

Classification

This is a confusing area. Traditional classifications relied on morphology alone. The World Health Organisation Classification (2008) which includes immunohistochemistry and genetic data is now generally accepted. A simplified version follows:

a) High Grade Tumours Diffuse large B cell lymphoma. Burkitt’s lymphoma.	Anaplastic large cell lymphomas (some do not express T cell markers).
b) Low Grade Tumours Small lymphocytic lymphoma (equivalent to B-CELL, p.442). Lymphoplasmacytic lymphoma. Mantle cell lymphoma. Follicular lymphoma. Extranodal marginal zone B-cell lymphoma Myeloma/Plasmacytoma.	T cell prolymphocytic leukaemia Peripheral T cell lymphoma. Adult T cell leukaemia. Angioimmunoblastic T cell lymphoma. Enteropathy-type T cell lymphoma. Mycosis fungoides (skin).

According to this classification each type is considered separately for treatment purposes.

The classification relies on cell surface markers which distinguish the B and T cells and their subsets. B and T cell monoclonality can be detected by gene rearrangements, and some types of lymphoma have characteristic translocations.

Subtyping of lymphoma

Immunohistochemistry

Molecular Genetic Analysis

In B cell differentiation different immunoglobulin genes are found by gene rearrangements, while in T cell differentiation a similar process occurs with T cell antigen receptors (p.92).

Follicular Lymphoma

This is one of the commonest forms – a ‘B’ cell lymphoma.

The neoplastic follicles are the malignant equivalent of normal germinal centres.

The tumour cells express the anti-apoptosis gene bcl–2 which immortalises them. There is often a translocation t (14;18) which joins the bcl–2 gene to the immunoglobulin heavy chain gene.

The lymphoma is slowly progressive but often eventually fatal. Sometimes it transforms into a diffuse large ‘B’ cell lymphoma (DLBCL).

Burkitt’s Lymphoma

This is a high grade lymphoma found mainly in Equatorial Africa, New Guinea and other malaria endemic areas. Similar tumours are seen in AIDS patients. The relationship with Epstein–Barr virus has already been discussed (p.81) – as has the translocation t (8;14) which activates the c-myc oncogene (p.152). The histological appearance is typical.

Endemic (African type) Burkitt’s lymphoma typically presents with lesions in the jaw, or in the abdomen, e.g. ovary, liver, gastrointestinal tract. The less common cases in the West typically involve lymph node, marrow and gut.

The disease may respond dramatically to aggressive treatment.

Diffuse Large B-Cell Lymphoma (DLBCL)

This is the commonest type of lymphoma and may arise in nodes or extra-nodal sites. They rapidly disseminate. They are composed of sheets of large round cells. About 50% of patients survive.

Solitary Plasmacytoma

A very small number of plasma cell tumours are solitary at presentation, e.g. in a long bone, the nasopharynx, a lymph node, alimentary tract. In 50% of cases, multiple myeloma occurs within 10 years.

Multiple Myeloma (Myelomatosis)

The disease has an incidence of 10/100 000 per annum with an age peak in the 7th decade.

The median survival, even with treatment, is 3 years.

Pathological effects are considered under two main headings:

1. Tumour growth and its effects

2. Synthesis of immunoglobulin and its effects

Myeloma, being a monoclonal tumour, will produce a single Ig. The common heavy chains are G (60%) and A (20%). In Waldenström’s macroglobulinaemia the immunoglobulin is always IgM.

Light chains of κ type are encountered more frequently than λ.

The presence of a single type of Ig is reflected in the electrophoretic pattern

The other normal immunoglobulins are always decreased.

In most cases high levels of ‘M’ protein cause:

1. High ESR

2. Marked rouleaux formation

3. Depending on type of Ig – increased blood viscosity.

Release of Light Chains

In many myelomas, some of the Ig molecules are incompletely formed, and unattached light chains are released with important effects. Because of their low molecular weight the light chains:

(1) Pass through the glomerular filtrate,

(a) appearing in the urine as Bence Jones protein (precipitates during heating – redissolves between 90 °–100 °C).

(b) During passage through the tubules the protein precipitates as casts and also damages the epithelial cells.

(2) Pass through capillaries

Infections, often of opportunistic type, and particularly pneumonias are common because the immune response is deficient since the high levels of ‘M’ protein are non-functioning.

Mycosis Fungoides

This is a primary T cell (CD4) lymphoma of the skin occurring usually in middle age. It presents as a scaly, red macule progressing to skin plaques and then nodules.

After many years the lymphoma may become generalised or exfoliate into the blood (Sézary syndrome).

Anaplastic Large Cell Lymphoma

The large malignant T cells (usually expressing surface marker CD30) often spread within the sinuses of the node and may mimic the cells in Hodgkin’s disease.

Although three-quarters of cases are of T cell type, the remainder are ‘null’ cell type, i.e. they express neither T nor B cell markers, causing further diagnostic difficulties. The cells express ALK-1 which resolves these difficulties.

The tumour shows 2 age peaks.

Cell Kinetics

Even in acute leukaemias, increased cell longevity beyond the normal (due to failure of response to mechanisms controlling ageing and destruction) is as important as proliferative rates in increasing the marrow cell population.

Pathology

Other organs may be involved: Splenomegaly, hepatomegaly and lymph node enlargement. More specific pathological clinical complications are seen in 2 subtypes.

Myelofibrosis

When haemopoietic cellular proliferation is overshadowed by progressive fibrosis of the marrow, splenomegaly (often of massive proportions) due to extramedullary haemopoiesis is often found.

Marrow: ordinary marrow puncture usually results in a ‘dry tap’, therefore trephine or cutting needle biopsy is required

2. Myelodysplastic Syndromes (MDS)

These are primary disorders of stem cells associated with several different chromosomal abnormalities. They lead to ineffective haemopoiesis of varying types, with the appearance in the peripheral blood of abnormal cells. A significant number progress to leukaemia.