Textbook of Pathology, 6th Edition

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Figure 12.2 A normal bone marrow in an adult as seen in a section CHAPTER 12
after trephine biopsy. Bony trabeculae support the marrow-containing
tissue. Approximately 50% of the soft tissue of the bone consists of
haematopoietic tissue and 50% is fatty marrow.

The stem cells, after a series of divisions, differentiate into
two types of progenitors—lymphoid (immune system) stem
cells, and non-lymphoid or myeloid (trilineage) stem cells. The
former develop into T, B and NK cells while the latter
Figure 12.1 Sites of haematopoiesis in the bone marrow in the
adult. differentiate into 3 types of cell lines—granulocyte-monocyte
progenitors (producing neutrophils, eosinophils, basophils
poiesis in certain pathologic conditions. The spleen and liver and monocytes), erythroid progenitors (producing red cells),
can also resume their foetal haematopoietic role in certain and megakaryocytes (as the source of platelets). The develop-
pathologic conditions and is called extramedullary ment of mature cells (i.e. poiesis)—red cells (erythropoiesis),
haematopoiesis. granulocytes (granulopoiesis), monocytes, lymphocytes
In the bone marrow, developing blood cells are situated (lymphopoiesis) and platelets (thrombopoiesis) are
outside the marrow sinuses, from where after maturation considered in detail later in relevant sections.
they enter the marrow sinuses, the marrow microcirculation Myeloid haematopoiesis or myelopoiesis includes
and thence released into circulation. differentiation and maturation of granulocytes, monocytes,
erythroid cells and megakaryocytes (Fig. 12.3). The Introduction to Haematopoietic System and Disorders of Erythroid Series
HAEMATOPOIETIC STEM CELLS differentiation and maturation of each series of these cells
from stem cells are regulated by endogenous glycoproteins
Haematopoieisis involves two stages: mitotic division or called as growth factors, cytokines and hormones. These are as
proliferation, and differentiation or maturation. under:
It is widely accepted that blood cells develop from a small
population of common multipotent haematopoietic stem Erythropoietin
cells. The stem cells express a variety of cell surface proteins Granulocyte colony-stimulating factor (G-CSF)
such as CD34 and adhesion proteins which help the stem Granulocyte-macrophage colony-stimulating factor (GM-
cells to “home” to the bone marrow when infused. The stem CSF)
cells have the appearance of small or intermediate-sized Thrombopoietin
lymphocytes and their presence in the marrow can be Each of these growth factors acts on specific receptors
demonstrated by cell culture techniques by the growth of for growth factor to initiate further cell events as shown
colony-forming units (CFU) pertaining to different cell lines. schematically in Fig. 12.3.
The stem cells have the capability of maintaining their
progeny by self-replication. The bone marrow provides a BONE MARROW EXAMINATION
suitable environment for growth and development of stem
cells. For instance, if haematopoietic stem cells are infused Examination of the bone marrow provides an invaluable
intravenously into a suitably-prepared recipient, they seed diagnostic help in some cases, while in others it is of value in
the marrow successfully but do not thrive at other sites. This confirming a diagnosis suspected on clinical examination or
principle forms the basis of bone marrow (or stem cell) trans- on the blood film. A peripheral blood smear examination,
plantation performed for various haematologic diseases. however, must always precede bone marrow examination.

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SECTION II



























Figure 12.3 Schematic representation of differentiation of multipotent stem cells into blood cells.

Bone marrow examination may be performed by two conditions, fungi (e.g. histoplasmosis) and parasites (e.g.
Haematology and Lymphoreticular Tissues
methods—aspiration and trephine biopsy. A comparison of the malaria, leishmaniasis, trypanosomiasis). Estimation of the
two methods is summarised in Table 12.1. proportion of cellular components in the marrow, however,
BONE MARROW ASPIRATION. The method involves can be provided by doing a differential count of at least 500
suction of marrow via a strong, wide bore, short-bevelled cells (myelogram, Table 12.2). In some conditions, the
needle fitted with a stylet and an adjustable guard in order marrow cells can be used for more detailed special tests such
to prevent excessive penetration; for instance Salah bone as cytogenetics, microbiological culture, biochemical
marrow aspiration needle (Fig. 12.4,A). Smears are prepared analysis, and immunological and cytological markers.
immediately from the bone marrow aspirate and are fixed
in 95% methanol after air-drying. The usual Romanowsky TREPHINE BIOPSY. Trephine biopsy is performed by a
technique is employed for staining and a stain for iron is simple Jamshidi trephine needle by which a core of tissue from
performed routinely so as to assess the reticuloendothelial periosteum to bone marrow cavity is obtained (Fig. 12.4,B).
stores of iron. The tissue is then fixed, soft decalcified and processed for
The marrow film provides assessment of cellularity, histological sections and stained with haematoxylin and
details of developing blood cells (i.e. normoblastic or eosin and for reticulin. Trephine biospy is useful over
megaloblastic, myeloid, lymphoid, macrophages and aspiration since it provides an excellent view of the overall
megakaryocytic), ratio between erythroid and myeloid cells, marrow architecture, cellularity, and presence or absence of
storage diseases, and for the presence of cells foreign to the infiltrates, but is less valuable than aspiration as far as
marrow such as secondary carcinoma, granulomatous individual cell morphology is concerned.

287
  TABLE 12.1: Comparison of Bone Marrow Aspiration and Trephine Biopsy.
Feature Aspiration Trephine
1. Site Sternum, posterior iliac crest; tibial head in infants Posterior iliac crest
2. Instrument Salah BM aspiration needle Jamshidi trephine needle
3. Stains Romanowsky, Perls’ reaction for iron on smears Haematoxylin and eosin, reticulin on tissue sections
4. Time Within 1-2 hours Within 1-7 days
5. Morphology Better cellular morphology of aspiration smears Better marrow architectural pattern but cell morphology
but marrow architecture is indistinct is not as distinct since tissue sections are examined
and not smears
6. Indications Anaemias, suspected leukaemias, neutropenia Additional indications are:
thrombocytopenia, polycythaemia, myeloma, myelosclerosis, aplastic anaemia and in
lymphomas, carcinomatosis, lipid storage cases with ‘dry tap’ on aspiration.
diseases, granulomatous conditions, parasites,
fungi, and unexplained enlargements of liver,
spleen or lymph nodes. CHAPTER 12




















Figure 12.4 The Salah bone marrow aspiration needle (A), Jamshidi trephine needle (B).


RED BLOOD CELLS cell, 15-20 μm in diameter having deeply basophilic
cytoplasm and a large central nucleus containing nucleoli.
ERYTHROPOIESIS The deep blue colour of the cytoplasm is due to high content
of RNA which is associated with active protein synthesis.
Although the stem cells which eventually form the mature As the cells mature, the nuclei lose their nucleoli and become
erythrocytes of the peripheral blood cannot be recognised
morphologically, there is a well-defined and readily smaller and denser, while the cytoplasm on maturation leads Introduction to Haematopoietic System and Disorders of Erythroid Series
recognisable lineage of nucleated red cells (i.e. the erythroid to replacement of dense blue colour progressively by pink-
series) in the marrow. staining haemoglobin. Each proerythroblast undergoes 4-5
replications and forms 16-32 mature RBCs.
Erythroid Series
2. BASOPHILIC (EARLY) ERYTHROBLAST. It is a round
The cells in this series are as under (Fig. 12.5): cell having a diameter of 12-16 μm with a large nucleus which
is slightly more condensed than the proerythroblast and
1. PROERYTHROBLAST. The earliest recognisable cell in
the marrow is a proerythroblast or pronormoblast. It is a large contains basophilic cytoplasm. Basophilic erythroblast
undergoes rapid proliferation.
  TABLE 12.2: Normal Adult Bone Marrow Counts (Myelogram). 3. POLYCHROMATIC(INTERMEDIATE) ERYTHRO-
BLAST. Next maturation stage has a diameter of 12-14 μm.
Fat/cell ratio : 50:50
Myeloid/erythroid (M/E) ratio : 2-4:1 (mean 3:1) The nucleus at this stage is coarse and deeply basophilic.
Myeloid series: 30-45% (37.5%) The cytoplasm is characteristically polychromatic i.e.
• Myeloblasts : 0.1-3.5% contains admixture of basophilic RNA and acidophilic
• Promyelocytes: 0.5-5% haemoglobin. The cell at this stage ceases to undergo
Erythroid series: 10-15% (mean 12.5%) proliferative activity.
Megakaryocytes: 0.5% 4. ORTHOCHROMATIC (LATE) ERYTHROBLAST. The
Lymphocytes: 5-20% final stage in the maturation of nucleated red cells is the
Plasma cells: < 3% orthochromatic or late erythroblast. The cell at this stage is
Reticulum cells: 0.1-2% smaller, 8-12 μm in diameter, containing a small and pyknotic

288











Figure 12.5 The erythroid series. There is progressive condensation of the nuclear chromatin which is eventually extruded from the cell at the
late erythroblast stage. The cytoplasm contains progressively less RNA and more haemoglobin.


nucleus with dark nuclear chromatin. The cytoplasm is Erythropoietin
characteristically acidophilic with diffuse basophilic hue due
to the presence of large amounts of haemoglobin. Erythropoietic activity in the body is regulated by the
hormone, erythropoietin, which is produced in response to
5. RETICULOCYTE. The nucleus is finally extruded from anoxia. The principal site of erythropoietin production is the
the late erythroblast within the marrow and a reticulocyte kidney though there is evidence of its extra-renal production
results. The reticulocytes are juvenile red cells devoid of in certain unusual circumstances. Its levels are, therefore,
nuclei but contain ribosomal RNA so that they are still able lowered in chronic renal diseases, while a case of renal cell
SECTION II
to synthesise haemoglobin. A reticulocyte spends 1-2 days carcinoma may be associated with its increased production
in the marrow and circulates for 1-2 days in the peripheral and erythrocytosis. Erythropoietin acts on the marrow at the
blood before maturing in the spleen, to become a biconcave various stages of morphologically unidentifiable as well as
red cell. The reticulocytes in the peripheral blood are distin- identifiable erythroid precursors.
guished from mature red cells by slightly basophilic hue in There is an increased production of erythropoietin in
the cytoplasm similar to that of an orthochromatic various types of anaemias but in anaemia of chronic diseases
erythroblast. Reticulocytes can be counted in the laboratory (e.g. in infections and neoplastic conditions) there is no such
by vital staining with dyes such as new methylene blue or enhancement of erythropoietin. In polycythaemia rubra vera,
brilliant cresyl blue. The reticulocytes by either of these there is erythrocytosis but depressed production of
staining methods contain deep blue reticulofilamentous erythropoietin. This is because of an abnormality of the stem
material (Fig. 12.6). While erythroblasts are not normally cell class which is not under erythropoietin control.
present in human peripheral blood, reticulocytes are found Immunoassay of erythropoietin in plasma or serum can
normally in the peripheral blood. Normal range of be done by sensitive techniques (ELISA and radio-
reticulocyte count in health is 0.5-2.5% in adults and 2-6% in immunoassay) due to its quite low values; normal values
infants. Their percentage in the peripheral blood is a fairly are 10-25 U/L.
accurate reflection of erythropoietic activity. Their proportion
is increased in conditions of rapid red cell regeneration e.g. The Red Cell
after haemorrhage, haemolysis and haematopoietic response The mature erythrocytes of the human peripheral blood are
of anaemia to treatment.
non-nucleated cells and lack the usual cell organelles. The
normal human erythrocyte is a biconcave disc, 7.2 μm in
diameter, and has a thickness of 2.4 μm at the periphery and
Haematology and Lymphoreticular Tissues
1 μm in the centre. The biconcave shape renders the red cells
quite flexible so that they can pass through capillaries whose
minimum diameter is 3.5 μm. More than 90% of the weight
of erythrocyte consist of haemoglobin. The lifespan of red
cells is 120 + 30 days.

NORMAL VALUES AND RED CELL INDICES. Range of
12
normal red cell count in health is 5.5 ± 1.0 × 10 /L in men
12
and 4.8 ± 1.0 × 10 /L in women. The packed cell volume (PCV)
or haematocrit is the volume of erythrocytes per litre of whole
blood indicating the proportion of plasma and red cells and
ranges 0.47 ± 0.07 L/L (40-54%) in men and 0.42 ± 0.05 L/L
(37-47%) in women. The haemoglobin content in health is 15.5
± 2.5 g/dl (13-18 g/dl) in men and 14.0 ± 2.5 g/dl (11.5-16.5
g/dl) in women. Based on these normal values, a series of
Figure 12.6 Reticulocytes in blood as seen in blood stained by absolute values or red cell indices can be derived which have
supravital dye, new methylene blue. diagnostic importance. These are as under:

1. Mean corpuscular volume (MCV) = Vitamin B (pyridoxine), vitamin E (tocopherol) and 289
6
PCV in L/L riboflavin are the other essential vitamins required in the
_________________ synthesis of red cells.
RBC count/L
3. Amino acids. Amino acids comprise the globin compo-
The normal value is 85 ± 8 fl (77-93 fl)*. nent of haemoglobin. Severe amino acid deficiency due to

2. Mean corpuscular haemoglobin (MCH) = protein deprivation causes depressed red cell production.
4. Hormones. As discussed above, erythropoietin plays a
Hb/L
________________ significant regulatory role in the erythropoietic activity.
RBC count/L Besides erythropoietin, androgens and thyroxine also appear
The normal range is 29.5 ± 2.5 pg (27-32 pg)*. to be involved in the red cell production.
3. Mean corpuscular haemoglobin concentration HAEMOGLOBIN. Haemoglobin consists of a basic protein,
(MCHC) = globin, and the iron-porphyrin complex, haem. The molecular
weight of haemoglobin is 68,000. Normal adult haemoglobin
Hb/dl
_________________ (HbA) constitutes 96-98% of the total haemoglobin content
PCV in L/L and consists of four polypeptide chains, α β . Small quantities CHAPTER 12
2 2
The normal value is 32.5 ± 2.5 g/dl (30-35 g/dl). of 2 other haemoglobins present in adults are: HbF containing
α γ globin chains comprising 0.5-0.8% of total haemoglobin,
2 2
Since MCHC is independent of red cell count and size, it and HbA having α δ chains and constituting 1.5-3.2% of
2 2
2
is considered to be of greater clinical significance as compared total haemoglobin. Most of the haemoglobin (65%) is
to other absolute values. It is low in iron deficiency anaemia synthesised by the nucleated red cell precursors in the
but is usually normal in acrostic anaemia.
marrow, while the remainder (35%) is synthesised at the
RED CELL MEMBRANE. The red cell membrane is a reticulocyte stage.
trilaminar structure having a bimolecular lipid layer Synthesis of haem occurs largely in the mitochondria by
interposed between two layers of proteins. The important a series of biochemical reactions summarised in Fig. 12.7.
proteins in red cell membrane are band 3 protein (named on Coenzyme, pyridoxal-6-phosphate, derived from pyridoxine
the basis of the order in which it migrates during (vitamin B ) is essential for the synthesis of amino levulinic
6
electrophoresis), glycophorin and spectrin; important lipids acid (ALA) which is the first step in the biosynthesis of
are glycolipids, phospholipids and cholesterol; and protoporphyrin. The reaction is stimulated by erythropoietin
carbohydrates form skeleton of erythrocytes having a lattice- and inhibited by haem. Ultimately, protoporphyrin combines
like network which is attached to the internal surface of the with iron supplied from circulating transferrin to form haem.
membrane and is responsible for biconcave form of the Each molecule of haem combines with a globin chain
erythrocytes. synthesised by polyribosomes. A tetramer of 4 globin chains,
A number of inherited disorders of the red cell membrane each having its own haem group, constitutes the
and cytoskeletal components produce abnormalities of the haemoglobin molecule (Fig. 12.8, A).
shape such as: spherocytosis (spherical shape from loss of part RED CELL FUNCTIONS. The essential function of the red
of the membrane), ovalocytosis (oval shape from loss of cells is to carry oxygen from the lungs to the tissue and to
elasticity of cytoskeleton), echinocytosis (spiny processes from transport carbon dioxide to the lungs. In order to perform Introduction to Haematopoietic System and Disorders of Erythroid Series
external surface due to metabolic abnormalities of red cells), these functions, the red cells have the ability to generate
and stomatocytosis (bowl-shaped red cells from expansion of energy as ATP by anaerobic glycolytic pathway (Embden-
inner membrane on one side). Meyerhof pathway). This pathway also generates reducing
power as NADH and NADPH by the hexose monophosphate
NUTRITIONAL REQUIREMENTS FOR ERYTHRO- (HMP) shunt.
POIESIS. New red cells are being produced each day for
which the marrow requires certain essential substances. 1. Oxygen carrying. The normal adult haemoglobin, HbA,
These substances are as under: is an extremely efficient oxygen-carrier. The four units of
tetramer of haemoglobin molecule take up oxygen in
1. Metals. Iron is essential for red cell production because succession, which, in turn, results in stepwise rise in affinity
it forms part of the haem molecule in haemoglobin. Its
deficiency leads to iron deficiency anaemia. Cobalt and of haemoglobin for oxygen. This is responsible for the sigmoid
shape of the oxygen dissociation curve.
manganese are certain other metals required for red cell The oxygen affinity of haemoglobin is expressed in term
production.
of P value which is the oxygen tension (pO ) at which 50%
50
2
2. Vitamins. Vitamin B and folate are essential for bio- of the haemoglobin is saturated with oxygen. Pulmonary
12
synthesis of nucleic acids. Deficiency of B or folate causes capillaries have high pO and, thus, there is virtual saturation
12
2
megaloblastic anaemia. Vitamin C (ascorbic acid) plays an of available oxygen-combining sites of haemoglobin. The
indirect role by facilitating the iron turnover in the body. tissue capillaries, however, have relatively low pO and, thus,
2
part of haemoglobin is in deoxy state. The extent to which
–1
*For conversions, the multiples used are as follows: ‘deci (d) = 10 , milli oxygen is released from haemoglobin at pO , in tissue
2
–6
–9
–3
(m) = 10 , micro (μ) = 10 , nano (n) = 10 , pico (p) = 10 –12 , femto (f) = capillaries depends upon 3 factors—the nature of globin
10 –15

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SECTION II
Figure 12.7 Schematic diagram of haemoglobin synthesis in the developing red cell.

chains, the pH, and the concentration of 2,3- 2. CO transport. Another important function of the red cells
2
biphosphoglycerate (2,3-BPG) as follows (Fig 12.8, B): is the CO transport. In the tissue capillaries, the pCO is
2
2
Normal adult haemoglobin (HbA) has lower affinity for high so that CO enters the erythrocytes where much of it is
2
oxygen than foetal haemoglobin and, therefore, releases converted into bicarbonate ions which diffuse back into the
greater amount of bound oxygen at pO of tissue capillaries. plasma. In the pulmonary capillaries, the process is reversed
2
A fall in the pH (acidic pH) lowers affinity of and bicarbonate ions are converted back into CO . Some of
2
oxyhaemoglobin for oxygen, so called the Bohr effect, thereby the CO produced by tissues is bound to deoxyhaemoglobin
2
causing enhanced release of oxygen from erythrocytes at the forming carbamino-haemoglobin. This compound dissociates
lower pH in tissue capillaries. in the pulmonary capillaries to release CO .
2
A rise in red cell concentration of 2,3-BPG, an intermediate RED CELL DESTRUCTION. Red cells have a mean lifespan
product of Embden-Meyerhof pathway, as occurs in anaemia of 120 days, after which red cell metabolism gradually
and hypoxia, causes decreased affinity of HbA for oxygen. deteriorates as the enzymes are not replaced. The destroyed
This, in turn, results in enhanced supply of oxygen to the red cells are removed mainly by the macrophages of the
tissue. reticuloendothelial (RE) system of the marrow, and to some






Haematology and Lymphoreticular Tissues




















Figure 12.8 A, Normal adult haemoglobin molecule (HbA) consisting of α 2 β 2 globin chains, each with its own haem group in oxy and deoxy
state. The haemoglobin tetramer can bind up to four molecules of oxygen in the iron containing sites of the haem molecules. As oxygen is bound,
salt bridges are broken, and 2,3-BPG and CO 2 are expelled. B, Hb-dissociation curve. On dissociation of oxygen from Hb molecule i.e. on release
of oxygen to the tissues, salt bridges are formed again, and 2,3-BPG and CO 2 , are bound. The shift of the curve to higher oxygen delivery is affected
by acidic pH, increased 2,3-BPG and HbA molecule while oxygen delivery is less with high pH, low 2,3-BPG and HbF.

extent by the macrophages in the liver and spleen (Fig.12.9). normal haemoglobin is taken as 13.0 g/dl for males and 291
The breakdown of red cells liberates iron for recirculation 11.5 g/dl for females. Newborn infants have higher
via plasma transferrin to marrow erythroblasts, and haemoglobin level and, therefore, 15 g/dl is taken as the
protoporphyrin which is broken down to bilirubin. Bilirubin lower limit at birth, whereas at 3 months the normal lower
circulates to the liver where it is conjugated to its level is 9.5 g/dl. Although haemoglobin value is employed
diglucuronide which is excreted in the gut via bile and as the major parameter for determining whether or not
converted to stercobilinogen and stercobilin excreted in the anaemia is present, the red cell counts, haematocrit (PCV)
faeces. Part of stercobilinogen and stercobilin is reabsorbed and absolute values (MCV, MCH and MCHC) provide
and excreted in the urine as urobilinogen and urobilin. A alternate means of assessing anaemia.
small fragment of protoporphyrin is converted to carbon
monoxide and excreted in expired air from the lungs. Globin Pathophysiology of Anaemia
chains are broken down to amino acids and reused for protein
synthesis in the body. Subnormal level of haemoglobin causes lowered oxygen-
carrying capacity of the blood. This, in turn, initiates
compensatory physiologic adaptations such as follows:
ANAEMIA—GENERAL CONSIDERATIONS
increased release of oxygen from haemoglobin;
Anaemia is defined as a haemoglobin concentration in blood increased blood flow to the tissues; CHAPTER 12
below the lower limit of the normal range for the age and maintenance of the blood volume; and
sex of the individual. In adults, the lower extreme of the redistribution of blood flow to maintain the cerebral blood
supply.
Eventually, however, tissue hypoxia develops causing
impaired functions of the affected tissues. The degree of
functional impairment of individual tissues is variable
depending upon their oxygen requirements. Tissues with
high oxygen requirement such as the heart, CNS and the
skeletal muscle during exercise, bear the brunt of clinical
effects of anaemia.

Clinical Features of Anaemia

The haemoglobin level at which symptoms and signs of
anaemia develop depends upon 4 main factors:
1. The speed of onset of anaemia: Rapidly progressive anaemia
causes more symptoms than anaemia of slow-onset as there
is less time for physiologic adaptation.
2. The severity of anaemia: Mild anaemia produces no
symptoms or signs but a rapidly developing severe anaemia
(haemoglobin below 6.0 g/dl) may produce significant Introduction to Haematopoietic System and Disorders of Erythroid Series
clinical features.
3. The age of the patient: The young patients due to good
cardiovascular compensation tolerate anaemia quite well as
compared to the elderly. The elderly patients develop cardiac
and cerebral symptoms more prominently due to associated
cardiovascular disease.
4. The haemoglobin dissociation curve: In anaemia, the affinity
of haemoglobin for oxygen is depressed as 2,3-BPG in the
red cells increases. As a result, oxyhaemoglobin is dissociated
more readily to release free oxygen for cellular use, causing
a shift of the oxyhaemoglobin dissociation curve to the right.
SYMPTOMS. In symptomatic cases of anaemia, the
presenting features are: tiredness, easy fatiguability,
generalised muscular weakness, lethargy and headache. In
older patients, there may be symptoms of cardiac failure,
angina pectoris, intermittent claudication, confusion and
visual disturbances.
SIGNS. A few general signs common to all types of anaemias
Figure 12.9 Normal red cell destruction in the RE system. are as under:

292 1. Pallor. Pallor is the most common and characteristic sign evaluated in an area where there is neither Rouleaux
which may be seen in the mucous membranes, conjunctivae formation nor so thin as to cause red cell distortion. Such an
and skin. area can usually be found at junction of the body with the
2. Cardiovascular system. A hyperdynamic circulation may tail of the film, but not actually at the tail. The following
be present with tachycardia, collapsing pulse, cardiomegaly, abnormalities in erythroid series of cells are particularly
midsystolic flow murmur, dyspnoea on exertion, and in the looked for in a blood smear:
case of elderly, congestive heart failure. 1. Variation in size (Anisocytosis). Normally, there is slight
3. Central nervous system. The older patients may develop variation in diameter of the red cells from 6.7-7.7 μm (mean
symptoms referable to the CNS such as attacks of faintness, value 7.2 μm). Increased variation in size of the red cell is
giddiness, headache, tinnitus, drowsiness, numbness and termed anisocytosis. Anisocytosis may be due to the presence
tingling sensations of the hands and feet. of cells larger than normal (macrocytosis) or cells smaller than
normal (microcytosis). Sometimes both microcytosis and
4. Ocular manifestations. Retinal haemorrhages may occur macrocytosis are present (dimorphic).
if there is associated vascular disease or bleeding diathesis.
Macrocytes are classically found in megaloblastic anaemia;
5. Reproductivesystem. Menstrual disturbances such as other causes are aplastic anaemia, other dyserythropoietic
amenorrhoea and menorrhagia and loss of libido are some anaemias, chronic liver disease and in conditions with
of the manifestations involving the reproductive system in increased erythropoiesis.
anaemic subjects. Microcytes are present in iron deficiency anaemia,
6. Renal system. Mild proteinuria and impaired concen- thalassaemia and spherocytosis. They may also result from
SECTION II
trating capacity of the kidney may occur in severe anaemia. fragmentation of erythrocytes such as in haemolytic anaemia.
7. Gastrointestinalsystem. Anorexia, flatulence, nausea, 2. Variation in shape (Poikilocytosis). Increased variation
constipation and weight loss may occur. in shape of the red cells is termed poikilocytosis. The nature
In addition to the general features, specific signs may be of the abnormal shape determines the cause of anaemia.
associated with particular types of anaemia which are Poikilocytes are produced in various types of abnormal
described later together with discussion of specific types of erythropoiesis e.g. in megaloblastic anaemia, iron deficiency
anaemias. anaemia, thalassaemia, myelosclerosis and microangiopathic
haemolytic anaemia.
Investigations of the Anaemic Subject 3. Inadequate haemoglobin formation (Hypochromasia).

After obtaining the full medical history pertaining to different Normally, the intensity of pink staining of haemoglobin in a
general and specific signs and symptoms, the patient is Romanowsky-stained blood smear gradually decreases from
examined for evidence of anaemia. Special emphasis is placed the periphery to the centre of the cell. Increased central pallor
on colour of the skin, conjunctivae, sclerae and nails. Changes is referred to as hypochromasia. It may develop either from
in the retina, atrophy of the papillae of the tongue, rectal lowered haemoglobin content (e.g. in iron deficiency
examination for evidence of bleeding, and presence of anaemia, chronic infections), or due to thinness of the red
hepatomegaly, splenomegaly, lymphadenopathy and bony cells (e.g. in thalassaemia, sideroblastic anaemia). Unusually
tenderness are looked for. deep pink staining of the red cells due to increased haemo-
In order to confirm or deny the presence of anaemia, its globin concentration is termed hyperchromasia and may be
type and its cause, the following plan of investigations is found in megaloblastic anaemia, spherocytosis and in
generally followed, of which complete blood counts (CBC) neonatal blood.
with reticulocyte count is the basic test. 4. Compensatory erythropoiesis. A number of changes are
Haematology and Lymphoreticular Tissues
associated with compensatory increase in erythropoietic
A. HAEMOGLOBIN ESTIMATION. The first and foremost activity. These are as under:
investigation in any suspected case of anaemia is to carry i) Polychromasia is defined as the red cells having more than
out a haemoglobin estimation. Several methods are available one type of colour. Polychromatic red cells are slightly larger,
but most reliable and accurate is the cyanmethaemoglobin generally stained bluish-grey and represent reticulocytes and,
(HiCN) method employing Drabkin’s solution and a thus, correlate well with reticulocyte count.
spectrophotometer. If the haemoglobin value is below the ii) Erythroblastaemia is the presence of nucleated red cells in
lower limit of the normal range for particular age and sex, the peripheral blood film. A small number of erythroblasts
the patient is said to be anaemic. In pregnancy, there is (or normoblasts) may be normally found in cord blood at
haemodilution and, therefore, the lower limit in normal birth. They are found in large numbers in haemolytic disease
pregnant women is less (10.5 g/dl) than in the non-pregnant of the newborn, other haemolytic disorders and in
state.
extramedullary erythropoiesis. They may also appear in the
B. PERIPHERAL BLOOD FILM EXAMINATION. The blood in various types of severe anaemias except in aplastic
haemoglobin estimation is invariably followed by anaemia. Erythroblastaemia may also occur after
examination of a peripheral blood film for morphologic splenectomy.
features after staining it with the Romanowsky dyes (e.g. iii) Punctate basophilia or basophilic stippling is diffuse and
Leishman’s stain, May-Grünwald-Giemsa’s stain, Jenner- uniform basophilic granularity in the cell which does not
Giemsa’s stain, Wright’s stain etc). The blood smear is stain positively with Perls’ reaction (in contrast to

293





























Figure 12.10 Some of the common morphologic abnormalities of red cells (The serial numbers in the illustrations correspond to the order in CHAPTER 12
which they are described in the text).

Pappenheimer bodies which stain positively). Classical vii) Acanthocytosis is the presence of coarsely crenated red
punctate basophilia is seen in aplastic anaemia, thalassaemia, cells. Acanthocytes are found in large number in blood film
myelodysplasia, infections and lead poisoning. made from splenectomised subjects, and in chronic liver
iv) Howell-Jolly bodies are purple nuclear remnants, usually disease.
found singly, and are larger than basophilic stippling. They viii) Burr cells are cell fragments having one or more spines.
are present in megaloblastic anaemia and after splenectomy. They are particularly found in uraemia.
5. Miscellaneous changes. In addition to the morphologic ix) Stomatocytosis is the presence of stomatocytes which have
changes of red cells described above, several other abnormal central area having slit-like or mouth-like appearance. They
red cells may be found in different haematological disorders. are found in hereditary stomatocytosis, or may be seen in
Some of these are as follows (Fig. 12.10): chronic alcoholism.
i) Spherocytosis is characterised by presence of spheroidal x) Ovalocytosis or elliptocytosis is the oval or elliptical shape
rather than biconcave disc-shaped red cells. Spherocytes are of red cells. Their highest proportion (79%) is seen in
seen in hereditary spherocytosis, autoimmune haemolytic hereditary ovalocytosis and elliptocytosis; other conditions
anaemia and in ABO haemolytic disease of the newborn. showing such abnormal shapes of red cells are megaloblastic Introduction to Haematopoietic System and Disorders of Erythroid Series
ii) Schistocytosis is identified by fragmentation of erythro- anaemia and hypochromic anaemia.
cytes. Schistocytes are found in thalassaemia, hereditary C. RED CELL INDICES. An alternative method to diagnose
elliptocytosis, megaloblastic anaemia, iron deficiency and detect the severity of anaemia is by measuring the red
anaemia, microangiopathic haemolytic anaemia and in cell indices:
severe burns. In iron deficiency and thalassaemia, MCV, MCH and
iii) Irregularly contracted red cells are found in drug and MCHC are reduced.
chemical induced haemolytic anaemia and in unstable In anaemia due to acute blood loss and haemolytic
haemoglobinopathies. anaemias, MCV, MCH and MCHC are all within normal
iv) Leptocytosis is the presence of unusually thin red cells. limits.
Leptocytes are seen in severe iron deficiency and In megaloblastic anaemias, MCV is raised above the
thalassaemia. Target cell is a form of leptocyte in which there normal range.
is central round stained area and a peripheral rim of
haemoglobin. Target cells are found in iron deficiency, D. LEUCOCYTE AND PLATELET COUNT. Measurement
thalassaemia, chronic liver disease, and after splenectomy. of leucocyte and platelet count helps to distinguish pure
v) Sickle cells or drepanocytes are sickle-shaped red cells found anaemia from pancytopenia in which red cells, granulocytes
and platelets are all reduced. In anaemias due to haemolysis
in sickle cell disease. or haemorrhage, the neutrophil count and platelet counts
vi) Crenated red cells are the erythrocytes which develop are often elevated. In infections and leukaemias, the leucocyte
numerous projections from the surface. They are present in counts are high and immature leucocytes appear in the blood.
blood films due to alkaline pH, presence of traces of fatty
substances on the slides and in cases where the film is made E. RETICULOCYTE COUNT. Reticulocyte count (normal
from blood that has been allowed to stand overnight. 0.5-2.5%) is done in each case of anaemia to assess the marrow

294 erythropoietic activity. In acute haemorrhage and in A. Cytoplasmic maturation defects
haemolysis, the reticulocyte response is indicative of 1. Deficient haem synthesis: iron deficiency anaemia
impaired marrow function. 2. Deficient globin synthesis: thalassaemic syndromes
F. ERYTHROCYTE SEDIMENTATION RATE. The ESR B. Nuclear maturation defects
is a non-specific test used as a screening test for anaemia. It Vitamin B and/or folic acid deficiency: megalo-
12
usually gives a clue to the underlying organic disease but blastic anaemia
anaemia itself may also cause rise in the ESR.
C. Haematopoietic stem cell proliferation and differentiation
G. BONE MARROW EXAMINATION. Bone marrow abnormality e.g.
aspiration is done in cases where the cause for anaemia is 1. Aplastic anaemia
not obvious. The procedures involved for marrow aspiration 2. Pure red cell aplasia
and trephine biopsy and their relative advantages and D. Bone marrow failure due to systemic diseases (anaemia of
disadvantages have already been discussed (page 285). chronic disorders) e.g.
In addition to these general tests, certain specific tests 1. Anaemia of inflammation/infections, disseminated
are done in different types of anaemias which are described malignancy
later under the discussion of specific anaemias. 2. Anaemia in renal disease
3. Anaemia due to endocrine and nutritional deficiencies
Classification of Anaemias (hypometabolic states)
Several types of classifications of anaemias have been 4. Anaemia in liver disease
SECTION II
proposed. Two of the widely accepted classifications are E. Bone marrow infiltration e.g.
based on the pathophysiology and morphology (Table 12.3). 1. Leukaemias
2. Lymphomas
PATHOPHYSIOLOGIC CLASSIFICATION. Depending 3. Myelosclerosis
upon the pathophysiologic mechanism, anaemias are 4. Multiple myeloma
classified into 3 groups:
F. Congenital anaemia e.g.
I. Anaemia due to blood loss. This is further of 2 types:
A. Acute post-haemorrhagic anaemia 1. Sideroblastic anaemia
B. Anaemia of chronic blood loss 2. Congenital dyserythropoietic anaemia.
The term hypoproliferative anaemias is also used to denote
II. Anaemia due to impaired red cell formation. A impaired marrow proliferative activity and includes 2 main
disturbance due to impaired red cell production from various groups: hypoproliferation due to iron deficiency and that due
causes may produce anaemia. These are as under: to other hypoproliferative disorders; the latter category
includes anaemia of chronic inflammation/infection, renal
 TABLE 12.3: Classification of Anaemias. disease, hypometabolic states, and causes of bone marrow
A. PATHOPHYSIOLOGIC failure.
I. Anaemia due to increased blood loss III. Anaemia due to increased red cell destruction (haemo-
a) Acute post-haemorrhagic anaemia lytic anaemias). This is further divided into 2 groups:
b) Chronic blood loss
A. Intracorpuscular defect (hereditary and acquired).
II. Anaemias due to impaired red cell production B. Extracorpuscular defect (acquired haemolytic anaemias).
a) Cytoplasmic maturation defects
1. Deficient haem synthesis: MORPHOLOGIC CLASSIFICATION. Based on the red cell
Iron deficiency anaemia size, haemoglobin content and red cell indices, anaemias are
Haematology and Lymphoreticular Tissues
2. Deficient globin synthesis: classified into 3 types:
Thalassaemic syndromes 1. Microcytic, hypochromic: MCV, MCH, MCHC are all
b) Nuclear maturation defects
Vitamin B and/or folic acid deficiency: reduced e.g. in iron deficiency anaemia and in certain non-
12
Megaloblastic anaemia iron deficient anaemias (sideroblastic anaemia, thalassaemia,
c) Defect in stem cell proliferation and differentiation anaemia of chronic disorders).
1. Aplastic anaemia 2. Normocytic, normochromic: MCV, MCH, MCHC are all
2. Pure red cell aplasia
d) Anaemia of chronic disorders normal e.g. after acute blood loss, haemolytic anaemias, bone
e) Bone marrow infiltration marrow failure, anaemia of chronic disorders.
f) Congenital anaemia 3. Macrocytic: MCV is raised e.g. in megaloblastic anaemia
III. Anaemias due to increased red cell destruction (Haemo- due to deficiency of vitamin B or folic acid.
12
lytic anaemias) (Details in Table 12.9) With these general comments on anaemias, a discussion
A. Extrinsic (extracorpuscular) red cell abnormalities of the specific types of anaemias is given in the following
B. Intrinsic (intracorpuscular) red cell abnormalities
pages.
B. MORPHOLOGIC
I. Microcytic, hypochromic ANAEMIA OF BLOOD LOSS
II. Normocytic, normochromic Depending upon the rate of blood loss due to haemorrhage,
III. Macrocytic, normochromic
the effects of post-haemorrhagic anaemia appear.

ACUTE BLOOD LOSS. When the loss of blood occurs ABSORPTION. The average Western diet contains 10-15 mg 295
suddenly, the following events take place: of iron, out of which only 5-10% is normally absorbed. In
i) Immediate threat to life due to hypovolaemia which may pregnancy and in iron deficiency, the proportion of
result in shock and death. absorption is raised to 20-30%. Iron is absorbed mainly in
ii) If the patient survives, shifting of interstitial fluid to the duodenum and proximal jejunum. Iron from diet containing
intravascular compartment with consequent haemodilution haem is better absorbed than non-haem iron. Absorption of non-
with low haematocrit. haem iron is enhanced by factors such as ascorbic acid
iii) Hypoxia stimulates production of erythropoietin resul- (vitamin C), citric acid, amino acids, sugars, gastric secretions
and hydrochloric acid. Iron absorption is impaired by factors
ting in increased marrow erythropoiesis.
like medicinal antacids, milk, pancreatic secretions, phytates,
LABORATORY FINDINGS phosphates, ethylene diamine tetra-acetic acid (EDTA) and
i) Normocytic and normochromic anaemia tannates contained in tea.
Non-haem iron is released as ferrous or ferric form but
ii) Low haematocrit is absorbed almost exclusively as ferrous form; reduction of
iii) Increased reticulocyte count in peripheral blood ferric to ferrous form when required takes place at the
(10-15% after one week) reflecting accelerated marrow intestinal brush border by ferric reductase. Transport across
erythropoiesis. the membrane is accomplished by divalent metal trans- CHAPTER 12
porter 1 (DMT 1). Once inside the gut cells, ferric iron may
CHRONIC BLOOD LOSS. When the loss of blood is slow be either stored as ferritin or further transported to transferrin
and insidious, the effects of anaemia will become apparent by two vehicle proteins—ferroportin and hephaestin. The
only when the rate of loss is more than rate of production mechanism of dietary haem iron absorption is not clearly
and the iron stores are depleted. This results in iron deficiency understood yet but it is through a different transport than
anaemia as seen in other clinical conditions discussed below. DMT 1. After absorption of both non-haem and haem forms
of iron, it comes into mucosal pool.
HYPOCHROMIC ANAEMIA Major mechanism of maintaining iron balance in the body
Hypochromic anaemia due to iron deficiency is the is by intestinal absorption of dietary iron. When the demand
commonest cause of anaemia the world over. It is estimated for iron is increased (e.g. during pregnancy, menstruation,
that about 20% of women in child-bearing age group are iron periods of growth and various diseases), there is increased
deficient, while the overall prevalence in adult males is about iron absorption, while excessive body stores of iron cause
2%. It is the most important, though not the sole, cause of reduced intestinal iron absorption (Fig. 12.12,A page 297).
microcytic hypochromic anaemia in which all the three red TRANSPORT. Iron is transported in plasma bound to a
cell indices (MCV, MCH and MCHC) are reduced and occurs β-globulin, transferrin, synthesised in the liver. Transferrin-
due to defective haemoglobin synthesis. Hypochromic bound iron is made available to the marrow where the
anaemias, therefore, are classified into 2 groups: developing erythroid cells having transferring receptors utilise
I. Hypochromic anaemia due to iron deficiency. iron for haemoglobin synthesis. It may be mentioned here
II. Hypochromic anaemias other than iron deficiency. that tranferrin receptors are present on cells of many tissues
The latter category includes 3 groups of disorders— of the body but their number is greatest in the developing
sideroblastic anaemia, thalassaemia and anaemia of chronic erythroblasts. Transferrin is reutilised after iron is released Introduction to Haematopoietic System and Disorders of Erythroid Series
disorders. from it. A small amount of transferrin iron is delivered to
other sites such as parenchymal cells of the liver. Normally,
IRON DEFICIENCY ANAEMIA transferrin is about one-third saturated. But in conditions
where transferrin-iron saturation is increased, parenchymal
The commonest nutritional deficiency disorder present iron uptake is increased. Virtually, no iron is deposited in
throughout the world is iron deficiency but its prevalence is the mononuclear-phagocyte cells (RE cells) from the plasma
higher in the developing countries. The factors responsible transferrin-iron but instead these cells derive most of their
for iron deficiency in different populations are variable and iron from phagocytosis of senescent red cells. Storage form
are best understood in the context of normal iron metabolism. of iron (ferritin and haemosiderin) in RE cells is normally
not functional but can be readily mobilised in response to
Iron Metabolism increased demands for erythropoiesis. However, conditions
such as malignancy, infection and inflammation interfere
The amount of iron obtained from the diet should replace with the release of iron from iron stores causing ineffective
the losses from the skin, bowel and genitourinary tract. These erythropoiesis.
losses together are about 1 mg daily in an adult male or in a
non-menstruating female, while in a menstruating woman EXCRETION. The body is unable to regulate its iron content
there is an additional iron loss of 0.5-1 mg daily. The iron by excretion alone. The amount of iron lost per day is
required for haemoglobin synthesis is derived from 2 primary 0.5-1 mg which is independent of iron intake. This loss is
sources—ingestion of foods containing iron (e.g. leafy nearly twice more (i.e. 1-2 mg/day) in menstruating women.
vegetables, beans, meats, liver etc) and recycling of iron from Iron is lost from the body in both sexes as a result of
senescent red cells (Fig. 12.11). desquamation of epithelial cells from the gastrointestinal

296


























SECTION II






















Figure 12.11 Daily iron cycle. Iron on absorption from upper small intestine circulates in plasma bound to transferrin and is transported to the
bone marrow for utilisation in haemoglobin synthesis. The mature red cells are released into circulation, which on completion of their lifespan of 120
days, die. They are then phagocytosed by RE cells and iron stored as ferritin and haemosiderin. Stored iron is mobilised in response to increased
demand and used for haemoglobin synthesis, thus completing the cycle (M = males; F = females).



tract, from excretion in the urine and sweat, and loss via hair cells of the spleen, liver and bone marrow and in the
and nails. Iron excreted in the faeces mainly consists of parenchymal cells of the liver.
Haematology and Lymphoreticular Tissues
unabsorbed iron and desquamated mucosal cells.
Pathogenesis
DISTRIBUTION. In an adult, iron is distributed in the body
as under: Iron deficiency anaemia develops when the supply of iron is
inadequate for the requirement of haemoglobin synthesis.
1. Haemoglobin—present in the red cells, contains most of Initially, negative iron balance is covered by mobilisation
the body iron (65%). from the tissue stores so as to maintain haemoglobin
2. Myoglobin—comprises a small amount of iron in the synthesis. It is only after the tissue stores of iron are exhausted
muscles (3.5%). that the supply of iron to the marrow becomes insufficient
3. Haem and non-haem enzymes—e.g. cytochrome, for haemoglobin formation and thus a state of iron deficiency
catalase, peroxidases, succinic dehydrogenase and anaemia develops. The development of iron deficiency
flavoproteins constitute a fraction of total body iron (0.5%). depends upon one or more of the following factors:
4. Transferrin-bound iron—circulates in the plasma and 1. Increased blood loss
constitutes another fraction of total body iron (0.5%). 2. Increased requirements
3. Inadequate dietary intake
All these forms of iron are in functional form.
4. Decreased intestinal absorption.
5. Ferritin and haemosiderin—are the storage forms of excess The relative significance of these factors varies with the
iron (30%). They are stored in the mononuclear-phagocyte age and sex of the patient (Table 12.4). Accordingly, certain

297








































Figure 12.12 Contrasting pathways of absorption and transport of iron, vitamin B and folic acid. CHAPTER 12
12

groups of individuals at increased risk of developing iron
 TABLE 12.4: Etiology of Iron Deficiency Anaemia.
deficiency can be identified (see below). In general, in
I. INCREASED BLOOD LOSS developed countries the mechanism of iron deficiency is
1. Uterine e.g. excessive menstruation in reproductive years, usually due to chronic occult blood loss, while in the
repeated miscarriages, at onset of menarche, post-menopausal developing countries poor intake of iron or defective
uterine bleeding.
absorption are responsible for iron deficiency anaemia.
2. Gastrointestinal e.g. peptic ulcer, haemorrhoids, hookworm
infestation, cancer of stomach and large bowel, oesophageal
varices, hiatus hernia, chronic aspirin ingestion, ulcerative colitis, Etiology Introduction to Haematopoietic System and Disorders of Erythroid Series
diverticulosis. Iron deficiency anaemia is always secondary to an underlying
3. Renal tract e.g. haematuria, haemoglobinuria. disorder. Correction of the underlying cause, therefore, is
4. Nose e.g. repeated epistaxis. essential part of its treatment. Based on the above-mentioned
5. Lungs e.g. haemoptysis.
pathogenetic mechanisms, following etiologic factors are
II. INCREASED REQUIREMENTS involved in development of iron deficiency anaemia at
1. Spurts of growth in infancy, childhood and adolescence. different age and sex (Table 12.4):
2. Prematurity.
3. Pregnancy and lactation. 1. FEMALES IN REPRODUCTIVE PERIOD OF LIFE. The
highest incidence of iron deficiency anaemia is in women
III. INADEQUATE DIETARY INTAKE
during their reproductive years of life. It may be from one or
1. Poor economic status. more of the following causes:
2. Anorexia e.g. in pregnancy.
i) Blood loss. This is the most important cause of anaemia in
3. Elderly individuals due to poor dentition, apathy and women during child-bearing age group. Commonly, it is due
financial constraints.
to persistent and heavy menstrual blood loss such as occurs
IV. DECREASED ABSORPTION in various pathological states and due to insertion of IUCDs.
1. Partial or total gastrectomy Young girls at the onset of menstruation may develop mild
2. Achlorhydria anaemia due to blood loss. Significant blood loss may occur
3. Intestinal malabsorption such as in coeliac disease. as a result of repeated miscarriages.

298 ii) Inadequate intake. Inadequate intake of iron is prevalent 4. INFANTS AND CHILDREN. Iron deficiency anaemia
in women of lower economic status. Besides diet deficient in is fairly common during infancy and childhood with a peak
iron, other factors such as anorexia, impaired absorption and incidence at 1-2 years of age. The principal cause for anaemia
diminished bioavailability may act as contributory factors. at this age is increased demand of iron which is not met by
iii) Increased requirements. During pregnancy and the inadequate intake of iron in the diet. Normal full-term
adolescence, the demand of body for iron is increased. During infant has sufficient iron stores for the first 4-6 months of
a normal pregnancy, about 750 mg of iron may be siphoned life, while premature infants have inadequate reserves
off from the mother—about 400 mg to the foetus, 150 mg to because iron stores from the mother are mainly laid down
the placenta, and 200 mg is lost at parturition and lactation. during the last trimester of pregnancy. Therefore, unless the
If several pregnancies occur at short intervals, iron deficiency infant is given supplemental feeding of iron or iron-
anaemia certainly follows. containing foods, iron deficiency anaemia develops.
2. POST-MENOPAUSAL FEMALES. Though the Clinical Features
physiological demand for iron decreases after cessation of
menstruation, iron deficiency anaemia may develop in post- As already mentioned, iron deficiency anaemia is much more
common in women between the age of 20 and 45 years than
menopausal women due to chronic blood loss. Following are in men; at periods of active growth in infancy, childhood
among the important causes during these years: and adolescence; and is also more frequent in premature
i) Post-menopausal uterine bleeding due to carcinoma of the infants. Initially, there are usually no clinical abnormalities.
uterus. But subsequently, in addition to features of the underlying
SECTION II
ii) Bleeding from the alimentary tract such as due to carcinoma disorder causing the anaemia, the clinical consequences of
of stomach and large bowel and hiatus hernia. iron deficiency manifest in 2 ways—anaemia itself and
3. ADULT MALES. It is uncommon for adult males to epithelial tissue changes.
develop iron deficiency anaemia in the presence of normal 1. ANAEMIA. The onset of iron deficiency anaemia is
dietary iron content and iron absorption. The vast majority generally slow. The usual symptoms are weakness, fatigue,
of cases of iron deficiency anaemia in adult males are due to dyspnoea on exertion, palpitations and pallor of the skin,
chronic blood loss. The cause for chronic haemorrhage may mucous membranes and sclerae. Older patients may develop
lie at one of the following sites: angina and congestive cardiac failure. Patients may have
i) Gastrointestinal tract is the usual source of bleeding which unusual dietary cravings such as pica. Menorrhagia is a
may be due to peptic ulcer, haemorrhoids, hookworm common symptom in iron deficient women.
infestation, carcinoma of stomach and large bowel,
oesophageal varices, hiatus hernia, chronic aspirin ingestion 2. EPITHELIAL TISSUE CHANGES. Long-standing
chronic iron deficiency anaemia causes epithelial tissue
and ulcerative colitis. Other causes in the GIT are changes in some patients. The changes occur in the nails
malabsorption and following gastrointestinal surgery. (koilonychia or spoon-shaped nails), tongue (atrophic
ii) Urinary tract e.g. due to haematuria and haemoglobinuria. glossitis), mouth (angular stomatitis), and oesophagus
iii) Nose e.g. in repeated epistaxis. causing dysphagia from development of thin, membranous
iv) Lungs e.g. in haemoptysis from various causes. webs at the postcricoid area (Plummer-Vinson syndrome).




Haematology and Lymphoreticular Tissues
























Figure 12.13 Laboratory findings in iron deficiency anaemia.

299





























Figure 12.14 Iron deficiency anaemia. A, PBF showing microcytic hypochromic anaemia. There is moderate microcytosis and hypochromia. CHAPTER 12
B, Examination of bone marrow aspirate showing micronormoblastic erythropoiesis.


Laboratory Findings iii) Reticulocyte count. The reticulocyte count is normal
The development of anaemia progresses in 3 stages: or reduced but may be slightly raised (2-5%) in cases after
Firstly, storage iron depletion occurs during which iron haemorrhage.
reserves are lost without compromise of the iron supply iv) Absolute values. The red cell indices reveal a
for erythropoiesis. diminished MCV (below 50 fl), diminished MCH (below
The next stage is iron deficient erythropoiesis during 15 pg), and diminished MCHC (below 20 g/dl).
which the erythroid iron supply is reduced without the v) Leucocytes. The total and differential white cell counts
development of anaemia. are usually normal.
The final stage is the development of frank iron vi) Platelets. Platelet count is usually normal but may be
deficiency anaemia when the red cells become microcytic slightly to moderately raised in patients who have had
and hypochromic. recent bleeding.
The following laboratory tests can be used to assess the
varying degree of iron deficiency (Fig. 12.13): 2.BONE MARROW FINDINGS. Bone marrow exami-
1.BLOOD PICTURE AND RED CELL INDICES. The nation is not essential in such cases routinely but is done Introduction to Haematopoietic System and Disorders of Erythroid Series
degree of anaemia varies. It is usually mild to moderate in complicated cases so as to distinguish from other
but occasionally it may be marked (haemoglobin less than hypochromic anaemias. The usual findings are as follows
6 g/dl) due to persistent and severe blood loss. The salient (Fig.12.14,B):
haematological findings in these cases are as under. i) Marrow cellularity. The marrow cellularity is increased
i) Haemoglobin. The essential feature is a fall in due to erythroid hyperplasia (myeloid-erythroid ratio
haemoglobin concentration up to a variable degree. decreased).
ii) Red cells. The red cells in the blood film are hypo- ii)Erythropoiesis. There is normoblastic erythropoiesis
chromic and microcytic, and there is anisocytosis and with predominance of small polychromatic normoblasts
poikilocytosis (Fig. 12.14,A). Hypochromia generally (micronormoblasts). These normoblasts have a thin rim
precedes microcytosis. Hypochromia is due to poor filling of cytoplasm around the nucleus and a ragged and
of the red cells with haemoglobin so that there is increased irregular cell border. The cytoplasmic maturation lags behind
central pallor. In severe cases, there may be only a thin so that the late normoblasts have pyknotic nucleus but
rim of pink staining at the periphery. Target cells, elliptical persisting polychromatic cytoplasm (compared from
forms and polychromatic cells are often present. megaloblastic anaemia in which the nuclear maturation
Normoblasts are uncommon. RBC count is below normal lags behind, page 307).
but is generally not proportionate to the fall in
haemoglobin value. When iron deficiency is associated iii) Other cells. Myeloid, lymphoid and megakaryocytic
cells are normal in number and morphology.
with severe folate or vitamin B deficiency, a dimorphic
12
blood picture occurs with dual population of red cells— iv) Marrow iron. Iron staining (Prussian blue reaction)
macrocytic as well as microcytic hypochromic. carried out on bone marrow aspirate smear shows

300 deficient reticuloendothelial iron stores and absence of of iron stores is desired such as in women with severe
siderotic iron granules from developing normoblasts. anaemia a few weeks before expected date of delivery.
Parenteral iron therapy is hazardous and expensive when
3.BIOCHEMICAL FINDINGS. In addition to blood and compared with oral administration. The haematological
bone marrow examination, the following biochemical tests response to parenteral iron therapy is no faster than the
are of value: administration of adequate dose of oral iron but the stores
i) The serum iron level is low (normal 40-140 μg/dl); it is are replenished much faster. Before giving the parenteral
often under 50 μg/dl. When serum iron falls below iron, total dose is calculated by a simple formula by multi-
15 μg/dl, marrow iron stores are absent. plying the grams of haemoglobin below normal with 250
ii) Total iron binding capacity (TIBC) is high (normal 250- (250 mg of elemental iron is required for each gram of deficit
450 μg/dl) and rises to give less than 10% saturation haemoglobin), plus an additional 500 mg is added for
(normal 33%). In anaemia of chronic disorders, however, building up iron stores. A common preparation is iron
serum iron as well as TIBC are reduced. dextran which may be given as a single intramuscular
iii) Serum ferritin is very low (normal 30-250 ng/ml) injection, or as intravenous infusion after dilution with
indicating poor tissue iron stores. The serum ferritin is dextrose or saline. The adverse effects with iron dextran
raised in iron overload and is normal in anaemia of chronic include hypersensitivity or anaphylactoid reactions,
disorders. haemolysis, hypotension, circulatory collapse, vomiting and
iv) Red cell protoporphyrin is very low (normal 20-40 muscle pain. Newer iron complexes such as sodium ferric
μg/dl) as a result of insufficient iron supply to form haem. gluconate and iron sucrose can be administered as repeated
SECTION II
v) Serum transferrin receptor protein which is normally intravenous injections with much lower side effects.
present on developing erythroid cells and reflects total
red cell mass, is raised in iron deficiency due to its release SIDEROBLASTIC ANAEMIA
in circulation (normal level 4-9 μg/L as determined by The sideroblastic anaemias comprise a group of disorders of
immunoassay). diverse etiology in which the nucleated erythroid precursors
in the bone marrow, show characteristic ‘ringed sideroblasts.’
Treatment
Siderocytes and Sideroblasts
The management of iron deficiency anaemia consists of
2 essential principles: correction of disorder causing the Siderocytes and sideroblasts are erythrocytes and
anaemia, and correction of iron deficiency. normoblasts respectively which contain cytoplasmic granules
of iron (Fig.12.15).
1. CORRECTION OF THE DISORDER. The underlying
cause of iron deficiency is established after thorough check- SIDEROCYTES. These are red cells containing granules of
up and investigations. Appropriate surgical, medical or non-haem iron. These granules stain positively with Prussian
preventive measures are instituted to correct the cause of blue reaction as well as stain with Romanowsky dyes when
blood loss. they are referred to as Pappenheimer bodies. Siderocytes are
normally not present in the human peripheral blood but a
2. CORRECTIONOF IRON DEFICIENCY. The lack of small number may appear following splenectomy. This is
iron is corrected with iron therapy as under: because the reticulocytes on release from the marrow are
i) Oral therapy. Iron deficiency responds very effectively finally sequestered in the spleen to become mature red cells.
to the administration of oral iron salts such as ferrous sulfate, In the absence of spleen, the final maturation step takes place
ferrous fumarate, ferrous gluconate and polysaccharide iron. in the peripheral blood and hence siderocytes make their
Haematology and Lymphoreticular Tissues
These preparations have varying amount of elemental iron appearance in the blood after splenectomy.
in each tablet ranging from 39 mg to 105 mg. Optimal SIDEROBLASTS. These are nucleated red cells (normo-
absorption is obtained by giving iron fasting, but if side- blasts) containing siderotic granules which stain positively
effects occur (e.g. nausea, abdominal discomfort, diarrhoea)
iron can be given with food or by using a preparation of lower
iron content (e.g. ferrous gluconate containing 39 mg
elemental iron). Oral iron therapy is continued long enough,
both to correct the anaemia and to replenish the body iron
stores. The response to oral iron therapy is observed by
reticulocytosis which begins to appear in 3-4 days with a
peak in about 10 days. Poor response to iron replacement
may occur from various causes such as: incorrect diagnosis,
non-compliance, continuing blood loss, bone marrow
suppression by tumour or chronic inflammation, and
malabsorption.
ii) Parenteral therapy. Parenteral iron therapy is indicated
in cases who are intolerant to oral iron therapy, in GIT Figure 12.15 A siderocyte containing Pappenheimer bodies, a
disorders such as malabsorption, or a rapid replenishment normal sideroblast and a ring sideroblast.

spontaneously in middle-aged and older individuals of both 301
sexes. The disorder has its pathogenesis in disturbed growth
and maturation of erythroid precursors at the level of
haematopoietic stem cell, possibly due to reduced activity
of the enzyme, ALA synthetase. The anaemia is of moderate
to severe degree and appears insidiously. The bone marrow
cells commonly show chromosomal abnormalities, neutro-
penia and thrombocytopenia with associated bleeding
diathesis. The spleen and liver may be either normal or mildly
enlarged, while the lymph nodes are not enlarged. Unlike
other types of sideroblastic anaemia, this type is regarded as
a myelodysplastic disorder in the FAB (French-American-
British) classification and thus, can be a preleukaemic
disorder (page 361). About 10% of individuals with refractory
acquired sideroblastic anaemia develop acute myelogenous
leukaemia. CHAPTER 12
B. Secondary acquired sideroblastic anaemia. Acquired
Figure 12.16 Sideroblastic anaemia bone marrow aspirate smear sideroblastic anaemia may develop secondary to a variety
in Perls’ stain shows marked excess of reticular iron and a ringed of drugs, chemicals, toxins, haematological and various other
sideroblast (arrow) showing Prusian blue granules in the cytoplasm.
diseases.
with Prussian blue reaction. Depending upon the number, 1. Drugs, chemicals and toxins: Isoniazid, an anti-tuberculous
size and distribution of siderotic granules, sideroblasts may drug and a pyridoxine antagonist, is most commonly
be normal or abnormal (Fig. 12.16). associated with development of sideroblastic anaemia by
Normal sideroblasts contain a few fine, scattered cytoplasmic producing abnormalities in pyridoxine metabolism. Other
granules representing iron which has not been utilised for drugs occasionally causing acquired sideroblastic anaemia
haemoglobin synthesis. These cells comprise 30-50% of are: cycloserine, chloramphenicol and alkylating agents (e.g.
normoblasts in the normal marrow but are reduced or absent cyclophosphamide). Alcohol and lead also cause
in iron deficiency. sideroblastic anaemia. All these agents cause reversible
sideroblastic anaemia which usually resolves following
Abnormal sideroblasts are further of 2 types: removal of the offending agent.
One type is a sideroblast containing numerous, diffusely
scattered, coarse cytoplasmic granules and are seen in 2. Haematological disorders: These include myelofibrosis,
conditions such as dyserythropoiesis and haemolysis. In this polycythaemia vera, acute leukaemia, myeloma, lymphoma
type, there is no defect of haem or globin synthesis but the and haemolytic anaemia.
percentage saturation of transferrin is increased. 3. Miscellaneous: Occasionally, secondary sideroblastic
The other type is ringed sideroblast in which haem anaemia may occur in association with a variety of inflam-
synthesis is disturbed as occurs in sideroblastic anaemias. matory, neoplastic and autoimmune diseases such as
Ringed sideroblasts contain numerous large granules, often carcinoma, myxoedema, rheumatoid arthritis and SLE. Introduction to Haematopoietic System and Disorders of Erythroid Series
forming a complete or partial ring around the nucleus. The
ringed arrangement of these granules is due to the presence Laboratory Findings
of iron-laden mitochondria around the nucleus. Sideroblastic anaemias usually show the following
haematological features:
Types of Sideroblastic Anaemias
1. There is generally moderate to severe degree of anaemia.
Based on etiology, sideroblastic anaemias are classified into
hereditary and acquired types. The acquired type is further 2.The blood picture shows hypochromic anaemia which
divided into primary and secondary forms: may be microcytic, or there may be some normocytic red
cells as well (dimorphic).
I. HEREDITARY SIDEROBLASTIC ANAEMIA. This is 3. Absolute values (MCV, MCH and MCHC) are reduced
a rare X-linked disorder associated with defective enzyme in hereditary type but MCV is often raised in acquired
activity of aminolevulinic acid (ALA) synthetase required for type.
haem synthesis. The affected males have moderate to marked
anaemia while the females are carriers of the disorder and 4. Bone marrow examination shows erythroid hyperplasia
do not develop anaemia. The condition manifests in with usually macronormoblastic erythropoiesis. Marrow
childhood or in early adult life. iron stores are raised and pathognomonic ring sideroblasts
are present.
II. ACQUIRED SIDEROBLASTIC ANAEMIA. The
acquired sideroblastic anaemias are classified into primary 5. Serum ferritin levels are raised.
and secondary types. 6. Serum iron is usually raised with almost complete
saturation of TIBC.
A. Primary acquired sideroblastic anaemia. Primary, idio-
pathic, or refractory acquired sideroblastic anaemia occurs 7. There is increased iron deposition in the tissue.

302
  TABLE 12.5: Laboratory Diagnosis of Hypochromic Anaemias.
Test Iron Deficiency Chronic Disorders Thalassaemia Sideroblastic Anaemia
1. MCV, MCH, MCHC Reduced Low normal-to-reduced Very low Very low
(except MCV raised in aquired type)
2. Serum iron Reduced Reduced Normal Raised
3. TIBC Raised Low-to-normal Normal Normal
4. Serum ferritin Reduced Raised Normal Raised
(complete saturation)
5. Marrow-iron stores Absent Present High High
6. Iron in normoblasts Absent Absent Present Ring sideroblasts
7. Hb electrophoresis Normal Normal Abnormal Normal



Treatment tions, the anaemia is complicated by other causes such as
iron, B and folate deficiency, hypersplenism, renal failure
12
The treatment of secondary sideroblastic anaemia is with consequent reduced erythropoietic activity, endocrine
primarily focussed on removal of the offending agent. No abnormalities etc. However, in general, 2 factors appear to
definite treatment is available for hereditary and idiopathic play significant role in the pathogenesis of anaemia in chronic
SECTION II
types of sideroblastic anaemias. However, pyridoxine is
administered routinely to all cases of sideroblastic anaemia disorders. These are: defective red cell production and reduced
(200 mg per day for 2-3 months). Blood transfusions and other red cell lifespan.
supportive therapy are indicated in all patients. 1. Defective red cell production. Though there is abun-
Differential diagnosis of various types of hypochromic dance of storage iron in these conditions but the amount of
anaemias by laboratory tests is summarised in Table 12.5. iron available to developing erythroid cells in the marrow is
subnormal. The mononuclear phagocyte system is
ANAEMIA OF CHRONIC DISORDERS hyperplastic which traps all the available free iron due to
the activity of iron binding protein, lactoferrin. A defect in
One of the commonly encountered anaemia is in patients of the transfer of iron from macrophages to the developing
a variety of chronic systemic diseases in which anaemia erythroid cells in the marrow leads to reduced availability
develops secondary to a disease process but there is no actual of iron for haem synthesis despite adequate iron stores,
invasion of the bone marrow. A list of such chronic systemic elevating serum ferritin levels. The defect lies in suppression
diseases is given in Table 12.6. In general, anaemia in chronic of erythropoieitn by inflammatory cytokines at some stage
disorders is usually normocytic normochromic but can have in erythropoiesis, and hepcidin which is the key iron
mild degree of microcytosis and hypochromia unrelated to regulatory hormone. These inflammatory cytokines include
iron deficiency. The severity of anaemia is usually directly TNF and IFN-β released in bacterial infections and tumours,
related to the primary disease process. The anaemia is and IL-1 and IFN-γ released in patients of rheumatoid
corrected only if the primary disease is alleviated.
arthritis and autoimmune vasculitis (Fig. 12.17).
Pathogenesis 2. Reduced red cell lifespan. Decreased survival of
circulating red cells in chronic renal disease is attributed to
A number of factors may contribute to the development of hyperplastic mononuclear phagocyte system.
Haematology and Lymphoreticular Tissues
anaemia in chronic systemic disorders, and in many condi-
Laboratory Findings
 TABLE 12.6: Anaemias Secondary to Chronic Systemic
Disorders. The characteristic features of anaemia in these patients
1. ANAEMIA IN CHRONIC INFECTIONS/INFLAMMATION uncomplicated by other deficiencies are as under:
a. Infections e.g. tuberculosis, lung abscess, pneumonia, osteomyelitis, i) Haemoglobin. Anaemia is generally mild to moderate.
subacute bacterial endocarditis, pyelonephritis. A haemoglobin value of less than 8 g/dl suggests the
b. Non-infectious inflammations e.g. rheumatoid arthritis, SLE, presence of additional contributory factors.
vasculitis, dermatomyositis, scleroderma, sarcoidosis, Crohn’s
disease. ii) Blood picture. The type of anaemia in these cases is
c. Disseminated malignancies e.g. Hodgkin’s disease, disseminated generally normocytic normochromic but may have slight
carcinomas and sarcomas. microcytosis and hypochromia.
2. ANAEMIA OF RENAL DISEASE e.g. iii) Absolute values. Red cell indices indicate that in spite
uraemia, renal failure of normocytic normochromic anaemia, MCHC is slightly
3. ANAEMIA OF HYPOMETABOLIC STATE e.g. low.
endocrinopathies (myxoedema, Addison's disease, hyperthyroidism,
hypopituitarism, Addison’s disease), protein malnutrition, scurvy and iv) Reticulocyte count. The reticulocyte count is generally
pregnancy, liver disease. low.

macrophages. These proteins include γ-globulin, C3, 303
haptoglobin, α -antitrypsin and fibrinogen. Elevation of
1
these proteins is responsible for raised ESR commonly
present in these patients.

MEGALOBLASTIC ANAEMIA

The megaloblastic anaemias are disorders caused by
impaired DNA synthesis and are characterised by a distinc-
tive abnormality in the haematopoietic precursors in the bone
marrow in which the maturation of the nucleus is delayed relative
to that of the cytoplasm. Since cell division is slow but
cytoplasmic development progresses normally, the nucleated
red cell precursors tend to be larger which Ehrlich in 1880
termed megaloblasts. Megaloblasts are both morphologically
and functionally abnormal with the result that the mature CHAPTER 12
red cells formed from them and released into the peripheral
blood are also abnormal in shape and size, the most
prominent abnormality being macrocytosis.
The underlying defect for the asynchronous maturation
of the nucleus is defective DNA synthesis due to deficiency
of vitamin B (cobalamin) and/or folic acid (folate). Less
12
Figure 12.17 Pathogenesis of anaemia of chronic disorders through common causes are interference with DNA synthesis by
suppression of erythropoiesis by cytokines.
congenital or acquired abnormalities of vitamin B or folic
12
acid metabolism. Before considering the megaloblastic
v) Red cell survival. Measurement of erythrocyte survival anaemia, an outline of vitamin B and folic acid metabolism
12
generally reveals mild to moderate shortening of their is given for a better understanding of the subject.
lifespan. The salient nutritional aspects and metabolic functions
vi) Bone marrow. Examination of the marrow generally of vitamin B and folic acid are summarised in Table12.7.
12
reveals normal erythroid maturation. However, the red
cell precursors have reduced stainable iron than normal, Vitamin B Metabolism
12
while macrophages in the marrow usually contain BIOCHEMISTRY. Vitamin B or cobalamin is a complex
12
increased amount of iron. Cases of chronic infection often organometallic compound having a cobalt atom situated
have myeloid hyperplasia and increase in plasma cells. within a corrin ring, similar to the structure of porphyrin
vii) Serum iron and TIBC. Serum iron is characteristically from which haem is formed. In humans, there are
reduced in this group of anaemias while TIBC is low-to- 2 metabolically active forms of cobalamin—methyl-
normal (in contrast to iron deficiency where there is cobalamin and adenosyl-cobalamin, which act as
reduction in serum iron but high TIBC, see Table12.5). coenzymes. The therapeutic vitamin B preparation is Introduction to Haematopoietic System and Disorders of Erythroid Series
12
viii) Serum ferritin. Serum ferritin levels are increased in called cyanocobalamin.
these patients and is the most distinguishing feature SOURCES. The only dietary sources of vitamin B are foods
12
between true iron-deficiency anaemia and iron-deficient of animal protein origin such as kidney, liver, heart, muscle
erythropoieisis in anemia of chronic diseases. meats, fish, eggs, cheese and milk. In contrast to folate, fruits
ix)Other plasma proteins. In addition, certain other and vegetables contain practically no vitamin B unless
12
plasma proteins called ‘phase reactants’ are raised in contaminated with bacteria. Cooking has little effect on its
patients with chronic inflammation, probably under the activity. Vitamin B is synthesised in the human large bowel
12
stimulus of interleukin-1 released by activated by microorganisms but is not absorbed from this site and,


 TABLE 12.7: Salient Features of Vitamin B 12 and Folate Metabolism.
Feature Vitamin B 12 Folate
1. Main foods Animal proteins only Green vegetables, meats
2. Cooking Little effect Easily destroyed
3. Daily requirements 2-4 μg 100-200 μg
4. Daily intake 5-30 μg 100-500 μg
5. Site of absorption Ileum Duodenum and jejunum
6. Mechanism of absorption Intrinsic factor Conversion to methyl-THF
7. Body stores 2-3 mg (enough for 2-4 yrs) 10-12 mg (enough for 4 months)

304 thus, the humans are entirely dependent upon dietary
sources. The average daily requirement for vitamin B is
12
2-4 μg.
ABSORPTION. After ingestion, vitamin B in food is
12
released and forms a stable complex with gastric R-binder.
R-binder is a form of glycoprotein found in various secretions
(e.g. saliva, milk, gastric juice, bile), phagocytes and plasma.
On entering the duodenum, the vitamin B -R-binder
12
complex is digested releasing vitamin B which then binds
12
to intrinsic factor (IF). The IF is a glycoprotein of molecular
weight 50,000 produced by the parietal cells of the stomach
and its secretion roughly parallels that of hydrochloric acid.
The vitamin B -IF complex, on reaching the distal ileum,
12
binds to the specific receptors on the mucosal brush border,
thereby enabling the vitamin to be absorbed. The IF,
therefore, acts as cell-directed carrier protein similar to
transferrin. The receptor-bound vitamin B -IF complex is
12
taken into the ileal mucosal cells where after several hours
the IF is destroyed, vitamin B released and is transferred Figure 12.18 Biochemical basis of megaloblastic anaemia (THF =
SECTION II
12
to another transport protein, transcobalamin (TC) II. The tetrahydrofolate; DHF = dihydrofolate; PGA = pteroyl glutamic acid; dUMP
= deoxy uridylate monophosphate; dTMP = deoxy thymidylate monophos-
vitamin B -TC II complex is finally secreted into the portal phate).
12
circulation from where it is taken by the liver, bone marrow
and other cells. There are 2 major vitamin B binding Lack of adenosyl B leads to large increase in the level
12
12
proteins—TC I and TC II, and a minor protein TC III. TC I is of methyl malonyl CoA and its precursor, propionyl CoA.
not essential for vitamin B transport but functions primarily This results in synthesis of certain fatty acids which are
12
as a storage protein while TC III is similar to TC II and binds incorporated into the neuronal lipids. This biochemical
a small amount of vitamin B (see Fig. 12.12,B). abnormality may contribute to the neurologic complications
12
TISSUE STORES. Normally, the liver is the principal storage of vitamin B deficiency.
12
site of vitamin B and stores about 2 mg of the vitamin, while
12
other tissues like kidney, heart and brain together store about Folate Metabolism
2 mg. The body stores of vitamin B are adequate for 2-4
12
years. Major source of loss is via bile and shedding of BIOCHEMISTRY. Folate or folic acid, a yellow compound,
intestinal epithelial cells. A major part of the excreted vitamin is a member of water-soluble B complex vitamins with the
B is reabsorbed in the ileum by the IF resulting in chemical name of pteroyl glutamic acid (PGA). Folic acid does
12
enterohepatic circulation. not exist as such in nature but exists as folates in
polyglutamate form (conjugated folates). For its metabolic
FUNCTIONS. Vitamin B plays an important role in general action as co-enzyme, polyglutamates must be reduced to
12
cell metabolism, particulary essential for normal dihydro- and tetrahydrofolate forms.
haematopoiesis and for maintenance of integrity of the
nervous system. Vitamin B acts as a co-enzyme for 2 main SOURCES. Folate exists in different plants, bacteria and
12
biochemical reactions in the body: animal tissues. Its main dietary sources are fresh green leafy
Haematology and Lymphoreticular Tissues
Firstly, as methyl cobalamin (methyl B ) in the methylation vegetables, fruits, liver, kidney, and to a lesser extent, muscle
12
of homocysteine to methionine by methyl tetrahydrofolate meats, cereals and milk. Folate is labile and is largely
destroyed by cooking and canning. Some amount of folate
(THF). The homocysteine-methionine reaction is closely synthesised by bacteria in the human large bowel is not
linked to folate metabolism (Fig. 12.18):
available to the body because its absorption takes place in
the small intestine. Thus, humans are mainly dependent
Methyl B
Homocysteine 12 Methionine upon diet for its supply. The average daily requirement is
100-200 μg.
When this reaction is impaired, folate metabolism is ABSORPTION AND TRANSPORT. Folate is normally
deranged and results in defective DNA synthesis responsible absorbed from the duodenum and upper jejunum and to a
for megaloblastic maturation. lesser extent, from the lower jejunum and ileum. However,
Secondly, as adenosyl cobalamin (adenosyl B ) in propionate absorption depends upon the form of folate in the diet.
12
metabolism for the conversion of methyl malonyl co-enzyme Polyglutamate form in the foodstuffs is first cleaved by the
A to succinyl co-enzyme A: enzyme, folate conjugase, in the mucosal cells to mono- and
diglutamates which are readily assimilated. Synthetic folic
Adenosyl B 12 acid preparations in polyglutamate form are also absorbed
Propionyl CoA → Methyl malonyl CoA → Succinyl CoA as rapidly as mono- and diglutamate form because of the

absence of natural inhibitors. Mono- and diglutamates with the synthesis of DNA. Deficiency of vitamin B traps 305
12
undergo further reduction in the mucosal cells to form folate as its transport form, methyl-THF, thereby resulting
tetrahydrofolate (THF), a monoglutamate. THF circulates in in reduced formation of the active form, methylene-THF,
the plasma as methylated compound, methyl THF, bound needed for DNA synthesis. This is referred to as methyl-folate
to a protein. Once methyl THF is transported into the cell by trap hypothesis. An alternative hypothesis of inter-relationship
a carrier protein, it is reconverted to polyglutamate (see of B and folate is the formate-saturation hypothesis. According
12
Fig. 12.12,C). to this hypothesis, the active substrate is formyl-THF.
Vitamin B deficiency results in reduced supply of formate
12
TISSUE STORES. The liver and red cells are the main storage to THF causing reduced generation of the active compound,
sites of folate, largely as methyl THF polyglutamate form. formyl THF.
The total body stores of folate are about 10-12 mg enough
for about 4 months. Normally, folate is lost from the sweat, Etiology and Classification of Megaloblastic Anaemia
saliva, urine and faeces.
FUNCTIONS. Folate plays an essential role in cellular The etiology of megaloblastic anaemia varies in different
metabolism. It acts as a co-enzyme for 2 important bio- parts of the world. As outlined in Table 12.8,megaloblastic
chemical reactions involving transfer of 1-carbon units (viz. anaemia is classified into 3 broad groups: vitamin B 12
methyl and formyl groups) to various other compounds. deficiency, folate deficiency, and deficiency from other CHAPTER 12
These reactions are as under: causes.
Thymidylate synthetase reaction. Formation of deoxy 1. VITAMIN B DEFICIENCY. In Western countries,
12
thymidylate monophosphate (dTMP) from its precursor deficiency of vitamin B is more commonly due to pernicious
12
form, deoxy uridylate monophosphate (dUMP). (Addisonian) anaemia. True vegetarians like traditional
Methylation of homocysteine to methionine. This reaction is Indian Hindus and breast-fed infants have dietary lack of
linked to vitamin B metabolism (Fig. 12.18). vitamin B . Gastrectomy by lack of intrinsic factor, and small
12
12
These biochemical reactions are considered in detail intestinal lesions involving distal ileum where absorption of
below together with biochemical basis of the megaloblastic vitamin B occurs, may cause deficiency of the vitamin.
12
anaemia. Deficiency of vitamin B takes at least 2 years to develop
12
when the body stores are totally depleted.
Biochemical Basis of Megaloblastic Anaemia
2. FOLATE DEFICIENCY. Folate deficiency is more often
The basic biochemical abnormality common to both vitamin due to poor dietary intake. Other causes include
B and folate deficiency is a block in the pathway of DNA
12
synthesis and that there is an inter-relationship between
vitamin B and folate metabolism in the methylation reaction  TABLE 12.8: Etiologic Classification of Megaloblastic
12
of homocysteine to methionine (Fig. 12.18). Anaemia.
As stated above, folate as co-enzyme methylene THF, is I. VITAMIN B 12 DEFICIENCY
required for transfer of 1-carbon moieties (e.g. methyl and A. Inadequate dietary intake e.g. strict vegetarians, breast-fed infants.
formyl) to form building blocks in DNA synthesis. These
1-carbon moieties are derived from serine or formimino- B. Malabsorption
glutamic acid (FIGLU). Two of the important folate- 1. Gastric causes: pernicious anaemia, gastrectomy, congenital lack Introduction to Haematopoietic System and Disorders of Erythroid Series
dependent (1-carbon transfer) reactions for formation of of intrinsic factor.
building blocks in DNA synthesis are as under: 2. Intestinal causes: tropical sprue, ileal resection, Crohn’s disease,
intestinal blind loop syndrome, fish-tapeworm infestation.
1. Thymidylate synthetase reaction. This reaction involves II. FOLATE DEFICIENCY
synthesis of deoxy thymidylate monophosphate (dTMP)
from deoxy uridylate monophosphate (dUMP). The methyl A. Inadequate dietary intake e.g. in alcoholics, teenagers, infants, old
age, poverty.
group of dUMP → dTMP reaction is supplied by the co-
enzyme, methylene-THF. After the transfer of 1-carbon from B. Malabsorption e.g. in tropical sprue, coeliac disease, partial
methylene-THF, dihydrofolate (DHF) is produced which gastrectomy, jejunal resection, Crohn’s disease.
must be reduced to active THF by the enzyme DHF-reductase C. Excess demand
before it can participate in further 1-carbon transfer reaction. 1. Physiological: pregnancy, lactation, infancy.
Drugs like methotrexate (anti-cancer) and pyrimethamine 2. Pathological : malignancy, increased haematopoiesis, chronic
(antimalarial) are inhibitory to the enzyme, DHF-reductase, exfoliative skin disorders, tuberculosis, rheumatoid arthritis.
thereby inhibiting the DNA synthesis. D. Excess urinary folate loss e.g. in active liver disease, congestive
heart failure.
2. Homocysteine-methionine reaction. Homocysteine is
converted into methionine by transfer of a methyl group from III. OTHER CAUSES
methylene-THF. After transfer of 1-carbon from methylene- A. Impaired metabolism e.g. inhibitors of dihydrofolate (DHF) reductase
THF, THF is produced. This reaction requires the presence such as methotrexate and pyrimethamine; alcohol, congenital
of vitamin B (methyl-B ). enzyme deficiencies.
12
12
Deficiency of folate from any cause results in reduced B. Unknown etiology e.g. in Di Guglielmo’s syndrome, congenital
supply of the coenzyme, methylene-THF, and thus interferes dyserythropoietic anaemia, refractory megaloblastic anaemia.

306 malabsorption, excess folate utilisation such as in pregnancy 4. Others. In addition to the cardinal features mentioned
and in various disease states, chronic alcoholism, and excess above, patients may have various other symptoms. These
urinary folate loss. Folate deficiency arises more rapidly than include: mild jaundice, angular stomatitis, purpura, melanin
vitamin B deficiency since the body’s stores of folate are pigmentation, symptoms of malabsorption, weight loss and
12
relatively low which can last for up to 4 months only. anorexia.
Patients with tropical sprue are often deficient in both
vitamin B and folate. Combined deficiency of vitamin B 12 Laboratory Findings
12
and folate may occur from severe deficiency of vitamin B 12 The investigations of a suspected case of megaloblastic
because of the biochemical interrelationship with folate anaemia are aimed at 2 aspects:
metabolism. A. General laboratory investigations of anaemia which include
3. OTHER CAUSES. In addition to deficiency of vitamin blood picture, red cell indices, bone marrow findings, and
B and folate, megaloblastic anaemias may occasionally be biochemical tests.
12
induced by other factors unrelated to vitamin deficiency. B. Special tests to establish the cause of megaloblastic anaemia
These include many drugs which interfere with DNA as to know whether it is due to deficiency of vitamin B 12
synthesis, acquired defects of haematopoietic stem cells, and or folate.
rarely, congenital enzyme deficiencies. Based on these principles, the following scheme of
investigations is followed:
Clinical Features
A. General Laboratory Findings
Deficiency of vitamin B and folate may cause following
SECTION II
12
clinical manifestations which may be present singly or in 1. BLOOD PICTURE AND RED CELL INDICES. Esti-
combination and in varying severity: mation of haemoglobin, examination of a blood film and
1. Anaemia. Macrocytic megaloblastic anaemia is the evaluation of absolute values are essential preliminary
cardinal feature of deficiency of vitamin B and/or folate. investigations (Fig. 12.19):
12
The onset of anaemia is usually insidious and gradually i) Haemoglobin. Haemoglobin estimation reveals values
progressive. below the normal range. The fall in haemoglobin
2. Glossitis. Typically, the patient has a smooth, beefy, red concentration may be of a variable degree.
tongue. ii) Red cells. Red blood cell morphology in a blood film
3. Neurologic manifestations. Vitamin B deficiency, parti- shows the characteristic macrocytosis. However,
12
cularly in patients of pernicious anaemia, is associated with macrocytosis can also be seen in several other disorders
significant neurological manifestations in the form of such as: haemolysis, liver disease, chronic alcoholism,
subacute combined, degeneration of the spinal cord and hypothyroidism, aplastic anaemia, myeloproliferative
peripheral neuropathy (Chapter 30), while folate deficiency disorders and reticulocytosis. In addition, the blood smear
may occasionally develop neuropathy only. The underlying demonstrates marked anisocytosis, poikilocytosis and
pathologic process consists of demyelination of the peripheral presence of macroovalocytes. Basophilic stippling and
nerves, the spinal cord and the cerebrum. Signs and occasional normoblast may also be seen (Fig. 12.20, A).
symptoms include numbness, paraesthesia, weakness, iii) Reticulocyte count. The reticulocyte count is generally
ataxia, poor finger coordination and diminished reflexes. low to normal in untreated cases.



Haematology and Lymphoreticular Tissues
























Figure 12.19 General laboratory findings in megaloblastic anaemia.

307






























Figure 12.20 Megaloblastic anaemia. A, PBF showing prominent macrocytosis of red cells and hypersegmented neutrophils. B, Examination CHAPTER 12
of bone marrow aspirate showing megaloblastic erythropoiesis.


iv) Absolute values. The red cell indices reveal an elevated iii) Other cells. Granulocyte precursors are also affected
MCV (above 120 fl) proportionate to the severity of to some extent. Giant forms of metamyelocytes and band
macrocytosis, elevated MCH (above 50 pg) and normal cells may be present in the marrow. Megakaryocytes are
or reduced MCHC. usually present in normal number but may occasionally
v)Leucocytes. The total white blood cell count may be be decreased and show abnormal morphology such as
reduced. Presence of characteristic hypersegmented hypersegmented nuclei and agranular cytoplasm.
neutrophils (having more than 5 nuclear lobes) in the iv)Marrow iron. Prussian blue staining for iron in the
blood film should raise the suspicion of megaloblastic marrow shows an increase in the number and size of iron
anaemia. An occasional myelocyte may also be seen. granules in the erythroid precursors. Ring sideroblasts are,
vi)Platelets. Platelet count may be moderately reduced however, rare. Iron in the reticulum cells is increased.
in severely anaemic patients. Bizarre forms of platelets v)Chromosomes. Marrow cells may show variety of
may be seen. random chromosomal abnormalities such as chromosome
2. BONE MARROW FINDINGS. The bone marrow breaks, centromere spreading etc. Introduction to Haematopoietic System and Disorders of Erythroid Series
examination is very helpful in the diagnosis of 3.BIOCHEMICAL FINDINGS. In addition to the general
megaloblastic anaemia. Significant findings of marrow blood and marrow investigations and specific tests to
examination are as under (Fig. 12.20,B):
determine the cause of deficiency (described below), the
i) Marrow cellularity. The marrow is hypercellular with following biochemical abnormalities are observed in cases
a decreased myeloid-erythroid ratio. of megaloblastic anaemia:
ii) Erythropoiesis. Erythroid hyperplasia is due to i) There is rise in serum unconjugated bilirubin and LDH as
characteristic megaloblastic erythropoiesis. Megaloblasts a result of ineffective erythropoiesis causing marrow cell
are abnormal, large, nucleated erythroid precursors, breakdown.
having nuclear-cytoplasmic asynchrony i.e. the nuclei are ii) The serum iron and ferritin may be normal or elevated.
less mature than the development of cytoplasm. The nuclei
are large, having fine, reticular and open chromatin that B. Special Tests for Cause of Specific Deficiency
stains lightly, while the haemoglobinisation of the
cytoplasm proceeds normally or at a faster rate i.e. nuclear In evaluating a patient of megaloblastic anaemia, it is
maturation lags behind that of cytoplasm (compared from iron important to determine the specific vitamin deficiency by
deficiency anaemia in which cytoplasmic maturation lags assay of vitamin B and folate. In sophisticated clinical
12
behind, page 299). Megaloblasts with abnormal mitoses laboratories, currently automated multiparametric,
may be seen. Features of ineffective erythropoiesis such random access analysers are employed based on separa-
as presence of degenerated erythroid precursors may be tion techniques by chemiluminescence and enzyme-linked
present. fluorescence detection systems which have largely

308
replaced the traditional microbiologic assays for vitamin If the 24-hour urinary excretion of ‘hot’ B is now
12
B and folate. Traditional tests are briefly described below. normal, the low value in first stage of the test was due to
12
IF deficiency (i.e. pernicious anaemia).
TESTS FOR VITAMIN B 12 DEFICIENCY. The normal
range of vitamin B in serum is 280-1000 pg/ml. Values Patients with pernicious anaemia have abnormal test
12
less than 100 pg/ml indicate clinically deficient stage. even after treatment with vitamin B due to IF deficiency.
12
Traditional tests employed to establish vitamin B 12 However, abnormal 24-hour urinary excretion of ‘hot’ B 12
deficiency are serum vitamin B assay, Schilling (24-hour is further investigated in stage III for a cause in intestinal
12
12
urinary excretion) test and serum enzyme levels. malabsorption of ‘hot’ B’ .
Stage III: Test for malabsorption of vitamin B . Some
1. SERUM VITAMIN B ASSAY. Assay of vitamin B in 12
12
12
12
blood can be done by 2 methods—microbiological assay patients absorb vitamin B in water as was stipulated in
and radioassay. the original Schilling test. Modified Schilling test employs
the use of protein-bound vitamin B . In conditions
12
i) Microbiological assay. In this test, the serum sample to causing malabsorption, the test is repeated after a course
be assayed is added to a medium containing all other of treatment with antibiotics or anti-inflammatory drugs.
essential growth factors required for a vitamin
B -dependent microorganism. The medium along with 3. SERUM ENZYME LEVELS. Besides Schilling test,
12
microorganism is incubated and the amount of vitamin another way of distinguishing whether megaloblastic
B is determined turbimetrically which is then compared anaemia is due to cobalamine or folate is by serum
12
SECTION II
with the growth produced by a known amount of vitamin determination of methylmalonic acid and homocysteine
B . Several organisms have been used for this test such by sophisticated enzymatic assays. Both are elevated in
12
cobalamine deficiency, while in folate deficiency there is
as Euglena gracilis, Lactobacillus leichmannii, Escherichia coli only elevation of homocysteine and not of methylmalonic
and Ochromonas malhamensis. E. gracilis is, however, consi-
dered more sensitive and accurate. The addition of anti- acid.
biotics to the test interferes with the growth and yields TESTS FOR FOLATE DEFICIENCY. The normal range
false low result. of serum folate is 6-18 ng/ml. Values of 4 ng/ml or less
are generally considered to be diagnostic of folate
ii) Radioassay. Assays of serum B by radioisotope
12
dilution (RID) and radioimmunoassay (RIA) have been deficiency. Measurement of formiminoglutamic acid
(FIGLU) urinary excretion after histidine load was used
developed. These tests are more sensitive and have the formerly for assessing folate status but it is less specific
advantage over microbiologic assays in that they are and less sensitive than the serum assays. Currently, there
simpler and more rapid, and the results are unaffected by are 3 tests used to detect folate deficiency—urinary
antibiotics and other drugs which may affect the living excretion of FIGLU, serum and red cell folate assay.
organisms.
1. URINARY EXCRETION OF FIGLU. Folic acid is
2. SCHILLING TEST (24 HOUR URINARY EXCRETION required for conversion of formiminoglutamic acid
TEST). Schilling test is done to detect vitamin B 12 (FIGLU) to glutamic acid in the catabolism of histidine.
deficiency as well as to distinguish and detect lack of IF Thus, on oral administration of histidine, urinary excretion
and malabsorption syndrome. The results of test also of FIGLU is increased if folate deficiency is present.
depend upon good renal function and proper urinary 2. SERUM FOLATE ASSAY. The folate in serum can be
58
collection. Radioisotope used for labeling B is either Co estimated by 2 methods—microbiological assay and
12
57
or Co. The test is performed in 3 stages as under:
radioassay.
Haematology and Lymphoreticular Tissues
Stage I: Without IF. The patient after an overnight fasting i) Microbiological assay. This test is based on the principle
is administered oral dose of 1 μg of radioactively labelled that the serum folate acid activity is mainly due to the
vitamin B (‘hot’ B ) in 200 ml of water. At the same presence of a folic acid co-enzyme, 5-methyl THF, and
12
12
time, 1 mg of unlabelled vitamin B (‘cold’ B ) is given that this compound is required for growth of the
12
12
by intramuscular route; this ‘cold’ B will saturate the microorganism, Lactobacillus casei. The growth of L. casei
12
serum as well as the tissue binding sites. The patient is is inhibited by addition of antibiotics.
kept fasting for a further period of 2 hours, following ii) Radioassay. The principle and method of radioassay
which urinary excretion of B is estimated: by radioisotope dilution (RID) test are similar to that for
12
In normal individuals, 24-hour urinary excretion is serum B assay. The test employs labelled pteroyl-
>10% of the oral dose of ‘hot’ B . 12
12 glutamic acid or methyl-THF. Commercial kits are
Patients with IF deficiency excrete lower quantity of available which permit simultaneous assay of both
‘hot’ B which is further confirmed by repeating the test vitamin B and folate.
12
as in stage II given below. 12
3. RED CELL FOLATE ASSAY. Red cells contain 20-50
Stage II: With IF. If the 24-hour urinary excretion of ‘hot’ times more folate than the serum; thus red cell folate assay
B is low, the test is repeated using the same procedure is more reliable indicator of tissue stores of folate than
12
as in stage I but in addition high oral dose of IF is serum folate assay. Microbiological radioassay and
administered along with ‘hot’ B . protein-binding assay methods can be used for estimation
12

of red cell folate. Red cell folate values are decreased in MORPHOLOGIC FEATURES. The most characteristic 309
patients with megaloblastic anaemia as well as in patients pathologic finding in PA is gastric atrophy affecting the
with pernicious anaemia. acid- and pepsin-secreting portion of the stomach and
sparing the antrum (Chapter 20). Gastric epithelium may
Treatment show cellular atypia. About 2-3% cases of PA develop
carcinoma of the stomach. Other pathologic changes are
Most cases of megaloblastic anaemia need therapy with secondary to vitamin B deficiency and include
12
appropriate vitamin. This includes: hydroxycobalamin as megaloblastoid alterations in the gastric and intestinal
intramuscular injection 1000 μg for 3 weeks and oral folic epithelium and neurologic abnormalities such as
acid 5 mg tablets daily for 4 months. Severely-anaemic peripheral neuropathy and spinal cord damage.
patients in whom a definite deficiency of either vitamin
cannot be established with certainty are treated with both Clinical Features
vitamins concurrently. Blood transfusion should be avoided
since it may cause circulatory overload. Packed cells may, The disease has insidious onset and progresses slowly. The
however, be infused slowly. clinical manifestations are mainly due to vitamin B 12
Treatment of megaloblastic anaemia is quite gratifying. deficiency. These include: anaemia, glossitis, neurological
The marrow begins to revert back to normal morphology abnormalities (neuropathy, subacute combined degeneration CHAPTER 12
within a few hours of initiating treatment and becomes of the spinal cord, retrobulbar neuritis), gastrointestinal
manifestations (diarrhoea, anorexia, weight loss, dyspepsia),
normoblastic within 48 hours of start of treatment. Reticulo- hepatosplenomegaly, congestive heart failure and
cytosis appears within 4-5 days after therapy is started and haemorrhagic manifestations. Other autoimmune diseases
peaks at day 7. Haemoglobin should rise by 2-3 g/dl each such as autoimmune thyroiditis may be associated.
fortnight. The peripheral neuropathy may show some
improvement but subacute combined degeneration of the Diagnostic Criteria
spinal cord is irreversible.
Since diagnosis of PA requires the patient to receive
lifelong parenteral B therapy, the diagnosis of PA is made
12
PERNICIOUS ANAEMIA by combined clinical and laboratory evaluation as per
Pernicious anaemia (PA) was first described by Addison in following diagnostic criteria:
1855 as a chronic disorder of middle-aged and elderly I. Major criteria:
individual of either sex in which intrinsic factor (IF) secretion i) Low serum B level in presence of normal renal function
12
ceases owing to atrophy of the gastric mucosa. The condition ii) Megaloblastic anaemia in bone marrow examination,
is, therefore, also termed Addisonian megaloblastic anaemia. which should not be due to folate deficiency
The average age at presentation is 60 years but rarely it can iii) Positive test for IF antibody
be seen in children under 10 years of age (juvenile pernicious II. Minor laboratory criteria:
anaemia). PA is seen most frequently in individuals of i) Macrocytosis in PBF
northern European descent and African Americans and is ii) Anaemia of variable degree
uncommon in South Europeans and Orientals. iii) Hypergastrinaemia
iv) Positive gastric parietal cell antibody
Pathogenesis v) Raised plasma homocysteine level Introduction to Haematopoietic System and Disorders of Erythroid Series
There is evidence to suggest that the atrophy of gastric vi) Gastric pH above 6
mucosa in PA resulting in absence or low level of IF is caused III. Minor clinical criteria:
by an autoimmune reaction against gastric parietal cells. The i) Neurologic features of parasthaesia, numbness or ataxia
evidences in support of immunological abnormalities in ii) Hypothyroidism
pernicious anaemia are as under: iii) Family history of PA or hypothyroidism
1. The incidence of PA is high in patients with other auto- iv) Vitiligo
immune diseases such as Graves’ disease, myxoedema, IV. Reference standard criteria:
thyroiditis, vitiligo, diabetes and idiopathic adrenocortical i) Schilling test showing malabsorption of oral cyano-
insufficiency.
cobalamin corrected by simultaneous administration of
2. Patients with PA have abnormal circulating autoantibodies IF.
such as anti-parietal cell antibody (90% cases) and anti-
intrinsic factor antibody (50% cases). Treatment
3. Relatives of patients with PA have an increased incidence Patients of PA are treated with vitamin B in the following
12
of the disease or increased presence of autoantibodies. way:
4. Corticosteroids have been reported to be beneficial in 1. Parenteral vitamin B replacement therapy.
12
curing the disease both pathologically and clinically. 2. Symptomatic and supportive therapy such as
5. PA is more common in patients with agammaglobulinaemia physiotherapy for neurologic deficits and occasionally blood
supporting the role of cellular immune system in destruction transfusion.
of parietal cells. 3. Follow-up for early detection of cancer of the stomach.

310


























SECTION II

Figure 12.21 Diagrammatic representation of classification of haemolytic anaemias based on principal mechanisms of haemolysis.

Most of the abnormalities due to vitamin B deficiency Extravascular haemolysis is more common than the
12
can be corrected except the irreversible damage to the spinal former. One or more factors may be involved in the
cord. Corticosteroid therapy can improve the gastric lesion pathogenesis of various haemolytic anaemias.
with a return of acid secretion but the higher incidence of Haemolytic anaemias are broadly classified into 2 main
gastric polyps and cancer of the stomach in these patients categories:
can only be detected by frequent follow-up. I. Acquired haemolytic anaemias caused by a variety of
extrinsic environmental factors (extracorpuscular).
HAEMOLYTIC ANAEMIAS
II. Hereditary haemolytic anaemias are usually the result of
GENERAL ASPECTS intrinsic red cell defects (intracorpuscular).
A simplified classification based on these mechanisms is
Definition and Classification given in Table 12.9 and diagrammatically represented in
Fig. 12.21.
Haemolytic anaemias are defined as anaemias resulting from
an increase in the rate of red cell destruction. Normally, effete Features of Haemolysis
red cells undergo lysis at the end of their lifespan of 120+30
days within the cells of reticuloendothelial (RE) system in A number of clinical and laboratory features are shared by
the spleen and elsewhere (extravascular haemolysis), and various types of haemolytic anaemias. These are briefly
haemoglobin is not liberated into the plasma in appreciable described below:
Haematology and Lymphoreticular Tissues
amounts. The red cell lifespan is shortened in haemolytic GENERAL CLINICAL FEATURES. Some of the general
anaemia i.e. there is accelerated haemolysis. However, clinical features common to most congenital and acquired
shortening of red cell lifespan does not necessarily result in haemolytic anaemias are as under:
anaemia. In fact, compensatory bone marrow hyperplasia
may cause 6 to 8-fold increase in red cell production without 1. Presence of pallor of mucous membranes.
causing anaemia to the patient, so-called compensated 2. Positive family history with life-long anaemia in patients
with congenital haemolytic anaemia.
haemolytic disease.
The premature destruction of red cells in haemolytic 3. Mild fluctuating jaundice due to unconjugated
anaemia may occur by 2 mechanisms: hyperbilirubinaemia.
4. Urine turns dark on standing due to excess of
Firstly, the red cells undergo lysis in the circulation and
release their contents into plasma (intravascular haemolysis). urobilinogen in urine.
In these cases the plasma haemoglobin rises substantially 5. Splenomegaly is found in most chronic haemolytic
and part of it may be excreted in the urine (haemoglobinuria). anaemias, both congenital and acquired.
6. Pigment gallstones are found in some cases.
Secondly, the red cells are taken up by cells of the RE
system where they are destroyed and digested (extravascular LABORATORY EVALUATION OF HAEMOLYSIS.
haemolysis). In extravascular haemolysis, plasma Pathways by which haemoglobin derived from effete red cells
haemoglobin level is, therefore, barely raised. is metabolised is already discussed on page 290. The

 TABLE 12.9: Classification of Haemolytic Anaemias. 4. Serum haptoglobin (α-globulin binding protein) is reduced 311
or absent.
I. ACQUIRED (EXTRACORPUSCULAR) 5. Plasma lactic dehydrogenase is raised.
A. Antibody: Immunohaemolytic anaemias 6. Evidences of intravascular haemolysis in the form of haemo-
1. Autoimmune haemolytic anaemia (AIHA) globinaemia, haemoglobinuria, methaemoglobinaemia and
i) Warm antibody AIHA haemosiderinuria.
ii) Cold antibody AIHA
II. Tests of increased red cell production:
2. Drug-induced immunohaemolytic anaemia
1. Reticulocyte count reveals reticulocytosis which is
3. Isoimmune haemolytic anaemia (page 340)
B. Mechanical trauma: Microangiopathic haemolytic anaemia generally early and is hence most useful initial test of marrow
erythroid hyperplasia.
C. Direct toxic effects: Malaria, bacterial, infection and other agents 2. Routine blood film shows macrocytosis, polychromasia and
D. Acquired red cell membrane abnormalities: paroxysmal presence of normoblasts.
nocturnal haemoglobinuria (PNH)
E. Splenomegaly 3. Bone marrow shows erythroid hyperplasia with usually
raised iron stores.
II. HEREDITARY (INTRACORPUSCULAR) 4. X-ray of bones shows evidence of expansion of marrow
A. Abnormalities of red cell membrane space, especially in tubular bones and skull. CHAPTER 12
1. Hereditary spherocytosis
2. Hereditary elliptocytosis (hereditary ovalocytosis) III. Tests of damage to red cells:
3. Hereditary stomatocytosis 1. Routine blood film shows a variety of abnormal
morphological appearances of red cells described on page
B. Disorders of red cell interior
366 and illustrated in Fig.12.10 already. A summary of
1. Red cell enzyme defects (Enzymopathies) contributory features of morphology of RBCs in arriving at
i) Defects in the hexose monophosphate shunt: G6PD the diagnosis of haemolytic anaemia and its cause is given
deficiency
in Table 12.10.
ii) Defects in the Embden-Meyerhof (or glycolytic)
pathway: pyruvate kinase deficiency 2. Osmotic fragility is increased or decreased.
2. Disorders of haemoglobin (Haemoglobinopathies) 3. Autohaemolysis test with or without addition of glucose.
i) Structurally abnormal haemoglobins: sickle syndromes, 4. Coombs’ antiglobulin test.
other haemoglobinopathies 5. Electrophoresis for abnormal haemoglobins.
ii) Reduced globin chain synthesis: thalassaemias 6. Estimation of HbA .
2
7. Estimation of HbF.
investigations of a patient suspected to have haemolytic 8. Tests for sickling.
anaemia should provide answers to 3 vital questions: 9. Screening test for G6PD deficiency and other enzymes (e.g.
1. Is there evidence of haemolysis? Heinz bodies test).
2. What is the type of haemolytic mechanism? IV.Tests for shortened red cell lifespan. A shortened red
3. What is the precise diagnosis? cell survival is best tested by Cr labelling method. Normal
51
The laboratory findings are conveniently divided into the RBC lifespan of 120 days is shortened to 20-40 days in
following 4 groups: moderate haemolysis and to 5-20 days in severe haemolysis. Introduction to Haematopoietic System and Disorders of Erythroid Series
I. Tests of increased red cell breakdown:
1. Serum bilirubin—unconjugated (indirect) bilirubin is I. ACQUIRED (EXTRACORPUSCULAR)
HAEMOLYTIC ANAEMIAS
raised.
2. Urine urobilinogen is raised but there is no bilirubinuria. Acquired haemolytic anaemias are caused by a variety of
3. Faecal stercobilinogen is raised. extrinsic factors, namely: antibody (immunohaemolytic


 TABLE 12.10: Red Cell Morphologic Features in Various Types of Haemolytic Anaemias.
Feature Etiology Type of Haemolytic Anaemia
1. Spherocytes Loss of spectrin from membrane Hereditary spherocytosis
AIHA
2. Target cells (Leptocytes) Increased ratio of surface area: volume Thalassaemias
Liver disease
HbS disease
HbC disease
3. Schistocytes Traumatic damage to red cell membrane Microangiopathy
4. Sickle cells Polymerisation of HbS Sickle syndromes
5. Acanthocytes (Spur cells) Abnormality in membrane lipids Severe liver disease
6. Heinz bodies Precipitated Hb Unstable Hb

312 anaemia), mechanical factors (microangiopathic haemolytic  TABLE 12.11: Conditions Predisposing to Autoimmune
anaemia), direct toxic effect (in malaria, clostridial infection Haemolytic Anaemia (AIHA).
etc), splenomegaly, and certain acquired membrane A. WARM ANTIBODY AIHA
abnormalities (paroxysmal nocturnal haemoglobinuria). 1. Idiopathic (primary)
These are discussed below:
2. Lymphomas-leukaemias e.g. non-Hodgkin’s lymphoma,
CLL, Hodgkin's disease.
A. IMMUNOHAEMOLYTIC ANAEMIAS 3. Collagen vascular diseases e.g SLE
Immunohaemolytic anaemias are a group of anaemias 4. Drugs e.g. methyldopa, penicillin, quinidine group
occurring due to antibody production by the body against 5. Post-viral
its own red cells. Immune haemolysis in these cases may be B. COLD ANTIBODY AIHA
induced by one of the following three types of antibodies: 1. Cold agglutinin disease
1. Autoimmune haemolytic anaemia (AIHA) characterised by a) Acute: Mycoplasma infection, infectious mononucleosis
formation of autoantibodies against patient’s own red cells. b) Chronic: Idiopathic, lymphomas
Depending upon the reactivity of autoantibody, AIHA is 2. PCH (Mycoplasma infection, viral flu, measles, mumps,
further divided into 2 types: syphilis)
i) ‘Warm’ antibody AIHA in which the autoantibodies are
reactive at body temperature (37°C). 1. Chronic anaemia of varying severity with remissions and
ii) ‘Cold’ antibody AIHA in which the autoantibodies react relapses.
SECTION II
better with patient’s own red cells at 4°C. 2. Splenomegaly.
3. Occasionally hyperbilirubinaemia.
2. Drug-induced immunohaemolytic anaemia.
Treatment of these cases consists of removal of the cause
3. Isoimmune haemolytic anaemia in which the antibodies are whenever present, corticosteroid therapy, and in severe cases
acquired by blood transfusions, pregnancies and haemolytic blood transfusions. Splenectomy is the second line of therapy
disease of the newborn. in this disorder.
An important diagnostic tool in all cases of immuno-
haemolytic anaemias is Coombs’ antiglobulin test for LABORATORY FINDINGS. The haematological and
detection of incomplete Rh-antibodies in saline directly biochemical findings in such cases are as under:
(direct Coombs’) or after addition of albumin (indirect 1. Mild to moderate chronic anaemia.
Coombs’). 2. Reticulocytosis.
3. Prominent spherocytosis in the peripheral blood film.
Autoimmune Haemolytic Anaemia (AIHA)
4. Positive direct Coombs’ (antiglobulin) test for presence
‘WARM’ ANTIBODY AIHA of warm antibodies on the red cell, best detected at 37°C.
5. A positive indirect Coombs’ (antiglobulin) test at 37°C
PATHOGENESIS. Warm antibodies reactive at body may indicate presence of large quantities of warm
temperature and coating the red cells are generally IgG class antibodies in the serum.
antibodies and occasionally they are IgA. Little is known 6. Unconjugated (indirect) hyperbilirubinaemia.
about the origin of these acquired red blood cell antibodies
in AIHA but the mechanism of destruction of red cells coated 7. Co-existent immune thrombocytopenia alongwith
with IgG is better understood. Human red cells coated with occasional venous thrombosis may be present (termed
IgG antibodies are bound to the surface of RE cells, especially Evans’ syndrome).
Haematology and Lymphoreticular Tissues
splenic macrophages. A part of the coated cell membrane is 8. In more severe cases, haemoglobinaemia and
lost resulting in spherical transformation of the red cells haemoglobinuria may be present.
(acquired spherocytosis). Red cells coated with IgG along
with C3 on the surface further promote this red cell-leucocyte ‘COLD’ ANTIBODY AIHA
interaction, accounting for more severe haemolysis. The PATHOGENESIS. Antibodies which are reactive in the cold
spleen is particularly efficient in trapping red cells coated (4°C) may induce haemolysis under 2 conditions: cold
with IgG antibodies. It is, thus, the major site of red cell agglutinin disease and paroxysmal cold haemoglobinuria.
destruction in warm antibody AIHA.
1. Cold agglutinin disease. In cold agglutinin disease, the
CLINICAL FEATURES. Warm antibody AIHA may occur antibodies are IgM type which bind to the red cells best at
at any age and in either sex. The disease may occur without 4°C. These cold antibodies are usually directed against the I
any apparent cause (idiopathic) but about a quarter of antigen on the red cell surface. Agglutination of red blood
patients develop this disorder as a complication of an cells by IgM cold agglutinins is most profound at very low
underlying disease affecting the immune system such as SLE, temperature but upon warming to 37°C or above,
chronic lymphocytic leukaemia, lymphomas and certain disagglutination occurs quickly. Haemolytic effect is
drugs such as methyl DOPA, penicillin etc (Table 12.11). mediated through fixation of C3 to the red blood cell surface
The disease tends to have remissions and relapses. The and not by agglutination alone. Most cold agglutinins affect
usual clinical features are as follows: juvenile red blood cells.

The etiology of cold antibody remains unknown. It is seen In each type of drug-induced immunohaemolytic 313
in the course of certain infections (e.g. Mycoplasma anaemia, discontinuation of the drug results in gradual
pneumonia, infectious mononucleosis) and in lymphomas. disappearance of haemolysis.
2. Paroxysmal cold haemoglobinuria (PCH). In PCH, cold
antibody is an IgG antibody (Donath-Landsteiner antibody) Isoimmune Haemolytic Anaemia
which is directed against P blood group antigen and brings Isoimmune haemolytic anaemias are caused by acquiring
about complement-mediated haemolysis. Attacks of PCH are isoantibodies or alloantibodies by blood transfusions,
precipitated by exposure to cold. pregnancies and in haemolytic disease of the newborn. These
PCH is uncommon and may be seen in association with antibodies produced by one individual are directed against
tertiary syphilis or as a complication of certain infections such red blood cells of the other. These conditions are considered
as Mycoplasma pneumonia, flu, measles and mumps. on page 340.

CLINICAL FEATURES. The clinical manifestations are due
to haemolysis and not due to agglutination. These include B. MICROANGIOPATHIC HAEMOLYTIC ANAEMIA
the following: Microangiopathic haemolytic anaemia is caused by
1. Chronic anaemia which is worsened by exposure to cold. abnormalities in the microvasculature. It is generally due to CHAPTER 12
2. Raynaud’s phenomenon. mechanical trauma to the red cells in circulation and is
3. Cyanosis affecting the cold exposed regions such as tips characterised by red cell fragmentation (schistocytosis). There
of nose, ears, fingers and toes. are 3 different ways by which microangiopathic haemolytic
4. Haemoglobinaemia and haemoglobinuria occur on anaemia results:
exposure to cold. 1. EXTERNAL IMPACT. Direct external trauma to red
Treatment consists of keeping the patient warm and blood cells when they pass through microcirculation, espe-
treating the underlying cause. cially over the bony prominences, may cause haemolysis
during various activities e.g. in prolonged marchers, joggers,
LABORATORY FINDINGS. The haematologic and karate players etc. These patients develop haemoglobi-
biochemical findings are somewhat similar to those found naemia, haemoglobinuria (march haemoglobinuria), and
in warm antibody AIHA except the thermal amplitude. sometimes myoglobinuria as a result of damage to muscles.
These findings are as follows:
1. Chronic anaemia. 2. CARDIAC HAEMOLYSIS. A small proportion of
patients who received prosthetic cardiac valves or artificial
2. Low reticulocyte count since young red cells are affected grafts develop haemolysis. This has been attributed to direct
more. mechanical trauma to the red cells or shear stress from
3. Spherocytosis is less marked. turbulent blood flow.
4. Positive direct Coombs’ test for detection of C3 on the
red cell surface but IgM responsible for C3 coating on red 3. FIBRIN DEPOSIT IN MICROVASCULATURE.
cells is not found. Deposition of fibrin in the microvasculature exposes the red
5. The cold antibody titre is very high at 4°C and very low cells to physical obstruction and eventual fragmentation of
red cells and trapping of the platelets. Fibrin deposits in the
at 37°C (Donath-Landsteiner test). IgM class cold antibody small vessels may occur in the following conditions:
has specificity for I antigen, while the rare IgG class i) Abnormalities of the vessel wall e.g. in hypertension, Introduction to Haematopoietic System and Disorders of Erythroid Series
antibody of PCH has P blood group antigen specificity.
eclampsia, disseminated cancers, transplant rejection,
haemangioma etc.
Drug-induced Immunohaemolytic Anaemia ii) Thrombotic thrombocytopenic purpura.
Drugs may cause immunohaemolytic anaemia by 3 different iii) Haemolytic-uraemic syndrome.
mechanisms: iv) Disseminated intravascular coagulation (DIC)
v) Vasculitis in collagen diseases.
1. αα α α α-METHYL DOPA TYPE ANTIBODIES. A small All these conditions are described in relevant sections
proportion of patients receiving α-methyl dopa develop separately.
immunohaemolytic anaemia which is identical in every
respect to warm antibody AIHA described above.
C. HAEMOLYTIC ANAEMIA FROM
2. PENICILLIN-INDUCED IMMUNOHAEMOLYSIS. DIRECT TOXIC EFFECTS
Patients receiving large doses of penicillin or penicillin-type Haemolysis may result from direct toxic effects of certain
antibiotics develop antibodies against the red blood cell-drug agents. These include the following examples:
complex which induces haemolysis.
1. Malaria by direct parasitisation of red cells (black-water
3. INNOCENT BYSTANDER IMMUNOHAEMOLYSIS. fever) (Fig. 12.22).
Drugs such as quinidine form a complex with plasma 2. Bartonellosis by direct infection of red cells by the
proteins to which an antibody forms. This drug-plasma microorganisms.
protein-antibody complex may induce lysis of bystanding 3. Septicaemia with Clostridium welchii by damaging the red
red blood cells or platelets. cells.

314 ii) Pancytopenia (mild granulocytopenia and thrombo-
cytopenia frequent).
iii) Intermittent clinical haemoglobinuria; acute haemo-
lytic episodes occur at night identified by passage of
brown urine in the morning.
iv) Haemosiderinuria very common.
v) Venous thrombosis as a common complication.
The presence of inordinate sensitivity of red blood cells,
leucocytes and platelets to complement in PNH can be
demonstrated in vitro by Ham’s test using red cell lysis at
acidic pH or by sucrose haemolysis test.
About 20% cases of PNH may develop myeloproli-
ferative or myelodysplastic disorder and some even
develop acute myeloid leukaemia.

E. HAEMOLYTIC ANAEMIA IN SPLENOMEGALY
Haemolytic anaemia is common in splenic enlargement from
SECTION II
Figure 12.22 Malarial parasite, Plasmodium falciparum, in the any cause (Chapter 14). Normally, the spleen acts as a filter
peripheral blood showing numerous ring stages and a crescent of and traps the damaged red blood cells, destroys them and
gametocyte. The background shows a normoblast. the splenic macrophages phagocytose the damaged red cells.
A normal spleen poses no risk to normal red blood cells. But
4. Other microorganisms such as pneumococci, staphylococci splenomegaly exaggerates the damaging effect to which the
and Escherichia coli. red cells are exposed. Besides haemolytic anaemia,
5. Copper by direct haemolytic effect on red cells in Wilson’s splenomegaly is usually associated with pancytopenia.
disease and patients on haemodialysis. Splenectomy or reduction in size of spleen by appropriate
6. Lead poisoning shows basophilic stippling of red blood therapy relieves the anaemia as well as improves the
cells. leucocyte and platelet counts.
7. Snake and spider bites cause haemolysis by their venoms.
8. Extensive burns. II. HEREDITARY (INTRACORPUSCULAR)
HAEMOLYTIC ANAEMIA
D. PAROXYSMAL NOCTURNAL
HAEMOGLOBINURIA (PNH) Hereditary haemolytic anaemias are usually the result of
intracorpuscular defects. Accordingly, they are broadly
PNH is a rare acquired disorder of red cell membrane in classified into 2 groups (see Table 12.9):
which there is chronic intravascular haemolysis due to undue Hereditary abnormalities of red cell membrane.
sensitivity of red blood cells to complement due to defective Hereditary disorders of the interior of the red cells.
synthesis of a red cell membrane protein. The defect affects
all the cells of myeloid progenitor lineage (RBCs, WBCs, A. HEREDITARY ABNORMALITIES OF
platelets) suggesting a deficient haematopoiesis. The disorder RED CELL MEMBRANE
generally presents in adult life.
The abnormalities of red cell membrane are readily identified
Haematology and Lymphoreticular Tissues
PATHOGENESIS. PNH is considered as an acquired clonal on blood film examination. There are 3 important types of
disease of the cell membrane while normal clone also inherited red cell membrane defects: hereditary
continues to proliferate. The defect is a mutation in the stem spherocytosis, hereditary elliptocytosis (hereditary
cells affecting myeloid progenitor cells that is normally ovalocytosis) and hereditary stomatocytosis.
required for the biosynthesis of glycosyl phosphatidyl
inositol (GPI) essential for anchoring of the cell; the mutant Hereditary Spherocytosis
form of the gene is an X-linked gene called PIG-A
(phosphatidyl inositol glycan). Thus, as a result of mutation, Hereditary spherocytosis is a common type of hereditary
there is partial or complete deficiency of anchor protein. Out haemolytic anaemia of autosomal dominant inheritance in
of about 20 such proteins described so far, the lack of two of which the red cell membrane is abnormal.
the proteins—decay accelerating factor (DAF, CD55) and a PATHOGENESIS. The molecular abnormality in hereditary
membrane inhibitor of reactive lysis (MIRL, CD59), makes the spherocytosis is a defect in proteins which anchor the lipid
RBCs unduly sensitive to the lytic effect of complement. bilayer to the underlying cytoskeleton. These protein
abnormalities are as under and are schematically illustrated
CLINICAL AND LABORATORY FINDINGS. Clinical in Fig.12.23:
and laboratory findings are as under: 1. Spectrin deficiency. Almost all cases have deficiency in
i) Haemolytic anaemia. the structural protein of the red cell membrane, spectrin.

315
























Figure 12.23 Diagrammatic representation of pathogenesis of hereditary spherocytosis. A, Normal red cell with biconcave surface and normal CHAPTER 12
size. B, Red cell membrane as seen in cross section in hereditary spherocytosis. Mutations in membrane proteins—α-spectrin, β-spectrin and
ankyrin, result in defect in anchoring of lipid bilayer of the membrane to the underlying cytoskeleton. C, This results in spherical contour and small
size so as to contain the given volume of haemoglobin in the deformed red cell. D, During passage through the spleen, these rigid spherical cells
lose their cell membrane further. This produces a circulating subpopulation of hyperspheroidal spherocytes while splenic macrophages in large
numbers phagocytose defective red cells causing splenomegaly.


Spectrin deficiency correlates with the severity of anaemia. LABORATORY FINDINGS. The usual haematological
Mutation in spectrin by recessive inheritance called α−spectrin and biochemical findings are as under:
causes more severe form of anaemia, while mutation by 1. Anaemia of mild to moderate degree.
dominant inheritance forming β-spectrin results in mild form 2. Reticulocytosis, usually 5-20%.
of the disease. 3. Blood film shows the characteristic abnormality of
2. Ankyrin abnormality.About half the cases of hereditary erythrocytes in the form of microspherocytes (Fig.12.24).
spherocytosis have defect in ankyrin, protein that binds
protein 3 and spectrin. Homozygous state with recessive 4. MCV is usually normal or slightly decreased but MCHC
is increased.
inheritance pattern has severe anaemia while heterozygotes 5. Osmotic fragility test is helpful in testing the spheroidal
with more common dominant inheritance pattern have nature of red cells which lyse more readily in solutions of
milder anaemia.
Inherited mutation in spectrin or ankyrin causes defect low salt concentration i.e. osmotic fragility is increased
in anchoring of lipid bilayer cell membrane. Red cells with (Fig.12.25).
such unstable membrane but with normal volume, when 6. Autohaemolysis test is similar to osmotic fragility test after
released in circulation, lose their membrane further, till they incubation and shows increased spontaneous Introduction to Haematopoietic System and Disorders of Erythroid Series
can accommodate the given volume. This results in formation autohaemolysis (10-15% red cells) as compared to normal
of spheroidal contour and smaller size of red blood cells, red cells (less than 4%). Autohaemolysis is correctable by
termed microspherocytes. These deformed red cells are not addition of glucose.
flexible, unlike normal biconcave red cells. These rigid cells 7. Direct Coombs’ (antiglobulin) test is negative so as to
are unable to pass through the spleen, and in the process distinguish this condition from acquired spherocytosis of
they lose their surface membrane further. This produces a AIHA in which case it is positive.
subpopulation of hyperspheroidal red cells in the peripheral Spherocytes may also be seen in blood film in acquired
blood which are subsequently destroyed in the spleen. immune haemolytic anaemia and following red cell
transfusion.
CLINICAL FEATURES. The disorder may be clinically
apparent at any age from infancy to old age and has equal Hereditary Elliptocytosis (Hereditary Ovalocytosis)
sex incidence. The family history may be present. The major Hereditary elliptocytosis or hereditary ovalocytosis is another
clinical features are as under: autosomal dominant disorder involving red cell membrane
1. Anaemia is usually mild to moderate. protein spectrin. Some patients have an inherited defect in
2. Splenomegaly is a constant feature. erythrocyte membrane protein 4.1 that interconnects spectrin
3. Jaundice occurs due to increased concentration of with actin in the cytoskeleton. The disorder is similar in all
unconjugated (indirect) bilirubin in the plasma (also termed respects to hereditary spherocytosis except that the blood
congenital haemolytic jaundice). film shows oval or elliptical red cells and is clinically a milder
4. Pigment gallstones are frequent due to increased bile disorder than hereditary spherocytosis.
pigment production. Splenectomy offers the only reliable Acquired causes of elliptocytosis include iron deficiency
mode of treatment. and myeloproliferative disorders.

316
























Figure 12.24 Peripheral blood film findings in hereditary spherocytosis.
SECTION II
Hereditary Stomatocytosis asymptomatic. Several variants of G6PD have been
Stomatocytes are cup-shaped RBCs having one surface described. The normal G6PD variant is designated as type B
concave and the other side as convex. This causes a central but blacks have normally A+ (positive) type G6PD variant.
slit-like or mouth-like appearance of red cells. The underlying The most common and significant clinical variant is A–
defect is in membrane protein, stomatin, having autosomal (negative) type found in black males. Like the HbS gene, the
dominant pattern of inheritance. The stomatocytes are A–type G6PD variant confers protection against malaria.
swollen red cells (overhydrated red cells) due to increased Individuals with A– G6PD variant have shortened red cell
permeability to sodium and potassium. The affected patients lifespan but without anaemia. However, these individuals
have mild anaemia and splenomegaly. develop haemolytic episodes on exposure to oxidant stress
such as viral and bacterial infections, certain drugs
B. HEREDITARY DISORDERS OF RED CELL INTERIOR (antimalarials, sulfonamides, nitrofurantoin, aspirin, vitamin
K), metabolic acidosis and on ingestion of fava beans
Inherited disorders involving the interior of the red blood (favism).
cells are classified into 2 groups:
PATHOGENESIS. Normally, red blood cells are well
1. Red cell enzyme defects (Enzymopathies): These cause protected against oxidant stress because of adequate
defective red cell metabolism involving 2 pathways generation of reduced glutathione via the hexose mono-
(Fig. 12.26): phosphate shunt. Individuals with inherited deficiency of
i) Defects in the hexose monophosphate shunt: Common G6PD, an enzyme required for hexose monophosphate
example is glucose-6-phosphate dehydrogenase (G6PD) shunt for glucose metabolism, fail to develop adequate
deficiency. levels of reduced glutathione in their red cells. This results
ii) Defects in the Embden-Meyerhof (glycolytic) pathway:
Haematology and Lymphoreticular Tissues
Example is pyruvate kinase (PK) deficiency.
2. Disorders of haemoglobin (haemoglobinopathies):
These are divided into 2 subgroups:
i) Structurally abnormal haemoglobin: Examples are sickle
syndromes and other haemoglobinopathies.
ii) Reduced globin chain synthesis: Common examples are
various types of thalassaemias.
These disorders are discussed below.
Red Cell Enzyme Defects (Enzymopathies)
G6PD DEFICIENCY
Among the defects in hexose monophosphate shunt, the most
common is G6PD deficiency. It affects millions of people
throughout the world. G6PD gene is located on the X
chromosome and its deficiency is, therefore, a sex (X)-linked Figure 12.25 Osmotic fragility test in hereditary spherocytosis
trait affecting males, while the females are carriers and are showing increased fragility.

317



























Figure 12.26 Abbreviated pathways of anaerobic glycolysis (Embden-Meyerhof) and hexose monophosphate (HMP) shunt in the metabolism CHAPTER 12
of erythrocyte. The two red cell enzyme defects, glucose-6 phosphate dehydrogenase (G6PD) and pyruvate kinase, are shown bold.


in oxidation and precipitation of haemoglobin within the PK DEFICIENCY
red cells forming Heinz bodies. Besides G6PD deficiency, Pyruvate kinase (PK) deficiency is the only significant
deficiency of various other enzymes involved in the hexose enzymopathy of the Embden-Meyerhof glycolytic
monophosphate shunt may also infrequently cause clinical pathway.The disorder is inherited as an autosomal recessive
problems. pattern. Heterozygote state is entirely asymptomatic, while
CLINICAL FEATURES. The clinical manifestations are those the homozygous individual presents during early childhood
of an acute haemolytic anaemia within hours of exposure to with anaemia, jaundice and splenomegaly.
oxidant stress. The haemolysis is, however, self-limiting even
if the exposure to the oxidant is continued since it affects the LABORATORY FINDINGS: These are as under:
older red cells only. Haemoglobin level may return to normal 1. Normocytic and normochromic anaemia.
when the older population of red cells has been destroyed 2. Reticulocytosis.
and only younger cells remain. Some patients may have only 3. Blood film shows bizarre red cells.
darkening of the urine from haemoglobinuria but more 4. Osmotic fragility is usually normal but after incubation
severely affected ones develop constitutional symptoms it is increased.
including jaundice. Treatment is directed towards the 5. Autohaemolysis is increased, but unlike hereditary Introduction to Haematopoietic System and Disorders of Erythroid Series
prevention of haemolytic episodes such as stoppage of spherocytosis, is not corrected by addition of glucose.
offending drug. Blood transfusions are rarely indicated. 6. Direct specific enzyme assay on red cells is the only
method of establishing the diagnosis.
LABORATORY FINDINGS. These are as under:
1.During the period of acute haemolysis, there is rapid Haemoglobinopathies
fall in haematocrit by 25-30%, features of intravascular Haemoglobin in RBCs may be abnormally synthesised due
haemolysis such as rise in plasma haemoglobin, to inherited defects. These disorders may be of two types:
haemoglobinuria, rise in unconjugated bilirubin and fall Qualitative disorders in which there is structural
in plasma haptoglobin. Formation of Heinz bodies is abnormality in synthesis of haemoglobin e.g. sickle cell
visualised by means of supravital stains such as crystal syndrome, other haemoglobinopathies.
violet, also called Heinz body haemolytic anaemia. However, Quantitative disorders in which there quantitatively
Heinz bodies are not seen after the first one or two days decreased globin chain synthesis of haemoglobin e.g.
since they are removed by the spleen, leading to formation thalassaemias.
of ‘bite cells’ and fragmented red cells. Both these groups of disorders may occur as homozygous
2. Between the crises, the affected patient generally has state in which both genes coding for that character are
no anaemia. The red cell survival is, however, shortened. abnormal, or heterozygous when one gene is abnormal and
The diagnosis of G6PD enzyme deficiency is made by the other gene is normal. However, there are examples of
one of the screening tests (e.g. methaemoglobin reduction combined disorders too in which there are two different
test, fluorescent screening test, ascorbate cyanide mutations in loci of two corresponding genes (i.e. double
screening test), or by direct enzyme assay on red cells. heterozygous) e.g. HbS gene from one parent and β-thal from
S thal
the other parent resulting in β β . These disorders may vary

318 Patients with HbS are relatively protected against falciparum
malaria. Sickle syndromes occur in 3 different forms:
1. As heterozygous state for HbS: sickle cell trait (AS).
2. As homozygous state for HbS: sickle cell anaemia (SS).
3. As double heterozygous states e.g. sickle β-thalassaemia,
sickle-C disease (SC), sickle-D disease (SD).

Heterozygous State:Sickle Cell Trait
Sickle cell trait (AS) is a benign heterozygous state of HbS in
which only one abnormal gene is inherited. Patients with
AS develop no significant clinical problems except when they
become severely hypoxic and may develop sickle cell crises.

LABORATORY FINDINGS. These patients have no
anaemia and have normal appearance of red cells. But in
hypoxic crisis, sickle cell crises develop. The diagnosis is
Figure 12.27 The geographic distribution of major haemo- made by 2 tests:
globinopathies and thalassaemias. Thalassaemia and HbD are the 1. Demonstration of sickling done under condition of
haemoglobin disorders common in India.
reduced oxygen tension by an oxygen consuming reagent,
SECTION II
sodium metabisulfite.
in presentation from asymptomatic laboratory abnormalities
to intrauterine death. 2. Haemoglobin electrophoresis reveals 35-40% of the total
There are geographic variations in the distribution of haemoglobin as HbS.
various haemoglobinopathies world over as shown in Homozygous State:Sickle Cell Anaemia
Fig. 12.27. Major examples of these disorders are described
below. Sickle cell anaemia (SS) is a homozygous state of HbS in the
red cells in which an abnormal gene is inherited from each
parent. SS is a severe disorder associated with protean clinical
STRUCTURALLY ABNORMAL HAEMOGLOBINS
manifestations and decreased life expectancy.
SICKLE SYNDROMES PATHOGENESIS. Following abnormalities are observed
The most important and widely prevalent type of haemo- (Fig. 12.28):
globinopathy is due to the presence of sickle haemoglobin 1.Basic molecular lesion: In HbS, basic genetic defect is the
(HbS) in the red blood cells. The red cells with HbS develop single point mutation in one amino acid out of 146 in
‘sickling’ when they are exposed to low oxygen tension. haemoglobin molecule— there is substitution of valine for
Sickle syndromes have the highest frequency in black race glutamic acid at 6-residue position of the β-globin, producing
and in Central Africa where falciparum malaria is endemic. Hb α β .
s
2 2





Haematology and Lymphoreticular Tissues
























Figure 12.28 Pathogenesis of sickle cell anaemia. A, Basic molecular defect. B, Mechanism of polymerisation and consequent sickling of red
cells containing HbS. C, Mechanism of sickling on oxygenation-deoxygenation.

2.Mechanism of sickling: During deoxygenation, the red 319
cells containing HbS change from biconcave disc shape to
an elongated crescent-shaped or sickle-shaped cell. This
process termed sickling occurs both within the intact red cells
and in vitro in free solution. The mechanism responsible for
sickling upon deoxygenation of HbS-containing red cells is
the polymerisation of deoxygenated HbS which aggregates
to form elongated rod-like polymers. These elongated fibres
align and distort the red cell into classic sickle shape.
3. Reversible-irreversible sickling: The oxygen-dependent
sickling process is usually reversible. However, damage to
red cell membrane leads to formation of irreversibly sickled
red cells even after they are exposed to normal oxygen
tension.
4.Factors determining rate of sickling: Following factors
determine the rate at which the polymerisation of HbS and CHAPTER 12
consequent sickling take place:
i) Presence of non-HbS haemoglobins: The red cells in patients
of SS have predominance of HbS and a small part consists of Figure 12.29 Sickle cell anaemia. PBF shows crescent shaped
non-HbS haemoglobins, chiefly HbF (2-20% of the total elongated red blood cells, a few target cells and a few erythroblasts.
haemoglobin). HbF-containing red cells are protected from
sickling while HbA-containing red cells participate readily growth and development and increased susceptibility to
in co-polymerisation with HbS. infection due to markedly impaired splenic function.
ii) Intracellular concentration of HbS.
LABORATORY FINDINGS. The diagnosis of SS is
iii) Total haemoglobin concentration.
considered high in blacks with haemolytic anaemia. The
iv) Extent of deoxygenation.
laboratory findings in these cases are as under (Fig. 12.29):
v) Acidosis and dehydration.
1. Moderate to severe anaemia (haemoglobin concen-
vi) Increased concentration of 2, 3-BPG in the red cells. tration 6-9 g/dl).
CLINICAL FEATURES. The clinical manifestations of homo- 2. The blood film shows sickle cells and target cells and
zygous sickle cell disease are widespread. The symptoms features of splenic atrophy such as presence of Howell-
begin to appear after 6th month of life when most of the HbF Jolly bodies.
is replaced by HbS. Infection and folic acid deficiency result 3. A positive sickling test with a reducing substance such
in more severe clinical manifestations. These features are as as sodium metabisulfite (described above).
under: 4. Haemoglobin electrophoresis shows no normal HbA
1. Anaemia. There is usually severe chronic haemolytic but shows predominance of HbS and 2-20% HbF.
anaemia (primarily extravascular) with onset of aplastic crisis Double Heterozygous States Introduction to Haematopoietic System and Disorders of Erythroid Series
in between. The symptoms of anaemia are generally mild
since HbS gives up oxygen more readily than HbA to the Double heterozygous conditions involving combination of
tissues. HbS with other haemoglobinopathies may occur. Most
S thal
2. Vaso-occlusive phenomena. Patients of SS develop common among these are sickle-β-thalassaemia (β β ),
recurrent vaso-occlusive episodes throughout their lives due sickle C disease (SC), and sickle D disease (SD). All these
to obstruction to capillary blood flow by sickled red cells disorders behave like mild form of sickle cell disease. Their
upon deoxygenation or dehydration. Vaso-obstruction diagnosis is made by haemoglobin electrophoresis and
affecting different organs and tissues results in infarcts which separating the different haemoglobins.
may be of 2 types: OTHER STRUCTURAL HAEMOGLOBINOPATHIES
i) Microinfarcts affecting particularly the abdomen, chest, Besides sickle haemoglobin, about 400 structurally different
back and joints and are the cause of recurrent painful crises abnormal human haemoglobins have been discovered in
in SS. different parts of the world. Some of them are associated with
ii) Macroinfarcts involving most commonly the spleen clinical manifestations, while others are of no consequence.
(splenic sequestration, autosplenectomy), bone marrow A few important and common variants are briefly described
(pains), bones (aseptic necrosis, osteomyelitis), lungs below:
(pulmonary infections), kidneys (renal cortical necrosis), CNS
(stroke), retina (damage) and skin (ulcers), and result in HbC Haemoglobinopathy
anatomic and functional damage to these organs. HbC haemoglobinopathy is prevalent in West Africa and in
3. Constitutional symptoms. In addition to the features of American blacks. The molecular lesion in HbC is substitution
anaemia and infarction, patients with SS have impaired of lysine for glutamic acid at β-6 globin chain position. The

320 disorder of HbC may occur as benign homozygous HbC chain synthesis. Thalassaemias were first described in people
disease, or as asymptomatic heterozygous HbC trait, or as of Mediterranean countries (North Africa, Southern Europe)
double heterozygous combinations such as sickle-HbC from where it derives its name ‘Mediterranean anaemia.’ The
disease and HbC-β thalassaemia. Word ‘thalassa’ in Greek means ‘the sea’ since the condition
was found commonly in regions surrounding the
HbD Haemoglobinopathy Mediterranean sea. The condition also occurs in the Middle
HbD occurs in North-West India, Pakistan and Iran. About East, Indian subcontinent, South-East Asia and, in general
3% of Sikhs living in Punjab are affected with HbD in blacks (see Fig. 12.27).
haemoglobinopathy (called HbD Punjab, also known as Hb-
Los Angeles). HbD Punjab arises from the substitution of GENETICS AND CLASSIFICATION
glutamine for glutamic acid at β-121 globin chain position. Thalassaemias are genetically transmitted disorders.
Normally, an individual inherits two β-globin genes located
HbE Haemoglobinopathy one each on two chromosomes 11, and two α-globin genes
HbE is predominantly found in South-East Asia, India, one each on two chromosomes 16, from each parent i.e.
Burma and Sri Lanka. HbE arises from the substitution of normal adult haemoglobin (HbA) is α β *. Depending upon
2 2
lysine for glutamic acid at β-26 globin chain position. Like whether the genetic defect or deletion lies in transmission of
other abnormal haemoglobins, HbE haemoglobinopathy may α- or β-globin chain genes, thalassaemias are classified into
also occur as asymptomatic heterozygous HbE trait, α- and β- thalassaemias. Thus, patients with α-thalassaemia
compensated haemolytic homozygous HbE disease, or as have structurally normal α-globin chains but their production
SECTION II
double heterozygous states in combination with other is impaired. Similarly, in β-thalassaemia, β-globin chains are
haemoglobinopathies such as HbE-β thalassaemia and HbE- structurally normal but their production is decreased. Each
α thalassaemia. of the two main types of thalassaemias may occur as
heterozygous (called α- and β-thalassaemia minor or trait), or
Haemoglobin O-Arab Disease as homogygous state (termed α- and β-thalassaemia major).
Hb O-Arab disease was first identified in an Arab family The former is generally asymptomatic, while the latter is a
but has now been detected in American blacks too. The severe congenital haemolytic anaemia.
homozygous form of the disease appears as mild haemolytic A classification of various types of thalassaemias
anaemia with splenomegaly. alongwith the clinical syndromes produced and salient
laboratory findings are given in Table 12.12.
Unstable-Hb Haemoglobinopathy
The unstable haemoglobins are those haemoglobin variants PATHOPHYSIOLOGY OF ANAEMIA IN
which undergo denaturation and precipitation within the red THALASSAEMIA
cells as Heinz bodies. These give rise to what is known as A constant feature of all forms of thalassaemia is the presence
congenital non-spherocytic haemolytic anaemia or congenital of anaemia which occurs from following mechanisms:
Heinz body haemolytic anaemia. These disorders have either
autosomal dominant inheritance or develop from sponta- α-Thalassaemia: In α-thalassaemia major, the obvious cause
neous mutations. The unstable haemoglobins arise from of anaemia is the inability to synthesise adult haemoglobin,
either a single amino acid substitution in the globin chain or while in α-thalassaemia trait there is reduced production of
due to deletion of one or more amino acids within the β- normal adult haemoglobin.
globin chain so that the firm bonding of the haem group β-Thalassaemia: In β-thalassaemia major, the most impor-
within the molecule is disturbed leading to formation of tant cause of anaemia is premature red cell destruction
Haematology and Lymphoreticular Tissues
methaemoglobin and precipitation of globin chains as Heinz brought about by erythrocyte membrane damage caused by
bodies. the precipitated α-globin chains. Other contributory factors
Over 100 unstable haemoglobins have been described. are: shortened red cell lifespan, ineffective erythropoiesis,
They are named according to the place where they are and haemodilution due to increased plasma volume. A
encountered. For instance: Hb-Koln, Hb-Hammersmith, Hb- deficiency of β-globin chains in β-thalassaemia leads to large
Zurich, Hb-Sydney, and so on. The diagnosis of unstable Hb excess of α-chains within the developing red cells. Part of
disease is made by test for Heinz bodies and by haemoglobin these excessive α-chains are removed by pairing with γ-globin
electrophoresis. chains as HbF, while the remainder unaccompanied α-chains
precipitate rapidly within the red cell as Heinz bodies. The
REDUCED GLOBIN CHAIN SYNTHESIS: precipitated α-chains cause red cell membrane damage.
THALASSAEMIAS During their passage through the splenic sinusoids, these
red cells are further damaged and develop pitting due to
DEFINITION removal of the precipitated aggregates. Thus, such red cells
The thalassaemias are a diverse group of hereditary disorders
in which there is reduced synthesis of one or more of the *In a normal adult, distribution of haemoglobin is as under: HbA (α β )
2 2
globin polypeptide chains. Thus, thalassaemias, unlike = 95-98%, HbA2 (α 2 δ 2 ) (a minor variant of HbA) = 1.5-3.5%, HbF (α 2 γ 2 ) =
haemoglobinopathies which are qualitative disorders of less than 1%. But the level of HbF in children under 6 months is slightly
haemoglobin, are quantitative abnormalities of polypeptide globin higher.

321
  TABLE 12.12: Classification of Thalassaemias.
Type Hb Hb-electrophoresis Genotype Clinical Syndrome
α-THALASSAEMIAS
1. Hydrops foetalis 3-10 gm/dl Hb Barts (g 4 ) (100%) Deletion of four α-genes Fatal in utero or in early infancy
2. Hb-H disease 2-12 gm/dl HbF (10%), HbH (2-4%) Deletion of three α-genes Haemolytic anaemia
3. α-Thalassaemia trait 10-14 gm/dl Almost normal Deletion of two α-genes Microcytic hypochromic blood
picture but no anaemia
β-THALASSAEMIAS
1. β-Thalassaemia major < 5 gm/dl HbA (0-50%), β thal /β thal Severe congenital haemolytic
HbF(50-98%) anaemia, requires blood transfusions

2. β-Thalassaemia intermedia 5-10 gm/dl Variable Multiple mechanisms Severe anaemia, but regular blood
transfusions not required
A
3. β-Thalassaemia minor 10-12 gm/dl HbA 2 (4-9%) β /β thal Usually asymptomatic CHAPTER 12
HbF (1-5%)


are irreparably damaged and are phagocytosed by the RE 1. Severe anaemia (haemoglobin below 6g/dl).
cells of the spleen and the liver causing anaemia, hepato- 2. Blood film show marked anisopoikilocytosis,
splenomegaly, and excess of tissue iron stores. Patients with hypochromia, microcytosis, polychromasia, basophilic
β-thalassaemia minor, on the other hand, have very mild stippling, numerous normoblasts and target cells.
ineffective erythropoiesis, haemolysis and shortening of red 3. Reticulocyte count is high.
cell lifespan. 4. Serum bilirubin level is elevated.
5. Haemoglobin electrophoresis shows 80-90% Hb-Bart’s
α α α α α-THALASSAEMIA and a small amount of Hb-H and Hb-Portland but no
MOLECULAR PATHOGENESIS. α-thalassaemias are HbA, HbA or HbF.
2
disorders in which there is defective synthesis of α-globin HbH Disease
chains resulting in depressed production of haemoglobins
that contain α-chains i.e. HbA, HbA and HbF. The α- Deletion of three α-chain genes produces HbH which is a β-
2
thalassaemias are most commonly due to deletion of one or globin chain tetramer (β ) and markedly impaired α-chain
4
more of the α-chain genes located on short arm of chromo- synthesis. HbH is precipitated as Heinz bodies within the
some 16. Since there is a pair of α-chain genes, the clinical affected red cells. An elongated α-chain variant of HbH
manifestations of α-thalassaemia depend upon the number disease is termed Hb Constant Spring.
of genes deleted. Accordingly, α-thalassaemias are classified CLINICAL FEATURES. HbH disease is generally present
into 4 types: as a well-compensated haemolytic anaemia. The features are
1. Four α-gene deletion: Hb Bart’s hydrops foetalis. intermediate between that of β-thalassaemia minor and Introduction to Haematopoietic System and Disorders of Erythroid Series
2. Three α-gene deletion: HbH disease. major. The severity of anaemia fluctuates and may fall to
3. Two α-gene deletion: α-thalassaemia trait. very low levels during pregnancy or infections. Majority of
4. One α-gene deletion: α-thalassaemia trait (carrier). patients have splenomegaly and may develop cholelithiasis.

Hb Bart’s Hydrops Foetalis LABORATORY FINDINGS. These are as follows:
When there is deletion of all the four α-chain genes 1. Moderate anaemia (haemoglobin 8-9 g/dl).
(homozygous state) it results in total suppression of α-globin 2. Blood film shows severe microcytosis, hypochromia,
chain synthesis causing the most severe form of α- basophilic stippling, target cells and normoblasts.
thalassaemia called Hb Bart’s hydrops foetalis. Hb Bart’s is 3. Mild reticulocytosis.
a gamma globin chain tetramer (γ ) which has high oxygen 4. HbH inclusions as Heinz bodies can be demonstrated
4
affinity leading to severe tissue hypoxia. in mature red cells with brilliant cresyl blue stain.
5. Haemoglobin electrophoresis shows 2-4% HbH and
CLINICAL FEATURES. Hb Bart’s hydrops foetalis is the remainder consists of HbA, HbA and HbF.
incompatible with life due to severe tissue hypoxia. The 2
condition is either fatal in utero or the infant dies shortly after α α α α α-Thalassaemia Trait
birth. If born alive, the features similar to severe Rh α-thalassaemia trait may occur by the following molecular
haemolytic disease are present (page 340).
pathogenesis:
LABORATORY FINDINGS. Infants with Hb Bart’s By deletion of two of the four α-chain genes in
hydrops foetalis born alive may have the following homozygous form called homozygous α-thalassaemia, or in
laboratory findings: double heterozygous form termed heterozygous α-thalassaemia.

322 By deletion of a single α-chain gene causing heterozygous
α-thalassaemia trait called heterozygous α-thalassaemia.
CLINICAL FEATURES. α-thalassaemia trait due to two
α-chain gene deletion is asymptomatic. It is suspected in a
patient of refractory microcytic hypochromic anaemia in
whom iron deficiency and β-thalassaemia minor have been
excluded and the patient belongs to the high-risk ethnic
group. One gene deletion α-thalassaemia trait is a silent
carrier state.
LABORATORY FINDINGS. The patients of α-thalas-
saemia trait may have the following haematological
findings:
1. Haemoglobin level normal or mildly reduced.
2. Blood film shows microcytic and hypochromic red cell
morphology but no evidence of haemolysis or anaemia.
3. MCV, MCH and MCHC may be slightly reduced.
4. Haemoglobin electrophoresis reveals small amount of
SECTION II
Hb-Bart’s in neonatal period (1-2% in α-thalassaemia 2
and 5-6% in α-thalassaemia 1) which gradually disappears
by adult life. HbA is either normal or slightly decreased
2
(contrary to the elevated HBA levels in β-thalassaemia
2
trait).
Figure 12.31 Pathogenesis of β-thalassaemia major.
β β β β β-THALASSAEMIAS
ii) Translation defect: Mutation in the coding sequence causing
MOLECULAR PATHOGENESIS. β-thalassaemias are stop codon (chain termination) interrupting β-globin
caused by decreased rate of β-chain synthesis resulting in messenger RNA. This would result in no synthesis of β-globin
reduced formation of HbA in the red cells. The molecular chain i.e. β° thalassaemia.
pathogenesis of the β-thalassaemias is more complex than iii) mRNA splicing defect: Mutation leads to defective mRNA
that of α-thalassaemias. In contrast to α-thalassaemia, gene processing forming abnormal mRNA that is degraded in the
deletion rarely ever causes β-thalassaemia and is only seen nucleus. Depending upon whether part of splice site remains
in an entity called hereditary persistence of foetal haemoglobin intact or is totally degraded, it may result in β thalassaemia
+
(HPFH). Instead, most of β-thalassaemias arise from different or β° thalassaemia.
types of mutations of β−globin gene resulting from single Depending upon the extent of reduction in β-chain
base changes. The symbol β° is used to indicate the complete synthesis, there are 3 types of β-thalassaemia:
+
absence of β-globin chain synthesis while β denotes partial
synthesis of the β-globin chains. More than 100 such 1. Homozygous form: β-Thalassaemia major. It is the most
mutations have been described affecting the preferred sites severe form of congenital haemolytic anaemia. It is further
in the coding sequences e.g. in promoter region, termination of 2 types (Fig. 12.31):
region, splice junctions, exons, introns. Some of the important i) β° thalassaemia major characterised by complete absence
Haematology and Lymphoreticular Tissues
ones having effects on β-globin chain synthesis are as under of β-chain synthesis.
+
(Fig. 12.30): ii) β thalassaemia major having incomplete suppression of
i) Transcription defect: Mutation affecting transcriptional β-chain synthesis.
promoter sequence causing reduced synthesis of β-globin 2. β-Thalassaemia intermedia: It is β-thalassaemia of
chain. Hence the result is partially preserved synthesis i.e. intermediate degree of severity that does not require regular
β thalassaemia. blood transfusions. These cases are genetically heterozygous
+
+
(β°/β or β /β).
3. Heterozygous form: β-Thalassaemia minor (trait). It is
a mild asymptomatic condition in which there is moderate
suppression of β-chain synthesis.
Besides β-thalassaemia minor, a few uncommon globin
chain combinations resulting in β-thalassaemia trait are as
under:
i) δβ-thalassaemia minor in which there is total absence of
both β and δ chain synthesis and is characterised by elevated
Figure 12.30 Schematic representation of sites of β-globin gene HbF level but unlike β-thalassaemia minor there is normal
mutation in chromosome 11 giving rise to β-thalassaemia. or reduced HbA level.
2

β β β β β-Thalassaemia Major 323
β-thalassaemia major, also termed Mediterranean or Cooley’s
anaemia is the most common form of congenital haemolytic
anaemia. β-thalassaemia major is a homozygous state with
either complete absence of β-chain synthesis (β° thalassaemia
+
major) or only small amounts of β-chains are formed (β thalas-
saemia major). These result in excessive formation of alternate
haemoglobins, HbF (α γ ) and HbA (α δ ).
2
2 2
2
2
CLINICAL FEATURES. Clinical manifestations appear
insidiously and are as under (Fig. 12.32):
1. Anaemia starts appearing within the first 4-6 months of
life when the switch over from γ-chain to β-chain production
occurs.
2. Marked hepatosplenomegaly occurs due to excessive red
cell destruction, extramedullary haematopoiesis and iron CHAPTER 12
overload.
3. Expansion of bones occurs due to marked erythroid
hyperplasia leading to thalassaemic facies and malocclusion
of the jaw.
4. Iron overload due to repeated blood transfusions causes
damage to the endocrine organs resulting in slow rate of
growth and development, delayed puberty, diabetes mellitus
and damage to the liver and heart.

LABORATORY FINDINGS. The haematological
investigations reveal the following findings:
1. Anaemia, usually severe.
Figure 12.32 Major clinical features of β-thalassaemia major. 2. Blood film shows severe microcytic hypochromic red cell
morphology, marked anisopoikilocytosis, basophilic
ii) Hb Lepore syndrome characterised by nonhomologous stippling, presence of many target cells, tear drop cells
fusion of β- and δ-genes forming an abnormal haemoglobin and normoblasts (Fig. 12.33).
called Hb Lepore (named after a family called Lepore). There 3. Serum bilirubin (unconjugated) is generally raised.
is total absence of normal β-chain synthesis. 4. Reticulocytosis is generally present.
An individual may inherit one β-chain gene from each
parent and produce heterozygous, homozygous, or double 5. MCV, MCH and MCHC are significantly reduced.
heterozygous states. Statistically, 25% of offsprings born to 6. WBC count is often raised with some shift to left of the
two heterozygotes (i.e. β-thalassaemia trait) will have the neutrophil series, with presence of some myelocytes and Introduction to Haematopoietic System and Disorders of Erythroid Series
homozygous state i.e. β-thalassaemia major. metamyelocytes.




























Figure 12.33 Peripheral blood film findings in β-thalassaemia major.

324 7. Gene therapy of thalasaemia involving genetic
manipulation in haematopoieitc stem cells may become an
option for future.
Since these patients require multiple blood transfusions,
they are at increased risk of developing AIDS. In general,
patients with β-thalassaemia major have short life
expectancy. The biggest problem is iron overload and conse-
quent myocardial siderosis leading to cardiac arrhythmias,
congestive heart failure, and ultimately death.
β β β β β-Thalassaemia Minor
The β-thalassaemia minor or β-thalassaemia trait, a
heterozygous state, is a common entity characterised by
moderate reduction in β-chain synthesis.
CLINICAL FEATURES. Clinically, the condition is usually
Figure 12.34 Osmotic fragility testing β-thalassaemia major asymptomatic and the diagnosis is generally made when the
showing decreased fragility.
patient is being investigated for mild chronic anaemia. The
spleen may be palpable.
7. Platelet count is usually normal but may be reduced in LABORATORY FINDINGS. These are as under:
SECTION II
patients with massive splenomegaly.
8. Osmotic fragility characteristically reveals increased 1. Mild anaemia; mean haemoglobin level is about 15%
lower than in normal person for the age and sex.
resistance to saline haemolysis i.e. decreased osmotic fragility 2. Blood film shows mild anisopoikilocytosis, microcytosis
(Fig. 12.34). and hypochromia, occasional target cells and basophilic
9. Haemoglobin electrophoresis shows presence of increased stippling.
amounts of HbF, increased amount of HbA , and almost 3. Serum bilirubin may be normal or slightly raised.
2
complete absence or presence of variable amounts of HbA.
The increased level of HbA has not been found in any 4. Mild reticulocytosis is often present.
2
other haemoglobin abnormality except β-thalassaemia. 5. MCV, MCH and MCHC may be slightly reduced.
The increased synthesis of HbA is probably due to 6. Osmotic fragility test shows increased resistance to
2
increased activity at both δ-chain loci. haemolysis i.e. decreased osmotic fragility.
10. Bone marrow aspirate examination shows normoblastic 7. Haemoglobin electrophoresis is confirmatory for the
erythroid hyperplasia with predominance of intermediate diagnosis and shows about two-fold increase in HbA and
2
and late normoblasts which are generally smaller in size a slight elevation in HbF (2-3%).
than normal. Iron staining demonstrates siderotic granules
in the cytoplasm of normoblasts, increased reticuloendo- Treatment
thelial iron but ring sideroblasts are only occasionally seen. Patients with β-thalassaemia minor do not require any
treatment. But they should be explained about the genetic
Treatment implications of the disorder, particularly to those of child-
bearing age. If the two subjects of β-thalassaemia trait marry,
Treatment of β-thalassaemia major is largely supportive. there is a 25% chance of developing thalassaemia major in
1. Anaemia is generally severe and patients require regular offsprings. Patients with β-thalassaemia minor have normal
Haematology and Lymphoreticular Tissues
blood transfusions (4-6 weekly) to maintain haemoglobin life expectancy but those who are treated under the mistaken
above 8 g/dl. diagnosis of iron deficiency may develop siderosis and its
2. In order to maintain increased demand of hyperplastic complications.
marrow, folic acid supplement is given. Prevention of Thalassaemia
3. Splenectomy is beneficial in children over 6 years of age
since splenic sequestration contributes to shortened red cell Finally, since thalassaemia is an inheritable disease, its
prevention is possible by making an antenatal diagnosis. This
lifespan. is done by chorionic villous biopsy or cells obtained by
4. Prevention and treatment of iron overload is done by amniocentesis and foetal DNA studied by PCR amplification
chelation therapy (desferrioxamine). Oral chelation with technique for presence of genetic mutations of thalassaemias.
kelfer or deferiprone is also available now.
5. Bone marrow transplantation from HLA-matched donor APLASTIC ANAEMIA AND OTHER
that provides stem cells which can form normal haemoglobin PRIMARY BONE MARROW DISORDERS
is being done in many centres with fair success rate, especially ‘Bone marrow failure’ is the term used for primary disorders
when done at an early stage before end-organ damage has of the bone marrow which result in impaired formation of
supervened.. the erythropoietic precursors and consequent anaemia. It
6. Some workers have found success with cord blood includes the following disorders:
transfusion. 1. Aplastic anaemia, most importantly.

2. Other primary bone marrow disroders such as: myelophthisic  TABLE 12.14: Causes of Pancytopenia. 325
anaemia, pure red cell aplasia, and myelodysplastic
syndromes. I. Aplastic anaemia (Table 12.13)
These disorders are examples of hypoproliferative II. Pancytopenia with normal or increased marrow cellularity e.g.
anaemias in which anaemia is not the only finding but the 1. Myelodysplastic syndromes
major problem is pancytopenia i.e. anaemia, leucopenia and 2. Hypersplenism
thrombocytopenia. While myelodysplastic syndromes are 3. Megaloblastic anaemia
discussed in Chapter 14, other conditions are considered III. Paroxysmal nocturnal haemoglobinuria (page 314)
below. IV. Bone marrow infiltrations e.g.
1. Haematologic malignancies (leukaemias, lymphomas, myeloma)
APLASTIC ANAEMIA 2. Non-haematologic metastatic malignancies
3. Storage diseases
Aplastic anaemia is defined as pancytopenia (i.e. 4. Osteopetrosis
simultaneous presence of anaemia, leucopenia and 5. Myelofibrosis
thrombocytopenia) resulting from aplasia of the bone
marrow. The underlying defect in all cases appears to be Dose-related aplasia of the bone marrow occurs with
sufficient reduction in the number of haematopoietic antimetabolites (e.g. methotrexate), mitotic inhibitors (e.g. CHAPTER 12
pluripotent stem cells which results in decreased or total daunorubicin), alkylating agents (e.g. busulfan), nitroso urea
absence of these cells for division and differentiation. and anthracyclines. In such cases, withdrawal of the drug
ETIOLOGY AND CLASSIFICATION. Based on the usually allows recovery of the marrow elements.
etiology, aplastic anaemia is classified into 2 main types: Idiosyncratic aplasia is depression of the bone marrow due
primary and secondary. Various causes that may give rise to qualitatively abnormal reaction of an individual to a drug
to both these types of aplastic anaemia are summarised in when first administered. The most serious and most common
Table 12.13 and briefly considered below: example of idiosyncratic aplasia is associated with
A. Primary aplastic anaemia. Primary aplastic anaemia chloramphenicol. Other such common drugs are sulfa drugs,
includes 2 entities: a congenital form called Fanconi’s oxyphenbutazone, phenylbutazone, chlorpromazine, gold
anaemia and an immunologically-mediated acquired form. salts etc.
1. Fanconi’s anaemia. This has an autosomal recessive 2. Toxic chemicals. These include examples of industrial,
inheritance and is often associated with other congenital domestic and accidental use of substances such as benzene
anomalies such as skeletal and renal abnormalities, and derivatives, insecticides, arsenicals etc.
sometimes mental retardation. 3. Infections. Aplastic anaemia may occur following viral
2. Immune causes. In many cases, suppression of haemato- hepatitis, Epstein-Barr virus infection, AIDS and other viral
poietic stem cells by immunologic mechanisms may cause illnesses.
aplastic anaemia. The observations in support of autoimmune 4. Miscellaneous. Lastly, aplastic anaemia has been reported
mechanisms are the clinical response to immuno- in association with certain other illnesses such as SLE, and
suppressive therapy and in vitro marrow culture experiments. with therapeutic X-rays.
B. Secondary aplastic anaemia. Aplastic anaemia may occur CLINICAL FEATURES. The onset of aplastic anaemia may
secondary to a variety of industrial, physical, chemical, occur at any age and is usually insidious. The clinical
iatrogenic and infectious causes: manifestations include the following: Introduction to Haematopoietic System and Disorders of Erythroid Series
1. Drugs. A number of drugs are cytotoxic to the marrow 1. Anaemia and its symptoms like mild progressive
and cause aplastic anaemia. The association of a drug with weakness and fatigue.
aplastic anaemia may be either predictably dose-related or 2. Haemorrhage from various sites due to thrombo-
an idiosyncratic reaction. cytopenia such as from the skin, nose, gums, vagina, bowel,
and occasionally in the CNS and retina.
3. Infections of the mouth and throat are commonly present.
  TABLE 12.13: Causes of Aplastic Anaemia. 4. The lymph nodes, liver and spleen are generally not
enlarged.
A. PRIMARY APLASTIC ANAEMIA
1. Fanconi’s anaemia (congenital) LABORATORY FINDINGS. The diagnosis of aplastic
2. Immunologically-mediated (acquired) anaemia is made by a thorough laboratory evaluation and
B. SECONDARY APLASTIC ANAEMIA excluding other causes of pancytopenia (Table 12.14). The
1. Drugs following haematological features are found:
i) Dose-related aplasia e.g. with antimetabolites (methotrexate), mitotic 1. Anaemia. Haemoglobin levels are moderately reduced.
inhibitors (daunorubicin), alkylating agents (busulfan), nitroso urea, The blood picture generally shows normocytic
anthracyclines. normochromic anaemia but sometimes macrocytosis may
ii) Idiosyncratic aplasia e.g. with chloramphenicol, sulfa drugs, be present. The reticulocyte count is reduced or zero.
oxyphenbutazone, phenylbutazone, chlorpromazine, gold salts.
2. Toxic chemicals e.g. benzene derivatives, insecticides, arsenicals. 2. Leucopenia. The absolute granulocyte count is
3. Infections e.g. infectious hepatitis, EB virus infection, AIDS, other particularly low (below 1500/μl) with relative lympho-
viral illnesses. cytosis. The neutrophils are morphologically normal but
4. Miscellaneous e.g. association with SLE and therapeutic X-rays their alkaline phosphatase score is high.

326 3.Thrombocytopenia. Platelet count is always reduced. marrow transplantation are: acute leukaemias—AML in first
4.Bone marrow examination. A bone marrow aspirate remission and ALL in second remission, thalassaemia major
may yield a ‘dry tap’. A trephine biopsy is generally and combined immunodeficiency disease). Complications of
essential for making the diagnosis which reveals patchy bone marrow transplantation such as severe infections, graft
cellular areas in a hypocellular or aplastic marrow due to rejection and graft-versus-host disease (GVHD) may occur
replacement by fat. There is usually a severe depression (Chapter 4).
of myeloid cells, megakaryocytes and erythroid cells so Severe aplastic anaemia is a serious disorder terminating
that the marrow chiefly consists of lymphocytes and in death within 6-12 months in 50-80% of cases. Death is
plasma cells (Fig. 12.35). Haematopoieitc stem cells usually due to bleeding and/or infection.
bearing CD34 marker are markedly reduced or absent. MYELOPHTHISIC ANAEMIA

Treatment Development of severe anaemia may result from infiltration
of the marrow termed as myelophthisic anaemia. The causes
The patients of mild aplasia may show spontaneous recovery, for marrow infiltrations include the following (Table12.14):
while the management of severe aplastic anaemia is a most Haematologic malignancies (e.g. leukaemia, lymphoma,
challenging task. In general, younger patients show better myeloma).
response to proper treatment. The broad outlines of the Metastatic deposits from non-haematologic malignancies
treatment are as under: (e.g. cancer breast, stomach, prostate, lung, thyroid).
A. General management: It consists of the following: Advanced tuberculosis.
SECTION II
1. Identification and elimination of the possible cause. Primary lipid storage diseases (Gaucher’s and Niemann-
2. Supportive care consisting of blood transfusions, platelet Pick’s disease).
concentrates, and treatment and prevention of infections. Osteopetrosis and myelofibrosis may rarely cause
B. Specific treatment: The specific treatment has been myelophthisis.
attempted with varying success and includes the following: The type of anaemia in myelophthisis is generally
1. Marrow stimulating agents such as androgen may be normocytic normochromic with some fragmented red cells,
administered orally. basophilic stippling and normoblasts in the peripheral blood.
2. Immunosuppressive therapy with agents such as anti- Thrombocytopenia is usually present but the leucocyte count
thymocyte globulin and anti-lymphocyte serum has been is increased with slight shift-to-left of myeloid cells i.e. a
tried with 40-50% success rate. Very high doses of picture of leucoerythroblastic reaction consisting of immature
glucocorticoids or cyclosporine may yield similar responses. myeloid cells and normoblasts is seen in the peripheral blood.
But splenectomy does not have any role in the management Treatment consists of reversing the underlying pathologic
of aplastic anaemia. process.
3. Bone marrow transplantation is considered in severe cases PURE RED CELL APLASIA
under the age of 40 years where the HLA and mixed culture- Pure red cell aplasia (PRCA) is a rare syndrome involving a
matched donor is available. (Other indications for bone selective failure in the production of erythroid elements in
the bone marrow but with normal granulopoiesis and mega-
karyocytopoiesis. Patients have normocytic normochromic
anaemia with normal granulocyte and platelet count.
Reticulocytes are markedly decreased or are absent.
PRCA exists in the following forms:
Transient self-limited PRCA: It is due to temporary marrow
Haematology and Lymphoreticular Tissues
failure in aplastic crisis in haemolytic anamias and in acute
B19 parvovirus infection and in transient erythroblastopenia
in normal children.
Acquired PRCA: It is seen in middle-aged adults in asso-
ciation with some other diseases, most commonly thymoma;
others are connective tissue diseases (SLE, rheumatoid
arthritis), lymphoid malignancies (lymphoma, T-cell
chronic lymphocytic leukaemia) and solid tumours.
Chronic B19 parvovirus infections: PRCA may occur from
chronic B19 parvovirus infection in children and is common
and treatable. B19 parvovirus produces cytopathic effects on
the marrow erythroid precursor cells and are charac-
Figure 12.35 Bone marrow trephine biopsy in aplastic anaemia teristically seen as giant pronomoblasts.
contrasted against normal cellular marrow. A, normal marrow biopsy Congenital PRCA (Blackfan-Diamond syndrome) is a rare
shows about 50% fatty spaces and about 50% is haematopoietic marrow chronic disorder detected at birth or in early childhood. It
which contains a heterogeneous mixture of myeloid, erythroid and
lymphoid cells. B, In aplastic anaemia, the biopsy shows suppression of occurs due to mutation in a ribosomal RNA processing gene
myeloid and erythroid cells and replacement of haematopoetic elements termed as RPS19. The disorder is corrected by glucocorticoids
by fat. There are scanty foci of cellular components composed chiefly of and marrow transplantation.
lymphoid cells.
❑

Disorders of Platelets, 327

Chapter 13
Chapter 13 Bleeding Disorders and

Basic Transfusion Medicine





THROMBOPOIESIS granules. Platelets are formed from pseudopods of
megakaryocyte cytoplasm which get detached into the blood
As outlined in previous chapter and illustrated in Fig. 12.3, stream. Each megakaryocyte may form up to 4000 platelets.
the trilineage myeloid stem cells in the bone marrow The formation of platelets from the stem cell takes about
differentiate into erythroid progenitor, granulocyte- 10 days.
monocyte progenitor, and megakaryocyte progenitor cells. PLATELETS. Platelets are small (1-4 μm in diameter),
Platelets are formed in the bone marrow by a process of discoid, non-nucleate structures containing red-purple CHAPTER 13
fragmentation of the cytoplasm of megakaryocytes. Platelet granules. The normal platelet count ranges from 150,000-
production is under the control of thrombopoietin, the nature 400,000/μl and the lifespan of platelets is 7-10 days. About
and origin of which are not yet established. The stages in 70% of platelets are in circulation while remaining 30% lie
platelet production are: megakaryoblast, promegakaryocyte, sequestered in the spleen. Newly-formed platelets spend
megakaryocyte, and discoid platelets (Fig. 13.1).
24-36 hours in the spleen before being released into
MEGAKARYOBLAST. The earliest precursor of platelets circulation but splenic stasis does not cause any injury to the
in the bone marrow is megakaryoblast. It arises from platelets normally. Factors such as stress, epinephrine and
haematopoietic stem cell by a process of differentiation. exercise stimulate platelet production.
The main functions of platelets is in haemostasis which
PROMEGAKARYOCYTE. A megakaryoblast undergoes includes two closely linked processes:
endo-reduplication of nuclear chromatin i.e. nuclear
chromatin replicates repeatedly in multiples of two without 1. Primary haemostasis. This term is used for platelet plug
division of the cell. Ultimately, a large cell containing up to formation at the site of injury. It is an immediate phenomenon
32 times the normal diploid content of nuclear DNA appearing within seconds of injury and is responsible for
(polyploidy) is formed when further nuclear replication cessation of bleeding from microvasculature. Primary
ceases and cytoplasm becomes granular. haemostasis involves three steps: platelet adhesion, platelet
granule release and platelet aggregation which are regulated
MEGAKARYOCYTE. A mature megakaryocyte is a large by changes in membrane phospholipids, and calcium
cell, 30-90 μm in diameter, and contains 4-16 nuclear lobes (Fig. 13.2). At molecular level, these important events are
having coarsely clumped chromatin. The cytoplasm is depicted diagrammatically in Fig. 13.3 and briefly outlined
abundant, light blue in colour and contains red-purple below: Disorders of Platelets, Bleeding Disorders and Basic Transfusion Medicine






























Figure 13.1 Thrombopoiesis.

328





















Figure 13.2 Main events in primary haemostasis—platelet adhesion, Figure 13.3 Molecular mechanisms in platelet adhesion and release
release (activation) and aggregation. reaction (Gp = glycoprotein).

Platelet adhesion: Platelets adhere to collagen in the IV. Haemorrhagic diathesis due to fibrinolytic defects.
SECTION II
subendothelium due to presence of receptor on platelet V. Combination of all these as occurs in disseminated
surface, glycoprotein (Gp) Ia-IIa which is an integrin. The intravascular coagulation (DIC).
adhesion to the vessel wall is further stabilised by von Before discussing the bleeding disorders, it is considered
Willebrand factor, an adhesion glycoprotein. This is achieved desirable to briefly review the broad outlines of scheme of
by formation of a link between von Willebrand factor and investigations to be carried out in such a case.
another platelet receptor, GpIb-IX complex.
Platelet release: After adhesion, platelets become INVESTIGATIONS OF HAEMOSTATIC FUNCTION
activated and release three types of granules from their
cytoplasm: dense granules, α-granules and lysosomal Normal haemostatic mechanism and thrombogenesis were
vesicles. Important products released from these granules discussed in Chapter 5. In general, the haemostatic
are: ADP, ATP, calcium, serotonin, platelet factor 4, factor mechanisms have 2 primary functions:
V, factor VIII, thrombospondin, platelet-derived growth To promote local haemostasis at the site of injured blood
factor (PDGF), von Willebrand factor (vWF), fibronectin, vessel.
fibrinogen, plasminogen activator inhibitor –1 (PAI-1) and To ensure that the circulating blood remains in fluid state
thromboxane A2. while in the vascular bed i.e. to prevent the occurrence of
Platelet aggregation: This process is mediated by fibrinogen generalised thrombosis.
which forms bridge between adjacent platelets via Formation of haemostatic plug is a complex mechanism
glycoprotein receptors on platelets, GpIIb-IIIa. and involves maintenance of a delicate balance among at least
2. Secondary haemostasis. This involves plasma coagu- 5 components (Fig. 13.4): (i) blood vessel wall; (ii) platelets;
lation system resulting in fibrin plug formation and takes (iii) plasma coagulation factors; (iv) inhibitors; and (v) fibrino-
several minutes for completion. This is discussed in detail in lytic system.
Chapter 5. Anything that interferes with any of these components
Haematology and Lymphoreticular Tissues
results in defective haemostasis with abnormal bleeding. In
BLEEDING DISORDERS order to establish a definite diagnosis in any case suspected
to have abnormal haemostatic functions, the following
(HAEMORRHAGIC DIATHESIS) scheme is followed:
Bleeding disorders or haemorrhagic diatheses are a group A. Comprehensive clinical evaluation, including the patient’s
of disorders characterised by defective haemostasis with history, family history and details of the site, frequency and
abnormal bleeding. The tendency to bleeding may be character of haemostatic defect.
spontaneous in the form of small haemorrhages into the skin B. Series of screening tests for assessing the abnormalities in
and mucous membranes (e.g. petechiae, purpura, various components involved in maintaining haemostatic
ecchymoses), or there may be excessive external or internal balance.
bleeding following trivial trauma and surgical procedure (e.g. C. Specific tests to pinpoint the cause.
haematoma, haemarthrosis etc). A brief review of general principles of tests used to
The causes of haemorrhagic diatheses may or may not investigate haemostatic abnormalities is presented below and
be related to platelet abnormalities. These causes are broadly summarised in Table 13.1.
divided into the following groups:
I. Haemorrhagic diathesis due to vascular abnormalities. A. Investigation of Disordered Vascular Haemostasis
II. Haemorrhagic diathesis related to platelet abnormalities. Disorders of vascular haemostasis may be due to increased
III. Disorders of coagulation factors. vascular permeability, reduced capillary strength and failure

diastolic and systolic for 5 minutes. After deflation, the 329
number of petechiae appearing in the next 5 minutes in
2
3 cm area over the cubital fossa are counted. Presence of
more than 20 petechiae is considered a positive test. The test
is positive in increased capillary fragility as well as in
thrombocytopenia.

B. Investigation of Platelets and Platelet Function
Haemostatic disorders are commonly due to abnormalities
in platelet number, morphology or function.

1. SCREENING TESTS. The screening tests carried out for
assessing platelet-related causes are as under:
i) Peripheral blood platelet count.
ii) Skin bleeding time.
iii) Examination of fresh blood film to see the morphologic CHAPTER 13
Figure 13.4 The haemostatic balance.
abnormalities of platelets.
2. SPECIAL TESTS. If these screening tests suggest a
to contract after injury. Tests of defective vascular function disorder of platelet function, the following platelet function
are as under: tests may be carried out:
1. BLEEDING TIME. This simple test is based on the i) Platelet adhesion tests such as retention in a glass bead
principle of formation of haemostatic plug following a column, and other sophisticated techniques.
standard incision on the volar surface of the forearm and the ii) Aggregation tests which are turbidometric techniques
time the incision takes to stop bleeding is measured. The test using ADP, collagen or ristocetin.
is dependent upon capillary function as well as on platelet iii) Granular content of the platelets and their release can be
number and ability of platelets to adhere to form aggregates. assessed by electron microscopy or by measuring the
Normal range is 3-8 minutes. A prolonged bleeding time may substances released.
be due to following causes: iv) Platelet coagulant activity is measured indirectly by
i) Thrombocytopenia. prothrombin consumption index.
ii) Disorders of platelet function.
iii) von Willebrand’s disease. C. Investigation of Blood Coagulation
iv) Vascular abnormalities (e.g. in Ehlers-Danlos syndrome).
v) Severe deficiency of factor V and XI. The normal blood coagulation system consists of cascade of
activation of 13 coagulation factors. These form intrinsic,
2. HESS CAPILLARY RESISTANCE TEST (TOURNI- extrinsic and common pathways which culminate in
QUET TEST). This test is done by tying sphygmomanometer formation of thrombin that acts on fibrinogen to produce
cuff to the upper arm and raising the pressure in it between fibrin. Fibrin clot so formed is strengthened by factor XIII Disorders of Platelets, Bleeding Disorders and Basic Transfusion Medicine


 TABLE 13.1: Screening Tests for Haemostasis (Coagulation Tests).
Laboratory Test Factor/Function Measured Associated Disorders
1. Bleeding time Platelet function, vascular integrity i) Qualitative disorders of platelets
ii) von Willebrand’s disease
iii) Quantitative disorders of platelets
iv) Acquired vascular disorders
2. Platelet count Quantification of platelets i) Thrombocytopenia
ii) Thrombocytosis
3. Prothrombin time Evaluation of extrinsic and common pathway i) Oral anticoagulant therapy
(deficiency of factors I, II, V, VII, X) ii) DIC
iii) Liver disease
4. Partial thromboplastin time Evaluation of intrinsic and common pathway i) Parenteral heparin therapy
(deficiency of factors I, II, V, VIII, IX, X, XI, XII) ii) DIC
iii) Liver disease
5. Thrombin time Evaluation of common pathway i) Afibrinogenaemia
ii) DIC
iii) Parenteral heparin therapy

330


























SECTION II















Figure 13.5 Pathways of blood coagulation, fibrinolytic system and participation of platelets in activation of the cascade and their role in
haemostatic plug formation.

which itself gets activated by thrombin. The process of III. One-stage prothrombin time (PT). PT measures the
fibrinolysis or clot dissolution and the role of platelets in extrinsic system factor VII as well as factors in the common
activation of cascade and formation of haemostatic plug are pathway. In this test, tissue thromboplastin (e.g. brain extract)
illustrated in Fig. 13.5. and calcium are added to the test. The normal PT in this test
is 10-14 seconds. The common causes of prolonged one-stage
1. SCREENING TESTS. Tests for blood coagulation sys- PT are as under:
tem include a battery of screening tests. These are as under: i) Administration of oral anticoagulant drugs.
ii) Liver disease, especially obstructive liver disease.
Haematology and Lymphoreticular Tissues
I. Whole blood coagulation time. The estimation of whole
blood coagulation time done by various capillary and tube iii) Vitamin K deficiency.
methods is of limited value since it is an insensitive and iv) Disseminated intravascular coagulation.
nonspecific test. Normal range is 4-9 minutes at 37°C. IV. Measurement of fibrinogen. The screening tests for
fibrinogen deficiency are semiquantitative fibrinogen titre
II. Activated partial thromboplastin time (APTT) or partial and thrombin time (TT). The normal value of thrombin time
thromboplastin time with kaolin (PTTK). This test is used is under 20 seconds, while a fibrinogen titre in plasma
to measure the intrinsic system factors (VIII, IX, XI and XII) dilution up to 32 is considered normal. Following are the
as well as factors common to both intrinsic and extrinsic common causes for higher values in both these tests:
systems (factors X, V, prothrombin and fibrinogen). The test i) Hypofibrinogenaemia (e.g. in DIC).
consists of addition of 3 substances to the plasma—calcium, ii) Raised concentration of FDP.
phospholipid and a surface activator such as kaolin. The iii) Presence of heparin.
normal range is 30-40 seconds. The common causes of a
prolonged PTTK (or APTT) are as follows: 2. SPECIAL TESTS. In the presence of an abnormality in
i) Parenteral administration of heparin. screening tests, detailed investigations for the possible cause
ii) Disseminated intravascular coagulation. are carried out. These include the following:
iii) Liver disease. i) Coagulation factor assays. These bioassays are based on
iv) Circulating anticoagulants. results of PTTK or PT tests and employ the use of substrate

plasma that contains all other coagulation factors except the source of frequent episodes of bleeding from the nose and 331
one to be measured. The unknown level of the factor activity gastrointestinal tract.
is compared with a standard control plasma with a known 2. Inherited disorders of connective tissue matrix. These
level of activity. Results are expressed as percentage of include Marfan’s syndrome, Ehlers-Danlos syndrome and
normal activity.
pseudoxanthoma elasticum, all of which have inherited
ii) Quantitative assays. The coagulation factors can be defect in the connective tissue matrix and, thus, have fragile
quantitatively assayed by immunological and other chemical skin vessels and easy bruising.
methods.
B. Acquired Vascular Bleeding Disorders
D. Investigation of Fibrinolytic System
Several acquired conditions are associated with vascular
Increased levels of circulating plasminogen activator are purpuras. These are as under:
present in patients with hyperfibrinolysis. Following
screening tests are done to assess these abnormalities in 1. Henoch-Schönlein purpura. Henoch-Schönlein or
fibrinolytic system: anaphylactoid purpura is a self-limited type of hyper-
1. Estimation of fibrinogen. sensitivity vasculitis occurring in children and young adults.
2. Fibrin degradation products (FDP) in the serum. Circulating immune complexes are deposited in the vessel CHAPTER 13
3. Ethanol gelation test. wall consisting of IgA, C3 and fibrin, and in some cases,
4. Euglobin or whole blood lysis time. properdin suggesting activation of alternate complement
More specific tests include: functional assays, immuno- pathway as the trigger event. The hypersensitivity vasculitis
logical assays by ELISA, and chromogenic assays of produces purpuric rash on the extensor surfaces of arms, legs
plasminogen activators, plasminogen, plasminogen activator and on the buttocks, as well as haematuria, colicky
inhibitor, and FDP. abdominal pain due to bleeding into the GIT, polyarthralgia
With this background knowledge on work up of a case and acute nephritis. In spite of these haemorrhagic features,
of haemostatic abnormality, we now turn to discuss the all coagulation tests are normal.
common specific haemorrhagic disorders under the 2. Haemolytic-uraemic syndrome. Haemolytic-uraemic
following headings: syndrome is a disease of infancy and early childhood in which
I. Haemostatic diatheses due to vascular disorders. there is bleeding tendency and varying degree of acute renal
II. Haemostatic diatheses due to platelet disorders. failure. The disorder remains confined to the kidney where
III. Coagulation disorders. hyaline thrombi are seen in the glomerular capillaries.
IV. Haemostatic diatheses due to fibrinolytic defects. 3. Simple easy bruising (Devil’s pinches). Easy bruising
V. Disseminated intravascular coagulation (DIC) of unknown cause is a common phenomenon in women of
child-bearing age group.
HAEMORRHAGIC DIATHESES DUE TO
VASCULAR DISORDERS 4. Infection. Many infections cause vascular haemorrhages
either by causing toxic damage to the endothelium or by DIC.
Vascular bleeding disorders, also called non-thrombo- These are especially prone to occur in septicaemia and severe
cytopenic purpuras or vascular purpuras, are normally mild measles.
and characterised by petechiae, purpuras or ecchymoses 5. Drug reactions. Certain drugs form antibodies and Disorders of Platelets, Bleeding Disorders and Basic Transfusion Medicine
confined to the skin and mucous membranes. The produce hypersensitivity (or leucocytoclastic) vasculitis
pathogenesis of bleeding is poorly understood since majority responsible for abnormal bleeding.
of the standard screening tests of haemostasis including the
bleeding time, coagulation time, platelet count and platelet 6. Steroid purpura. Long-term steroid therapy or Cushing’s
function, are usually normal. Vascular purpuras arise from syndrome may be associated with vascular purpura due to
damage to the capillary endothelium, abnormalities in the defective vascular support.
subendothelial matrix or extravascular connective tissue that 7. Senile purpura. Atrophy of the supportive tissue of
supports the blood vessels, or from formation of abnormal cutaneous blood vessels in old age may cause senile atrophy,
blood vessels. especially in the dorsum of forearm and hand.
Vascular bleeding disorders may be inherited or acquired.
8. Scurvy. Deficiency of vitamin C causes defective colla-
gen synthesis which causes skin bleeding as well as bleeding
A. Inherited Vascular Bleeding Disorders
into muscles, and occasionally into the gastrointestinal and
A few examples of hereditary vascular disorders are given genitourinary tracts.
below:
HAEMORRHAGIC DIATHESES DUE TO
1. Hereditary haemorrhagic telangiectasia (Osler-Weber- PLATELET DISORDERS
Rendu disease). This is an uncommon inherited autosomal
dominant disorder. The condition begins in childhood and Disorders of platelets produce bleeding disorders by one of
is characterised by abnormally telangiectatic (dilated) the following 3 mechanisms:
capillaries. These telangiectasias develop particularly in the A. Due to reduction in the number of platelets i.e. various forms
skin, mucous membranes and internal organs and are the of thrombocytopenias.

332 B. Due to rise in platelet count i.e. thrombocytosis. antimetabolites), certain antibiotics (sulfonamides, PAS,
C. Due to defective platelet functions. rifampicin, penicillins), drugs used in cardiovascular diseases
(digitoxin, thiazide diuretics), diclofenac, acyclovir, heparin
A. THROMBOCYTOPENIAS and excessive consumption of ethanol.
Clinically, the patient presents with acute purpura. The
Thrombocytopenia is defined as a reduction in the peripheral platelet count is markedly lowered, often below 10,000/μl
blood platelet count below the lower limit of normal i.e. below and the bone marrow shows normal or increased number of
150,000/μl. Thrombocytopenia is associated with abnormal megakaryocytes.
bleeding that includes spontaneous skin purpura and The immediate treatment is to stop or replace the
mucosal haemorrhages as well as prolonged bleeding after suspected drug with instruction to the patient to avoid taking
trauma. However, spontaneous haemorrhagic tendency the offending drug in future. Occasional patients may require
becomes clinically evident only after severe depletion of the temporary support with glucocorticoids, plasmapheresis or
platelet count to level below 20,000/μl. platelet transfusions.
Thrombocytopenia may result from 4 main groups of
causes: Heparin-induced Thrombocytopenia
1. Impaired platelet production.
2. Accelerated platelet destruction. Thrombocytopenia due to administration of heparin is
distinct from that caused by other drugs in following ways:
3. Splenic sequestration. i) Thrombocytopenia is generally not so severe to fall to level
4. Dilutional loss. below 20,000/μl.
SECTION II
A list of causes of thrombocytopenia is given in ii) Unlike drug-induced thrombocytopenia, heparin-induced
Table 13.2. Three of the common and important causes— thrombocytopenia is not associated with bleeding but instead
drug-induced thrombocytopenia, idiopathic thrombocyto- these patients are more prone to develop thrombosis.
penic purpura (ITP), and thrombotic thrombocytopenic The underlying mechanism of heparin-induced
purpura (TTP), are discussed below. thrombocytopenia is formation of antibody against platelet
factor 4 (PF-4)-heparin complex. This specific antibody
Drug-induced Thrombocytopenia activates the endothelial cells and initiates thrombus
Many commonly used drugs cause thrombocytopenia by formation. It occurs in a small proportion of cases after the
depressing megakaryocyte production. In most cases, an patient has received heparin for 5-10 days.
immune mechanism by formation of drug-antibody comp- Diagnosis is made by a combination of laboratory and
lexes is implicated in which the platelet is damaged as an clinical features with 4 Ts: thrombocytopenia, thrombosis,
‘innocent bystander’. Drug-induced thrombocytopenia is time of fall of platelet count, absence of other causes of
associated with many commonly used drugs and includes: thrombocytopenia.
chemotherapeutic agents (alkylating agents, anthracyclines,
Immune Thrombocytopenic Purpura (ITP)

Idiopathic thrombocytopenic purpura or immune
 TABLE 13.2: Causes of Thrombocytopenia.
thrombocytopenic purpura (ITP), is characterised by
I. IMPAIRED PLATELET PRODUCTION immunologic destruction of platelets and normal or increased
1. Generalised bone marrow failure e.g. megakaryocytes in the bone marrow.
Aplastic anaemia, leukaemia, myelofibrosis, megaloblastic PATHOGENESIS. On the basis of duration of illness, ITP is
anaemia, marrow infiltrations (carcinomas, lymphomas, multiple
Haematology and Lymphoreticular Tissues
myeloma, storage diseases). classified into acute and chronic forms, both of which have
different pathogenesis.
2. Selective suppression of platelet production e.g.
Drugs (quinine, quinidine, sulfonamides, PAS, rifampicin, Acute ITP. This is a self-limited disorder, seen most
anticancer drugs, thiazide diuretics), (heparin, diclofenac, frequently in children following recovery from a viral illness
acyclovir), alcohol intake. (e.g. hepatitis C, infectious mononucleosis, CMV infection,
HIV infection) or an upper respiratory illness. The onset of
II. ACCELERATED PLATELET DESTRUCTION
1. Immunologic thrombocytopenias e.g. acute ITP is sudden and severe thrombocytopenia but
recovery occurs within a few weeks to 6 months. The
ITP (acute and chronic), neonatal and post-transfusion
(isoimmune), drug-induced, secondary immune ‘thrombo- mechanism of acute ITP is by formation of immune complexes
cytopenia (post-infection, SLE, AIDS, CLL, lymphoma). containing viral antigens, and by formation of antibodies
against viral antigens which crossreact with platelets and
2. Increased consumption e.g. lead to their immunologic destruction.
DIC, TTP, giant haemangiomas, microangiopathic haemolytic
anaemia. Chronic ITP. Chronic ITP occurs more commonly in adults,
particularly in women of child-bearing age (20-40 years). The
III. SPLENIC SEQUESTRATION
Splenomegaly disorder develops insidiously and persists for several years.
Though chronic ITP is idiopathic, similar immunologic
IV. DILUTIONAL LOSS thrombocytopenia may be seen in association with SLE, AIDS
Massive transfusion of old stored blood to bleeding patients.
and autoimmune thyroiditis. The pathogenesis of chronic ITP

333



























CHAPTER 13
















Figure 13.6 Laboratory findings of ITP contrasted with those found
in a normal individual. A, Peripheral blood in ITP shows presence of
reduced number of platelets which are often large. B, Bone marrow in
ITP shows characteristically increased number of megakaryocytes with
single non-lobulated nuclei and reduced cytoplasmic granularity (inbox
on right photomicrograph).



is explained by formation of anti-platelet autoantibodies, LABORATORY FINDINGS. The diagnosis of ITP can be Disorders of Platelets, Bleeding Disorders and Basic Transfusion Medicine
usually by platelet-associated IgG humoral antibodies suspected on clinical features after excluding the known
synthesised mainly in the spleen. These antibodies are causes of thrombocytopenia and is supported by the
directed against target antigens on the platelet glycoproteins, following haematologic findings:
Gp IIb-IIIa and Gp Ib-IX complex. Some of the antibodies 1. Platelet count is markedly reduced, usually in the range
directed against platelet surface also interfere in their of 10,000-50,000/μl.
function. The mechanism of platelet destruction is similar to
that seen in autoimmune haemolytic anaemias. Sensitised 2. Blood film shows only occasional platelets which are
platelets are destroyed mainly in the spleen and rendered often large in size (Fig. 13.6,A).
susceptible to phagocytosis by cells of the reticuloendothelial 3. Bone marrow shows increased number of mega-
system. karyocytes which have large non-lobulated single nuclei
and may have reduced cytoplasmic granularity and
CLINICAL FEATURES. The clinical manifestation of ITP presence of vacuoles (Fig. 13.6, B).
may develop abruptly in cases of acute ITP, or the onset may 4. With sensitive techniques, anti-platelet IgG antibody can
be insidious as occurs in majority of cases of chronic ITP. be demonstrated on platelet surface or in the serum of
The usual manifestations are petechial haemorrhages, easy patients.
bruising, and mucosal bleeding such as menorrhagia in 5. Platelet survival studies reveal markedly reduced platelet
women, nasal bleeding, bleeding from gums, melaena and lifespan, sometimes less than one hour, as compared with
haematuria. Intracranial haemorrhage is, however, rare. normal lifespan of 7-10 days.
Splenomegaly and hepatomegaly may occur in cases with
chronic ITP but lymphadenopathy is quite uncommon in TREATMENT. Spontaneous recovery occurs in 90% cases
either type of ITP. of acute ITP, while only less than 10% cases of chronic ITP

334 recover spontaneously. Treatment is directed at reducing the B. THROMBOCYTOSIS
level and source of autoantibodies and reducing the rate of Thrombocytosis is defined as platelet count in excess of
destruction of sensitised platelets. This is possible by 4,00,000/μl. While essential or primary thrombocytosis or
corticosteroid therapy, immunosuppressive drugs (e.g. thrombocythaemia is discussed under myeloproliferative
vincristine, cyclophosphamide and azathioprine) and disorders in the next chapter, secondary or reactive
splenectomy. Beneficial effects of splenectomy in chronic ITP thrombocytosis can occur following massive haemorrhage,
are due to both removal of the major site of platelet iron deficiency, severe sepsis, marked inflammation,
destruction and the major source of autoantibody synthesis. disseminated cancers, haemolysis, or following splenectomy.
Platelet transfusions are helpful as a palliative measure only Thrombocytosis causes bleeding or thrombosis but how it
in patients with severe haemorrhage. produces is not clearly known.
As such, transitory and secondary thrombocytisis does
Thrombotic Thrombocytopenic Purpura (TTP) and not require any separate treatment other than treating the
Haemolytic-Uraemic Syndrome (HUS) cause.
Thrombotic thrombocytopenic purpura (TTP) and haemo-
lytic-uraemic syndrome (HUS) are a group of thrombotic C. DISORDERS OF PLATELET FUNCTIONS
microangiopathies which are essentially characterised by Defective platelet function is suspected in patients who show
triad of thrombocytopenia, microangiopathic haemolytic anaemia skin and mucosal haemorrhages and have prolonged
and formation of hyaline fibrin microthrombi within the micro- bleeding time but a normal platelet count. These disorders
SECTION II
vasculature throughout the body. These are often fulminant may be hereditary or acquired.
and lethal disorders occurring in young adults. The intra-
vascular microthrombi are composed predominantly of Hereditary Disorders
platelets and fibrin. The widespread presence of these platelet Depending upon the predominant functional abnormality,
microthrombi is responsible for thrombocytopenia due to inherited disorders of platelet functions are classified into
increased consumption of platelets, microangiopathic haemo- the following 3 groups:
lytic anaemia and protean clinical manifestations involving
different organs and tissues throughout the body. 1. DEFECTIVE PLATELET ADHESION. These are as
under:
PATHOGENESIS. Unlike DIC, a clinicopathologically i) Bernard-Soulier syndrome is an autosomal recessive
related condition, activation of the clotting system is not the disorder with inherited deficiency of a platelet membrane
primary event in formation of microthrombi. TTP is initiated glycoprotein which is essential for adhesion of platelets to
by endothelial injury followed by release of von Willebrand vessel wall.
factor and other procoagulant material from endothelial cells,
leading to the formation of microthrombi. Trigger for the ii) In von Willebrand’s disease, there is defective platelet
endothelial injury comes from immunologic damage by adhesion as well as deficiency of factor VIII (page 336).
diverse conditions such as in pregnancy, metastatic cancer, 2. DEFECTIVE PLATELET AGGREGATION. In thromba-
high-doze chemotherapy, HIV infection, and mitomycin C. sthenia (Glanzmann’s disease), there is failure of primary
platelet aggregation with ADP or collagen due to inherited
CLINICAL FEATURES. The clinical manifestations of TTP deficiency of two of platelet membrane glycoproteins.
are due to microthrombi in the arterioles, capillaries and
venules throughout the body. Besides features of thrombo- 3. DISORDERS OF PLATELET RELEASE REACTION.
cytopenia and microangiopathic haemolytic anaemia, These disorders are characterised by normal initial
Haematology and Lymphoreticular Tissues
characteristic findings include fever, transient neurologic aggregation of platelets with ADP or collagen but the
deficits and renal failure. The spleen may be palpable. subsequent release of ADP, prostaglandins and 5-HT is
defective due to complex intrinsic deficiencies.
LABORATORY FINDINGS. The diagnosis can be made
from the following findings: Acquired Disorders
1. Thrombocytopenia. Acquired defects of platelet functions include the following
2. Microangiopathic haemolytic anaemia with negative clinically significant examples:
Coombs’ test. 1. ASPIRIN THERAPY. Prolonged use of aspirin leads to
3. Leucocytosis, sometimes with leukaemoid reaction. easy bruising and abnormal bleeding time. This is because
4. Bone marrow examination reveals normal or slightly aspirin inhibits the enzyme cyclooxygenase, and thereby
increased megakaryocytes accompanied with some suppresses the synthesis of prostaglandins which are
myeloid hyperplasia. involved in platelet aggregation as well as release reaction.
5. Diagnosis is, however, established by examination of The anti-platelet effect of aspirin is clinically applied in
biopsy (e.g. from gingiva) which demonstrates typical preventing major thromboembolic disease in recurrent
microthrombi in arterioles, capillaries and venules, myocardial infarction.
unassociated with any inflammatory changes in the vessel 2. OTHERS. Several other acquired disorders are associated
wall.
with various abnormalities in platelet functions at different