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Introduction to Haematology
Introduction to
Haematology
Chapter Outline
• Introduction
• Components of Blood and Their Functions
▪ Erythrocytes
▪ Leukocytes
▪ Thrombocytes
▪ Plasma and serum (liquid components)
• Haematopoietic System of the Body
▪ Erythropoiesis
▪ Leukopoiesis
▪ Thrombopoiesis
• Review Questions
229
8
Anuradha Chakravarthy
IntroductIon
Blood has been considered the essence of life for centuries. Since the invention of microscope,
the cellular elements of blood have come to be recognized. It is pumped by heart and circulates
through the body in the vascular system. After performing a thorough history and physical
examination, testing the blood allows for the best way to diagnose diseases.
Haematology (haem–blood, logos–study) is the study of blood and is concerned primar-
ily with the study of formed elements of the blood. These include erythrocytes or red
blood cells (RBC), leukocytes or white blood cells (WBC), and thrombocytes or platelets
(PLT). Thus, the haematology laboratory routinely reports the number of cells in circula-
tion, haemoglobin concentration and dierential count of leukocytes based on the study
of the stained blood smear.
Study of the blood smear under microscope also helps in detecting the morphological
abnormalities of various cells seen in the peripheral blood circulation. This test may be done
as part of a general health exam to help diagnose many illnesses. Blood smears are made on
microscope slides and stained before they are examined under the microscope. The smear
shows the number and type of white blood cells (dierential), abnormally-shaped blood cells,
and gives a rough estimate of white blood cell and platelet counts.
Another aspect of the haematology laboratory is to investigate the causes of bleeding
disorders. The technician will need whole blood to investigate the disorders. This is discussed
separately in another section of this book (Chapters 13 and 14).
The blood specimen is collected by a phlebotomist (technician who collects the blood
from blood vein). The blood sample then comes to the laboratory and receives a permanent
identication number, preferably through a bar code. The entire process may be automated to
230
expedite the process. Technicians working in the haematology laboratory should be acquainted with the structure, function, production and development of various cellular elements of
circulating blood in order to understand the clinical signicance of dierent haematological
tests and to identify technical errors.
Medical Laboratory Technology: Volume 1
components of Blood and theIr functIons
Blood ows through the vascular channels and transports vital nutrients and waste products
of the body. Other functions of the components of blood include defending the body through
immunologic reactions, stoppage of bleeding and maintenance of body temperature.
The blood has two major components—cellular and uid. The cellular component consists
of erythrocytes, leukocytes and thrombocytes (platelets). Thrombocytes are the smallest
component (1–4 μm in diameter), erythrocytes are middle-sized (6.7 to 7.7 μm in diameter)
and leukocytes are the largest with a wide range of size (9 to 20 μm). Red blood cells (RBCs)
constitute the highest number of formed elements in the circulation (5.5 × 106/μL.), followed
by platelets (300 × 103/μL) and leukocytes (7.5 × 103/μL) [1L = 106 µL and 1 µL = 1 cubic mm or
mm3]. We have used units that are in common usage in laboratories of developing countries.
These formed elements oat in the uid component of blood, called plasma, and are thus
able to perform their functions. The plasma has a soluble protein in it, called brinogen.
During the cloing of blood, brinogen is removed from plasma as brin and the clear uid
left behind is called serum. Thus, in order to obtain plasma for diagnostic tests, blood should
not be allowed to clot. On the other hand, if serum is needed (contains all chemicals), allow
the blood to clot and then centrifuge. The uid component above the clot is the serum.
Erythrocytes
Erythrocytes (erythros–red, cytos–cell) are non-nucleated cells. They contain haemoglobin (a
conjugated protein) and are the heaviest of all the formed elements of blood. The shape of
the erythrocyte is that of a biconcave disc, aached back-to-back on two sides, thus making it
thicker at the edge and thinner at the centre (Figure 8.1). Hence, the at erythrocyte seen in the
peripheral blood appears to have a paler area at the centre. Haemoglobin is responsible for
carrying oxygen from the lungs to the tissue, and brings back to the lung the gaseous waste
products (carbon dioxide) of cellular metabolism. Internal uid concentration of erythrocytes
is equivalent to about 0.85% sodium chloride. If erythrocytes are dropped in water, they swell
due to endosmosis (water moving into the cell), which leads to the rupture of the membrane.
This is called haemolysis (haem–blood, lysis–destruction). Conversely, if erythrocytes are
dropped in concentrated solutions of sodium chloride (e.g., 2% NaCl), they shrink due to
exosmosis (water moving out of the cell). This is why physiological saline injected into the
body through the intravenous route (IV) has a concentration of 0.85%) NaCl. This is also
called normal saline because it is equal to the normal solute concentration of the body.
Figure 8.1 Morphology of red blood cells
Introduction to Haematology
231
Leukocytes
The leukocytes (leukos–white, cytos–cell) or white blood cells (WBC) are a group of nucleated
cells that assist in the defence mechanism of the body against invasion of pathogenic
microbes. The three well-recognized types of leukocytes found in blood circulation
are granulocytes, lymphocytes and monocytes (Figure 8.2). Granulocytes are further
subdivided into neutrophils, eosinophils and basophils. Neutrophils are sometimes called
polymorphonuclear cells or PMN (in short, polys or polymorphs). They ingest (phagocytose)
bacteria within the cells. This is the defence mechanism the body utilizes against any
foreign particle like bacteria. Hence leucocytosis (increased number of white blood cells) is
commonly seen in case of bacterial infections. Eosinophils and basophils protect the body
by releasing chemical substances (histamine and heparin), which set o a chain of reactions
ultimately leading to an inammatory response at the site of infection. Allergic reactions are
also produced in the same way. Thus, eosinophilia and basophilia are commonly seen in many
allergic reactions as well as parasitic infections.
Figure 8.2 The haematopoietic system of the body
Lymphocytes, on the other hand, are involved in the immunologic response of the body.
This is one of the major defence systems by which the body ghts infections. Immunologic
232
response implies killing the infective micro-organisms either directly through chemical mediators or by raising antibodies against antigens (foreign proteins) present in these microorganisms. Antibodies are produced by lymphocytes.
Monocytes are the largest cells found in the peripheral blood. Cells with similar function
and belonging to the same lineage (family) are present in the tissue. These are referred to
as macrophages and belong to the reticuloendothelial system (RES). Their main function is
to remove particulate maer, dead and dying cells, cellular debris, senescent red cells and
antigen-antibody complexes. RES cells are located throughout the body in dierent organs
and tissues and are present in most parts of the bloodstream. Their location often dictates
their size and precise shape, and sometimes even warrants a special name. For example,
Kuper cells are found in the liver; in the brain, they are known as microglia; and when
found in the lymph nodes, bone marrow, and spleen, they are referred to as reticular cells.
Tissue histiocytes or macrophages are those located in the subcutaneous tissues.
Medical Laboratory Technology: Volume 1
Thrombocytes
Thrombocytes (thrombos–clot, cytos–cell) or platelets in collaboration with coagulation
factors in the plasma assist in the stoppage of bleeding. This process is called haemostasis
(haem–blood, stasis–stoppage). They are sticky in character and form the haemostatic plug
which eectively seals the tom blood vessel and thus stops the haemorrhage.
Plasma and Serum (Liquid Components)
Plasma is the principal vehicle for the transportation of materials from one part of the body
to another through the blood vessels (veins and arteries). Lymphatic ducts also participate in
transportation but only to a limited extent. The plasma carries various materials—nutrients,
metabolites, waste products, regulatory substances and others. The ow of plasma helps to
control the body temperature. As mentioned earlier, the soluble protein brinogen, present
in plasma, is one of the important coagulation factors. When coagulation of plasma occurs,
brinogen is converted to brin and the brin threads, along with platelets, enmesh and glue
together the various cellular components of blood. This is called a blood clot and the clear
uid left after the clot is removed is called serum. The serum contains most of the chemicals
present in the plasma except brinogen and some of the coagulation factors which are either
labile or used up during the cloing process.
An adult of 60-kg body weight has about 4.5 L of blood in circulation. The loss of blood
during blood sampling is not harmful unless large amounts of blood are taken and the patient
is seriously ill or is a neonate. In a healthy adult, donation of 500 mL of blood does not aect
health. The body readily replenishes such losses.
haematopoIetIc system of the Body
Haematopoiesis (haem–blood, poiesis–synthesis) is the production, development and
maturation of cellular elements of blood (Figure 8.2). Under normal conditions, the production
of blood cells is carried out by the bone marrow (medullary haematopoiesis); while in the
foetus, extramedullary haematopoiesis is normal until the bone marrow is matured and
functional. Therefore, the development of extramedullary haematopoiesis in children and
adults is a pathologic phenomenon.
Haematopoiesis consists of production of erythrocytes (erythropoiesis), leukocytes
(leukopoiesis) and thrombocytes (thrombopoiesis). Leukopoiesis is further subdivided
into granulopoiesis (production of neutrophils, eosinophils, basophils and monocytes) and
lymphopoiesis (production of lymphocytes).
Introduction to Haematology
233
The progenitor (parent cell) for all of these cells is the stem cell (Figure 8.2). There is an
extensive experimental evidence for its presence but its morphological identity has not been
established. The totipotent stem cell is a common precursor for erythrocytes, granulocytes,
megakaryocytes and lymphocytes. The-morphologically identiable cells for each of these series are referred to as proerythroblasts (pronormoblasts), myeloblasts, megakaryoblasts and
lymphoblasts, respectively.
The blast cells through several stages of division and dierentiation develop into mature
cells of each series. Cellular division requires formation of new deoxyribonucleic acid (DNA),
which in turn needs vitamin B
and folic acid. Therefore, deciency of these two nutrients
12
results in a decrease in the number of mature cells in the circulation.
In a healthy individual, only mature cells are seen in the peripheral blood. In stained
smears, both mature and immature cells are recognized by their morphology (size, colour,
cytoplasmic granulation, nuclear segmentation, shape of the nucleus, cell margins and other
characteristics).
Erythropoiesis
The development of a mature erythrocyte requires several stages of division and dierentiation:
proerythroblast, basophilic normoblast, polychromatophilic normoblast, orthochromatic
normoblast and reticulocyte (Figure 8.2). Only erythrocytes and reticulocytes are found in the
peripheral blood of individuals. Factors required for erythropoiesis include erythropoietin,
vitamin B12, folic acid and iron.
Erythropoietin is secreted largely by the kidney and is the most important regulator of
erythropoiesis. Its decient production results in aplasia of the bone marrow.
Vitamin B12 and folic acid are necessary for DNA synthesis and hence for cell division.
Their deciency results in decrease in DNA synthesis and delay in nuclear maturation. The
rate of cytoplasmic maturation remains normal. Hence, the nuclear maturations lag behind
the cytoplasmic maturation; the cells become large in size and are referred to as megaloblasts.
Erythrocytes formed from these megaloblasts are larger in size and are called macrocytes.
Anaemia caused by the deciency of vitamin B12 and/or folic acid is called megaloblastic
macrocytic anaemia. In India, the most common cause of megaloblastic anaemia is nutritional
deciency of either one or both these vitamins. Pernicious anaemia is a type of megaloblastic
anaemia due to deciency of intrinsic factor (normally present in the gastric juice) which in
turn causes malabsorption and hence deciency of vitamin B12.
Iron is an important constituent of the haemoglobin molecule. Deciency of iron results in
poor haemoglobin synthesis in erythrocytes, and consequently, the red cells become smaller
(microcytic) and paler (hypochromic). Anaemia with these characteristics is called microcytic
hypochromic.
Reticulocytes are juvenile erythrocytes that contain some cytoplasmic remnants, which can
be stained by dyes such as new methylene blue and brilliant cresyl blue. They are originally
seen at the site of their formation, the bone marrow, and take 2–3 days to mature, of which the
last 24 h are in circulation. Only about 1–2% of the circulating erythrocytes are reticulocytes.
Increased number of reticulocytes in circulation (reticulocytosis) indicates increased erythropoietic activity in the bone marrow.
The normal life span of RBCs is 120 days (±20 days) at the end of which senescent
erythrocytes are removed by macrophages in the reticuloendothelial organs such as the
spleen by the process of phagocytosis. As a result, haemoglobin is released which is degraded into its constituents. Most of the constituents are reutilized by the body (iron from
haem and amino acids from globin) but the protoporphyrin component of haem leads to
the synthesis of bilirubin.
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Medical Laboratory Technology: Volume 1
Leukopoiesis
Leukopoiesis is the production of white blood cells or leukocytes. It has three independent lines
or series, which lead to three dierent types of white cells—the granulocytes, lymphocytes
and monocytes. The life span of leukocytes, unlike that of erythrocytes, is quite variable from
a few hours (neutrophils) to several years (T-lymphocytes).
Granulopoiesis
Granulopoiesis begins at the myeloblast, which by a process of division and dierentiation
passes through stages of a promyelocyte, myelocyte, metamyelocyte and stab (band)
cell ultimately forming a mature granulocyte (Figure 8.2). The granulocytic series is
subdivided into three lines resulting in the formation of neutrophils, eosinophils and
basophils. The granules appear at the last stage of myelocyte formation so that from this
stage onwards, neutrophilic, eosinophilic and basophilic cells can be recognized. These
cells can be dierentiated easily by their morphology as seen in the peripheral blood
smear stained by any of the popular Romanowsky stains such as the Wright stain or
Leishman stain. Neutrophils average 12 μm in diameter and are smaller than eosinophils
and monocytes and slightly larger than basophils. The nucleus is segmented into 3–5 lobes
and stains deeply basophilic. Mature neutrophils are called segmented neutrophils. The
cytoplasm has very ne granules with stains tan to pink with Wright stain. Eosinophils
generally have a bilobed nucleus and coarse, bright red cytoplasmic granules and are
thus easily recognized. Basophils are the least numerous of leukocytes and have slightly
lobulated or indented nucleus; granules are abundant and larger, stain deep purple and
overcrowd the nucleus so as to obscure it from view. The immature forms are not found
in the peripheral blood under normal conditions. If seen, it is considered pathological and
must be reported.
Lymphopoiesis or lymphocytic series
Lymphopoiesis is not totally limited to the bone marrow. The thymus, lymph nodes and other
lymphopoietic organs are also involved in the maturation of lymphocytes. Lymphocytes are
of two types—В and T. These lymphocytes are related to humoral and cellular immunity,
respectively. These will be discussed separately in chapters on Immunology. Mature
lymphocytes are classied as large and small lymphocytes. The large lymphocytes are the
size of polymorphonuclear cells (12 μm), whereas the small lymphocytes are about the size
of a normal erythrocyte (8 μm).
Monocytic series
The monocytic series of leukopoiesis follows the same paern as other leukocytes: stem
cells promonocyte → mature monocyte. The nuclei of monocytes are not segmented and are
indented in shape (reniform or kidney-like), the cytoplasm is not granulated and the cell size
is the largest (15 μm) of all leukocytes seen in circulation. When the monocyte transforms into
a macrophage it becomes larger (30 μm).
Thrombopoiesis
Thrombopoiesis is dierent from other haematopoietic systems. The blast cell, called
megakaryoblast, gives rise to the giant multinucleated megakaryocytes. The cytoplasm of
megakaryocytes breaks away in fragments in the bone marrow giving rise to non-nucleated
thrombocytes. They stay in circulation for about 10 days.
Introduction to Haematology
235
revIew QuestIons
1. List the formed elements of the blood and state their functions.
2. What is the dierence between serum and plasma? How is serum prepared in the labo-
ratory?
3. What is haemoglobin? Where is it located? What is its function?
4. Why do the red blood cells lyse in water? How can this be prevented?
5. Dene the following terms:
Haemostasia, Haemostatic plug, Haematopoiesis, Phagocytosis, Medullary haemato-
poiesis, Reticulocytosis and Intrinsic factor
6. What are the causes of pernicious anaemia, microcytic hypochromic anaemia and meg-
aloblastic anaemia?
7. What is the normal life span of red blood cells?
8. Arrange the following according to their approximate size (increasing order):
Neutrophils, Monocytes, Mature lymphocytes (large), Immature lymphocytes (small),
Erythrocytes and Thrombocytes
9. If you centrifuge an anticoagulated blood specimen, which of the formed elements of
blood will be at the boom of the tube and which ones will be at the top of the packed
column of cells?
10. What are reticulocytes? How are they formed in the body? What would you infer in case
of increased circulation of reticulocyte in the body?
11. Which are the cells involved in the process of granulopoiesis?
12. What are the constituents of haem? How is bilirubin formed in the body?
13. Name the mineral, which is essential in the synthesis of haemoglobin.
14. What do red cells look like in the seing of vitamin B12 deciency?
15. What is a megakaryoblast? How is this related to thrombocytes?
16. How long do reticulocytes circulate in the body?
17. Name the circulating cells in blood which are non-nucleated.
18. Name some clinical conditions that lead to reticulocytosis.
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Medical Laboratory Technology: Volume 1
Basic Laboratory Procedures in Haematology
Chapter Outline
• Overview
• Collection and Processing of Blood Specimen
▪ Preparation of plasma
▪ Preparation of serum
• Preparation of Blood Films
▪ Procedure for preparing thick blood lm
▪ Procedure for preparing thin blood lm
• Cleaning of Laboratory Glassware in Haematology
• Review Questions
9
Anuradha Chakravarthy
Overview
This chapter will focus on the basic laboratory procedures of the haematology laboratory. This
includes the processing of blood specimens and procedures for maintaining and cleaning of
laboratory supplies.
COlleCtiOn and PrOCessing Of BlOOd sPeCimen
As described in the previous chapter, blood samples are collected by phlebotomists in the
specimen collection area or at the bed side. These are then submied to the haematology
laboratory. Anticoagulated blood specimens are submied to the haematology laboratory
for analysis of cellular components, microscopic study of the cells and chemical analysis
of haemoglobin.
Blood specimens collected by venepuncture are occasionally processed in the collection
area but most often are sent directly to the laboratory for further processing and testing.
Early processing or separation of blood components is recommended for more reliable results. Plasma is the liquid portion of anticoagulated blood, while serum is the liquid portion
of the cloed blood. Plasma contains brinogen along with soluble ingredients of the uid
component of blood. Serum, on the other hand, contains all chemicals of plasma uid, except
brinogen which is used in clot formation.
Preparation of Plasma
1. Collect blood in a tube with dry anticoagulant. Make sure the tube is not wet, otherwise
this will cause haemolysis.
2. Mix instantly by gentle inversion. This yields whole blood.
Basic Laboratory Procedures in Haematology
237
3. Whole blood is centrifuged at 2500 rpm for 10 min. This separates the cellular component
of the blood, which goes to the boom as a red- coloured uid, from its liquid component
that stays at the top.
4. Take out the liquid portion (plasma) into a separate tube for further use. Plasma is
commonly used in coagulation laboratory and in blood grouping (Figure 9.1).
Figure 9.1 Plasma components of whole blood (anticoagulated) when left undisturbed
Preparation of Serum
A. With centrifugation
1. Collect blood in tube without any anticoagulant.
2. Allow the blood to clot for 30–60 min at room temperature.
3. Release the clot from the wall of the tube by means of an applicator stick.
4. Centrifuge at 2500 rpm for 10 min.
5. The liquid portion at the top is the serum (Figure 9.2c). Transfer the serum into another
test tube for further testing.
Note If electric centrifuge is not available, one can obtain serum with the help of a hand centrifuge.
B. Without centrifugation
Under some circumstances, when the blood is collected in containers which cannot be
centrifuged, the following method may be applicable to obtain the serum (Figure 9.2).
Figure 9.2 Blood collected in a tube (a) without anticoagulant, (b) will clot in few minutes, and (c)
when centrifuged will yield serum