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SECTION VII

Haematology

by
Parvez Ahmed, Ch Altaf Hussain, Hamid Saeed Malik, Ayesha Khurshid

Acknowledgement
Maj. Ali Ahmed
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No Chapter Page

37. Basic Bruch Up …………………………………………………..

38. The basic methods used in haematology ………………………….
39. Blood cell morphology ……………………………………………….
40. The examination of bone marrow ……………………………..
41. Blood cell cytochemistry …………………………………………….
42. Haemoglobin disorders ………………………………………………
43. Enzymopathies and membrane defects …………………………...
44. Diagnostic methods in bleeding disorders ………………………
45. Molecular Medicine ……………………………………………………
46. Transfusion medicine ………………………………………………..
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37. Basic Brush Up

HAEMOPOIESIS and progenitor cells that can only differentiate on
specific lines. These are also called CFUs and include
The blood consists of a fluid part called plasma and CFU-T (for T-lymphocytes), CFU-B (for B-
the formed elements called cells. The blood cells are lymphocytes), CFU-GM (for granulocytes and
of three types: red blood cells (RBC), white blood monocytes), CFU-Eo (for eosinophils), CFU-Meg
cells (WBC) and platelets (Plt). White blood cells are (for megakaryocytes), Burst Forming Units for
further divided into three main groups, granulocytes Erythroid cells (BFU-E) and CFU-E.
(neutrophils, eosinophils and basophils), monocytes
Gestational
and lymphocytes. Blood cells are continuously age
Phase of haemopoiesis Location
destroyed, either by ageing or as a result of their
Mesoblastic: Begins in yolk sac wall
functional activities, and are replaced by new cells.
2 weeks - 2 where small nest of blood cell Wall of yolk
There is a fine balance between the rates of formation
months production can be seen, referred to sac
and destruction of these cells in healthy people. The as blood islands
production of blood cells is termed as haemopoiesis.
Hepatic: Islands of blood cell
SITES OF BLOOD FORMATION 6 weeks –
development occur within liver
parenchyma. Dominant site for first Liver
In the first 19-20 days of the embryonic stage, blood birth
half of gestation, also occurs to
cells are formed in the wall of the yolk sac in ‘blood some extent within spleen
islands’. These cells are mesodermal in origin; hence Myeloid: Within bone marrow,
this phase of haemopoiesis is called the Mesoblastic begins in clavicle at 2.5
Phase. Mesoblastic haemopoiesis produces only RBCs, 2.5 months- months, continues to rise until Bone
which remain nucleated throughout their life span. The birth myeloid tissue becomes major site marrow
haemoglobin in these RBCs is also most primitive, of haemopoiesis in latter half of
called embryonic haemoglobin. The liver is the main gestation.
site of haemopoiesis in the foetus from the 5th to 30th
week of intra-uterine life. Some haemopoiesis continues The stem cells maintain their number by self-
in the liver even after birth, for 1-2 weeks. This is renewal. When the need arises, a stem cell divides
termed as the Hepatic Phase of haemopoiesis. All into two. One of the ‘daughter’ cells replaces the
types of blood cells are produced in the later part of this parent cell in a stem-cell pool while the other
phase. RBCs produced in this phase are larger than differentiates along the required cell line. All of this
adult’s RBCs, but are non-nucleated. These contain less takes place under the influence of certain proteins,
primitive haemoglobin called foetal haemoglobin. Bone which are called haemopoietic growth factors.
marrow gradually takes over the haemopoietic function These include interleukins (IL) and colony-
from the 5th month until term when it is the only major stimulating factors (CSF), which are secreted by
site for forming blood cells. Lymphocyte precursors are various cells in response to stimuli. Important
formed in the liver and bone marrow. The main sites for haemopoietic growth factors include IL-3, GM-CSF,
lymphocyte production are the spleen, lymph nodes and G-CSF and Erythropoietin (Epo). There are certain
other lymphoid tissue. Initially, haemopoiesis takes other proteins that have an inhibitory influence on
place in the marrow of all bones. After birth, it slowly haemopoiesis. The examples include Interferon (INF)
and gradually recedes to the marrow of flat bones and and Tumour Necrosis Factor (TNF).
vertebrae. At birth, bone marrow constitutes 1.5% of
body weight, which increases to 4.5% in the adults. THE STEPS OF BLOOD FORMATION
However in children, 75% of the total marrow is The formation of each type of blood cell is named
haemopoietic whereas in old age only 30-40% of the after the cell line. For RBCs, it is called
marrow is haemopoietic. In young adults 50% of the Erythropoiesis, for granulocytes, it is called
total marrow is haemopoietic. Non-haemopoietic Granulopoiesis, for platelets it is called
marrow consists of fat cells. Thrombopoiesis & for lymphocytes, it is called
Lymphopoiesis. The formation of blood cells and
THE ORIGIN OF BLOOD CELLS their delivery into the bloodstream involves three
processes, as described below:
All blood cells are formed from the undifferentiated
1. Multiplication/Proliferation which takes place
primitive cell, which resembles a large lymphocyte
by successive division of stem and progenitor
and is called pleuripotent or totipotent
cells by the process of mitosis.
haemopoietic stem cell. It gives rise to lineage
specific stem cells, termed colony-forming units 2. Maturation/Differentiation that occurs by the
lymphoid and spleen (CFU-L & CFU-S). These in progressive development of specific structural
turn differentiate into more committed stem cells and functional cell characteristics.
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3. The release of mature cells from the marrow 6. Red Blood Cell (RBC): The mature RBC is a
into the bloodstream. Some maturation normally non-nucleated cell. It is a biconcave disc that is
occurs after the release of cells, e.g. maturation about 7.2 µm in diameter. The cytoplasm is pink
of reticulocytes to RBCs. Immature forms may due to the presence of haemoglobin. There is no
be released into circulation under conditions of nucleus, no mitochondria and no ribosome.
stress
GRANULOPOIESIS
ERYTHROPOIESIS The earliest recognisable cell of the granulocytic
In normal marrow the proerythroblast is the first series in the bone marrow is the myeloblast. It
identifiable cell of the erythroid series. It divides and divides and matures into various granulocytes in
matures to a RBC through various stages. The stages. The process is characterised by:
process of normoblastic maturation is characterised • Change in the size of the cell.
by the following progressive changes: • Maturation and lobulation of the nucleus.
• Decrease in cell size • Production of specific granules in the cytoplasm.
• Haemoglobinization The time for maturation from myeloblast to mature
• Extrusion of the nucleus. granulocyte is about 4 days. The various stages in the
The time for maturation from pronormoblast to development of a granulocyte are:
mature red cell is about 7 days. The various stages in 1. Myeloblast: This is the first recognisable cell of
the development of a RBC are
this series. It has a large round or oval nucleus
1. Pronormoblast: It is a round cell with a which occupies most of the cell and contains 2-4
diameter of 12-20 µm. It has a large nucleus nucleoli. The cytoplasm is non-granular and deep
surrounded by a small amount of cytoplasm. The blue in colour. It divides and matures into a
cytoplasm is deep blue in colour. The nucleus is Promyelocyte.
round and consists of a network of uniformly 2. Promyelocyte: This is the next cell in the white-
distributed chromatin strands. It is reddish purple cell series. It resembles a myeloblast, but is
in colour and contains several nucleoli. It divides larger, has more cytoplasm, which contains
and matures to basophilic or early normoblast. purplish-red granules (azurophilic granules). The
2. Basophilic (Early) Normoblast: It is 10-16 µm nucleus still contains some nucleoli or their
in diameter. It has a large nucleus with thick remnants. It divides and matures into a
chromatin strands and no nucleoli. The Myelocyte.
cytoplasm is blue like the pronormoblasts. It 3. Myelocyte: The next stage in granulopoiesis is a
divides and matures into a polychromatic or myelocyte, which differs from the promyelocyte
intermediate normoblast. in two respects. First, the cytoplasmic granules
3. Polychromatic (Intermediate) Normoblast: It develop their specific character (purplish for
is 8-14 µm in diameter. The nucleus occupies a neutrophils, eosinophilic for eosinophils,
smaller part of the cell and stains deeply. The basophilic for basophils). Second, the nucleus
cytoplasm gives a reddish tinge and is not so has no nucleoli. The diameter of a myelocyte
blue in colour, due to the formation of may be up to 25 µm. The cytoplasm is light blue
haemoglobin. It divides and matures into in the early stages and acquires a pinkish colour
Orthochromatic or late normoblast. with maturation. A myelocyte does not divide
and only matures into a metamyelocyte.
4. Orthochromatic (Late) Normoblast: It varies
from 8 to 10 µm in diameter. The cytoplasm is 4. Metamyelocyte: The nucleus of this cell is
acidophilic (red) due to haemoglobinization. The small, eccentric and slightly indented. The
nucleus is small & appears as a deeply staining, cytoplasm is pinkish and contains specific
blue-black homogeneous mass (pyknotic). It granules. This cell is slightly smaller in size than
becomes eccentric in position and is finally the myelocyte. The specific granules are more
extruded out from the cell. Late normoblasts abundant.
cannot divide and only mature into reticulocytes 5. Band (Stab) form: It is a mature metamyelocyte,
by extrusion of the nucleus. which has a band-like nucleus adapted to a U
5. Reticulocyte: The reticulocyte is a flat disc- shape. The specific granules are abundant.
shaped cell. It has no nucleus and is slightly 6. Mature Granulocyte: Depending upon the type
larger than the mature red cell. It has a diffuse, of specific granules, these are of three types:
basophilic (bluish) tinge (polychromatic). With
supravital stains such as Brilliant Cresyl Blue, a. Neutrophil: It is 12-14 µm in diameter. The
the basophilic material, which is RNA, appears nucleus is lobulated having two to five lobes
in the form of a reticulum. The reticulocyte that are connected by thin chromatin strands.
becomes a mature red cell in about 1-4 days. The cytoplasm is pink and contains
Half of this time is spent in the spleen. numerous fine, purplish granules.
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b. Eosinophil: The mature eosinophil is a prominent nucleolus, usually centrally placed.
slightly larger than the mature neutrophil. Its Cytoplasm is variable.
average diameter is about 16 µm. The
3. Large lymphocyte: It is about 12-16 µm in
nucleus usually has two lobes. The
diameter. The cytoplasm is sky blue in colour
cytoplasm is packed with relatively larger
and contains few granules, which stain purplish
granules, which do not overlap the nucleus.
red. The nucleus is round or slightly indented.
The granules stain reddish- orange with
Nucleoli are absent.
Romanowsky Stains.
4. Small lymphocyte: The large lymphocyte
c. Basophil: The mature basophil has a lighter-
matures into a small lymphocyte and is 9-12 µm
staining nucleus than the neutrophil. It
in diameter. The cytoplasm is scanty and stains
seldom contains more than two lobes. The
blue. Purplish-red granules may be present. The
cytoplasm is pink and contains a number of
nucleus is round or slightly indented. Nucleoli
large oval or round, deeply-staining
are absent.
basophilic granules. They do not pack the
cytoplasm as do eosinophilic granules, but THROMBOPOIESIS
overlie the nucleus.
Platelets are formed from the cytoplasm of a large
MONOPOIESIS cell in the bone marrow known as megakaryocyte.
This also passes through various stages of
Monocytes are formed mainly in the bone marrow
development in the bone marrow. These are:
and migrate to the spleen, lymphoid and other tissues
1. Megakaryoblast: It is a large cell about 20-30
and organs of the body where these are transformed
µm in diameter. It has a large oval or indented
into macrophages. The various stages in its
nucleus that contains several nucleoli. The
development are as follows:
cytoplasm is blue, small in amount and contains
1. Monoblast: It is the earliest recognisable cell of
no granules. It may show budding.
the series. It is a large cell similar in structure to
2. Promegakaryocyte: This is formed from the
the myeloblast. Its nuclear outline is, however,
megakaryoblast and is larger than the
not as regular as in myeloblasts and it may show
megakaryoblast. It has a deep-blue cytoplasm
indentation or convolutions.
that contains azurophilic granules. The nucleus is
2. Promonocyte: It is a large cell about 20 µm in
non-lobulated or partly lobulated. From here
diameter. It has abundant cytoplasm, grey- blue
onwards, only the nucleus divides while the cell
in colour and may contain fine azurophilic
enlarges without division (Endomitosis).
granules. The nucleus is usually round or kidney-
3. Megakaryocyte: It is a large cell, from 30-90
shaped, giving a folded appearance, but it may
µm in diameter. It contains a single multi-
be lobulated.
lobulated or indented nucleus. The number of
3. Monocyte: It is slightly smaller than a
nuclear lobes varies from 4-16, depending upon
promonocyte. The other features are similar. Its
the number of divisions it has undergone. The
cytoplasm has a typical ‘ground glass’
cytoplasm is abundant and stains light blue. It
appearance. The nucleus is like a band folded
contains fine azurophilic granules. The margin is
upon itself to assume a spherical shape.
irregular and may show pseudopod formation.
LYMPHOPOIESIS 4. Platelet: It is a small discoid structure, 1-2 µm in
size. These are formed by the partitioning of the
Mature lymphocytes develop mainly in the lymphoid megakaryocyte cytoplasm into numerous
tissues of the body, namely the lymph nodes, spleen, structures that separate to form platelets.
gastrointestinal tract and tonsils. Bone marrow makes
only a small contribution to lymphocyte production.
CFU-L probably migrates to lymphoid tissue early in
ANAEMIAS
life. These also develop through stages. The Anaemia is defined as a decrease in haemoglobin
maturation of lymphocytes is characterised by: level (or total circulating red cell mass) for the age
• Maturation of the nucleus and cytoplasm. and sex of a person. The influence of gender is
• Adaptation to their function by an expression of important after puberty. The haemoglobin level in
specific proteins. adult females is lower as compared to adult males of
the same age group. This is due to the influence of
1. Lymphoblast: It is the earliest recognizable cell menstrual loss and the lack of androgens.
of the series. It measures 15-20 µm in diameter
and contains a large, round or oval nucleus. CLASSIFICATION AND AETIOLOGY
Nucleoli are present, usually 1-2 in number. The
cytoplasm is non-granular and deep blue in There are various criteria for classifying anaemia. Each
colour, forming a narrow rim around the nucleus. type of classification has certain advantages and
disadvantages. For routine laboratory work, the
2. Prolymphocyte: This is the next stage in the morphological classification is most useful. In this
formation of lymphocytes. The nucleus contains classification, anaemias are divided into three main
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groups depending upon the size of the RBCs and the HAEMATOLOGICAL ALIGNANCIES
amount of haemoglobin present in each cell. These
groups can be identified by measuring absolute values Haematological malignancies arise from an
as well as by examination of red cell morphology on uncontrolled, clonal proliferation of the cells of the
stained blood films. These groups are: haemopoietic system. These include:
• Leukaemias
1. Microcytic Hypochromic Anaemia: In this • Lymphomas
type of anaemia individual RBCs are smaller in • Myeloproliferative disorders
size than normal and contain a subnormal
• Myelodysplastic syndromes
amount of haemoglobin. All absolute values
• Plasma cell dyscrasias
(MCV, MCH, and MCHC) are below normal.
This type of anaemia is commonly seen in: • Malignant disorders of the monocyte
macrophage system
• Iron deficiency
• Thalassaemia LEUKAEMIAS
• Sideroblastic anaemia
• Anaemia of chronic disorders (some cases) Leukaemia can be defined as the malignant
2. Macrocytic Anaemia: In this type of anaemia proliferation, abnormal maturation and accumulation
individual RBCs are larger than normal, but the of various cells in the hierarchy of haemopoietic
cells. These can be divided into acute and chronic
amount of haemoglobin in each cell is usually
leukaemias, based on the clinical course of the
below normal. Absolute values show increased
MCV with usually normal MCH/MCHC. This disease & the state of maturation of the malignant
type of anaemia is commonly seen in: cells in the blood and bone marrow.
• Megaloblastic anaemia
Acute Leukaemias
• Aplastic anaemia
Acute leukaemias usually have a rapid onset and are
• Haemolytic anaemia characterised by the presence of 20% or more blast
• Liver disease cells in the bone marrow. The acute leukaemias
• Myxoedema have been classified, by a group of French, American
• Hypopituitarism and British haematologists, into various groups and
• Pregnancy sub-groups with well-defined morphological and
• Alcoholism cytochemical criteria (FAB classification). The two
main groups are acute myeloid leukaemias (AML)
3. Normocytic Normochromic Anaemia: In this and acute lymphoblastic leukaemias (ALL).
type of anaemia, although the haemoglobin
concentration in the blood is reduced, the Acute Myeloid Leukaemias: The acute myeloid
individual RBCs appear normal and absolute leukaemias, sometimes called acute non-lymphoblastic
values are also within normal limits. This type of leukaemias (ANLL), are sub-divided into 8 sub-groups:
anaemia is seen in: M0 to M7. The original FAB classification is based on
• Acute blood loss morphology of blasts in the bone marrow, stained with
• Leukaemia Romanowsky Stains, Sudan Black-B (SBB) or
Myeloperoxidase (MPO) Stains, except in cases of AML
• Bone marrow infiltration
M0 where an anti-myeloperoxidase antibody is used to
• Chronic renal failure
demonstrate MPO in the blast cells. There are two types
• Chronic infections (chronic disorders) of blast cells identified by FAB group. These are Type-I
and Type-II blasts. Type-I blasts have a high N/C ratio,
DIAGNOSIS an indistinct Golgi zone, no granules in the cytoplasm,
un-condensed chromatin and prominent nucleoli whereas
The following investigations are to be performed for Type-II blasts have the same morphological features but
diagnosing a case of anaemia: their cytoplasm contains Auer rods. Type-III blasts
• Estimation of Haemoglobin (Hb). contain granules which stain positively with Sudan Black-
• Estimation of Total Red Blood Cell Count B or Myeloperoxidase Stains.
(TRBC).
• Estimation of Haematocrit (Hct) or Packed Cell
Volume (PCV).
• Calculation of absolute values.
• Examination of peripheral blood film.
• Reticulocyte count.
After determining the morphological type of
anaemia, the patient is further investigated to
determine the cause of it. Figure1: Myeloblast in AML
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Classification type of AML is characterised by accumulation of
abnormal promyelocytes, sometimes called Type-III
There are two classification systems which are blasts, in the bone marrow. These are large, heavily-
followed. FAB (French American British) granulated promyelocytes with multiple Auer rods. In
classification which is based on morphology and some cells these Auer rods are so numerous that they
cytochemical stains only. WHO classification evolved form a mass called a faggot body. These stain intensely
which incorporated immunophenotyping along with positive with SBB/MPO. In a variant M3, granules, Auer
recurrent cytogenetic abnormalities which are better rods and faggot bodies are scanty.
predictor of prognosis. WHO classification of 2008
was followed but now in 2016 it has been revised. AML-M4 (Acute Myelomonocytic Leukaemia): This
FAB Classification type of AML also takes into account one feature in
peripheral blood as well i.e. absolute monocyte count,
The salient features of this classification are as under. which should be more than 1x109/L. In the bone marrow,
AML-M0 (Acute Myeloid Leukaemia without MPO the blasts constitute more than 20% of non-erythroid cells
expression): It is characterised by the presence of Type-I and the monocytic component is more than 20%.
blasts. These react positively with anti-MPO antibodies. AML-M5 (Acute Monocytic Leukaemia): Acute
Blasts constitute 20% or more of all nucleated cells in the monocytic leukaemia is characterised by the presence of
bone marrow. more than 20% Type-I blasts of non-erythroid cells in the
AML-M1 (Acute Myeloid Leukaemia without bone marrow but total monocytic component
maturation): This type of AML is characterised by the (monoblasts, promonocytes and monocytes) constitute
presence of Type-I and Type-II blasts which constitute more than 80%. Blasts are larger, nucleus is irregular,
20% or more of all nucleated cells in the bone marrow, sometimes giving a convoluted appearance and
but more than 90% of non-erythroid cells. An occasional cytoplasm has a ‘ground glass’ appearance.
cell shows an Auer rod. Three percent or more of blasts AML-M6 (Acute Erythroblastic Leukaemia): In this
are SBB/MPO positive. category, erythroid cells constitute more than 50% of all
nucleated cells in the bone marrow. These have
AML-M2 (Acute Myeloid Leukaemia with megaloblastic features i.e. these are larger than normal
maturation): This type of AML is similar to M1 with erythroblasts and have open chromatin. Sometimes, these
two exceptions. First, the blasts constitute less than 90% are binucleate or even multinucleate (gigantoblasts).
of non-erythroid cells in the bone marrow and second These cells show large, Periodic Acid Schiff (PAS)-
that the monocytic component in the bone marrow is less positive granules. There are also present Type-I or Type-
than 20%. Auer rods are more frequent. II blasts, which constitute more than 20% of the non-
AML-M3 (Acute Promyelocytic Leukaemia): This erythroid cells.

Table 1: French-American-British Classification of Acute Leukaemias

FAB Classification Description FAB Classification Description

Acute lymphoblastic Acute myelogenous
leukaemia leukaemia
L1 Lymphoblasts with uniform, round nuclei Mo with minimal differentiation
and scant cytoplasm
Maturation myeloblastic; no cytoplasmic
M1 granulation
L2 More variability of lymphoblasts; nuclei M2 Differentiated myeloblastic; few to many
may be irregular with more cytoplasm than cells may have sparse granulation
L1
L3 Lymphoblasts have finer nuclear chromatin M3 Promyelocytic; granulation typical of
and blue to deep blue cytoplasm with promyelocytic morphology
cytoplasmic vacuolisation
M4 Myelomonoblastic; mixed myeloblastic
and monocytoid morphology

M5 Monoblastic; pure monoblastic or

monocytoid morphology
M6 Erythroleukaemic; predominantly
immature erythroblastic morphology,
sometimes megaloblastic appearance
M7 Megakaryoblastic; cells have shaggy
borders that may show some budding
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chromatin is heterogeneous and the nucleoli are large &

AML-M7 (Acute Megakaryoblastic Leukaemia):
prominent. Many times, it is difficult to differentiate between
The blasts, in this type of AML, constitute more than
ALL-L1 and ALL-L2. To overcome this problem, a scoring
20% of all cells in the bone marrow and with at least
criteria has been suggested.
50% of megakaryocytic lineage. Some blasts show
budding of the cytoplasm into platelet-like structures, ALL-L3: Morphologically, this is the most distinct
which stain positively with PAS Stain. sub-group of ALL. Blasts are large but
heterogeneous. The nuclei are regular and oval-to-
WHO Classification of AML 2016 round in shape. The nuclear chromatin is
Acute myeloid leukaemia (AML) and related homogenous and finely stippled. Its nucleoli are
neoplasms prominent and vesicular. The cytoplasm is relatively
A. AML with recurrent genetic abundant, deeply basophilic and contains several
abnormalities vacuoles (in the cytoplasm).
AML with t(8;21)(q22;q22.1);RUNX1-RUNX1T1
AML with inv(16)(p13.1q22) or Table 2: Scoring System for ALL
t(16;16)(p13.1;q22);CBFB-MYH11
Cell Character Score
APL with PML-RARA
High nucleocytoplasmic ratio in at least +1
AML with t(9;11)(p21.3;q23.3);MLLT3-KMT2A
75% of cells
AML with t(6;9)(p23;q34.1);DEK-NUP214
Low nucleocytoplasmic ratio in at least 25% -1
AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2);
of cells
GATA2, MECOM
AML (megakaryoblastic) with No more than one and inconspicuous +1
t(1;22)(p13.3;q13.3);RBM15-MKL1 nucleolus in at least 75% of cells
Provisional entity: AML with BCR-ABL1 One or more prominent nucleoli in at least -1
AML with mutated NPM1 25% of cells
AML with biallelic mutations of CEBPA Irregular nuclear out line in at least 25% of -1
Provisional entity: AML with mutated RUNX1 cells
B. AML with myelodysplasia-related At least 50% cells are large (twice a normal -1
changes small lymphocyte)
C. Therapy-related myeloid neoplasms Score 0 to +2 = ALL-L1
D. AML, NOS Score –1 to –4 = ALL-L2
AML with minimal differentiation
AML without maturation Recently, the immunological classification of ALL
AML with maturation has gained popularity because of its correlation to the
Acute myelomonocytic leukaemia prognosis of the disease. The classification is based
Acute monoblastic/monocytic leukaemia on demonstration of lineage-specific antigens in the
Pure erythroid leukaemia cytoplasm or on the cell membrane of the blasts.
Acute megakaryoblastic leukaemia
Acute basophilic leukaemia
Acute panmyelosis with myelofibrosis
E. Myeloid sarcoma
F. Myeloid proliferations related to Down
syndrome
Transient abnormal myelopoiesis (TAM)
Myeloid leukaemia associated with Down syndrome
Acute Lymphoblastic Leukaemias: Acute
lymphoblastic leukaemias are divided into three sub-groups Figure 2: Lymphoblast in ALL
by the FAB group, based on morphology of blasts in the
bone marrow stained with Romanowsky Stains. These Chronic Leukaemias: Chronic Leukaemias are
types are ALL-L1, ALL-L2 and ALL-L3. The salient characterised by the chronic course of the disease and
features of these sub-groups are as under. the mature nature of the malignant cells. These
include:
ALL-L1: In this type of ALL, the blasts are small in size • Chronic granulocytic/myeloid leukaemia
with scanty cytoplasm. The nucleus is mostly regular in (CGL/CML).
shape with occasional cells showing a cleft or indentation • Chronic lymphocytic leukaemia (CLL)
in the nucleus. The chromatin is homogenous & nucleoli
• Chronic myelomonocytic leukaemia (CMML)
are inconspicuous.
• Hairy-cell leukaemia (HCL)
ALL-L2: In this type, the blasts are of heterogeneous size,
but predominantly large. Its nuclear shape is predominantly Of these, CGL is also classified with
irregular, showing frequent clefts or indentations. Nuclear myeloproliferative disorders but it is more
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appropriate to consider it under chronic leukaemias. splenomegaly and/or lymphadenopathy and a
Similarly, CMML is also classified under high total leucocyte count in the peripheral
Myelodysplastic Syndromes / Myeloproliferative blood. It is further sub-classified into CLL,
Neoplasms, which is more appropriate. Prolymphocytic Leukaemia (PLL) and a mixture
of the two (CLL/PLL) based on the stage of
1. Chronic Granulocytic/Myeloid Leukaemia: maturation of the majority of its malignant cells.
CGL is characterised by its chronic course, Three stages of the disease have been
splenomegaly and high total leucocyte count recognised, based on clinical and laboratory
in the peripheral blood. Differential leucocyte features. This is called Binet Staging and is
counts show all stages (blast to mature important from a management point of view.
granulocyte) of all types of granulocytes. This system takes into consideration Hb
Basophils are usually increased. There is a bi- concentration, platelet count & the number of
modal peak that is myelocytes and mature lymphoid areas involved. Five areas of lymphoid
forms are more abundant, whereas tissue are considered. These are: lymph nodes of
metamyelocytes are less in number. Being the head & neck, lymph nodes of the axilla,
abnormal cells, they have a very-low activity lymph nodes of the groin, spleen and liver. In
of normal enzymes, e.g. Leucocyte/Neutrophil
Alkaline Phosphatase (LAP/NAP). A scoring 3. Stage A, Hb is more than 11g/dl, platelet count
system based on NAP staining has been is more than 100x109/L and less than three
created to differentiate between leukemoid lymphoid areas are involved. In Stage B, Hb and
reactions and CGL. Philadelphia platelets are the same, but more than 3 lymphoid
Chromosome, t (9;22), can be demonstrated in areas are involved. In Stage C any number of
about 90% of cases, whereas the bcr/abl lymphoid areas may be involved, but either the
hybrid gene can demonstrated in almost 100% Hb is less than 11g/dl or the platelet count is less
of cases. CGL has three phases, each than 100x10/L, or both.
characterised by particular clinical and
laboratory features. These are chronic Table 3: CLL Classification
phase, accelerated phase and blast Rai classification
transformation. Almost every patient, if not Stage
treated with curative therapy, eventually 0 Absolute lymphocytosis >15 × 109 /L
develops blast transformations when the I As stage 0 + enlarged lymph nodes
leukaemia becomes acute. The accelerated (adenopathy)
phase is characterised by a worsening of the II As stage 0 + enlarged liver and/or spleen ±
(adenopathy)
clinical condition with the development of III As stage 0 + anaemia (Hb < 11 g /dl) ±
anaemia & thrombocytopenia, with or without (adenopathy) ± (organomegaly)
an increase in basophils to 20% or more. The IV As stage 0 + thrombocytopenia (platelets <
blast count, both in peripheral blood and bone 100 x 109/L ± (adenopathy) ±
marrow increases, but in the marrow it does (organomegaly)
not exceed beyond 20%. Fibrosis may increase
in the bone marrow and nucleated RBCs 4. Hairy-Cell Leukaemia: Hairy-cell leukaemia
appear in the peripheral blood. Blast (HCL) is characterised by old age, massive
transformations or crisis is characterised by a splenomegaly, pancytopenia in the peripheral
presence of more than 20% blasts in the bone blood and a presence of hairy cells in the
marrow in addition to the features of the peripheral blood and bone marrow. Hairy cells
accelerated phase. Both myeloid and lymphoid are of the size of a large lymphocyte with
blast transformations may occur but later it is inconspicuous nucleolus and the cytoplasm is
less common (one-third of cases). drawn out into hair-like processes. These cells
stain positively for Acid Phosphatase, which is
resistant to Tartrate (TRAP).

MYELODYSPLASTIC SYNDROMES
The MDS are a group of clonal BM neoplasms
characterized by ineffective hematopoiesis,
manifested by morphologic dysplasia in
hematopoietic cells and by peripheral cytopenia(s).
Cytopenias defined as: hemoglobin, <10 g/dL;
Figure 3: Bimodal Peak in CML platelet count, <100 x 109/L; and absolute neutrophil
count, < 1.8 x 109/L. Rarely, MDS may present with
2. Chronic Lymphocytic Leukaemia: CLL is mild anemia or thrombocytopenia above these levels.
characterised by its chronic course, PB monocytes must be, < 1 x 109/L.
276 | P a g e
Table 4: PB and BM findings and Cytogenetics in CML

MYELOPROLIFERATIVE DISORDERS and accumulation of histiocytes. These include

malignant histiocytosis of various types. The
These disorders are characterised by an uncontrolled disorders are not very common and their description
proliferation of myeloid progenitors in the haemopoietic is beyond the scope of this manual.
stem cell hierarchy, with an accumulation of mature
cells of the series. These disorders may ultimately LYMPHOMAS
transform into Acute Leukaemia. These include: Lymphomas are malignant neoplasms of lymphoid
tissue. These are broadly divided into Hodgkin's and
1. Polycythemia Vera (PV): In this disorder, Non-Hodgkin's Lymphomas. Hodgkin's
mature RBCs are increased with an increase in Lymphomas commonly known as Hodgkin’s disease
the absolute red-cell mass. (HD) is classified into the following sub-types:
2. Chronic Myeloid Leukaemia (CML): In this • Lymphocyte predominance
disorder, mature elements of granulocytic cell • Nodular sclerosis
series accumulate. This has been discussed in • Mixed cellularity
detail under Chronic Leukaemias.
• Lymphocyte depletion
3. Chronic Neutrophilic Leukaemia (CNL): In
this disorder, there is an increase in the number
Non-Hodgkin’s Lymphomas (NHL) have been
of mature neutrophils.
classified in several ways. Currently, the most
4. Essential Thrombocythemia (ET): In this
accepted is the International Working Formulation. It
disorder, there is an increase in the absolute
is reproduced below:
number of platelets.
5. Primary Myelofibrosis (PMF): In this disorder, 1. Low Grade
instead of a proliferation of haemopoietic cells, a. Malignant lymphoma, small lymphocytic
there is marked proliferation of fibroblasts in the b. Malignant lymphoma, follicular,
bone marrow, with increased reticulin formation predominantly small-cleaved cells
and collagenisation. This results in extra c. Malignant lymphoma, follicular, mixed
medullary haemopoiesis manifesting with the small-cleaved and large cells
leuco-erythroblastic blood picture.
2. Intermediate Grade
MALIGNANT DISORDERS OF THE a. Malignant lymphoma, follicular,
MONOCYTE MACROPHAGE SYSTEM predominantly large cells
b. Malignant lymphoma, diffuse, small-
In this group, there is an uncontrolled proliferation cleaved cells
277 | P a g e
c. Malignant lymphoma, diffuse, mixed small b. T-Cell Neoplasms
and large cells I. Precursor T-Cell neoplasms:
d. Malignant lymphoma, diffuse, large cells Precursor T-lymphoblastic Leukaemia/
Lymphoma
3. High Grade
II. Peripheral T-Cell and NK-Cell Neoplasms:
a. Malignant lymphoma, large cells,
immunoblastic
1. T-Cell chronic Lymphocytic Leukaemia/
b. Malignant lymphoma, lymphoblastic
Prolymphocytic Leukaemia
c. Malignant lymphoma, small, non-cleaved cells
2. Large granular lymphocytic Leukaemia
4. Miscellaneous (T-Cell Type & NK-Cell Type)
a. Composite Malignant Lymphoma 3. Mycosis fungoides/ Sezary Syndrome
b. Mycosis fungoides 4. Peripheral T-Cell Lymphoma
c. Extra-medullary plasmacytoma 5. Angio-immunoblastic T-cell Lymphoma
d. Histiocytic Lymphoma 6. Angiocentric Lymphoma
e. Unclassified 7. Intestinal T-Cell Lymphoma
f. Others 8. Adult T-Cell Lymphoma
The most recent classification is the Revised 9. Anaplastic Large-cell Lymphoma,
European-American Lymphoma (REAL) Group Ki-1 Lymphoma
classification. A WHO modification of this 10. Anaplastic Large-cell Lymphoma,
classification is under review. This classification (Hodgkin’s-like)
includes Hodgkin’s Disease and other lymphoid
c. Hodgkin’s Disease
malignancies as well. It is reproduced below:
1. Lymphocytic predominance
a. B-Cell Neoplasms - 2. Nodular sclerosis
I. Precursor B- cell Neoplasms 3. Mixed cellularity
Precursor B-lymphoblastic Lymphoma/ 4. Lymphocytic depletion
Leukaemia 5. Lymphocytic-rich classical HD
II. Peripheral B-Cell Neoplasms
1. B-cell chronic Lymphocytic MULTIPLE MYELOMA AND
Leukaemia/ prolymphocytic RELATED DISORDERS
Leukaemia/ small-cell Lymphocytic
Lymphoma
Mutiple myeloma is a neoplastic disease
2. Lymphoplasmacytoid Lymphoma/
characterized by plasma cell accumulation in the
immunocytoma
bone marrow, presence of monoclonal protein in the
3. Mantle-cell Lymphoma
serum and/or urine and in symptomatic patients
4. Follicular-centre Cell Lymphoma
related tissue damage.
5. Marginal Zone Lymphoma
Plasma cell neoplasms result from expansion of a
6. Splenic Marginal Zone Lymphoma
clone of a immunoglobulin (Ig) secreting, heavy
7. Hairy-Cell Leukaemia
chain class switched, terminaly differentiated B cells
8. Plasmacytoma/Plasma Cell Myeloma
that typically secrete a single monoclonal
9. Diffuse, Large B-Cell Lymphoma. Sub-
immunoglobulin called paraprotein.
Type Primary Mediastinal B-Cell
Lymphoma
10. High-Grade B-Cell Lymphoma Monoclonal gammopathy of undetermined
( significance (MGUS)
A serum paraprotein may sometimes be detected
B
without any evidence of myeloma or other underlying
u
disease and is termed as MGUS
r
k
i HAEMOSTASIS
t
t Haemostasis literally means “stoppage of blood flow”.
- There are three basic components of haemostasis:
extravascular, vascular and intravascular.
l
i The extravascular component is mainly the pressure
k exerted on blood vessels because of an accumulation
e of extravasated blood in the tissue space. The
) efficiency of this component depends upon the bulk
of surrounding tissue, type of tissue and tone of the
Table 5: Plasma Cell Neoplasms tissue.
278 | P a g e
The vascular component constitutes the blood Table 6: Characterstics of coagulation proteins
vessels themselves. The role played by blood Zymogens
vessels depends upon their size, amount of +GLA Prothrombin
smooth muscle in their wall and integrity of the Domain Factor VII
lining endothelium. On injury, a blood vessel Factor IX
Factor X
undergoes vasoconstriction as a neurogenic Protein C
response, thus decreasing the blood flow. Factor XI
Together with the extravascular component, it -GLA Factor XII
may stop the blood flow altogether. Domain Prekallikrin
Factor XIIIA
The injury exposes collagen and tissue factor that Factor XIIIB
initiate the participation of intravascular Factor XIII
TAFI
components of haemostasis. The key components
in intra-vascular haemostasis are platelets, Factor V
coagulation factors, anticoagulants and Cofactors Factor VIII
fibrinolytic factors. Platelets and coagulation Soluble VWF
factors promote the formation of a thrombus, Protein S
which occludes the injured site and results in the Protein Z
arrest of bleeding. Anticoagulant proteins help HK
Tissue factor
in limiting the thrombus formation at site of Thrombomodulin
injury, while fibrinolytic factors help in the Cellular EPCR
dissolution of thrombus. A fine balance between Fibrinogen
these keeps the blood in a fluid state. A tilt of the A α chain
balance to one side or the other may result in a Structural protein B β chain
failure of coagulation which leads to a bleeding γ chain
Antithrombin
disorder or an increased propensity to coagulation TFPI α
leading to a hypercoagulable state or thrombosis. Inhibitors ZPI
The exposure of collagen in the wall of blood
vessel (following the injury) provides a surface
for adhesion of platelets. The platelets that adhere The activation of the coagulation system
to this surface undergo a metamorphosis and a simultaneously brings into play another set of
release reaction which attracts more platelets, proteins that have an opposing effect. That is, these
leading to an aggregation of platelets that results obstruct the process of coagulation to prevent an
in the formation of a platelet plug. The numbers extension of clots beyond the required limit. The
as well as the functional integrity of these most important proteins of this system are Tissue
platelets affect this phase of haemostasis. Factor Pathway Inhibitor (TFPI), Antithrombin
(AT), Protein C and Protein S. Another group of
This primary platelet plug is strengthened by the proteins, which are collectively termed the
formation of fibrin threads and is converted into a fibrinolytic system, regulates the deposition &
thrombus. Fibrin formation is initiated in two removal of fibrin. The major protein of this system,
ways. First the injury to the vessel’s wall leads to plasmin, is produced by the action of plasminogen
an exposure of tissue factor (TF) or factor III activators on a protein called plasminogen, which is
with which combines a plasma protein, factor synthesised by the liver. The most important
VII, and initiates an extrinsic pathway of plasminogen activator is the Tissue Plasminogen
coagulation. The exposure of negatively-charged Activator (t-PA), released by the injured endothelium
elements of the vessel wall (collagen) activates of the vessel wall.
another protein, factor XII, which initiates the
intrinsic pathway of coagulation. The two DISORDERS OF HAEMOSTASIS
pathways converge on a common pathway,
Based on the physiology of haemostasis, the
activating factor X that, in turn, complexes with
disorders of haemostasis (as described above) can be
activated factor V.
grouped into those arising due to:
1. Vascular defects
This complex converts the prothrombin in the
2. Platelet defects
plasma into thrombin, which then polymerises the
3. Defects in the coagulation pathway
fibrinogen into fibrin threads. These threads are
4. Defects in the anticoagulant pathway
then stabilised by the action of activated factor
5. Defects in the fibrinolytic pathway
XIII. In this cascade, platelets also play a part by
6. Others
providing phospholipid. In all, there are 12
proteins and one metal ion (Ca ++), which
Each of these can be subdivided, based on clinical
participate in the coagulation process.
manifestations, into bleeding disorders and
279 | P a g e
hypercoagulable states or thrombophilia. Each sub- factors. Extensive damage to hepatocytes will
group can be further divided, based on aetiology, into result in a compromised synthesis of coagulation
hereditary/congenital or acquired disorders. factors, leading to their deficiency. Besides, the
liver produces bile (which is required for the
1. Vascular Defects: Hereditary, connective- tissue absorption of Vitamin K), which in turn is
disorders like Ehlers-Danlos Syndrome and needed for the synthesis of the active forms of
Psuedoxanthoma Elasticum are characterised factors II, VII, IX and X. Liver disease,
by weak vessel walls and an abnormal collagen particularly obstructive, will therefore also cause
that is unable to initiate platelet a qualitative deficiency of these coagulation
adhesion/coagulation, thus leading to easy factors and lead to a bleeding disorder. Also,
bruising and haemorrhagic state. A similar defect some quantitative and qualitative disorders of the
is acquired in old age (Senile Purpura) and proteins of this pathway also result in a
Vitamin C deficiency (Scurvy). Hereditary hypercoagulable state. The most important of
alterations in the vessels’ wall structure, e.g., these is a hereditary, qualitative defect of factor
Hereditary Haemorrhagic Telangiectasia and V, Factor V Leiden and Prothrombin Gene
Cavernous Haemangiomas lead to a bleeding Mutation G→A20210.
disorder due to weak vessel walls. A similar
weakness may also result from acquired diseases 4. Defects in the Anti-Coagulant Pathway: The
like diabetes mellitus and amyloidosis. A quantitative deficiencies of the proteins of this
bleeding disorder may also result from damage pathway result in a hypercoagulable state
to the blood vessels by an immune process, as in (thrombophilia). Thromophilia is used to
Henoch-Schonlein Purpura or in chronic describe inherited or acquired disorders of the
bacterial infections. A thrombotic disorder may haemostatic mechanisms that predispose to
result from a disease of the vessel walls, e.g., thrombosis. These can be hereditary or acquired.
atheroma formation and endothelial injury due to Common causes of hereditary thrombophilia include
toxins or viruses. Factor V Leiden gene mutation, Antithrombin,
Protein C and S deficiency, Prothrombin G20210A
2. Platelet Defects: Platelet defects may be Variant. Acquired causes are age, major trauma,
quantitative or qualitative. Thrombocytopenia major surgery, malignancy, lupus anticoagulant,
(decreased platelet count) is one of the most estrogen therapy etc.
common causes of a bleeding diathesis. This Thrombophilia Screening is mainly done by
may result either from decreased production or testing for Factor V Leiden mutation,
increased consumption. The most important Antithrombin, Protein C and S deficiency. PCR
causes of Thrombocytopenia are acquired and is also used for detecting Factor V Leiden
not hereditary. Of these the most common is mutation.
auto-immune or idiopathic thrombocytopenic Thrombophilia screening should be done before
purpura (ITP). The most important causes of starting anticoagulant therapy but therapy should
qualitative platelet defects are hereditary. These not be delayed to perform the test, ideally
include the Bernard Soulier Syndrome, screening is done 1 month after completion of
Glanzmann Thrombasthenia, and Storage Pool anticoagulant therapy.
defects. A similar disorder can also result from Intrepretation of test results should be done in
the repetitive ingestion of aspirin. correlation with age and clinical history of the
patient.
3. Defects in the Coagulation Pathway: Although
defects in this pathway, e.g. increased levels of 5. Defects in the Fibrinolytic Pathway: These
coagulation factors that may result in a defects most commonly result in a thrombotic
hypercoagulable state, more important are the tendency. These may be hereditary or acquired.
defects that result in a bleeding disorder. These
can be hereditary or acquired. Hereditary 6. Others: Some disorders that lead either to a
bleeding disorders constitute the most important tendency to bleed or a hypercoagulable state
group. These occur because of a quantitative or involves more than one of the above groups as
qualitative deficiency of coagulation factors. well as other elements. The most important of
Although a bleeding disorder may occur because these are Von Willebrand Disease (vWD), DIC
of a deficiency of any coagulation disorder, the and auto-immune diseases like SLE. vWD
most common are Haemophilia A (Factor-VIII results from an abnormality or a deficiency of
deficiency) and Haemophilia B (Christmas one part of the factor VIII complex, von
Disease because of factor IX-deficiency). Most Willebrand factor (VIII:vWF). This part is
important of the acquired bleeding disorders are independently produced by vascular endothelium
liver disease and disseminated intra-vascular and is required for platelet-vessel wall
coagulation (DIC). The liver is the site for interaction. It results in a bleeding disorder that
synthesizing the majority of the coagulation has the features of a disease due to both a platelet
280 | P a g e
defect and a coagulation- protein defect. This is a factors are produced and result in either a
hereditary defect. DIC is characterized by the bleeding or a hypercoagulable state. The
loss of localization or compensated control of production of lupus anti-coagulants results in a
intravascular activation of coagulation. prothrombotic state, whereas the production of
Causes include infections, malignancy, obstetrical factor VIII inhibitors results in a haemophilia-
complications, widespread tissue damage, like disorder.
hypersensitivity reactions, vascular
abnormalities, heat stroke, hypoxia atc. Bibliography:
The key event underlying DIC is increased 1. Dacie and Lewis. Practical Hematology 11th
activity of thrombin in circulation that Edition. S Mitchell Lewis, Barbra J Bain,
overwhelms its normal rate of removal by natural Imelda Bates eds. Churchill Livingstone
anticoagulants. DIC clinically manifests mainly London 2013
as a bleeding disorder with a component of the
thrombotic state. It results from the initiation of 2. Wintrobe’s Clinical Hematology 12th
an uncontrolled coagulation process, which edition. John P. Greer, John Forester, Gerge
results in a consumption of platelets and M. Rogers ,Friox Paraskevas, Bertil Glader,
coagulation proteins. This eventually leads to Danial A Arber, Robert T. Means, Jr.
the deficiency of coagulation factors as well as Wolters Kluver, Lippincott Williams &
thrombocytopenia, leading to a bleeding Wilkins London 2009
disorder. This is an acquired defect. 3. Post Graduate Haematology 7th Edition . A.
Victor Hoffbrand, Daniel Catovsky, Edward
7. In the course of some auto-immune diseases, G. D. Tuddenham eds. Blackwell
inhibitors of coagulation or anti-thrombotic Publishing London 2016
281 | P a g e

38. THE BASIC METHODS USED IN HAEMATOLOGY

ESTIMATION OF HAEMOGLOBIN (HB) 2. Cyanmethaemoglobin Reference Solution:

CONCENTRATION The cyanmethaemoglobin reference preparation
Whole-blood haemoglobin concentration can be is used for direct comparison with blood which is
estimated by a number of methods. The most also converted to HiCN. Solutions of different
commonly used are as follows: concentrations are commercially available and, if
• the Cyanmethaemoglobin Method unopened, are stable for many years. But once
• the Alkaline Haematin Method opened, they are only stable for a few hours. It is
• the Acid Haematin Method therefore recommended that a calibration curve
be prepared with the help of these solutions and
future readings should be taken from it. But it is
Each of these methods has its advantages and
disadvantages. Most commonly, the necessary that with each batch of tests or at least
Cyanmethaemoglobin method is used. The major a few times a day, the calibration is checked by a
advantage of this method is the availability of a stable fresh cyanmethaemoglobin reference solution or
and reliable standard preparation. This method, an internal reference prepared against it. The
manufacturer’s inset with the pack of standards
however, does not measure sulphhaemoglobin (SHb).
The Acid Haematin method has the advantage of gives the Hb g/L equivalent of the HiCN
being used without a colorimeter (Sahli's concentration of the standard.
haemoglobinometer), but it is the least accurate of all. Procedure:
The Alkaline Haematin method has the advantage Venous blood collected in EDTA or free-flowing
that it can measure carboxy-haemoglobin, capillary blood can be used. Measurements can be
methaemoglobin and sulph-haemoglobin, but it does carried out on blood that has been stored at 4°C for
not measure foetal haemoglobins (HbF and Hb several days, provided it is free from infection and
Barts'). contamination. 20 µl of blood is added to 4 ml of
diluent and well mixed by inverting the tube several
THE CYANMETHAEMOGLOBIN times. It is allowed to stand at room temperature for
METHOD 3-5 minutes so that all of the Hb is converted to
The principle of this method is that a blood sample is HiCN. The absorbance is then measured in the
diluted in a solution containing potassium cyanide spectrophotometer at 540 nm. The Hb level can be
and potassium ferricyanide (Drabkin's Solution). It directly read from a previously prepared calibration
converts haemoglobin (Hb) and methaemoglobin (Hi) curve or chart. Alternatively, the absorbance of a
to cyanmethaemoglobin (HiCN), which is a stable known standard is also read in the spectrophotometer
compound. The absorbance of the solution is with each batch of tests and the Hb is calculated by
measured in a photoelectric colorimeter with a the formula:
yellow-green filter or with a spectrophotometer at a
wavelength of 540 nm and is compared with a Abs. of test × Conc. of Std. (g/L)
Hb (g/L) =
standard solution of HiCN. Abs. of Std

Requirements: Preparation of a Calibration Curve/Chart:

1. Diluent (Drabkin's Solution) Commercially available standard solutions of HiCN are
Potassium ferricyanide 200 mg diluted in Drabkin's Solution so as to give
Potassium cyanide 50 mg concentrations equivalent to Hb concentrations of 2.0,
Potassium dihydrogen 140 mg
phosphate
4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0 and 20.0 g/dl.
Nonidet P40 (Sigma) 1 ml Pre-diluted standards are also commercially available.
Distilled water up to 1000ml The absorbance is read in a spectrophotometer at 540
The pH should be between 7.0-7.4 and the nm. These readings are converted into Hb concentration
solution should be clear and pale yellow in in g/dl with the help of conversion tables provided by
colour. It should give zero absorbance against the manufacturer of the standard. The absorbance is
water at 540 nm. The reagent is stored at room plotted against Hb concentration on linear graph paper,
temperature in a brown borosillicate glass bottle. with absorbance being on the vertical axis and Hb conc.
If Nonidet is not available, then the reaction time on the horizontal axis. All points must join in a straight
is to be increased, as haemolysis may be slow. line. A ready reference chart can be prepared from this
The reagent can be obtained in a prepared, curve.
concentrated form. If it is stored properly, the
reagent is fit for use for several months. The Precautions:
reagent should be discarded if it becomes turbid • The performance of the equipment and the
or the absorbance changes. calibration curve should be quality controlled by
282 | P a g e
simultaneously testing a commercial/ in in-house 4. Alternatively
lternatively prepare a 1/200 dilution of blood in
reference preparation with each batch of tests & diluent in a test tube by adding 20 µl of blood to
maintaining qualityity control charts. For details, 4 ml diluent.
see the ‘Quality Control’ chapter. 5. Place the cover slip firmly on the Neubauer
• If Nonidet has not been added to the diluent, then Chamber (the indication of correct placement is
10-15
15 extra minutes should be given for the that diffraction rings are seen on either side).
reaction to complete and a reading should be 6. Discard the first 4-5 5 drops from the RBC pipette
immediately taken. before charging the chamber. Blood diluted in a
• Abnormal plasma proteins and nd a high white
white- cell test tube can be used as such after mixing.
count may result in a turbid reaction mixture. This 7. Charge one side of the chamber by introducing a
should be centrifuged and the clear supernatant small drop of diluted blood at the edge of the
should be used for taking the reading. cover slip. The sample
ample will move under the cover
slip by capillary action.
Reference Ranges: 8. Wait for 2 minutes to allow the cells to settle.
9. Count the cells using a x40 objective in the
Adult female: 12.0 – 15.0 g/dl central, large, doubly-ruled ruled square of the
Adult male: 13.0 – 17.0 g/dl Neubauer Chamber. For counting, select 5 small
squares--four on the corners and one in the
centre. At least 500 cells should be counted. If
DETERMINATION OF THE TOTAL RED the cells are not sufficient in 5 small squares,
BLOOD CELL COUNT (TRBC) then include more squares for counting and
The number of erythrocytes present in one litre of modify the calculations accordingly.
blood is the total red blood cell count. The Calculation:
recommended reference method for counting RBCs The total ruled area of the Neubauer uer Chamber is 3x3 mm,
is by using an automated haematology analyser. divided into 9 large squares, each with an area of 1 mm2. The
Counting RBCs by the visual method is cumbersome central square is further divided into 25 squares.
and gives inaccurate results. Therefore, the absolute Depth of the chamber = 0.1 mm
values calculated from this count are also inaccurate Thus, the volume of the central square
and of little clinical value. The visual
ual method is = Length x breadth x height
described here to highlight the visual counting = 1 x 1 x 0.1 = 0.10. mm3
3
procedures and those of the automated haematology 1 mm = 1µl µl
analyser. The number of RBCs counted in the central square is N.
0.1 µl have N number of cells
Requirements: 1µll have N/ 0.1 cells, dilution used is 1 in 200
1. RBC pipette with a bulb containing red beads as in Number of cells per litre is:
haemocytometer or any automatic pipette capable = N x 200 x 106 = 2N x 109
of measuring 20 µll volumes and a test tube. 0.1
2. Improved Neubauer Chamber with a cover slip. (it
is a thick glass slide with H- shaped moats in it.
The area between
ween 2 limbs of H is 0.1 mm lower
than the area on the sides). When a cover slip is
fixed across these limbs, a depth of 0.1 mm is
provided in the centre. Above and below the
horizontal moat is the ruled area. The moat
prevents any mixing of the two sample
samples charged on
either side.
3. Red-Cell
Cell Diluting Fluid: this is prepared by
dissolving 3.2 g of sodium citrate and 1.0 ml
Figure 1: Neubauer chamber cell counting area
commercial formaldehyde solution in 100 ml
distilled water. Reference Ranges:
4. A microscope Adult male 5.5x1012/L
= 4.5-5.5x10
Adult female 4.8x1012/L
= 3.8-4.8x10
Procedure:
1. Draw well-mixed
mixed blood in a RBC
RBC-pipette up to
mark 0.5.
5. Care should be taken not to have any
DETERMINATION OF PACKED
PAC CELL
air bubbles in the blood column. Wipe clean the VOLUME (PCV) OR HAEMATOCRIT
ATOCRIT (HCT)
outer side of the pipette. When anti-coagulated
coagulated blood is centrifuged, RBCs are
2. Draw the RBC diluting fluid up to mark 201 packed at the bottom of the tube into a compact mass.
(1/200 dilution). These packed RBCs can be expressed as volume of
3. Gently rotate the pipette between the thumb and RBC per unit volume of centrifuged blood (L/L),
the forefinger, to mix well. termed as Packed Cell Volume (PCV).
( The packed
283 | P a g e
cells can also be expressed as a percentage (%) of the Sources of Error:
total volume of centrifuged blood, termed as • Sampling error
Haematocrit (Hct). These parameters can be • Incorrect concentration of the anti-coagulant
anti
determined by using automated equipment or by • Variation in the bore of the tube
manually using a centrifuge. Manually, the packed • Incorrect mixing
cell volume can be estimated either by the Macro • Storage for 6-8 hours
method or the Micro method. • Incorrect filling of the tube
• Incorrect centrifugation
THE MICRO METHOD • Haemolysis
• Incorrect reading
The International Council on Standardisation in
• Clots in the blood sample
Haematology (ICSH) recommends the Micro Method
for determining the PCV/Hct. • Variations in the internal diameter of tubes

Requirements: CALCULATION OF RED CELL

• Heparinised (for capillary blood) or plain (for INDICES (ABSOLUTE VALUES)
anti-coagulated
coagulated venous blood) Capillary Tubes
75 mm in length with a 1 mm bore. Mean Corpuscular Volume (MCV), Mean
• A Micro Haematocrit Centrifuge to provide a Corpuscular Haemoglobin (MCH) and Mean
centrifugal force of 12000g (Fig. 34.2) Corpuscular Haemoglobin Concentration (MCHC)
• A Micro Haematocrit Reader are generally referred to as Red Cell Indices or
• Plasticin Absolute Values.. A recent addition is the calculation
of RDW. These form the basis for the morphological
Procedure: classification of anaemias. Absolute values are best
1. Fill a suitable ble capillary tube with blood. determined by automated haematology analysers but
Preferably each sample should be run in can be calculated by using the following measured
duplicate as breakage and leakage of capillary parameters:
tubes is not uncommon. • Total red cell count (expressed as x 1012/L).
2. Seal one end of the tubes with plasticin and place • Packed cell volume (expressed as L/L)
these in the microhaematocrit centrifuge. • Haemoglobin conc. (expressed as g/L)
3. Centrifuge for 3-5 minutes.
4. Take out the tube and place it in the holder of a MEAN CORPUSCULAR VOLUME
VOL (MCV)
microhaematocrit reader in such a way that the This can be calculated by using the following
base of the packed red cells is in line with the formula if PCV and the TRBC are known:
base line (0 scale) of the PCV (L/L)
reader and the upper layer MCV in femtolitre s (fl) = × 1000
TRBC (× 10 12 /L)
of plasma is in line with
the slanting line (100 Reference Range:
scale). Adults (both genders): 83-101 fl
5. Now adjust the sliding line
so that it cuts between the MEAN CORPUSCULAR
SCULAR
red cell layer and the buffy HAEMOGLOBIN (MCH)
coat. Note the reading.
This is the packed cell This can be calculated by using the following
volume. formula if the Hb and TRBC are known:
Hb (g/L)
MCH in picograms (pg) =
Figure 2: Microhaematocrit Centrifuge TRBC (× 10 12 /L)
Advantages: Reference Range:
• A lesser amount of blood is required. Even Adults (both genders): 27.0-32.0
32.0 pg
capillary blood can be used for making the
method convenient for the screening of anaemia.
• Less time is consumed MEAN CORPUSCULAR
• Several samples can be run simultaneously HAEMOGLOBIN CONCENTRATION
CONCENTR
• Plasma trapping is less (MCHC)
• It is so accurate that it can be used for calibrating
automated blood counters. This can be calculated by using the following
formula if the Hb and PCV are known:
Disadvantage: Hb (g/L)
MCHC in g/dl =
Special equipment is required. PCV (L/L) × 10
284 | P a g e
Reference Range: Calculations:
Adults (both genders): 31.5-35.0 g/dl Area of the large square
= 1 mm2
Note- the MCH is more reliable when obtained from Depth of the Neubauer Chamber = 0.1 mm
an automated counter, as the RBC count & Hb Volume of one large square = 0.1 mm3 = 0.1 µl
are more accurate. On the other hand, the Dilution of blood 1 in 20
MCHC is more reliable in a manual system as Mean number of cells counted =N
this is calculated by Hb & Hct and both can be N × 20 × 10 6
TLC/L =
measured accurately by the manual method. 0.1
= N x 200 x 106
DETERMINATION OF TOTAL = N x 0.2 x 109
LEUCOCYTE COUNT (TLC) Reference Range:
Adults (both genders): 4-10x109/L
Total Leucocyte Count (TLC) per litre of blood is
also best estimated by an automated haematology Precautions
analyser. However, it can also be estimated by the 1. Pipette should be dry and clean.
visual method. The visual method can also be applied 2. Dilution should be correct.
for estimating cell counts in samples other than 3. If liquid flows into the moat, recharge the
whole blood, e.g., CSF, body fluids, cell cultures or chamber and count again.
cell concentrates, etc. 4. Debris of RBC should not be confused with WBC.
5. Cells sticking to debris should be recognised.
Requirements:
6. If nucleated RBC are present in differential
• WBC pipettes with a bulb containing white
leucocyte count then correct the TLC as follows:
beads, as in the haemocytometer or an automatic
• Count NRBC/100 WBC in DLC
pipette capable of measuring 50 µl fluid.
• Correct TLC by using following formula:
• An improved Neubauer Chamber with a cover
100 × Observed TLC
slip Corrected TLC =
100 + (NRBC/100W BC)
• WBC-diluting fluid prepared by mixing 4 ml
Glacial Acetic Acid and 10 drops of 0.3% DETERMINATION OF PLATELET
aqueous solution of Methylene Blue and making
the volume to 20 µl with distilled water. COUNT
Methylene Blue stains the nuclei of the WBCs, Like other formed elements of blood, platelets can
while Glacial Acetic Acid destroys the red blood also be counted by:
cells • an electronic particle counter
• A microscope • the direct visual method
Procedure: The direct visual method is quite reliable and all
1. Draw the blood in a WBC pipette up to the 0.5 abnormal platelet counts obtained from an electronic
mark. Wipe clean the outer side of the pipette. counter need to be confirmed by this method. The
2. Then, draw the diluting fluid up to mark 11. method that is recommended by the ICSH is
3. Mix gently by rotating the pipette between the described below in detail:
thumb and the forefinger. Requirements:
4. Alternatively draw 20 µl well-mixed, anti- 1. Diluting fluid of 1% Ammonium Oxalate is
coagulated blood in an automatic pipette and add it recommended. It is prepared by dissolving 1 g
to a test tube containing 0.38 ml of diluting fluid. dried ammonium oxalate in 100 ml glass- distilled
5. Place cover slip on the Neubauer Chamber and fix water. The solution is filtered through a micropore
it as described in the TRBC procedure. filter (0.22 µm) and stored in the refrigerator.
6. Charge the chamber after discarding 2-3 drops of 2. An improved Neubauer Chamber with a cover slip
diluted blood. 3. WBC-diluting Pipette or 20 µl and 1.9 ml
7. Let it stand for 5 minutes, so that the cells settle adjustable automatic pipettes.
down. 4. a test tube
8. Count the white blood cells by using a high, dry 5. a moist chamber or a Petri Dish with moist cotton
(x40) lens in the 4 large corner squares of the or tissue paper
Neubauer Chamber. Cells on the left and bottom
lines are counted, whereas the cells on the right and Procedure:
top lines are not. At least 100 cells should be 1. Make a 1 in 20 dilution of a whole-blood sample.
counted, even if the number of squares used for the If a WBC pipette is used, then the dilution is
counting is to be increased. made as in TLC. Otherwise, mix 20 µl of well-
9. Calculate the mean cell count in a single large mixed EDTA anti-coagulated blood with 380 µl
square by dividing the number of cells counted in of diluent in a suitable test tube to make a 1 in 20
four large squares by 4. dilution and mix well.
2. Fix a cover slip on a clean Neubauer Chamber
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and charge the chamber. filaments more deeply and uniformly and is preferred
3. Now place the counting chamber in a moist for use. The number of reticulocytes in the peripheral
chamber or a Petri Dish with moist cotton (to blood represents the erythropoietic activity.
avoid drying) for 20 minutes, so that the platelets
Requirements:
become settled.
4. Place under the microscope and count by using • Reticulocyte Stain: Take 1.0 g of New
the high, dry (x40 objective) lens of an ordinary Methylene Blue or Brilliant Cresyl Blue and
Light Microscope, with the condenser racked dissolve in 100 ml of Citrate Saline Solution
down and the diaphragm suitably narrowed. The (0.049 g Trisodium Citrate dissolved in 100 ml
platelets are seen as small, highly refractile discs. of normal saline). Filter the mixture--it is ready
5. Count the platelets in the central large square for use
(1 mm in area). The total number of platelets • Pasteur Pipettes
counted should be at least 200, even if more • 75x10 mm plastic Test Tube
squares are to be included in the counting. • Microscope glass slide
• An incubator or a Water Bath (37°C)
Calculations: • A Spreader
Area of large central square = 1 mm2
• A Microscope
Depth of Neubauer chamber = 0.1 mm
Volume of one large square = 0.1mm3 = 0.1 µl Procedure:
Dilution of blood 1 in 20 1. Deliver 2 or 3 drops of stain by means of a
Number of platelets counted =N Pasteur Pipette into a test tube. Add 2-3 drops of
the patient's EDTA, anti-coagulated blood to it.
N × 20 × 10 9 2. Incubate the mixture at 37°C in a water bath or
=
0.1 incubator for 15-20 minutes.
= N x 0.2 x 109 3. Re-suspend the cells by gentle mixing. Prepare
smears on glass slides and air dry.
Reference Range: 4. When the films are dry, examine under a
All ages and genders: 150-400x109 /L microscope using an oil-immersion lens.
Precautions: 5. Choose an area of the film where the cells are
• The water used for preparing the diluent must be not distorted or overlapping and are properly
particle-free and glass-distilled. stained. Count the reticulocytes and the RBCs in
• Glassware used must be scrupulously clean. the area. The field of counting can be narrowed
• The chamber and the cover slip must be clean either by using an eye piece provided with an
and scratch-free. adjustable diaphragm or by inserting a piece of
• The details of the procedure must be carefully paper or card-board in the centre of which a
followed. small square with sides about 4 mm is cut, into
the eye piece. At least 100 reticulocytes are
• Carefully fill and count the cells within the
counted.
chamber.
6. Calculate the percentage of reticulocytes. If the
• Carefully mix the blood and perform accurate
number of reticulocytes seen is 100 and total red
pipetting and counting of cells.
blood cells present are 2500 then the reticulocyte
DETERMINATION OF ABSOLUTE count is equal to:
EOSINOPHIL COUNT 100 × 100
= 4%
2500
The absolute eosinophil count is sometimes requested 7. This can be converted into an absolute
either in blood or in other body fluids and secretions. reticulocyte count, if TRBC is known, by using
Details of the method for counting are the same as for the following formula:
TLC. The diluent is however different. A suitable
% reticulocytes × TRBC (×1012 )
diluent is as under: Reticulocytes 109 /L =
Acetone 10 ml 100
Distilled water 90 ml 8. It is important to adjust the reticulocyte count
Eosin 01 g according to the degree of anaemia. This is
DETERMINING RETICULOCYTE known as the Adjusted Reticulocyte Count. For
this purpose, optimum haemoglobin is taken as
COUNTS 15 g/dl or a PCV of 0.45 L/L. Then Corrected
Reticulocytes are immature red cells. These contain Reticulocyte Count %
thread-like structures in the cytoplasm, which consist Observedcount (%) × Patient Hb (g/L)
of ribonucleic acid (RNA). RNA has the property of = OR
reacting with certain dyes such as Brilliant Cresyl
150
Blue or New Methylene Blue (supravital stains) to Observedcount (%) × Patient PCV (L/L)
=
form a blue or purple precipitate of granules or 0.45
filaments. New Methylene Blue stains the RNA
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Reference Ranges: the tube.
Adults (both genders) 0.2-2.5% 3. Fix the tube in the Westergren Stand and allow it
Infants 2-5% to stand there for exactly 1 hour.
4. At the end of the one hour, read the height of the
Precautions:
clear plasma to the nearest one mm
1. The reticulocyte count should be done on fresh blood
because, if the blood is stored, the reticulocytes will Precautions:
mature, leading to a false low count. • Westergren tubes must be scrupulously clean and
dry. After use, these should be thoroughly
2. At least 1000 red cells should be counted. washed with tap water, then rinsed with acetone
3. Reticulocytes should not be confused with HbH and allowed to dry.
inclusions found in HbH disease. HbH inclusions • The surface of the table on which the stand is
stain paler, are dot-like and occur in most of the placed, must be level and vibration-free.
red cells. If there is doubt, the reticulocyte count • The test should be protected from draught and
should be performed again after incubating the direct sunlight. The test should be carried out at
red cells and stain solution for another 2-4 hours. room temperature (18-25°C). Sedimentation is
If HbH inclusions are present, the count should accelerated at high temperatures.
not decrease.
4. Heinz Bodies appear as small dots present near Reference Ranges:
the cell membrane and should not be confused Females: (17-50 years) - up to 12 mm in 1 hour
with reticulocytes. Males: (17-50 years) - up to 10 mm in 1 hour
Newborns: ESR is usually low
DETERMINATION OF THE
SEDIMENTATION RATE OF THE PREPARATION AND STAINING
ERYTHROCYTES (ESR) OF BLOOD FILMS
If a column of anti-coagulated blood is allowed to
Examining a properly prepared and stained blood
stand vertically in a tube with a narrow bore, the red
film constitutes the most important investigation in
cells will settle down towards the bottom of the tube.
Haematology. It is performed for:
The rate at which the red cells settle is known as the
erythrocyte sedimentation rate (ESR). • Differential Leucocyte Counts (DLC).
• General assessment and verification of various
ESR can be performed either by Wintrobe's Method cell counts.
or by Westergren's Method. The Westergren Method • Study of RBC morphology for classifying
is recommended by the ICSH. In this method, various anaemias.
properly diluted blood sediments in an open-ended • Study of WBC morphology for diagnosing
glass tube mounted vertically on a stand. The leukaemias and other WBC disorders.
Westergren Method can be performed on blood that • Study of platelet morphology for diagnosing
has been collected either directly in liquid Tri- some platelet disorders.
Sodium Citrate anticoagulant or in powdered EDTA. • Study of parasites found in plasma or RBCs
Four volumes of venous blood are anti-coagulated (haemoparasites).
with 1 volume of 3.2 percent Trisodium Citrate. If the • Study of other defects like Rouleaux formations,
EDTA is used as an anticoagulant, then add 1 volume agglutination, fragmentation, RBC inclusions, WBC
of 3.2% Trisodium Citrate to 4 volumes of blood inclusions, platelet clumps and satellitism, etc.
before performing the test.
PREPARATION OF BLOOD FILMS:
Requirements:
1. A Westergren Tube is an open-ended tube, 30 Blood films can be made on cover slips (Cover Slip
cm in length and has a diameter of 2.55 mm. It is Method) or on glass slides (Wedge Technique).
marked from the bottom in mm up to 20 cm Although the Cover Slip Method provides superior
length. The bore must be uniform and smooth1. WBC distribution, it is not preferred because of
2. A Westergren Stand following disadvantages:
3. A rubber teat or a mechanical device for filling • Difficult to prepare because of the fragility and
the tube. the small size of the cover slips.
• Cover slips are difficult to handle, clean and label.
Procedure:
1. Take a Westergren tube and fill it with diluted • Platelets are unevenly distributed between two
blood to the zero mark with suction applied by a cover slips.
teat or mechanical device. • There are no specific areas to be examined.
2. Place a finger tip over the upper end of the Blood films prepared on glass slides using the
Westergren tube to hold the column of blood in Wedge Technique are preferred because:
• These are easy to prepare.
• Pre-cleaned slides are available.
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• Handling and labelling is easy. combines with anionic components of the cell, e.g.
• It is easy to find abnormal cells, as these DNA and stain these blue, whereas Eosin Y
tend to collect at the tail and on the edges of combines with the cationic components, various
the film. proteins and stains them red. Then there occurs a
It has some disadvantages as well, e.g., greater stain-stain interaction. This composition and mode
trauma to the cells and an uneven distribution of of action allows Romanowsky Stains to reveal the
white cells, which tend to collect at the tail. subtle differences in shades of the staining and
allows for a differential staining of granules. The
Requirements: pH of the staining mixture is extremely important
• Pre-cleaned (grease, dust and lint-free) glass for the differential staining. An alkaline pH
slides for microscopy. accentuates the basic dye staining. Therefore; an
• Spreader: A spreader is also a piece of glass (cover optimum pH is to be sought. A pH of 6.8 is
slip or glass slide). It should be narrower than the recommended for the optimal staining of all
glass slide. Its edge should be thin, smooth and components. The four most commonly used
polished. The tough cover slip of a Neubauer Romanowsky Stains are:
Chamber can serve as an excellent spreader.
Procedure: • Jenner's Stain
• Place a small drop of blood in the centre line of • Wright's Stain
the slide, one cm from one end. • Leishman Stain
• Immediately place a spreader in front of the • Giemsa Stain
blood drop at an angle of 45o. Move it back so
that it touches the drop of blood. Blood will Leishman Stain and May-Grunwald-Giemsa Stain are
spread along the margin in contact with slide of the most frequently used. The preparation & method
the spreader by capillary action. of using these stains is described below:
• Push the spreader forward along the length of the
slide with a rapid, but smooth and straight
movement. PREPARATION OF LEISHMAN STAIN
• Allow the film to dry in the air. Requirements:
Characteristics of a Good Blood Film: • Leishman Stain Powder of high (at least 80%)
• It covers at least half the length of the glass slide. purity, 0.2 g
• It is narrower than the slide. • Methanol (acetone free), 100 ml
• It is spread homogeneously with gradual • Conical flask
transition from thick to thin areas clearly • Funnel and filter paper
identifiable into a head (thick part near the blood • Mortar and pestle
drop), body (middle part) and a tail (a thin, Preparation:
terminal part). • Weigh 0.2 g of powder stain and transfer it to a
• It has no bubbles, streaks, troughs or holes. mortar.
• It terminates into a smooth, straight or slightly • Grind with about 25 ml of Methanol and allow it to
curved end. settle. Transfer the supernatant through a filter
• It is thin enough to yield to at least x10 low- paper to the flask.
power fields where RBCs do not overlap. • Add another 25 ml of Methanol to the mortar
Common Defects of Blood Films Their Causes: containing residual stain. Repeat the grinding,
1. A thick film results if the blood drop is too large, allow it to settle and transfer the supernatant to the
spreading is done too quickly or the angle of the flask.
spreader is too high. • Repeat the procedure until all of the Methanol has
2. A thin film results if the blood drop is too small, been used and most of the stain has been dissolved.
spreading has been too slow or the angle of the • Place the flask in a water bath at 50°C for 15
spreader is too low. minutes.
3. A gritty tail results if spreading has been too • Filter into a clean, brown, borosilicate glass bottle
slow, there was a delay in spreading, only a part for ripening.
of the blood drop was utilised or the spreader • Leave to mature for at least 2-3 days in the dark at
was not appropriate. In addition, some room temperature.
anticoagulants other than EDTA and a high TLC
can also give rise to gritty tail. A good practice is to initially make 2-3 bottles at one
time. When one bottle is finished, it should be replaced
STAINING OF BLOOD FILMS with freshly prepared stain and left to mature. In the
meantime, another bottle of stain is used. The required
The stains most commonly used for the staining of
volume of stain for daily use should be filtered into a
blood films are Romanowsky Stains. These stains
smaller dropping bottle every morning.
are composed of Azure B and Eosin Y. Azure B
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PREPARATION OF BUFFER STAINING OF BLOOD FILMS WITH
(SORENSEN’S 66 MMOL/L) MAY- GRUNWALD-GIEMSA STAIN
Preparation: Requirements:
1. Solution A: Dissolve carefully-weighed • Prepared May-Grunwald Stain
Potassium Dihydrogen Phosphate in one litre of • Prepared Giemsa Stain
distilled water in a conical flask. Transfer to a • Methanol (acetone-free)
clean glass bottle and store in the refrigerator. • Buffered Water (as in the Leishman staining)
2. Solution B: Dissolve and store Disodium • Staining Jars
Hydrogen Phosphate in one litre of distilled
water. Procedure:
3. To prepare a buffer of pH 6.8, mix 50.8 ml of • Place the air-dried blood film in a jar containing
Solution A with 49.2 ml of Solution B. Methanol, for 5-10 minutes.
• Transfer the film to a jar containing May-
PREPARATION OF MAY- Grunwald's Stain diluted with an equal amount
GRUNWALD-GIEMSA STAIN of water. Leave for 15-20 minutes.
• Now transfer the film to a jar containing
Requirements:
Giemsa's Stain diluted 1:10 with water. Leave
• May-Grunwald's Stain Powder of high (at least
for 10-15 minutes.
80%) purity 0.3 g
• Wash in 3-4 changes of buffered water (pH 6.8)
• Giemsa's Stain Powder of high (at least 80%)
and allow it to stand in a jar containing buffered
purity 0.3 g
water for 3-5 minutes, for differentiation to take
• Methanol (acetone-free) 200 ml
place.
• Conical flasks
• Drain and dry in a vertical position.
Preparation:
COMMON PROBLEMS IN STAINING
• In a conical flask, transfer the weighed May-
Grunwald's Stain powder. Add 100 ml of AND THEIR CAUSES:
Methanol to it and dissolve. 1. Too-red staining is caused if:
• In another conical flask, transfer the weighed • The stain is too acidic (pH <6.4)
Giemsa Stain Powder. Add 100 ml Methanol to • Buffer has been used in excess
it and dissolve. • Insufficient time has been allowed for
• Warm both flasks in a water bath at 50°C for 15 staining
minutes, at intervals shaking them.. • Excessive washing has been done
• The stains are filtered into clean bottles and • The blood film is very thin
stored in the dark at room temperature. • The water used is contaminated, particularly
THE STAINING OF BLOOD FILMS with chlorine.
• The stain is too old (the methanol converted
WITH LEISHMAN STAIN to fumeric acid)
Requirements: 2. Too-blue staining is caused if:
• Prepared Leishman Stain • The stain is too alkaline
• Too-little buffer has been added
• Buffered water: Dilute 50 ml of Sorensen's
• The staining time was too long
Buffer of pH 6.8 to one litre with distilled water. • The washing was inadequate
• A Staining Rack • The water was alkaline
• The blood film was thick
Procedure: • The blood film had been stored for a long
• Prepare the blood film and air-dry it. time
• Keep it on a staining rack and completely cover • The blood contained an increased quantity
of proteins
it with the stain.
• The blood contained heparin
• Leave it to stain for 2 minutes. • The TLC was very high
• Pour the buffered water onto the slide about • The haematocrit was too low
twice the amount of the stain. Mix by blowing • Too-short drying time of the blood film
3. Pale Staining
gently through a pipette. Leave for 5-7 minutes.
• Old staining solution
• Pour off the stain mixture. Wash in the buffer, • Overused staining solution
cleaning the underside of the slide with a cotton • Incorrect preparation of stock
swab or tissue paper. • Impure dyes, especially azure A and/or C
• Place vertically to drain and dry. • High ambient temperature
289 | P a g e
4. The film is washed off during staining if fixation • Lymphocytes
is not complete. • Monocytes
5. The deposit on the slide is seen when the stain is • Eosinophils
allowed to dry on the slide before adding the • Basophils
buffer or the buffer was not mixed properly with • Various maturation stages, e.g. blasts,
the stain. promyelocytes, metamyelocytes and band
forms.
DIFFERENTIAL LEUCOCYTE Maturation stages are not normally seen in
COUNT (DLC) peripheral blood. Band forms can be seen in
peripheral blood and if recorded separately these
Differential Leucocyte Count (DLC) provides the are normally not more than 6% of the counted
relative number of each type of leucocyte in the cells.
blood. It is performed on a well-spread and well-
Reference Ranges
stained blood film. This is of utmost importance, Count
because the even distribution of white cells depends Cells %
x109/L
very much upon the meticulous technique used to Neutrophils 2.0-7.0 40- 80%
prepare the blood film and the correct identification Lymphocytes 1.0 - 3.0 20- 40%
Monocytes 0.2-1.0 09 -10%
of the cells depends upon the quality of the staining. Eosinophils 0.02 -0.5 01-06%
If the edge of the spreader is rough, then many Basophils 0.02 -0.1 <1-2%
leucocytes, especially neutrophils, may accumulate at
the tail end. If the film is not well prepared or if it is Common Problems in Cell Identification and
too thin, neutrophils and monocytes predominate at Their Causes:
the margins and the tail and lymphocytes 1. Fewer than expected cells from the TLC in the
predominate in the middle of the film. A slight middle portion may result from an accumulation
difference in distribution is present even in a well- of cells at the tail. This results from a faulty
prepared film. spreader or an improper spreading technique.
2. Difficulty in identifying cells may result from:
Procedure: • Poor staining
1. Choose the middle portion of the film where the • De-naturation of the cells
cells are evenly spread when seen under the low 3. De-naturation of the cells occurs in:
power of the microscope. Place a drop of Cedar • Delay in preparing the smears (more than 5
Wood Oil and move the oil-immersion objective hours for normal cells and 1 hour for
in place. abnormal cells).
2. Identify and count each type of cell. Start • Improper anticoagulant concentration
counting from the tail of the film and move
• Blood mixed with IV fluid in the line
towards the head along a linear strip.
• Patient’s receipt of plasma expanders
3. When a single strip is completed, then the lens is
adjusted to another position, vertically upwards • Severe septicaemia
or downwards. The counting of the cells is 4. Activation of lymphocytes.
started again, now proceeding in the reverse 5. Vacuolation of monocytes.
direction. HESS TEST
4. This procedure is continued until 100 cells have
been counted. The Hess test also known as the Tourniquet test
5. The counting of cells can be done by: measures the capillary resistance (vascular fragility)
• Using a manual or electronic key counter. as well as any abnormality of the platelet number or
• Writing individual cells and recording the function. It is a non-specific test and may not always
numbers of each cell in a division of five. give positive results. This test is named after Alfred
6. If the count is very high, it is better to count 200- Fabian Hess.
500 cells in order to get an accurate idea of the Principle
relative number of cells. Impeding venous return raises the blood pressure in
7. If there are nucleated red cells present, these are the capillaries, resulting in small breaches. Normally
not included in the WBC. Instead, these are these are plugged by platelets but, if the breaches are
counted separately & reported as the number of more due to increased vascular fragility or if the
nucleated red cells/100 WBC. platelets are either less in number or defective in
8. If one basophil appears in 100 cells then another function, then blood extravasates and petechiae
100 cells should be counted to estimate their appear in greater numbers.
correct percentage.
9. DLC is commonly reported as a percentage or Requirements:
the absolute number calculated from the TLC of Sphygmomanometer
each type of cell, as under: Procedure:
• Neutrophils Apply the sphygmomanometer cuff to the arm, and
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inflate it to 80 mm Hg pressure. Maintain this • Clean the volar surface of the forearm with spirit
pressure for 5 minutes. Inspect the volar surface of swabs and choose an area of the skin that does
the forearm for the appearance of petechiae over the not have any visible veins.
antecubital fossa. Count the number of petechiae in a • Make two 4-8 mm long, 1 mm deep, separate
3 cm2 area. If there are 20 or more petechiae, the test punctures along the long axis of the forearm, 5-
is positive. 10 cm apart with a standard-depth lancet or with
Causes of Positive Result in Hess’s Test: a template.
• Thrombocytopenia • Let the blood flow out freely and start the
stopwatch.
• Platelet function defect
• Keep on blotting the oozing blood by gently
• Decrease in capillary resistance
touching it with the edge of circular filter paper
• Scurvy Dengue Fever.
at 30 second intervals, until the blood stops
flowing and no blood spot is left on filter paper.
BLEEDING TIME (BT) • Stop the stopwatch and note the time. This is the
Principle Bleeding Time.
When a standard incision is made on the volar surface of • If the Bleeding Time is more than 15 minutes &
the forearm all mechanisms involved in the arrest of blood is still oozing, stop the test and apply
bleeding are activated to stop the blood Loss. The time pressure until the bleeding is arrested. Write the
taken for the blood to stop flowing from the wound, result as ‘bleeding time more than 15 minutes’.
(without assistance) is known as the Bleeding Time. The Precautions:
Bleeding Time depends upon the number of platelets
• Check the platelet count before the test. If the
and the quality of their functioning. If the number of
count is less than 50x109/L, then the test
platelets is reduced below a critical level or these are
should not be performed.
functionally abnormal, the bleeding time is prolonged.
• There is always a tendency for the wound to
The Bleeding Time is also prolonged in von Willebrand
close. Therefore, a 1 mm-deep incision should be
Disease as the platelets’ functioning is disturbed due to
made. A superficial incision will result in
the absence of vWF.
erroneous results.
Requirements: • The blood pressure, number and size of incisions
• Sphygmomanometer must be standardised.
• Lancet or template • The area of skin that is selected for the puncture
• Circular filter paper should be clear of visible veins.
• Stopwatch Reference Ranges:
Method: Dukes' Method: 2 - 7 minutes
There are two methods by which the bleeding time Ivy's Method (lancet): 2 - 7 minutes
can be measured: Ivy's Method (template): 2.5 – 9.5 minutes
1. Duke's Method. This method is sometimes used Interpretations:
with infants and children. 1. A prolongation in BT commonly occurs in:
2. Ivy's Method. This is the standard method used.
• Thrombocytopenia
Duke's Method: • von Willebrand Disease
In this method, incisions are made in the ear lobe, the • Platelet-function defects
pulp of the finger or heel (while it is warm), as these • Aspirin ingestion
sites are rich in capillaries. • Afibrinogenaemia
• Clean the site with a spirit swab. 2. A short Bleeding Time commonly occurs when
• Allow the area to dry. the technique is faulty.
• With the help of a lancet, puncture deeply so that
blood flows out freely. Start the stopwatch. At half- WHOLE-BLOOD CLOTTING TIME
minute intervals, blot the drop of blood at the site of Principle
the puncture with the help of a filter paper. When blood obtained by a clean venepuncture is put in
• Keep on doing so until blood stops flowing and a glass tube, clotting mechanisms are activated and soon
there is no mark of blood left on the filter paper. a clot is formed. The time taken by the blood to clot in
At this point, stop the stopwatch and note the this way is called Whole-Blood Clotting Time (CT).
time. This is the Bleeding Time. Whole-Blood Clotting Time is an insensitive and non-
Ivy’s Method: specific test. It will be prolonged only in severe
This is the standard method - haemophilia or Christmas Disease, when the factor
• Apply the sphygmomanometer’s cuff to the arm levels are as low as 1 percent. It is some- times used as a
of a patient lying supine on a couch. bedside procedure to screen for a heparin effect and
circulating anticoagulants. The Lee and White Method
• Inflate the cuff to 40 mm Hg. This pressure
is commonly used.
should be maintained throughout the test.
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Requirements: PROTHROMBIN TIME (PT)
• Disposable plastic syringe
• Glass test tubes 75x12 mm (10 mm bore) Principle
Prothrombin Time measures the activity of the
• Water Bath at 37°C
extrinsic & common pathways of coagulation (factors
• Stop Watches (3)
II, V, VII, X and fibrinogen) under standardised
Procedure: conditions. When tissue thromboplastin and calcium
1. Place three glass test tubes in the water bath at are added to citrated plasma, this pathway is activated
37°C, to warm up. and a fibrin clot is formed. The time taken for this
2. Clean the venepuncture site with a spirit swab clot to form is called the Prothrombin Time.
and let it dry. The Preparation of Thromboplastin:
3. Using a disposable plastic syringe, collect 3 ml Thromboplastin is freely available commercially and
of blood. As the blood enters the syringe, start all is preferred as it is pre-standardised. However, it can
three of the stopwatches. also be prepared in the lab, from a rabbit’s brain. The
4. Put 01 ml of the blood in each of the three glass rabbit-brain preparation, however, is not as sensitive
tubes already placed in the water bath. as that of the human brain but, due to the danger of
AIDS, use of human brain has been abandoned. The
5. Initially tilt the tubes after 4 minutes and then method of preparation is as follows:
after every 30 seconds to see whether the blood
• Sacrifice a rabbit and take out its brain.
has clotted or not.
• Strip the membranes and the blood vessels from
6. When the blood clots in a tube, stop the the brain.
stopwatch for that tube. Note the time taken by • Remove the cerebellum and the brain stem and
the blood to clot for each tube. Take the mean of cut the cerebrum into very small pieces.
the three readings as result. This is the Clotting • Take about 50 ml of Acetone in a mortar and add
Time. about 200 g of the cerebrum to it.
Precautions: • Macerate the brain in the Acetone.
• The venepuncture should be clean and only those • Allow it to stand. Decant the supernatant
samples are to be dealt with which are obtained acetone, add fresh acetone and repeat the
after a single prick. This is because, due to procedure.
repeated trauma, more tissue factor is released • Keep on changing the acetone until a non-
and the clotting time may be shortened. granular powdery material is obtained.
• It is important to start the stopwatch as soon as • Collect this powdery material on clean filter
the blood enters the syringe. paper and let it dry in a desicator.
• The tubes should be of the specified bore (10 • Once dry, store in small amounts in stopper
mm), otherwise the results may vary. tubes at 4°C.
• It is to be freshly suspended in saline (300 mg in
Reference Range:
5 ml saline) for use. Warm at 37°C for 15 - 30
5-11 minutes
minutes and collect the supernatant for use.
Interpretations: • It is important to check the Prothrombin Time of
Clotting Time is prolonged in: control plasma by using the prepared
• Severe Haemophilia thromboplastin. If it is more than 14 seconds,
• Severe Christmas Disease then more powder is added until the time is
• Anticoagulant therapy, particularly with Heparin adjusted to 14 seconds. If it is less, then dilute
• Factor XII deficiency. with Isotonic Saline until the control plasma
• Circulating anticoagulants gives a time of 14 seconds.
Requirements:
• Patient’s platelet-poor plasma: Collect 9 volumes
of patient blood in one volume of Trisodium
Citrate (31.3 g/L trisodium dihydrate or 38 g/L
trisodium pentahydrate) in a plastic tube.
Centrifuge at 2000 g for 15 minutes, preferably
at 4°C. Collect the platelet-poor supernatant
plasma into a plastic tube for testing.
• Normal, ‘control’ plasma: Prepared by pooling
platelet-poor plasma obtained from 4-20 normal,
healthy individuals.
• Thromboplastin: Either commercial or in-house
prepared Thromboplastin can be used. The
Figure 3: Manual estimation of coagulation tests. reagents are commercially available and in some
292 | P a g e
of these, Thromboplastin and Calcium Chloride o Oral anticoagulant therapy (Vitamin K
have been combined. antagonists)
• Glass Tubes 75x12 mm o Obstructive jaundice
• Automatic Micropipettes of 100 µl volume o Liver disease
• A Water Bath set at 37°C o Haemorrhagic disease of the newborn
• Stop watches o Malabsorption
• A Table Lamp o Vitamin K deficiency
o Hereditary deficiency of concerned factors
Procedure: o DIC
• Set the table lamp behind the water bath in such a
way that the tubes can be seen against it but the eyes PARTIAL THROMBOPLASTIN TIME
of the technician are protected from direct light. WITH KAOLIN (PTTK)
• Place four plain glass tubes in the water bath to Principle
warm at 37°C. Platelet poor plasma is first incubated with a contact
• Deliver 100 µl of test plasma in a test tube and activator such as Kaolin Silica or ellagic acid for a set
wait for two minute. period. During this phase factor XIIa is produced that
• Deliver 200 µl of commercial tissue cleaves factor XI to factor Xla, but in the absence of
thromboplastin and start the stopwatch calcium, coagulation dose not proceed beyond this.
simultaneously. Mix the contents and leave. After recalcification factor XIa activates factor lX
• After 6-8 seconds, examine the tube against and coagulation follows that lead to a clot formation.
shielded light for clot formation, by tilting. Keep This measures the overall efficiency of the intrinsic
on doing so every 1-2 seconds by briefly taking pathway of coagulation. It also depends upon the
the tube out of the water. activity of factors II, V and X. Phospholipid is added
• Stop the stopwatch when a visible clot is formed to provide a standardised amount of platelet factor III
in the test tube and note the time. activity and then the mixture is clotted by the
• Repeat the procedure once again on the test addition of Calcium Chloride. The time taken for the
plasma. Take the mean of the two recorded fibrin clot to appear is noted.
times.
Preparation of Bell & Alton Platelet Substitute:
• Repeat the test on the control plasma as done for
• Take 1 g acetone-dried brain (prepared for
the patient’s plasma.
thromboplastin).
Precautions: • Dissolve in 20 ml Acetone and let it stand at
• The blood should be collected through a clean room temperature for 2 hours.
venepuncture and without much stasis. • Centrifuge and discard the supernatant.
• The proportion of anticoagulant and blood • Dry the deposit in a desicator.
should be precise and appropriate. • Dissolve in 20 ml Chloroform and leave at room
• The samples should not be allowed to stand at temperature for 2-4 hours, mixing it from time to
room temperature for too long. If a delay is time.
expected, these should be transported on crushed • Filter and evaporate the filtrate in a desicator at
ice. 37°C.
• The platelet-poor plasma should be separated as • Suspend the residue in 10 ml normal saline.
soon as possible. • Determine the PTTK of normal, pooled plasma
• The blood should be collected and processed with the prepared reagent, adjusting the
using disposable plastic syringes, tubes and concentration to a PTTK of 35 seconds (as in the
pipettes. thromboplastin preparation).
• The test should always be performed in clean .
glass tubes. Requirements:
• Test and control plasma prepared as for
Reference Range:
10-14 seconds Prothrombin Time.
The result is reported along with controls as below: • Platelet substitute - commercial or in-house
• Patient plasma 16 seconds prepared. (some commercial reagents are
pre-mixed with Kaolin).
• Control plasma 14 seconds
The results are also reported as a ratio between the • Kaolin in Barbitone Buffer at pH 7.4:
Sodium diethylbarbiturate 11.74 g
patient’s Prothrombin Time and that of the test Hydrochloric acid 430 ml.
plasma or as INR. Kaolin 2.15 g.
Interpretations: • Calcium Chloride (as for Prothrombin Time).
• Prothrombin Time is prolonged in deficiency of • Automatic Micropipettes of 100 and 200 µl
Factors II, V, VII and X. This can occur in the volumes
following conditions: • Test tubes 75x12 mm, both plastic and glass
293 | P a g e
• Stop watches. • Thrombin 50 NIH units/ml (commercially
• A timer. obtained).
• A table lamp. • Other requirements as for PT and PTTK.
• A water bath set at 37°C. Procedure:
Procedure: • Pre-warm a few glass tubes in a water bath set at
• Mix equal volumes of platelet substitute and the 37°C.
kaolin suspension and leave in the water bath to • Place 200 µl test plasma in a tube.
warm up. • Add 100 µl Thrombin. Start the stopwatch.
• Add Calcium Chloride into a glass tube placed in • Inspect for clot formation and note the time
a water bath to warm. when a clot appears.
• Place a few 75x2 mm glass tubes in the water • Repeat the procedure again and take the average
bath to warm.
of the two times.
• Place 100 µl test plasma in a pre-warmed tube.
• Add 100 µl platelet substitute-kaolin mixture to • Also observe the quality of the clot.
it. Start the timer and mix at intervals. • Repeat the procedure on the control plasma
• Leave in the water bath for 10 minutes..
• After exactly 10 minutes, add 100 µl Calcium Precautions:
Chloride, start the stop watch and mix. Examine As described for PT and PTTK
for clot formation at intervals as for Prothrombin
Reference Range:
Time. Stop the watch as soon as a fibrin clot
9-11 seconds;best to report with the control
appears and note the time.
• Repeat the procedure on the test plasma and take
the average of the two times. Interpretations:
• Repeat the procedure on the normal pooled Thrombin Time is prolonged in:
plasma as for the test plasma. • Heparin therapy
• Raised FDPs, Fibrinogen deficiency
Precautions: • Dysfibrinogenaemia (the clot is transparent and
bulky)
• As for prothrombin time, the instructions
• Multiple myeloma
provided by the manufacturer should be • Infancy
followed. • Hypoalbuminaemia
Reference Range: Bibliography:
25-43 seconds, It is better to report against the 1. Dacie and Lewis. Practical Hematology 11th
‘normal’ control as in PT. Each laboratory needs to Edition. S Mitchell Lewis, Barbra J Bain,
determine its own reference range. Imelda Bates eds. Churchill Livingstone
Interpretations: London 2013.
PTTK is prolonged in: 2. Wintrobe’s Clinical Hematology 12th edition.
• Deficiency of factors XII, XI, IX, VIII, X, V, II. John P. Greer, John Forester, Gerge M. Rogers,
• Anticoagulant therapy with Heparin Friox Paraskevas, Bertil Glader, Danial A
• Circulating anticoagulants Arber, Robert T. Means, Jr. Wolters Kluver,
Lippincott Williams & Wilkins London 2009.
• Massive transfusion of stored blood
3. Post Graduate Haematology 7th Edition . A.
• Liver disease
Victor Hoffbrand, Daniel Catovsky, Edward G.
• DIC
D. Tuddenham eds. Blackwell Publishing
THROMBIN TIME (TT) London 2016.
4. KOEPKE J.A; McLAREN C.E., WIJETUNGA
Principle A AND HOWEN B: ‘’A method to examine the
Thrombin acts directly on fibrinogen and converts it need for duplicate testing of common
to fibrin. The time it takes for a clot to form, after the coagulation tests’’ Am. J. Clin. Pathol.,102, 2 ,
addition of Thrombin, is called Thrombin Time. 242 – 246, 1994.
5. VASSAUT A. et al:. ‘’ Prolocole de validation
Requirements: de techniques’’ Ann. Biol. Clin, 44, 686 – 745,
• Test and control plasma as previously described. 1985.
294 | P a g e

39. BLOOD CELL MORPHOLOGY

Study of the morphology of blood cells in a well- endogenously (auto-antibodies) as in Cold
spread and well-stained blood film yields invaluable Haemagglutinin Disease or, rarely, introduced from
diagnostic information. Therefore, the blood film outside, e.g. an infusion of large amounts of
should be examined carefully and systematically. It is mismatched plasma. In cases of incompatible blood
preferable that the film be mounted with a cover glass transfusions, the agglutinates seen comprise of the
using a neutral mounting medium. It provides not only donor’s original cells
good refraction but also preserves the blood film. First,
it should be examined under a low-power (x10) ABNORMALITIES OF SIZE
objective. This will give an idea of the quality of the Anisocytosis: If the size of the RBCs varies (beyond
film, distribution & staining of the cells and the platelet normal limits) in the same blood film, it is termed
aggregates. It also gives an idea about Rouleaux anisocytosis. It is expressed as + to +++. This is a non-
formation, the presence of agglutinates, dimorphic specific feature of several haematological disorders.
populations of cells & the presence of some
haemoparasites, e.g. microfilariae. Then, select a Microcytosis: When the average size of RBCs in a
suitable area and switch to a dry, high-power (x40) blood film is less than normal, it is termed
objective. An oil-immersion (x100) objective should be microcytosis. The degree of microcytosis is directly
reserved for the study of the finer details of cells. There proportional to the decrease in MCV. It seldom occurs
are three types of cells in the blood: RBCs, WBCs and alone but is usually accompanied with hypochromia.
platelets. Each of these should be studied Microcytosis is commonly seen in iron-deficiency
systematically. anaemia and thalassaemia. Sometimes, small cells with
no central pale area are seen. These usually have
RED BLOOD CELL MORPHOLOGY normal MCV. These are termed spherocytes.
Normal red blood cells appear as circular discs of about Macrocytosis: When the average size of an RBC is
6-8.5 µm in diameter, roughly equal to the size of the more than normal, it is termed a macrocyte. The degree
nucleus of a small lymphocyte. They have a bright of macrocytosis is directly proportional to the increase
reddish colour (due to haemoglobin) at the periphery, in MCV. Common causes of macrocytosis are liver
which becomes pale towards the centre because of the disease, megaloblastic anaemia, aplastic anaemia,
bi-concave shape of the RBC. The central pale area refractory anaemia, obstructive airway disease, excess
normally does not exceed one third of the RBC’s total of alcohol, treatment with hydroxyurea and
area. In a normal blood film, RBCs lie separately in the hyperglycaemia. In patients whose marrow is
central area of the film. RBCs are examined for their responding by increased haematopoiesis and there are a
distribution, size, shape, colour (Hb content) and lot of polychromatic cells, these appear as macrocytes.
inclusions. Abnormalities in these characteristics may
be artefactual or may arise in disease because of: ABNORMALITIES OF COLOUR
1. Changes in plasma proteins and the development The only true variation in colour is the hypochromia. It
of antibodies to RBC-surface antigens. results from the decreased haemoglobinization of
2. Abnormal erythropoiesis RBCs, commonly seen in iron-deficiency anaemia and
3. Inadequate haemoglobin formation thalassaemia. The degree of hypochromia is
4. Damage to the red cells in circulation proportional to MCHC. Leptocytes may appear
hypochromic because of flattening. Spherocytes appear
5. Increased erythropoiesis hyperchromic because of their loss of the central pale
ABNORMALITIES OF DISTRIBUTION area and an increased thickness of the cell. Macrocytes
may also appear hyperchromic because of increased
Rouleaux Formation: Rouleaux Formation (the thickness.
stacking of RBCs on top of each other) is seen when
the fibrinogen concentration of blood is increased, e.g.
in infections, pregnancy and tissue necrosis. It is
characteristically seen in conditions
with abnormal globulin production, e.g. in Multiple
Myeloma. The degree of Rouleaux formation is
directly proportional to the ESR.
Agglutination: Agglutination is defined as the random
aggregation of RBCs. These form clusters of varying Figure 1: Microcytic hypochromic RBC morphology in iron
deficiency
numbers of cells. This results from a bridging of cells
by antibody molecules, particularly IgM against Target Cells have a central, haemoglobinized area,
antigens on the surface of RBCs that are circulating in surrounded by a pale ring and then a peripheral
the plasma. These may have been produced haemoglobinized area. These also result from an
295 | P a g e
increased membrane surface due to the increase in its hereditary defect of the membranes.
cholesterol and phospholipid content. These are
characteristically seen in thalassaemias, HbC Disease, Stomatocytes: When RBCs have a 'mouth'-like slit,
HbD Disease, HbE Disease, obstructive liver disease, these are called Stomatocytes. A few stomatocytes
post-splenectomy and iron-deficiency anaemia. If they are usually seen in normal blood films. Their
are artefacts, then these are confined to only a portion number is increased in alcoholism, liver disease and
of the blood film. Rh-null Disease. These are numerous in a hereditary,
membrane defect.

Schistocytes: These are fragmented red blood cells

of various shapes and sizes. Large cells from which
portions are fragmented sometimes appear as
‘helmets’ and are called helmet cells. Schistocytes
are increased in conditions like Iron-Deficiency
Anaemia, Megaloblastic Anaemia and Thalassaemia,
but are characteristically increased when RBCs are
Figure 2: Target cells exposed to mechanical trauma. This occurs when
RBCs are passing through meshes of fibrin as in
Dimorphism: This is the term used when two distinct DIC, or through narrowed vessels as in
populations of RBCs are seen in the blood film. One microangiopathy or through prosthesis.
population may be normal and the other abnormal,
particularly hypochromic microcytic or macrocytic. It Echinocytes and Burr Cells: Echinocytes or
is seen in Sideroblastic Anaemia, when a patient has crenated cells have evenly distributed, blunt spicules
been transfused or when a patient is receiving of uniform size on their surface. These are formed if
haematinics for treating a deficiency anaemia. anti-coagulated blood is allowed to stand for long
periods, e.g. overnight at room temperature or if the
ABNORMALITIES OF SHAPE film is prepared on a slide that has fatty material on
it or if the blood pH is raised. These are also seen in
Poikilocytosis: When the shape of RBCs varies more than patients who have Uraemia or have been on a
expected (for normal individuals), in the blood film, it is cardiopulmonary bypass. Burr Cells are also
termed as Poikilocytosis; the abnormally-shaped RBC is echinocytes, but their spicules are reversible.
termed a poikilocyte. Poikilocytosis is also a non-specific
feature seen in several haematological disorders: Acanthocytes: These are small, densely staining
abnormal erythropoiesis, megaloblastic anaemia, MDS, RBCs with thorn-like projections. Generally, the
iron-deficiency anaemia, thalassaemia, and myelofibrosis. projections are fewer, of varying sizes, variable in
However, specific types of poikilocytes are diagnostic of number and more blunt than echinocytes. These may
specific disorders. be hereditary or acquired. Hereditary causes include
Spherocytes: When RBCs are more spheroidal than McLeod Phenotype and disorders of lipid
normal, these are termed spherocytes. These may result metabolism. The acquired causes include Spur-Cell
from genetic defects of red-cell membranes (as in Anaemia and chronic liver disease.
hereditary spherocytosis), because of an interaction
Pyropoikilocytes: These are seen in a rare hereditary
between or complement-coated red cells with
disorder, Pyropoikilocytosis, and comprise
macrophages as in immune haemolytic anaemias, ABO
microspherocytes and fragments of RBCs. Their
haemolytic disease of newborns & from the action of
number greatly increases when blood is heated to 45°C.
certain bacterial toxins, e.g. Cl. perfringens. Spherical
forms may also be seen when anti-coagulated blood is Sickle Cells: These are thin, elongated, deeply staining
allowed to stand for a long time, e.g. banked blood. red cells with elongated ends. They may be straight,
Elliptocytes and Ovalocytes: About 10% of RBCs curved or of various other shapes. These are produced
in a normal blood film (particularly at the tail end), by the polymerisation of the HbS in Sickle-Cell
appear oval and, less commonly, elliptical in shape. Disease.
Their proportion is higher in Iron-Deficiency
Anaemia, Megaloblastic Anaemia & Myelofibrosis. INCLUSIONS IN THE RBCs
In iron deficiency, they are usually more elongated Hb Crystals: Some abnormal Hb, particularly C and S,
(pencil cells) whereas, in Megaloblastic Anaemia, polymerise to form crystals inside the RBCs. The
they are macrocytic as well (oval macrocytes). In polymerisation of the HbS gives a distinct shape to
Myelofibrosis, the ovalocytes are somewhat pointed RBCs: the ‘sickle’ cell. HbS and HbC occurring
on the narrow side (tear-drop cells). If this shape is together polymerise to form straight crystals with
seen in the vast majority of the cells and in the parallel sides and one blunt projecting end, or multiple
central area of the film, then the condition is termed crystals projecting from a common centre. HbC
Elliptocytosis or Ovalocytosis. This results from a crystals are hexagonal with blunt ends.
296 | P a g e
Howell-Jolly Bodies: These are small, rounded unsegmented neutrophils (stab forms). In more severe
fragments of the nucleus that stain reddish-blue to cases, metamyelocytes, myelocytes and even
blue-black resulting from an incomplete extrusion of promyelocytes & blasts may appear. Left shift is most
the nucleus. These contain DNA and are <1 µm in commonly seen in severe bacterial infections. Severe
diameter. They usually occur singly in RBCs but may left shift, together with an increased count is termed
be multiple. The most common cause is a splenectomy leukemoid reaction. This should be differentiated
or splenic atrophy, but these are also seen in from myeloproliferative disorders, e.g. Chronic
alcoholism, Sickle-Cell Anaemia & Megaloblastic Granulocytic Leukaemia. In leukemoid reactions,
Anaemia. leucocyte alkaline phosphatase (LAP) is
characteristically high. Neutrophils with an
Basophilic Stippling or Punctate Basophilia: These unsegmented nucleus or, at the most, a bi-lobed
are fine to coarse, deep blue to purple & small, but nucleus with clear staining of both acidophilic and
multiple inclusions of varying sizes. These represent basophilic contents are called Pelger Cells. These are
aggregated ribosomes and are seen in Thalassaemia, characteristically seen in a benign inherited disorder:
Megaloblastic Anaemia, liver disease, lead poisoning, Pelger-Huet Anomaly. Pseudo-Pelger cells are seen in
unstable Hb, Pyrimidine 5-Nucleotidase Deficiency MDS, AML and CML.
and infections.
Hypersegmentation: It is defined as an increase in the
Pappenheimer Bodies: These are small, dark staining, proportion of neutrophils with 4 or more lobes of
irregular granules composed of haemosiderin occurring nucleus. Normally, a 3-lobed nucleus is seen in 40-
near the periphery of the cells. Their presence is related 50% neutrophils, 4-lobed in 15-20% and 5-lobed in
to an overload of iron. These stain positively with <0.5%. It is a diagnostic feature of Megaloblastic
Perl’s Stain. They are seen in Sideroblastic Anaemia, Anaemia but may also be seen in Uraemia and
Dyserythropoietic Anaemia and Thalassaemia. treatment with cytotoxic drugs (Methotrexate and
Hydroxyurea).
Cabot Rings: This is a thin, reddish blue ring- like
structure occupying varying portions of the RBCs. It
may be twisted to form a figure of 8. Its origin is not
clear. These are commonly seen in severe anaemia of
any type but most commonly in Megaloblastic
Anaemia, lead poisoning and Dyserythropoietic
Anaemias. These may occur alone, but are usually
associated with Punctate Basophilia and Howell-Jolly
Bodies.
Parasites: These include malarial parasites and Figure 3: Neutrophilic hypersegmentation
Babesia. For details see the section on A small number of neutrophils with a pyknotic
‘PARASITOLOGY’. nucleus are seen--these are dying cells and their
number increases in infections. Normally, cytoplasm
WHITE BLOOD CELL MORPHOLOGY contains fine azurophilic granules that are evenly
NEUTROPHILS distributed. In toxic granulation, the granules are
larger, densely staining and may also be increased in
Neutrophils are normally the predominant type of number.
WBCs in peripheral blood. These are of uniform size,
around 13 um in diameter and have a segmented
nucleus. These are examined for:
1. Stage of maturation
2. The shape of the nucleus and the number of lobes
3. The presence and appearance of granules
4. Cytoplasmic inclusions, other than granules.
In normal blood there are hardly any immature forms
except up to 8% stab forms (with an unsegmented
nucleus). Normally, in peripheral blood, neutrophils Figure 4: Toxic granulation in neutrophil
have 2-5 lobes but the number of 3-4 lobed cells is Hypogranular cells are seen in myelodysplastic
more than those with 2 and 5 lobes. In females, 2-3% syndromes. In rare inherited disorders, characteristic
neutrophils show an appendage at a terminal nuclear granular abnormalities are seen. In Alder-Reilly
segment, called a drumstick. It represents the inactive Anomaly, the granules are very large, discrete, stain
X chromosome. The granules in the cytoplasm of deep red and may obscure the nucleus. In Chediak-
neutrophils are azurophilic, small and evenly dispersed. Higashi Syndrome, there are very large (giant), but
When immature forms appear in the peripheral blood, scanty azurophilic granules. Sometimes small, round or
it is called left shift. The simplest left shift is oval patches of blue colour are seen. These are called
evidenced by an increase in the percentage of Dohle Bodies. These are commonly seen in severe
297 | P a g e
bacterial infections. In a rare inherited disorder, May- lymphocytes or virocytes. Lymphocytes predominate
Hegglin Anomaly, such structures are also seen. In in the blood films of infants and young children. Turk
severe infections, vacuoles of varying sizes may be Cells are transformed lymphocytes that are seen in
seen. Bacteria may also be seen within these vacuoles. infections. These are slightly larger with a rounded,
eccentric nucleus and an abundant deeply-basophilic
EOSINOPHILS cytoplasm. In viral infections, larger cells with an
Eosinophils are slightly larger than neutrophils (12-17 irregular outline and a distinct, round, oval or kidney-
µm in diameter), have a bi-lobed nucleus and the shaped nucleus are presented.
cytoplasm is packed with spherical golden-orange
granules. Their number increases in allergic conditions
and parasitic infections. De-granulation, hyper- THE MORPHOLOGY OF PLATELETS
segmentation of the nucleus and vacuolation occur as a
reactive change. Abnormal granules are seen in CML, Platelets are small (1-3 µm) discoid cells which have
Myelodysplasia and AML. no nucleus but a central granulated area. Larger
platelets, even ribbons of platelets, are seen under
BASOPHILS conditions of stress (bleeding due to any reason).
The platelet count rises in acute inflammation or
These are of the same size as eosinophils. Large, dark stress. In myeloproliferative disorders, very large
blue granules of variable sizes obscure the nucleus. The platelets, giant platelets or even cytoplasmic
nucleus is usually folded upon itself and appears as fragments of megakaryocytes may be seen. In some
compact, irregular and dense. These tend to form individuals, platelets form rosettes around
vacuoles and de-granulate. They are less than 1% in neutrophils. This is called platelet satellitism and is
normal blood. Their number is increased in CML. an antibody-mediated phenomenon. In about 1%
MONOCYTES EDTA samples, platelets clump together.
Characteristic abnormalities may be seen in some
These are the largest or the leucocytes in circulation. rare inherited disorders. In Bernard Soulier
They have a bluish grey cytoplasm with a ground-glass Syndrome, platelets are large (giant), whereas in
appearance and a nucleus which appears to be folded Grey Platelet Syndrome, these appear grey, due to a
upon itself. Their number increases in chronic lack of granules.
infections and in some types of leukaemias. A reactive
change is defined by the appearance of vacuoles and a Bibliography:
spreading ability of the cell. Such cells are called Dacie and Lewis. Practical Hematology 11 th
Macrophages and may be seen on the margins of blood Edition. S Mitchell Lewis, Barbra J Bain, Imelda
films in severe bacterial infections. Bates eds. Churchill Livingstone London 2013
Wintrobe’s Clinical Hematology 12 th edition. John
LYMPHOCYTES P. Greer, John Forester, Gerge M. Rogers ,Friox
The majority (90%) of lymphocytes in peripheral blood Paraskevas, Bertil Glader, Danial A Arber, Robert
are small with a rounded nucleus and a thin rim of T. Means, Jr. Wolters Kluver, Lippincott Williams
cytoplasm occasionally containing a few reddish & Wilkins London 2009
granules. About 10% are large with more abundant Post Graduate Haematology 7 th Edition . A. Victor
cytoplasm and more frequent azurophilic granules in Hoffbrand, Daniel Catovsky, Edward G. D.
the cytoplasm. Open chromatin and, sometimes with Tuddenham eds. Blackwell Publishing London
visible nucleoli, may appear. These are called reactive 2016
298 | P a g e

40. THE EXAMINATION OF BONE MARROW

In many haematological conditions, particularly expected distribution of the disease process. The
leukaemias, examination of the bone marrow may be various sites for bone- marrow aspiration include:
the only procedure to arrive at a correct diagnosis. It
1. Posterior Superior Iliac Spine (PSIS): This is
provides the opportunity of directly examining the
the most suitable site in adults and in children
tissue which forms blood cells. The examination is
over two years of age. It has the ease of
easy to perform and safe, except in severe bleeding
puncturing multiple sites at one time as well as
disorders like haemophilia, and can be performed as
sampling large volumes of bone marrow. It is
an out-door procedure as many times as required.
safe and, as the patient cannot see it, it causes
There are two methods of examining the bone
less apprehension to her/him. A bone marrow
marrow:
trephine biopsy can also be performed from this
• Smears prepared from a needle aspirate
site, through the same skin puncture.
• Sections prepared from a trephine or open
surgical biopsy specimen of the bone marrow 2. The Sternum: The sternum is used in obese,
Smears show better morphological details of adult patients. It is punctured opposite the second
individual cells and are also used for cytochemical or third intercostal space, slightly to one side of
staining and immunological studies. However these the midline. The total thickness of the sternum is
do not show spatial distribution of normal and about 1.5 cm. Therefore, it is necessary that a
abnormal cells and their exact quantity. For this a guard be applied to the needle, so that it should
biopsy section is examined. not penetrate more than 0.5 cm of the bone.
Disadvantage is that trephine cannot be done due
THE ASPIRATION OF BONE to patient apprehension
MARROW
3. Spinous Process of the Vertebrae: The spinous
INDICATIONS processes can also be selected for bone-marrow
aspiration. However, it is necessary that these
A needle aspiration of the bone marrow is indicated
should be palpable. This is not the site of choice.
for the diagnosis of primary haematological diseases
as well as for the diagnosis of certain other illnesses. 4. The Tibia: In cases of children less than 2 years
It is also performed for determining effects of of age, the anteromedial surface of the tibia,
treatment given for some diseases. slightly below the tibial tuberosity, is the site of
choice.
a. Diagnostic -
• Megaloblastic Anaemia 5. Anterior Superior Iliac Spine: This site may be
• Aplastic Anaemia used in obese patients, but it is not convenient.
• Sideroblastic Anaemias First, it is more painful as here the skin here is
• Iron-Deficiency Anaemia richly supplied with sensory nerves. Secondly,
• Anaemia of chronic disorder the overlying cartilage is thick. Thirdly it is more
• Acute Leukaemias apprehensive for the patient
• Multiple Myeloma 6. Others: Occasionally, an aspiration may be
• Metastasis in the bone marrow performed directly from a lesion that is visible in
• Storage Disorders an X- ray, e.g. lytic lesions in the ribs and bones
• Visceral Leishmaniasis (Kala Azar) of the skull.
• To obtain haemopoietic cells for cytogenetic
studies, molecular genetic studies and Requirements:
immuno-phenotyping 1. Needles like salah, kilma and islam were used in
• Culture for Mycobacteria and other bacteria the past but now a days spinal needle of 14 – 16
in cases of PUO. G is used for marrow aspiration. Clean, grease-
free glass slides, preferably with a frosted end,
b. Prognostic - for easy labelling
• The staging of chronic leukaemias 2. A Spreader
• The staging of lymphomas 3. A large piece of filter paper
• To determine the response of treatment 4. Disposable Syringes of 10 ml
given for acute leukaemia and other 5. Disposable Syringes of 20-50 ml with a nozzle to
disorders. fit in the aspiration needle.
6. Antiseptic Lotion (0.5% Chlorhexidine in
SITES FOR ASPIRATION Ethanol)
Selecting a site for bone-marrow aspiration depends 7. Local Anaesthetic (2% Lignocaine)
upon the age of the patient, her/his physique and the 8. A surgical blade mounted on a handle
299 | P a g e
9. Surgical Towels with Romanowsky Stain and one with Prussian Blue
10. Disposable, Surgical Gloves Method. Smears that are well- spread and carry
11. Towel Clips enough fragments should be stained. The methods
12. Sponge Forceps and stains used for Romanowsky Staining are the
13. A Medicine Bowl same as described earlier for the staining of blood
14. Sterile, Surgical Gauze smears. The only difference is that the timings are
doubled when Leishman Stain is used, i.e., the slide
PROCDEDURE:
is covered with pure Leishman stain; it should be left
1. Prepare the tray or trolley with all of the in place for 4-5 minutes instead of two. The buffer is
requirements (listed above). then mixed on the slide and it is left for 15-16
2. Place a piece of filter paper and arrange 2-3 glass minutes, instead of 8 minutes. This is because bone-
slides on it in slanting position against a support. marrow smears are thicker than blood smears and the
3. Label at least 10 slides with the patient’s cells, being more in number, require more time for
identification and arrange for smear preparation. staining.
4. Draw about 5 ml of 2% Lignocaine in a
disposable syringe and keep aside for later use. PRUSSIAN BLUE STAINING
5. Wash your hands thoroughly with soap and This is required to stain the iron content of bone
water and put on the surgical gloves. marrow and both intracellular and extracellular iron
6. Explain the procedure to the patient and reassure is stained. The method utilises Perl's Reaction. In this
her/him. She/he should be particularly explained reaction, water-insoluble haemosiderin acquires a
about suction pain. blue colour when exposed to the acidic solution of
7. Position the patient, depending upon the site Potassium Ferrocyanide.
selected for the aspiration procedure.
8. Clean the site with an antiseptic solution. Clean Requirements:
an area that is larger than required, to prevent 1. Fixative: Absolute Methanol
infection. 2. Acidic Potassium Ferrocyanide 1%
9. Drape the area with surgical towels. a. Solution A - Potassium Ferrocyanide 2%:
10. Inject Lignocaine into the skin, subcutaneous Dissolve 2g Potassium Ferrocyanide in 100
tissues and the periosteum of the bone in an area ml of distilled water.
of about 1-2 cm. Wait for 3-5 minutes. b. Solution B: Hydrochloric Acid 0.2 mol/L:
11. Make a small, skin-deep nick with a blade at the Add 2 ml of concentrated HCl to 98 ml of
selected site. distilled water.
12. Introduce the aspiration needle with a gentle c. Working Solution: Mix equal volumes of
boring movement. When the bone marrow is Solutions A and B just before use.
entered, there is a feeling of ‘giving away’ of 3. Counter Stain: 0.1% Nuclear Fast-Red Solution.
resistance. Move the needle a little more 100 mg/100 ml in distilled water.
forward, until it is fixed. 4. Slide-staining jars (Coplin Jars)
13. Remove the stillet and attach a 20-30 ml
disposable syringe to the needle. Procedure:
14. Suck about 0.5 ml of marrow. (one of the 1. Air-dry the film and fix in absolute Methanol for
indications that the marrow has been penetrated 20 minutes.
satisfactorily is the suction pain). 2. When dry, place in the Working Solution for 10
15. Detach the syringe and replace the stillet. minutes.
16. Immediately start making the smears so that the 3. Wash well in running tap water for about 20
marrow does not clot by pouring the aspirated minutes. Rinse in distilled water.
marrow on the slanted slides so that the free 4. Counter-stain with 0.1 % Nuclear Fast-Red
blood drains while fragments remain stuck on aqueous solution for 5 minutes.
the slide, accomplishes this. Pick up the 5. Air-dry and mount.
fragments with the edge of the spreader and Result:
gently smear on the pre-arranged slides. The iron will stain as bright blue granules.
17. Put the remaining marrow in an EDTA bottle Now a days automated staining procedure is also
and mix it, so that more slides can be prepared (if available.
required).
18. Secure the haemostasis by firmly pressing the
puncture site for 5-10 minutes. EXAMINING BONE-MARROW SMEARS
19. Apply the dressing.
First, examine all of the stained slides with the naked
THE STAINING OF BONE-MARROW eye and choose one which has enough marrow
ASPIRATE SMEARS particles (fragments), is properly spread, is evenly
stained and is the best. A good smear has a reddish
In all cases, at least two smears should be stained blue colour. In some diseases (Multiple Myeloma,
300 | P a g e
Megaloblastic Anaemia, Auto-immune Disorders, with indistinct, ruffled borders and contain
etc.) the particles tend to clot during their several discrete, rounded nuclei. Osteoblasts
preparation. In Cold Haemagglutinin Disease, RBCs should not be confused with Plasma Cells. These
agglutinate if the slides and syringe were not warmed are oval, have a compact nucleus and the
before the collection of the sample & the preparation cytoplasm stains bluer, has no granules and has a
of the smears. Then, examine the selected slide under frayed border.
a low-power (x10) objective of the microscope and 5. Abnormal Cells: Examine for abnormal cells
note the following: such as Macrophages containing parasites (LD
1. Cellularity: This is noted for both fragments and Bodies), Histiocytes (with or without
trails. Normally, 1/3 to 1/2 a fragment of an haemophagocytosis), storage cells like Gaucher
adult’s bone marrow contains haemopoietic and Niemann Pick Cells and clumps of
cells, whereas the rest of it comprises of fat malignant cells. Now select a well-stained area
spaces. If the proportion of haemopoietic tissue and examine with a dry, high- power (x40)
is less than this, then the marrow is termed objective. Mounted slides are best viewed with
‘hypocellular’ and if it is more, then the marrow this objective. An oil- immersion (x100)
is termed ‘hypercellular’. Some- times, objective should only be used for differentiating
fragments are only composed of a thick mass of the finer details of the cells (if required).
cells. This is called packed marrow. Similarly, 6. Erythropoiesis: Note the quantity and quality of
the cellularity of the trails is noted. A trail is the erythropoiesis. Whether it is normal, reduced or
part of the smear that is left behind by a fragment increased, and whether it is normoblastic or
during spreading megaloblastic. Also look for dysplastic features,
e.g. cytoplasmic bridging, nuclear lobulation,
multi-nuclearity and fragmentation, etc.
7. Myelopoiesis: Note the quantity and quality of
myelopoiesis. Particularly look for any change in
the maturation sequence, granularity of the
cytoplasm (hypogranular), an excess of
eosinophilic or basophilic cells and the presence
of giant myelocytes and metamyelocytes.
8. Parasites: Examine for the presence of parasites
such as Plasmodium falciparum (which tend to
Figure 1: BM Fragment
sequestrate in the bone marrow) and Leishmania
3. Megakaryocytes: Both the number and donovani.
maturation stages are to be noted. Normally, 2-6
9. Myelogram: Now perform a differential count
megakaryocytes per low-power field are present of all nucleated cells in the bone marrow. This
and these contain a nucleus with 6-10 lobes or should include all stages of maturation of the
segments. Megakaryocytes tend to collect
WBCs. However, all stages of erythroblasts are
towards the tail end and may form masses if the
counted collectively. At least 500 cells should be
bone marrow has clotted prior to the preparation
counted. It is preferable that the differential
of the smear. count should be performed from more than one
randomly selected area. The differential count of
a bone-marrow smear is called a Myelogram.
Table 1: Normal ranges for differential counts on
aspirated bone marrow.
Myeloblasts 0–3
Promyelocytes (neutrophil) 2 – 13
Metamyelocytes 2–6
Neutrophils 22 – 46
Myelocytes (eosinophil) 0–3
Figure 2: BM Trail containing a megakaryocyte Eosinophils 0.3 – 4
Basophils 0 – 0.5
4. Other Cells: At this stage, some normal and
Lymphocytes 5 – 20
abnormal, large cells may also be seen.
Monocytes 0–3
Osteoclasts, Osteoblasts, Macrophages, Mast
Plasma cells 0 – 3.5
Cells and Endothelial Cells are normally seen in
small numbers, but their number may be Erythroblasts 5 – 35
increased in certain disease states. Osteoclasts Megakaryocytes 0–2
should not be confused with Megakaryocytes. Macrophages 0–2
These are much larger, have granular cytoplasm
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10. ME Ratio: Calculate the Myeloid-Erythroid BONE-MARROW SMEAR REPORTS

Ratio (ME Ratio), which is done by dividing the
total number of myeloid cells by the total A bone marrow report should include the patient’s
number of erythroid cells. Lymphocytes, plasma identification parameters, the date when the bone
cells and other non-myeloid and non-erythroid marrow aspiration was performed, date of reporting
cells are excluded. the bone marrow results, name of the pathologist
reporting the bone marrow results, site from where
11. Blasts: Normal marrow contains <2.5 %
the bone marrow was aspirated, consistency of the
blasts. if the marrow contains an excess of
bone (normal, hard or soft) & the force required to
blasts (≥5%), then give their morphological
aspirate (easy, difficult, bloody, dry). After this, all
description in order to differentiate between
observed facts should be reported sequentially. A
myeloblasts and lymphoblasts.
typical sequence is: the description of the
erythropoiesis, of the myelopoiesis, of the
megakaryocytes, of the lymphocytes and plasma
cells, of the abnormal cells (including blasts,
presence of parasites), of the iron status and, finally,
the ME Ratio. Results of cytochemical stains if used
should also be documented. It is highly preferable
that a myelogram is also included in the report.
Finally, the report should detail the conclusions
drawn, the most probable diagnosis and any
Figure 3: Myeloblasts in BM suggestions regarding further investigations, if
12. Iron: Now examine the Prussian Blue- required.
stained smear. First, (under low power) check
for visible haemosiderin in the fragments. BONE-MARROW TREPHINE BIOPSY
The slight colour of a few granules is
normally present. Note whether iron Bone marrow aspirate is complemented by bone
(haemosiderin) is increased or absent. Then marrow trephine biopsy.
examine under oil immersion for the quantity INDICATIONS:
and quality of Siderocytes and Sideroblasts. • Repeated dry/bloody tap
Siderocytes are mature RBCs that contain a • Aplastic Anaemia
few haemosiderin granules. Sideroblasts are • Myelosclerosis/Marrow Fibrosis
erythroblasts that contain haemosiderin • Multiple Myeloma
granules in the cytoplasm. Normally, in about
• Chronic Leukaemias
40% of the polychromatic erythroblasts, a
• Acute Megakaryoblastic Leukaemia (M7)
few, small siderotic granules are seen
scattered in the cytoplasm. Particularly look • The staging of lymphomas
for ring sideroblasts at the periphery of the • The staging of other tumours (metastasis)
fragments. These are erythroblasts in which • In the case of PUO, for granulomas
the haemosiderin granules are larger, more SITES FOR THE TREPHINE BIOPSY
numerous and arranged in the form of a ring Only two sites can be safely used. These are the
around the nucleus. These are only seen in posterior superior iliac spine and the anterior superior
disease states, e.g. Sideroblastic Anaemias & iliac spine. The first one is the preferred site.
MDS.
Requirements:
These are the same as for a bone-marrow aspiration,
except that a Trephine Biopsy Needle is required in
place of an aspiration needle and a bottle containing
fixative is required. Most commonly, the needles that
are used for bone marrow trephine biopsies are the
Jamshidi and Islam needles. Now a days a variety of
disposable sterilized needles are available These come
in three sizes: a standard adult size, paediatric size and a
large size for obese patients. The most commonly used
fixative is 10% buffered formal saline as used for other
Figure 4: Prussian blue Staining of BM Fragments surgical biopsies. A preferred fixative is Acetic Acid–
13. Cytochemistry: In cases of leukaemia, further Zinc–Formalin (AZI), which is prepared by dissolving
cytochemical stains may be required to 12.5 g Zinc Chloride, 150 ml concentrated Formalin, 7.5
differentiate between the various FAB types of ml Glacial Acetic Acid and water up to 1000 ml. The
leukaemias. specimen should be left in the fixative for 20-24 hours.
302 | P a g e
Procedure: THE EXAMINATION OF BIOPSY
1. Prepare the trolley and the patient as for bone SECTIONS OF BONE MARROW
marrow aspiration. A Trephine Biopsy may be
obtained at the same sitting as the aspiration. The First, scan the whole section with a scanner objective for
only precaution required is that the insertion site the relative distribution of cellular and fatty marrow. In
of the trephine biopsy needle (in the bone) normal adults, this ratio is 1:2 to 1:1. Then, examine for
should be slightly away from the site where the gross abnormalities like necrosis, granulomas,
aspiration needle was inserted. The needle, metastasis and lymphoid aggregates. Note any abnormal
however, can be introduced through the same infiltrate and its location. Switch to a x10 objective and
skin incision. note the number and distribution of the megakaryocytes.
2. After penetrating the periosteum & the cortical Also note the relative distribution of various
bone, when the needle is fixed, the stillet is haemopoietic elements. Switch to a x40 objective and
removed and firm, smooth, regular rotating note the morphology of both normal and abnormal
movements are performed with enough pressure to constituent cells. Examine for any parasites or other
further penetrate to a depth of about 1.5 to 2 cm. inclusions in the cells. Then examine the section stained
3. To detach the internal portion of the marrow, with the reticulin stain and note the amount of fibrosis.
clockwise and anti-clockwise movements are In a normal marrow, only a few scattered fine fibres are
performed several times without further seen, whereas in myelofibrosis, interlacing bundles of
penetration. After this, the needle is withdrawn thick fibres are seen. The fibrosis in between can be
with the same rotatory movement. graded from I to III, with the last being grade IV. If
4. The biopsy is dislodged onto a glass slide required, the sections that were stained with the May-
through the end that is opposite to the Grunwald-Giemsa Stain should be examined. These are
penetrating end, with the help of a stillet, to ideal for differentiating between megaloblasts and other
avoid a crushing effect. blasts as well as for identifying intracellular and
5. The cylindrical biopsy is gently rubbed against extracellular parasites.
the glass slide, with the help of another glass
slide, to make impression smears.
6. It is then put in a specimen bottle that contains
the fixative.
PROCESSING AND STAINING A
BONE- MARROW
Fixed bone-marrow biopsies are de-calcified,
dehydrated and impregnated with wax-like other
histopathology specimens. Then sections are cut and
stained as for other tissues. Figure 5: BM Trephine biopsy

Staining Requirements REPORTING BONE-MARROW

1. For bone marrow aspirates. Giemsa stain and TREPHINE BIOPSY SECTIONS
Leishman stains are used. First, the gross appearance and size of the biopsy
2. For trephine biopsy Haematoxylin-Eosin (H&E) specimen is reported. Trephine biopsy length should be
is used. > 2cm. All details should be clearly mentioned as shown
3. Reticulin stain is used for reticulin slides. in the proforma. Any abnormalities noted should be
4. Immunohistochemistry can be done on trephine highlighted. Finally, give the most likely diagnosis,
biopsy by using CD20, CD3, CD10, CD15, followed by suggestions regarding further
CD30 & others as recommended. investigations.
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41. BLOOD CELL CYTOCHEMISTRY

Cytochemical techniques can be applied to both red prepared directly and not from blood that
blood cells and white blood cells to demonstrate contains any anti-coagulant in it.
various chemical constituents in the cells. These
A positive control must be included with each batch
techniques are extremely useful in the diagnosis of
of a patient’s slides.
various haematological disorders. Their main use
however lies in the study of immature white cells LEUCOCYTE/ NEUTROPHIL
(blasts) to classify various types of leukaemias and in ALKALINE PHOSPHATASE
identifying maturation abnormalities in the
myelodysplastic syndromes and myeloproliferative (LAP/NAP)
disorders. Cytochemical techniques are applicable for LAP activity is found predominantly in mature
use with the Light Microscopic and the Electron neutrophils, with some activity in metamyelocytes
Microscopic. Some techniques are only available and in the reticulum cells of bone marrow. It is
with the use of an Electron Microscope, e.g. platelet associated with distinct tubular structures in the
peroxidase (PPO) activity in megakaryoblasts. cytoplasm. It is allowed to react upon a conjugated
substrate, Naphthol AS Phosphate, which produces
RED BLOOD CELL an insoluble coloured compound localised to the site
CYTOCHEMISTRY of the enzyme’s activity. There are several methods,
Cytochemical techniques are applied to both but the method described by Rutenberg et al gives
developing and mature erythroid cells to best results. However, it is seldom feasible to procure
demonstrate: and use individual reagents because of a limited
1. Iron-incorporation defects: Perl’s Reaction for workload. The reagents are available in kit form by
siderotic granules or haemosiderin several manufacturers. It is advisable to use these
2. Haemoglobin defects: HbF, HbH inclusions, kits. The method given in the literature (enclosed in
Heinz Bodies, etc. the kit) should be followed. With each test or batch of
3. Enzyme defects: Demonstration of G6PD tests, a positive control slide (prepared from the
deficiency blood obtained from a neonate or a patient with acute
These methods are described in detail in the relevant infection) must be stained. Blood films should be
chapters. made soon after the blood is collected, preferably
within 30 minutes, as LAP activity decreases rapidly
WHITE BLOOD CELL CYTOCHEMISTRY in EDTA, anti-coagulated blood.
The main use of cytochemistry in haematology
involves the leucocytes. It is used: Result: Discrete, bright blue granules represent the
sites of LAP activity.
1. to differentiate between normal and abnormal
neutrophils {Leucocyte/ Neutrophil Alkaline Scoring: The LAP/NAP activity is represented as a
Phosphatase (LAP/NAP)} in order to score in absolute numbers. The scoring of the activity
differentiate between a leukemoid reaction and a is graded as under:
myeloproliferative disorder.
2. To study any enzyme abnormalities of the 0 No granules at all
leucocytes 1 Very few granules
2 Few to moderately high number
3. To characterise cells in Lymphoproliferative of granules
Disorders {Acid Phosphatase (ACP), Tartrate- 3 Moderately high to numerous
Resistant Acid Phosphatase (TRAP)}. granules
4. To study patterns of differentiation of early 4 Cytoplasm packed with granules
granulocytic and monocytic cells
{Myeloperoxidase (MPO), Esterases, etc.} For scoring the activity, 100 consecutive mature
neutrophils are graded for activity under the high-
General precautions and instructions, applicable to all power/oil immersion lens of a microscope. The score
cytochemical staining procedures, are as under: is the sum of individual scores of 100 neutrophils.
Note: blood films should be made soon after blood
1. Top-quality reagents should be used. collection as NAP activity decreases rapidly in
EDTA, anti-coagulated blood.
2. All glassware that is used must be washed with
detergent and then thoroughly rinsed with ample
Reference Ranges:
water.
In neonates 150-300
3. Blood or bone marrow smears should be In children and adults 35-100
304 | P a g e
Significance: dropping bottles for 2-4 days. Add additional Sodium
1. High scores are found in: Nitroprusside if the staining becomes less distinct.
• As physiological in newborns, children and Solution-II to be prepared fresh each time by adding
pregnant females 4 drops of 3% analytical-grade Hydrogen Peroxide to
• Leukemoid reactions 25 ml of distilled water.
• Infections Procedure:
• Cirrhosis of the liver
1. Cut filter paper to a size about half an inch
• Polycythemia Vera
longer than the size of the slide and place it over
• Down’s Syndrome the smear.
• Active Hodgkin’s Disease
2. Drop Solution-I onto the filter paper until it is
• Blast transformation in CGL
just wet (approximately 8-10 drops). Let it stand
• Aplastic Anaemia
for one-half to one minute.
2. Low scores are found in: 3. Flood the slide with Solution-II. Gently blow to
• CGL mix the two solutions. Let it stand for one-half to
• PNH one minute.
4. Peel off the filter paper. The smear should be of
definite red colour. To remove any excess of
stain, hold the slide with forceps and wash in
running water.
5. Counter-stain with 1:10 diluted Giemsa Stain for
40 minutes.
6. Wash with tap water, dry and mount.
Result: Sites with enzyme activity will stain pink to red.

FIGURE 1: LAP score in leukemoid reaction Significance: The activity is seen with increasing
strength in all cells of the granulocytic series except
MYELOPEROXIDASE (MPO, POX)
very early myeloblasts, which may be negative.
Myeloperoxidase is an enzyme present in the Eosinophil granules stain strongly. Promonocytes and
azurophilic lysosomal granules of granulocytes and monocytes also show activity, whereas monoblasts
their precursor, in eosinophil granules and in and all stages of lymphoid cells are negative.
monocytes. In neutrophils, these granules are larger
and appear first, i.e. in the blast stage. In monocytes, SUDAN BLACK B (SBB) STAINING
these are small and appear late. It is also present in This is a lipophilic dye that binds irreversibly to an
the specific granules of eosinophils and basophils. unidentified granular component, most probably the
The enzyme acts upon Benzidine in the presence of phospholipid membrane of granules in granulocytes,
Hydrogen Peroxide to yield a coloured product that is
localised to the site of the enzyme activity. As
Benzidine is a carcinogenic substance, alternate
substrates may also be used. The substrate of choice
is 3,3’-Diamin Benzidine (DAB). Kits utilising this
substrate are commercially available and are
recommended for laboratories which have a large
workload. For smaller workloads, methods based on
Benzidine are cheap and easy to carry out. This Figure 2: Sudan Black positive in AML blasts
method is described below in detail:
eosinophils and some monocytes containing MPO
Reagents:
activity either directly or through an enzyme-linked
Solution-I
reaction. The reaction parallels the MPO activity in
Benzidine base 2.0 g
various cells. Being simpler than the MPO method, it
Basic fuchsin 1.2 g
is preferred by most laboratories. The FAB group that
Sodium nitroprusside (saturated solution) 4 ml
classifies leukaemias recommends it. The Sheehan
Ethyl alcohol (95 percent) 400 ml
and Storey Method has remained undisputed and is
Grind 2.0 g of Benzidine base in a mortar with a
described below:
small amount of Ethyl Alcohol. Add the rest of the
alcohol, mixing well in the mortar. Filter this solution Reagents:
into a bottle. To the filtered solution, add basic 1. Fixative: 40% Formaldehyde
Fuchsin and 3 ml of a saturated solution of Sodium 2. Solution A (Stain): prepared by dissolving 0.3g
Nitroprusside. Keep at room temperature in dark of Sudan Black-B in 100 ml of absolute Ethyl
305 | P a g e
Alcohol. The mixture is frequently shaken substrate (it is a commonly-used substrate), then the
vigorously for 1-2 days to dissolve all of the dye ACP activity is revealed by bright red granules.
and then it is filtered. Otherwise, the results are indicated in the method
3. Solution B (Buffer): 16 mg of pure Phenol sheet that is provided by the manufacturer.
Crystals are dissolved in 30 ml of absolute Ethyl
Significance:
Alcohol. Add it to 100 ml of 0.3% solution of
Granulocytes are strongly positive. In bone marrow,
Disodium Hydrogen Phosphate in distilled water.
the macrophages, plasma cells and megakaryocytes
Stir vigorously, to dissolve the phenol and filter.
are strongly positive. Monoblasts react more strongly
4. Sudan Black-B Staining Solution: 30 ml of
than Myeloblasts. T-lymphocytes of all stages show
Solution A is mixed with 20 ml of Solution B
ACP activity. In T-ALL, the reaction is localised to
and filtered through a double layer of filter
an area corresponding to the Golgi Zone (polar). The
paper. The mixture should be neutral or slightly
reaction is also positive in T-CLL, but not so
alkaline.
consistently. About two-thirds of cases of T-PLL also
5. Counter Stain: Giemsa Stain Stock Solution (as
show activity. In all of these, the reaction is inhibited
for staining a thick film for malarial parasites) is
by prior treatment with Tartrate. In Hairy-Cell
diluted 1/50 with distilled water.
Leukaemia, the reaction is not inhibited by Tartrate
Procedure: and, hence, is called Tartrate Resistant Acid
1. Fix air-dried smears in formalin vapour for 10 Phosphatase (TRAP). Some B-Prolymphocytes may
minutes. This is done by exposing smears to pure also show a weak positive reaction, which may also
formalin in a jar so that the formalin does not be resistant to tartrate.
come in contact with the smear.
2. Immerse the slides for I hour in the SBB PERIODIC ACID-SCHIFF REACTION
Staining Solution. (PAS)
3. Transfer the slides to a staining rack and
Glycogen is the stored energy source for several cells
immediately flood with 70% Alcohol. After 30
in the body. It is present in almost all cells of
seconds, tip the alcohol off and flood it again
haemopoietic tissue. However, its quantity and
with 70% Alcohol for another 30 seconds.
distribution inside various haemopoietic cells is
Repeat this three times.
different. These differences are utilised to
4. Counter-stain with diluted Giemsa Stain for 40
differentiate between various types of cells. Glycogen
minutes.
is a carbohydrate and reacts positively in a PAS
5. Wash, air dry and mount.
Reaction. It is differentiated from other carbohydrates
by the fact that when treated with Diastase, the
Result: the granules stain grey to black.
reaction becomes negative. In this reaction, the
Interpretation: As for MPO. The only notable
carbohydrate is liberated from the protein and is
difference is in the eosinophil granules, which have a
oxidised to the Aldehyde by the Schiff Reagent.
clear core when stained with SBB.
These are pink colour in subsequent reactions.
ACID PHOSPHATASE (ACP) STAINING Reagents:
Activity of the ACP enzyme is present in almost all More than 10 different chemicals are required to
haemopoietic cells. However, these cells differ in prepare the reagents in the laboratory. Some of these
quantity and distribution of this hydrolase in the cell. are very expensive and may not be easily available.
These differences are utilised in the differential For low-workload laboratories, therefore, the in-
diagnosis of malignant disorders of haemopoietic house preparation of these reagents may not be cost-
cells. Like other enzymes, its activity is also effective. All of these reagents are available
demonstrated by the conversion of a colourless commercially in the form of kits. It is advisable to
substrate to a stable, coloured compound that is procure the kit and follow the procedure that is
visible under a Light Microscope. recommended by the manufacturer.

Reagents and Procedure: Result:

At least 9 different chemicals are required to prepare Glycogen stains pink to bright red in an untreated smear,
the reagents in the laboratory. Some of these are very but this reaction disappears in a diastase-treated smear.
expensive and may not be easily available. For low- Other PAS-positive materials give positive reactions in
workload laboratories, therefore, the in-house both treated and untreated smears.
preparation of these reagents may not be cost- Interpretation:
effective. All of these reagents are available The cytoplasmic positivity may be diffuse or
commercially in the form of kits. It is advisable to granular. A diffuse positive reaction with few
procure the kit and follow the procedure that is granules is seen in myeloblasts and monoblasts. A
recommended by the manufacturer. negative reaction is seen in normal erythroblasts.
Result: Neutrophils react most strongly, whereas specific
If the kit utilises Naphthol-AS-BI Phosphate as the granules of eosinophils are negative with diffuse
306 | P a g e
cytoplasmic positivity. Megakaryocytic Cells and 1. The differentiation of M1 from M5
platelets are positive. In the common type of 2. The diagnosis of M6 and M7, in which the blasts
‘Childhood ALL’ (C-ALL), blasts may contain give positive reaction that is localised to the
blocks of PAS-positive material. The cells of chronic Golgi area. The reaction is sensitive to Fluoride.
B-lymphoproliferative disorders often have an 3. The diagnosis of T-ALL. The localised reaction
increased number of positive granules. Erythroblasts, is resistant to Fluoride.
in almost all diseased states, stain diffuse pink, 4. To differentiate between T-PLL and B-PLL.
whereas in AML-M6, there may be large blocks of Table 1: Differential Staining Characteristics in Acute
PAS-positive material in the cytoplasm. Myeloid (Non-Lymphoblastic) Leukaemia
REACTION M1 M2 M3 M4 M5 M6 M7
POX + to ++ ++ +++ + to ++ -/+ + -
ESTERASES SBB >3% In myeloblast
CAE blasts
These are group of 9 (1-9) hydrolases, best
demonstrated by Naphthol AS-D Chloroacetate as a ANAE - +/- - to + + to ++ +++ + Localised ++
NaFl S NaFl S Localised
substrate. These are called specific esterases and are
NaFl S
not inhibited by Sodium Fluoride. The remaining is
NASDA + + ++ + to ++ +++ ++ -
inhibited by Sodium Fluoride and is called non- NaFl S NaFl S NaFl S
specific esterases (NSE). These are identified by the ACP -/+ + + to ++ + to ++ +++ +/- ++
name of the substrate that is used to demonstrate PAS + + ++ + to ++ + to + + to ++
them. All important esterase stains are commercially Diffuse DiffuseDiffuse ++
available in the form of kits.
Table 2: Differential Staining Characteristics in Acute
Chloroacetate Esterase (CAE) Lymphoblastic Leukaemia
This is a specific esterase present in granulocytes and REACTION EARLY B- C-ALL T-ALL B-ALL
mast cells. The cytoplasmic CAE activity appears as ALL
myeloblasts mature to promyelocytes. Promyelocytes POX/SBB - - - -
PAS - to ++ + to ++ -/+ -
and myelocytes stain strongly. The enzyme is Coarse Coarse granular
optimally active at pH 7.0-7.6 and it is not inhibited granular
ACP -/+ -/+ ++ to +++ -
by Sodium Fluoride. It parallels that of MPO or SBB. ANAE -/+ -/+ ++ -
However, it is usually negative in monoblasts. It is Localised
used in combination with ANAE in demonstrating NaFl R
OIL RED O - - - +
monocytic and granulocytic precursors in the same
preparation.
Bibliography:
α Naphthol Acetate Esterase (ANAE) 1. Dacie and Lewis. Practical Haematology 11th
The reaction produced is diffuse red or brown in Edition. S Mitchell Lewis, Barbra J Bain, Imelda
colour. This hydrolase gives a distinct positive Bates eds. Churchill Livingstone London 2013
reaction in normal and leukaemic monocytic cells 2. Wintrobe’s Clinical Haematology 12th edition.
and T-lineage lymphoid cells. In monocytes, the John P. Greer, John Forester, Gerge M. Rogers
reaction is diffuse and is sensitive to Sodium ,Friox Paraskevas, Bertil Glader, Danial A
Fluoride, whereas in T-lymphoid cells it is localised Arber, Robert T. Means, Jr. Wolters Kluver,
as a dot and is resistant to Sodium Fluoride. Lippincott Williams & Wilkins London 2009
Megakaryocytes stain strongly and leukaemic 3. Post Graduate Haematology 7th Edition . A.
megakaryocytes may show focal and diffuse Victor Hoffbrand, Daniel Catovsky, Edward G.
positivity. Leukaemic erythroblasts may show focal D. Tuddenham eds. Blackwell Publishing
or diffuse positivity. Its value lies in: London 2016
307 | P a g e

42. HAEMOGLOBIN DISORDERS

Haemoglobin is the oxygen-carrying pigment of the red synthesis of α globin chains.
blood cells. It is a conjugated protein composed of four a. α-thalassaemia silent carrier state-(-α/ αα)
sub-units. Each sub-unit is composed of a globin chain b. α-thalassaemia trait-(-α/-α or --/αα)
and a haem group. Each haem group has a single iron c. HbH disease (--/-α)
atom in the form of ferrous ion. When red blood cells d. Hb Barts (hydrops foetalis syndrome)-(--/--)
pass through the lungs, they take up oxygen from the air, 2. β-thalassaemias: There is deficient or no
which combines with the ferrous iron of the haem. This synthesis of β globin chains.
reaction is not that of oxidation, but is of oxygenation i.e., a. β-thalassaemia trait – (β+/o/ β)
the ferrous form of iron is not converted to the ferric b. β-thalassaemia major – (β+/o/ β+/o)
form. Since there are four haem groups in one molecule c. Thalassaemia intermedia – (variable)
of haemoglobin, it can combine with four oxygen 3. δβ-thalassaemia: There is deficient synthesis of
molecules. There are various types of haemoglobins that both δ and β globin chains.
differ from each other with respect to the structure of their
globin chains. The haem moiety is identical in all types of QUALITATIVE OR STRUCTURAL
haemoglobins. The α-globin chain consists of 141 amino DISORDERS OF THE HAEMOGLOBIN
acids, whereas, the β-chain is composed of 146 amino
Structural disorders are further classified on the basis
acids. Normal adult blood contains three types of Hb
of the physical and chemical properties of an
major component is Hb A with molecular structure α2 abnormal Hb molecule into:
β2. Minor Hb contains γ (HbF) or δ (Hb A2) globin 1. Haemoglobins with altered solubility (HbS, C
chains instead of β chain. Foetal haemoglobin (HbF) is etc.)
the predominant haemoglobin in the intrauterine life. At 2. Unstable haemoglobins
birth 90% Hb is HbF. After birth HbF starts decreasing 3. Haemoglobins with altered oxygen affinity
and is replaced with HbA and HbA2. When the infant is 4. Thalassaemic structural variants (Hb Lepore,
six months of age, it is about 5%. The adult level of 1% is HbE, Hb Constant Spring)
reached at the end of the first year of life. In adults, MISCELLANEOUS HAEMOGLOBIN
haemoglobin consists of 97% HbA and 3% HbA2. ABNORMALITIES
THE CLASSIFICATION OF Some haemoglobins are neither structurally nor
HAEMOGLOBIN DISORDERS functionally abnormal and have little clinical
significance or implication. An example is Hereditary
These are broadly classified into quantitative and
Persistence of Foetal Haemoglobin (HPFH) where
qualitative disorders, as described below:
HbF persists into adult life.
1. Quantitative Disorders - In these there is
reduced synthesis of a structurally normal globin INVESTIGATIONS OF
chain. These are called Thalassaemias and are HAEMOGLOBIN DISORDERS
named after the deficient globin chain. For The following plan of investigations is suggested in
example in β-Thalassaemia there is a reduced clinically suspected cases of haemoglobin disorders:
synthesis of the β globin chains. 1. Basic Tests -
2. Qualitative Disorders: In this category, the a. Full blood count
globin chain being synthesised is structurally b. Red cell morphology
abnormal. This is due to the substitution of one c. Reticulocyte count
or more normal amino acids in any of the globin 2. First-line Identification Tests -
chains with different amino acids. In Sickle-Cell a. Hb electrophoresis on cellulose acetate
Anaemia, Valine substitutes Glutamic Acid at membrane
the sixth position of the β chain. 3. Second-line Identification Tests (based on the
results of Hb electrophoresis on cellulose acetate
QUANTITATIVE DISORDERS OF membrane) -
HAEMOGLOBIN SYNTHESIS a. Estimation of HbA2
(THALASSAEMIA) b. Estimation of HbF
c. Test for Sickling
Thalassaemias are inherited, quantitative disorders of 4. Other Tests -
globin-chain synthesis. These are classified on the a. PCR, for identifying mutations
basis of deficient or absent synthesis of the chains b. Electrophoresis on other media like Agar
involved. The following are the main types of Gel, Starch Gel, etc. in various buffers
Thalassaemias: c. Tests for an unstable Hb
1. α-thalassaemias: There is deficient or absent d. Tests for Methaemoglobin
308 | P a g e
e. Tests for altered-affinity
affinity haemoglobins buffer for routine haemoglobin studies and is
f. Isoelectric focusing prepared as under:
g. Estimation of the rate of globin--chain synthesis Boric acid 6.4 g
Only some of these tests are performed in a routine Tris aminomethane 5.1 g
laboratory. Disodium EDTA 0.3 g
Water to make 1 litre
HAEMOGLOBIN LECTROPH
LECTROPHORESIS
Cellulose Acetate Membrane is used for the initial 2. Tris-EDTA-Borate Buffer pHp 8.9: This is the
haemoglobin electrophoresis. It is a smooth, buffer for HbA2 estimation and is prepared as
homogeneous and strong medium on which under:
separation of the different types of haemoglobins is Tris aminomethane 14.4 g
excellent. For more precise results, Polyacrylamide Disodium EDTA 1.56 g
Gel, Starch Gel and Agar Gel are used. Before Boric acid 0.92 g
proceeding for haemoglobin electrophoresis it is Water to make 1 litre
necessary to prepare the haemolysate, i.
i.e. to break the
red cells so that haemoglobin is released from them. 3. Electrophoresis apparatus
4. Cellulose acetate membrane strips
5. Trichloracetic Acid 3%
6. Ponceau S Stain 0.2%
7. Acetic Acid 5%
8. Staining Trays
9. Scissors
Procedure:
Follow the procedure given in the section on
‘ELECTROPHORESIS’ and apply the following
modifications:
1. Apply the lysate near the cathode bridge towards
the right of the base line using a capillary tube.
2. Run at 200 V for 30-45 minutes.
Fig: 1 Cellulose acetate membrane electrophoresis. 3. Cellulose acetate strip wil then be immersed in
3% trichloroacetic acid for few seconds followed
Preparation of the Haemolysate: by immersion in 0.2% Ponceus stain for 15 min.
1. Any anticoagulant may be used, but EDTA is 4. After staining the strip, dry it between two layers
suitable for this purpose. of filter paper and then in an incubator at 37°C.
2. About 2 ml of anti-coagulated blood is taken and 5. It is essential to run normal and positive controls
three washings are given with Isotonic Saline. with each batch, for comparison.
This is done by adding normal saline 4 times of
blood volume, mixing, centrifuging and Result:
decanting the supernatant. The relative electrophor-
3. After the final wash, add a half volume of etic mobility of different
distilled water to the packed cells lls that are left haemoglobins is shown
behind and shake. This will cause the haemolysis in the figure.
of the cells.
ESTIMATION OF
4. Add an equal volume of Carbon Tetrachloride
and mix well. HbA2 Figure 2: Cellulose acetate strip
5. Centrifuge the mixture at about 3000 3000-rPM for 15 HbA2 can be estimated chromatographically using
minutes. The clear red lysate is then pipetted off columns or by electrophoresis. HbA2 columns are
into another test tube. available in kit form. This is a more accurate method but
6. The lysate can be stored at 4°C (if not immediately is expensive, unless the columns are prepared in-house.
in
used) and can be transported to another laboratory
(on ice), if facilities are ot available. Estimation of Hb A2 by Elution from cellulose
7. The Hb in the lysate should be about 10 g/dl. If it acetate
is more than that, add distilled water to adjust to Principle: Haemolysate is separated in to component
the
he required haemoglobin concentration. fractions by alkaline electrophoresis on cellulose
Cellulose Acetate Membrane Electrophoresis acetate membrane. The relative proportion of
Various buffers can be used for haemoglobin separated fraction is quantitated by spectrometry of
electrophoresis at different pH
H levels, using cellulose eluates of separated fractions.
acetate membrane strips. Requirements:
Requirements: 1. An electrophoresed strip of Hb
1. Tris-EDTA-Borate Buffer pH 7.9
7.9: This is the 2. Tris EDTA Borate Buffer of pHp 8.9
309 | P a g e
3. Test tubes anti-coagulated blood.
4. Pipettes 2. Fix the slides in 80 % Ethanol for 5 minutes.
5. a Spectrophotometer 3. Remove the slides from the fixative and wash in
running tap water.
Procedure:
4. Air-dry the slides.
1. Set up 3 tubes marked A, A2 and Blank (B).
5. Place the slides in the Elution Solution for 20
2. Put 2ml buffer in Tube A and 4 ml in each of
seconds. The pH level and the time are
Tubes A2 and B.
absolutely critical.
3. Cleanly cut the portions of the strip that bear the
6. Rinse immediately in tap water.
HbA and HbA2 bands.
7. Air-dry once again.
4. Place the cut portions of the strip of HbA in
8. Counter-stain with aqueous Eosin for 5 minutes.
Tube A, that of HbA2 in Tube A2 and a piece of
9. Dry the slides after rinsing in tap water.
clear strip in Tube B.
10. Examine under an oil immersion lens.
5. Allow them to elute for 30 minutes.
6. Read the absorbance of Tubes A and A2 against Results:
B in a spectrophotometer at 416 nm. The cells that contain HbF will stain pink, whereas
the cells that contain HbA will appear as clear ‘ghost’
Calculation:
cells.
Abs HbA2
% HbA2= × 100
Abs HbA2 + (Abs HbA × 5) SEMI-QUANTITATIVE ESTIMATION
Reference Range: 1.5-3.5% OF FOETAL BLOOD IN THE
MATERNAL CIRCULATION
Interpretation:
A HbA2 of >3.5% is diagnostic of the β Thalassaemia Kleihauer’s Test can also be used to roughly find
trait. out the volume of foetal blood that is entering the
maternal circulation. This is important because, in
ESTIMATION OF HbF feto-maternal incompatibility, the dose of anti-Rh D
HbF can be estimated qualitatively by staining in situ. immunoglobulin that is given to the mother depends
This is done by the Acid Elution Technique. upon the quantity of blood that has entered the
Otherwise, it is estimated quantitatively by the Alkali maternal circulation. If the loss is less than 4 ml, then
De-naturation Method. Both of these procedures are the usual dose of 100 µg is enough to prevent the
hereby described. mother from sensitisation. The procedure is as
follows:
ACID ELUTION METHOD 1. Prepare thin, uniform smears of maternal blood.
(KLEIHAUER’S TEST) The cells should be separate and uniformly
spread.
Principle 2. Stain as detailed above.
The test is based on the principle that HbF resists 3. Focus the stained film under low power.
acid elution to a greater extent than HbA. It is 4. Count the number of foetal cells (darkly
performed on smears of blood made on a glass slide. staining) per low-power field and also count the
Cells that contain HbA are cleared of their number of adult red cells (ghost cells).
haemoglobin, whereas cells which contain HbF retain 5. The volume (ml) of foetal red cells in the
their haemoglobin and hence, stain pink. maternal circulation can be calculated by the
Requirements: following formula:
1. Fixative: 80 % Ethyl Alcohol 2000× Foetalredcells×1.33
2. Elution Solution - Adultredcells
a. Solution A: Dissolve 7.5 g Haematoxylin in Where 2000 is the approximate maternal red
one litre of 90 % Ethanol. cells in 1 ml blood, 1.33 is the correction factor
b. Solution B: Dissolve 24 g Ferric Chloride in (because all the foetal red cells do not retain their
20 ml of 2.5 mol/L HCl and make the haemoglobin after acid elution). If, however,
volume to one litre with distilled water. there are less than 10 foetal red cells in 5 low-
c. Working Solution: Mix 5 volumes of power fields, then it can be safely assumed that
Solution A and 1 volume of Solution B. The less than 4 ml of foetal blood has crossed the
pH should be 1.5. The solution is stored at placental barrier.
4°C. It is important to filter the solution
before use, otherwise a deposit will be left ESTIMATION OF HbF - BETKE’S
on the stained slides. METHOD
3. Counter Stain: Dissolve 2.5 g Eosin in one litre Principle
of distilled water. HbF is more resistant than HbA to de-naturation by
Procedure: an alkaline solution of NaOH. This method detects
1. Prepare peripheral blood smears from EDTA HbF in the range of 0.5-50%.
310 | P a g e
Reagents: water) at 540 nm.
1. Haemolysate
Calculation:
2. Saturated Ammonium Sulphate Solution
3. Sodium Hydroxide 1.2 mol/L Abs Test
Hb F% = × 100
4. Drabkin's Solution Abs Std
5. Pipettes Reference Range:
6. Test Tubes and a Test Tube Stand
7. Filter paper After one year of age <1%
8. A Spectrophotometer
Procedure: HIGH PERFORMANCE LIQUID
1. Prepare a Cyanmethaemoglobin (HiCN) Solution CHROMATOGRAPHY (HPLC)
by adding 0.2 ml of haemolysate to 4 ml HPLC has following advantages:-
Drabkin's Solution. 1. Analyses are automated and thus utilize less staff
2. Take two test tubes and label them as ‘test’ and time and permit processing of large batches.
‘standard’. Place these in the stand. 2. Very small samples (5 µL) are sufficient for
3. In the test tube marked ‘test’, add: analysis.
a. Hi CN 2.8 ml 3. Quantification of normal/variant Hb is available
b. Na OH 0.2 ml for every sample.
(Wait for 2 minutes) 4. Provisional identification of large proportion of
c. Saturated Ammonium Sulphate 2.0 ml variant Hb can be made.
4. Mix and wait for 10 minutes.
5. After mixing thoroughly, filter the solution. Principle: HPLC depends on the interchange of
6. Make a 25% solution of standard by adding 0.7 charged groups on ion exchange material with
ml of the HiCN Solution to 4.3 ml of Drabkin's charged groups on Hb molecule. A typical Column
Solution in the test tube marked ‘standard’. packing is 5m spherical silica gel. The surface of
7. Read the absorbance of both (at 540 nm) against support is modified by carboxyl group to have
the distilled water. weakly cationic charge which allows the separation
of Hb molecules with different charges by ion
Calculation: exchange.
Abs test
% HbF = × 25 Interpretation & Comments:
Abs Std
1. Accurate results but as with every method of
ESTIMATION OF HbF - SINGER’S
hemoglobin analysis control should be run
METHOD
with every batch.
Principle
2. It should be noted that Hb A is separated into its
HbF is more resistant than HbA to de-naturation by
component fraction of Hb Ao and Hb A1. A1
an alkaline solution of NaOH. This method detects
fraction frequently subdivide in several peaks.
HbF over 50% as well.
3. HPLC usually separats Hb A, A2 F, S, C, D & G
Reagents: peaks.
1. Haemolysate 4. Retention time of glycosylated and other
2. Sodium Hydroxide 1.2 mol/L derivative of Hb S can be same as of Hb Ao and
3. Acidified 50% saturated ammonium sulphate A2.
(50% of saturated ammonium sulphate 800 ml, 5. HPLC is also applicable for quantification of Hb
10N HCl 2 ml) A1c for monitoring of diabetes mellitus.
4. Ammonia 0.04% V/V
DEMONSTRATION OF HbH
Procedure: INCLUSIONS
1. Take two test tubes and mark them as 'test’ and
‘standard’. Haemoglobin H (β4) is formed in the red cells of
2. To the tube marked ‘test’, add 3.2 ml of lysate patients with α-thalassaemia. It should be suspected
and 0.2 ml Sodium Hydroxide. when a patient has red cell indices suggestive of
3. Shake the mixture vigorously and start a thalassaemia, namely a low MCV, MCH and a high
stopwatch. red cell count, but does not have a raised HbA2 or
4. After exactly one minute, add 6.6 ml of acidified HbF and is not iron- deficient.
50% saturated Ammonium Sulphate.
Principle
5. Shake vigorously and filter.
Red cells that contain HbH, when exposed to supra
6. To the tube marked ‘standard’ add 0.2 ml of
vital stains (e.g., Brilliant Cresyl Blue as in the
original haemolysate and 4.8 ml of 0.04% (v/v)
reticulocyte preparations), form multiple blue-green
Ammonia Solution.
dots inside the red cells, giving a pitted, ‘golf ball’
7. Read the absorbance of both (against distilled
appearance.
311 | P a g e
Requirements: solution.
1. Brilliant Cresyl Blue 10 g/L in Citrate Saline.
Procedure:
2. Glass slides, Pasteur pipettes
1. Take 2 ml of the working solution and add 4
3. Test tubes
drops of EDTA anti-coagulated, whole blood to
4. Glass slides
it. Mix thoroughly.
5. A Microscope
2. Centrifuge at 1200 g for 5 minutes.
Procedure: 3. Remove the tube and note the appearance of the
1. Mix equal volumes of Brilliant Cresyl Blue solution.
Solution and EDTA anti-coagulated blood.
Interpretations:
2. Incubate at 37°C for 2 hours (better in a water
HbA is soluble in concentrated Phosphate Buffer,
bath).
hence it gives a uniform red colour without any
3. Make the films and allow them to dry.
precipitate. If there is no HbA, and the whole of the
4. See under an oil-immersion lens for typical HbH
Hb is HbS, then only a red precipitate will be formed,
inclusions. HbH precipitates as multiple pale-
whereas the rest of the fluid will be clear. In cases of
staining greenish blue, almost spherical bodies of
the sickle-cell trait, both a homogeneous red solution
varying sizes. They can be clearly differentiated
of HbA as well as a precipitate of HbS will be seen.
from the darker-staining, reticulo-filamentous
material of reticulocytes. They typically have a THE SICKLING TEST
‘golf ball’ appearance.
Principle
Precautions: This test is based on the decreased solubility of HbS at
HbH is an unstable Hb, therefore fresh blood should low-oxygen tension. For this purpose, a reducing reagent,
be used for the demonstration of HbH inclusions. e.g. Sodium Dithionite or Sodium Metabisulphite is
Interpretations: added by sealing the blood under a cover slip.
HbH inclusions are diagnostic of α thalassaemia. The Requirements:
number of cells containing HbH inclusions varies 1. Sodium Metabisulphite 2%: Dissolve 2 g in 100
according to the type of α thalassaemia. With an α ml distilled water.
thalassaemia trait, 0.01-1% of the red cells contain 2. Glass slides
inclusions. In HbH disease, at least 10% of the red 3. Cover slips
cells contain the inclusions. 4. Bunsen Burner
5. White petroleum jelly, or wax
DETECTION OF SICKLE 6. Microscope
HAEMOGLOBIN-HBS
Procedure
HbS is found in sickle-cell disease. In this abnormal 1. Add 5 drops of Sodium Metabisulphite to one
Hb, valine is substituted for glutamic acid at the sixth drop of EDTA anti-coagulated blood in a test
position of the β-globin chain. One of the properties tube and mix.
of HbS, which is responsible for the clinical 2. Put one drop from the mixture on a slide.
symptoms, is its conversion into insoluble crystals 3. Place a cover slip over the mixture.
when exposed to low-oxygen tension. Tubular 4. Take some wax or petroleum jelly on an iron rod
filaments are produced and the red cells become and soften it by heating it over the flame of a
sickle- shaped. HbS can be detected by the Bunsen Burner. Apply the jelly on the sides of
Qualitative Solubility Test, the Sickling Test and the cover slip so that no air can enter through it.
haemoglobin electrophoresis. Hb electrophoresis has 5. After sealing it completely, immediately look for
been described earlier, while the other two tests are sickling, again after 1-2 hours and, again, after
described below. 12 hours.
QUALITATIVE SOLUBILITY TEST Interpretations:
Immediate sickling indicates HbS disease. Sickling
Principle
that occurs after 1-2 hours & sometimes after 12
The test is based on the principle that HbS is
hours, is suggestive of the HbS trait.
relatively insoluble in a concentrated phosphate
buffer in the presence of reducing substances. DEMONSTRATION OF HEINZ
Requirements: BODIES
1. Phosphate Buffer, at pH 7.1 Heinz Bodies are precipitated globin chains which
a. Potassium Dihydrogen Phosphate 33.78 g may be seen as red cell inclusions.
b. Dipotassium Hydrogen Phosphate 59.33 g
c. Saponin 2.5g Principle
d. Water 250 ml Heinz Bodies are demonstrated by cytochemical
2. Dissolve 0.1 g of Sodium Metabisulphite in 10 staining but can also be seen as refractile objects in
ml of buffer, prior to use, to make a working unstained preparations by lowering the microscope
312 | P a g e
condenser, by dark-ground illumination or by phase- • Add 5 ml of the buffer and mix.
contrast microscopy. • Centrifuge at 1200 g for 20 minutes and remove
Requirements: the stroma with a pipette.
• Methyl Violet Solution: Dissolve 0.5 g Methyl • Take another set of similarly marked test tubes
Violet in 100 ml 9 g/L NaCl and filter. and transfer 5 ml of treated lysate from each tube
to the corresponding new tube.
• Pipettes
• Place both tubes in the water bath at 50°C for
• Test tubes
one hour. Examine periodically for turbidity and
• Glass Slides
flocculation. If the test sample contains an
• a Microscope unstable haemoglobin, then a precipitate will be
Procedure: seen. The control tube should remain clear.
• Mix 1 drop of EDTA anti-coagulated blood and • If a precipitate forms, then centrifuge the tubes and
4 drops of Methyl Violet Solution in a test tube. transfer 1 ml of the clear supernatant to another tube.
• Allow to stand for 10 minutes at room • Take, in another tube, 1 ml lysate from Step 7
temperature. (unheated).
• Prepare the films and allow them to dry. • Add 19 ml Drabkin's Reagent to both.
• See under an oil-immersion lens. • Read the absorbance of both at 280 nm.
Interpretations: Calculation:
Methyl Violet stains the Heinz Bodies as intense purple Abs sample - Abs heated sample
% Unstable Hb = × 100
inclusions in RBCs. Their size varies from 1-3 µm. One Abs unheated sample
or more may be present in a single cell, usually lying
close to the cell membrane. The presence of Heinz ISOPROPANOL PRECIPITATION
Bodies in the blood is a sign of chemical poisoning, TEST
drug intoxication, G6PD-deficiency or the presence of
an unstable haemoglobin, e.g. Hb Koln. These may also Principle
be produced by the action of some aromatic nitro and When a lysate that contains an unstable haemoglobin
amino compounds (such as inorganic oxidising agents) is incubated in the presence of Isopropanol, the
on the red cells. unstable haemoglobin precipitates while the normal
Hb does not.
HEAT INSTABILITY TEST Requirements:
Principle 1. Isopropanol Buffer:
When the haemolysate is exposed to heat (under • Tris 12.12 g
controlled conditions), unstable haemoglobins • HCl 1 mol/L 42 ml
precipitate while normal Hb does not. • Distilled water 1 L
• Isopropanol 170 ml
Requirements:
1. Tris-HCl Buffer, pH 7.4 (0.05 mol/L): Prepare Tris-HCl 0.1 mol/L Buffer of pH 7.4 by
dissolving Tris and HCl in distilled water,
• Tris 18.17 g
making the volume to one litre. Take 830 ml of
• HCl 1 mol/L 42 ml
this buffer and add to it 170 ml Isopropanol
Dissolve the Tris in about 500 ml distilled water.
(17% v/v). Store at 4°C.
Add HCl and make the volume to one litre with
2. Pipettes
distilled water.
3. Test Tubes with a Test Tube Stand
2. Drabkin’s Reagent
4. Centrifuge
3. Pipettes
4. Test Tubes and a Test Tube Stand Procedure:
5. a Water Bath set at 50°C • Wash ‘test’ RBCs and ‘control’ RBCs three
6. a Centrifuge times in normal saline and pack by centrifugation
7. a Spectrophotometer (as described earlier).
• Take 1 ml of each of the packed cells in two
Procedure:
tubes, marked ‘test’ and ‘control’. Add 1.5 ml
• Take two test tubes and mark as ‘test’ and
distilled water to both and shake vigorously.
‘standard’.
• Centrifuge at 1200 g for 20 minutes and remove
• Wash the red cells of freshly-taken blood (from
the stroma with a pipette.
the patient and a normal control). Wash the cells
3 times in saline and then pack. • Take two tubes marked ‘test’ and ‘control’
• Take 1 ml packed cells of the patient in the test • Place 2 ml pf buffer into each. Place these in a
tube marked ‘test’ and 1 ml of control packed water bath at 37°C, to warm for 5 minutes.
cells in the test tube marked ‘standard’. • Add 0.2 ml of ‘test’ and ‘control’ lysate to the
• Add 5 ml distilled water to both and shake corresponding tubes. Stopper the tubes and mix
vigorously, to lyse. by inversion.
313 | P a g e
• Re-place in the water bath and examine at 5, 20 2. Wintrobe’s Clinical Hematology 12th edition.
and 30 minute-intervals. In a positive test, a John P. Greer, John Forester, Gerge M. Rogers
precipitate will appear in the patient’s sample ,Friox Paraskevas, Bertil Glader, Danial A
tube in 5 minutes and will become flocculant in Arber, Robert T. Means, Jr. Wolters Kluver,
20 minutes. The ‘control’ tube will remain clear. Lippincott Williams & Wilkins London 2009
Bibliography: 3. Post Graduate Haematology 7th Edition . A.
1. Dacie and Lewis. Practical Hematology 11th Victor Hoffbrand, Daniel Catovsky, Edward G.
Edition. S Mitchell Lewis, Barbra J Bain, Imelda D. Tuddenham eds. Blackwell Publishing
Bates eds. Churchill Livingstone London 2013 London 2016
314 | P a g e

43. ENZYMOPATHIES AND MEMBRANE DEFECTS

• Sulphonamides
ENZYMOPATHIES • Nitrofurans
Like other cells of the body, red blood cells also contain • Water-soluble
soluble Vitamin K
a number of enzymes for their metabolic processes. 3. Foods, such as fava beans
However, red blood cells differ from other cells of the
body in that all of the enzymes required throughout their
life are produced before an extrusion of the nucleus and
this decay with the age of the cell, finally results in tthe
death of RBCs. The main metabolic pathways for which
enzymes are required are:
• Anaerobic glycolytic pathway, for energy
production (also called the Embden Meyerhof
Pathway).
• Pentose Phosphate Pathway,, which is aerobic
and is utilised for the maintenance of reduced Fig 1: Hexose monophosphate shunt
glutathione to overcome oxidant stress. There are two types of G-6-PD PD enzymes, B and A.
• the Trios Phosphate Pathway G6PD A is found only in African persons. The
• the Purine Metabolic Pathway abnormal variant of this type is A-,A but it is not very
• Pathway for the degradation of RNA severe. The most common type is G6PD B of which
(pyrimidine) several variants have been described. These may
produce either qualitative or quantitative or a mixed
A number of enzymes are involved in these abnormality of the enzyme which may result in one of
metabolic pathways. Quantitative or qualitative the following four clinical conditions:
defects of these enzymes results in an early death of • Congenital Nonspherocytic Haemolytic Anaemia
RBCs under certain circumstances. These • Neonatal Jaundice
abnormalities are collectively called enzymopathies
enzymopathies. • Favism
The abnormalities of almost all known enzymes are • Acute Intravascular Haemolysis
described, but the majority of these are very rare,
occurring only as one in 10,000 or more individuals. G-6-PD
PD SCREENING TESTS
The most commonly-occurring
occurring enzymopathies of
clinical significance are: Principle
G6PD is released from the lysed erythrocytes
erythroc and
• Glucose-6-phosphate
phosphate Dehydrogenase (G6PD)
Deficiency catalyses the conversion of Glucose-6-Phosphate
Glucose to 6-
Phosphogluconate with a conversion of NADP to
• Pyruvate Kinase (PK) Deficiency
NADPH. The production of NADPH can be detected by:
• Pyrimidine-5-Nucleotidase (P-5ND) 5ND) Deficiency
• its property of fluoresce in UV light
The following paragraphs
agraphs describe the tests for the
• conversion of Hi to Hb.
detection of these enzymopathies.
• De-colourisation
colourisation of a reducible dye
GLUCOSE-6-PHOSPHATE
DYE-REDUCTION
REDUCTION TEST
ENCY
DEHYDROGENASE DEFICIENCY
In this test, NADPH, in the presence of Phenazine
Glucose-6-Phosphate
Phosphate Dehydrogenase (G (G-6-PD) is an Methosulphate (PMS), reduces the blue dye
enzyme which takes part in the hexose hexose- (Dichlorophenol Indophenol) to a colourless form.
monophosphate shunt. It is required for the The rate at which the colour disappears in the
production of NADPH to keep glutathione in a reaction mixture is proportional to the amount of G-
reduced state. The metabolic pathways of red blood 6-PD
PD in the red cells. Reagents are difficult to
cells are shown here. Reduced glutathione is prepare in a routine laboratory. The test is available
important in bearing the brunt of the oxidative stress. in kit form designed for single-test
single use. The
A small amount of met Hb (Hi)’, produced all the procedure may differ for each kit and is provided
time, is converted back
ack to Hb by reduced glutathione accordingly. As such, the procedure and the
(GSH). Increased oxidant stress not only increases Hi instructions
tions given by the manufacturer of the kit
production but also results in the oxidation of many should be strictly adhered to for good results.
other components, particularly the cell membrane.
This results from: METHAEMOGLOBIN-REDUCTION
REDUCTION
1. Infections TEST
2. Drugs -
• Anti-malarials, e.g. chloroquine,
uine, quinine Principle:- Sodium nitrite converts Hb to Hi. When
no methylene blue is edit Hi persists but incubation of
315 | P a g e
sample with Hi allows stimulationation of pentose is suspected by:
phosphate pathway in subjects with normal G6PD • History - Autosomal recessive
levels. Hi is reduced during incubation period. In
G6PD deficient subjects block in pentose phosphate • Chronic Non-
pathway prevents this reduction. spherocytic
Haemolytic
Requirements: Anaemia
• Sodium Nitrite-Glucose Solution -
o Sodium Nitrite 1.25 g • Macrocytosis
o Glucose 5.0 g • Prickle cells in the Fig 2: Role of PK in
o Distilled water to 100 ml peripheral blood film embden-meyerholf
embden
• Methylene Blue - pathway
o Methylene Blue Chloride 3H2O 0.15 g
o Distilled water to 1 L PYRIMIDINE 5-NUCLEOTIDASE
NUCLEOTIDASE
• Test Tubes with a Test Tube Stand DEFICIENCY
• Incubator/water bath
• a Spectrophotometer This enzyme is required for the degradation of RNA
into soluble metabolites which diffuse out of the cell.
Procedure: In its absence, RNA precipitates into a small blue dot
1. It is best to take blood in ACD and use it like deposits causing basophilic stippling. The
immediately. However, if the test is put up enzyme is also inhibited by lead, thus causing
immediately, even EDTA anti-coagulated
coagulated blood can basophilic stippling in lead poisoning. The enzyme
be used. can only be assayed in specialised laboratories.
labora
2. Adjust the PCV to 0.4-0.5, 0.5, removing enough
plasma. MEMBRANOPATHIES
3. Take 3 test tubes and mark as Test (1), Positive
These are the third common cause of congenital
Reference (2) and Normal Reference (3).
haemolytic anaemias. The normal shape of a red blood
4.
cell depends upon it’s structurally and functionally-
functionally
Tube Tube Tube
(1) (2) (3)
normal cell membrane and cytoskeleton.
cytoskelet The cell
Reagent-1 0.1 ml 0.1 ml - membrane is a lipid bi-layer
layer in which certain proteins
Reagent-2 0.1 ml - - are inserted. These proteins are then anchored to the
Test blood 2 ml - - cytoskeleton. The cytoskeleton itself is composed of
Normal blood - 2 ml 2 ml various proteins. Abnormalities in these proteins result
(These tubes can be used fresh or they can be in various structural abnormalities
normalities of RBCs rendering
evaporated until dry and stored at 4°C for six them susceptible to lysis in response to osmotic,
months for future use) temperature and metabolic changes. The important
a. Mix well by inversion. abnormalities are:
5. Incubate at 37°C for 3 hours, without shaking. 1. Hereditary Spherocytosis
Dilute 0.1 ml from each tube with 10 ml distilled 2. Hereditary Elliptocytosis
water and visually compare after 22-10 minutes. 3. Hereditary Stomatocytosis
Interpretation: Normal blood produces colour
similar to that in normal reference tube (i.e clear The common tests used for the detection of these
red).
d). Blood from deficient subjects give brown abnormalities are described below.
color similar to that in deficient reference tube.
MODIFIED OSMOTIC FRAGILITY
Quantitative Estimation of G-6-PD TEST
This is mainly of academic interest and is seldom
required clinically. It is however useful in detecting 1. Make 1% saline stock solution by mixing 10 g
female carriers and deficient patients during or soon NaCl in 1 L DW.
after an episode of acute haemolysis. The kits are 2. Take two sets of clear glass tubes, 11 tubes in
available from various manufacturers and the each set. Label one set as normal control & other
instructions therein provided must be followed. test as test T.
3. Make saline dilutions using above 1% saline
PYRUVATE KINASE (PK) solution as follows.
DEFICIENCY 4. Add 50µl of patient blood to each tube of set
labeled T
This is an enzyme of the Embden-Meyerhof
Meyerhof Path
Pathway 5. Add 50µl of Normal blood to each tube of set
and its deficiency is the second most common after labeled NC
G-6-PD
PD deficiency. There is no screening test 6. Leave suspension for 30 min at room
available. The enzyme temperature. Mix again & then centrifuge
cent for 5
can be assayed in reference laboratories. Deficiency min at 1200g.
 316 | P a g e
7. Visually observe hemolysis in both sets and the refrigerator). Centrifuge 1 ml blood and save
compare. the serum.
DW in Saline in Tube label 5. Into the tube marked ‘G,’ add 50 µl of Glucose
tube tube Solution.
1. 5ml 0 ml 0% saline 6. Incubate all 4 tubes at 37°C for 48 hours, mixing
2. 4.5ml 0.5ml 0.1 saline gently after 24 hours.
7. Pool two plain tubes separately and two glucose
3. 4.0ml 1.0ml 0.2% saline
tubes separately.
4. 3.5ml 1.5ml 0.3% saline 8. Mix and determine the PCV & Hb on a portion
5. 3.0 2.0ml 0.4% saline of each.
6. 2.75ml 2.25ml 0.45% saline 9. Dilute a small amount of the saved blood in
7. 2.5ml 2.5 0.5% saline 1/100 in Drabkin’s Reagent.
8. 2.0ml 3.0ml 0.6% saline 10. Centrifuge the remaining blood and separate the
supernatant.
9. 1.5ml 3.5ml 0.7% saline 11. Dilute supernatants from each of the two tubes and
10. 1.0ml 4.0ml 0.8% saline the supernatant from the saved blood 1/10 in
11. 0.5ml 4.5ml 0.9% saline Drabkin’s Reagent. If there is marked haemolysis,
the dilution can be increased up to 1/50.
8. Remove supernatants & estimate amount of lysis in 12. Using the pre-incubation serum from Step 2
each using spectrometer at wavelength setting of dilution as ‘blank’ and the blood tube from Step
540 nm. Use as blank the supernatant for tube 11. 9 as ‘standard’, read all tubes at 625 nm in a
spectrophotometer.
assign value of 100% lysis to the reading
with supernatant of tube 1 (DW only) & Calculation:
express the reading from other tube as % of Rt Do
Lysis % = × × (I - PCVt)× 100
value of tube 1. Plot the results against NaCl Ro Dt
concentration.
Where:
Incubated Osmotic fragility test Ro =Abs of dilute whole blood
Blood samples are first incubated at 37°C for 24 Rt =Abs of dilute serum (after
hours and the test is performed as described above. incubation)
An additional tube of 12 g/L should also be included Do =Dilution of whole blood
in this test. Dt =Dilution of serum
PCVt =Packed cell volume
Reference Ranges:
Non-incubated MCF 4.0-4.45 g/L of NaCl Reference Ranges:
Incubated MCF 4.65-5.9 g/L of NaCl Without glucose 0.2-2.0 %
With glucose 0-0.9 %
THE AUTO-HAEMOLYSIS TEST
PAROXYSMAL NOCTURNAL
Principle HAEMOGLOBINURIA (PNH)
The test provides information about the metabolic
competence of the red cells and helps in It is an acquired clonal disorder in which RBCs are
differentiating between enzyme and membrane abnormally sensitive to the normal constituents of the
defects. Blood is incubated both with and without serum. Characteristically, it presents as
glucose at 37°C for 48 hours and the amount of haemoglobinuria during sleep, in haemosiderinuria
spontaneous haemolysis is measured calorimetrically. and in jaundice. The serum components which are
responsible for lysis, are those of normal
Requirements: complement. The screening tests for this condition
1. Glucose Solution 100 g/L are as follows:
2. Drabkin's Solution
3. Screw-capped Bottles/Tubes with a Stand HEAT-RESISTANCE TEST
4. Pipettes
Allow ‘test’ and ‘control’ samples of blood to clot at
5. a Spectrophotometer
37°C. The test is positive if free haemoglobin starts
Procedure: diffusing into the serum soon after clot formation.
1. It is essential to use a strict aseptic technique to The control serum remains clear.
avoid bacteria-induced haemolysis.
2. Six ml of a de-fibrinated blood sample is SUCROSE LYSIS TEST
required for this test. Principle
3. Put up 4 tubes, two marked Plain (P) and two Red cells absorb complement components at a low
marked Glucose (G). ionic strength (isotonic sucrose solution) and lyse if
4. Into each, put 1 ml blood and save 1 ml (store in the PNH defect is present.
317 | P a g e
Requirements: 5. Put up six tubes as shown in Table 1
1. A fresh solution of sucrose 92.4 g/L 6. Mix the contents carefully.
2. Normal saline 7. Incubate at 37°C for one hour. If the test is
3. Fresh serum collected from a normal, healthy positive, there is only trace haemolysis in Tube
person. 1, while Tube 2 shows +++ haemolysis. All of
4. Normal AB or group-compatible serum the other tubes remain clear.
5. Pipettes For quantitation, prepare:
6. Test Tubes with a Test Tube Stand • a ‘blank’ tube containing 0.5 ml serum.
7. A Centrifuge • a ‘standard’ tube containing 0.05 ml cell
suspension and 0.55 ml distilled water.
Procedure: • Six tubes marked correspondingly
• Wash the patient’s RBCs three times in normal containing 0.3 ml supernatant from each test
saline and pack them by centrifugation (as tube.
described earlier). • Add to each, 5 ml of Drabkin's Reagent.
• Prepare a 50% cell suspension. • Read all of the tubes against the ‘blank’ at
• Put up two tubes, one marked P (Plain) and one 540 nm.
marked S (Sucrose). • Calculate % lyses for each tube by following
• In both tubes, place 0.05 ml normal serum. formula:
• To tube P, add 0.85 ml normal saline. Abs of Test tube
Lysis % = × 100
• To tube G, add 0.85 ml sucrose solution. Abs of Std tube
• To both, add 0.1 ml cell suspension.
Result
• Incubate at 37°C for 30 minutes.
Normal result shows not more than 2% haemolysis.
• Centrifuge the tubes.
PNH shows 10-50 % haemolysis.
Results:
Table 1: Procedure of Acidified Serum Lysis
Increased lysis in the ‘sucrose’ tube as compared to
(HAM’s) Test
the ‘saline tube’ is a positive result.
Tube→
→ 1 2 3 4 5 6
Fresh normal serum (ml) 0.5 0.5 - 0.5 0.5 -
HAM’S TEST (ACIDIFIED SERUM Inactivated normal serum (ml) - - 0.5 - - 0.5
LYSIS TEST) HCl 0.2 mol (ml) - 0.05 0.05 - 0.05 0.05

Principle Patient RBC (ml) 0.05 0.05 0.05 - - -

When PNH cells are exposed at 37°C to a patient’s Normal RBC (ml) - - - 0.05 0.05 0.05
own or normal serum at pH 6.5-7.0, they show
abnormal lysis. Note: If test is positive, repeat the whole procedure
using patient's own serum to differentiate from
Requirements: HEMPAS. In later condition cells are not lysed in
• HCl 0.2 mol/L patient's own serum while in PNH test is positive
• Normal saline even with patient's own serum. The sensitivity of test
• Washed RBCs from a normal healthy person can be improved by adding 0.01 ml of Magnesium
• Normal fresh AB or group-compatible serum Chloride 250 mmol/L (23.7g/L) before incubation.
• Drabkin's Reagent
• Pipettes Bibliography:
• Test Tubes with a Test Tube Stand 1. Dacie and Lewis. Practical Hematology 11 th
• A Centrifuge Edition. S Mitchell Lewis, Barbra J Bain,
• A Water Bath Imelda Bates eds. Churchill Livingstone
London 2013
Procedure: 2. Wintrobe’s Clinical Hematology 12 th edition.
1. Separate normal and the patient’s serum from John P. Greer, John Forester, Gerge M. Rogers
freshly-collected, defibrinated blood. ,Friox Paraskevas, Bertil Glader, Danial A
2. Wash the normal and patient’s red cells three Arber, Robert T. Means, Jr. Wolters Kluver,
times with normal saline and pack by Lippincott Williams & Wilkins London 2009
centrifugation. 3. Post Graduate Haematology 7 th Edition . A.
3. Prepare a 50% suspension of both cells. Victor Hoffbrand, Daniel Catovsky, Edward
4. Inactivate portions of the patient's and normal sera G. D. Tuddenham eds. Blackwell Publishing
by heating in a water bath at 56°C for 30 minutes. London 2016
318 | P a g e

44. DIAGNOSTIC METHODS IN BLEEDING

DISORDERS
The functional tests of coagulation are based on Amount of anticoagul ant = 0.00185 × blood (ml) × (100 - PCV)
mimicking the in vivo conditions in the laboratory. 60 × 4.5
Amount of blood required =
However, the quantitative requirements of various 100 - PCV

coagulation factors to produce the end point in a 4. The blood sample must be collected through a
particular test may not be the same as they are in single, clean venepuncture so that a minimum of
vivo. Therefore, a gross discrepancy may be seen tissue thromboplastin is introduced in the sample.
between the laboratory result and the clinical 5. The samples must be collected in disposable
condition. It is best exemplified by a grossly- syringes and placed into glass tubes. For this
prolonged aPTT in factor XII deficiency, whereas the purpose, disposable plastic syringes and tubes are
clinical manifestations of this deficiency are economical.
extremely mild. The following are three types of 6. The samples should be kept cold, preferably on ice,
assays available to quantitate coagulation factors: until processed.
7. Platelet-poor plasma should be separated as soon as
1. Immunological Assays: These are quantitative possible. This is done by centrifuging the sample at
assays. These measure the coagulation protein, 2000 g for 15 minutes (preferably in a refrigerated
regardless of its functional capacity. centrifuge).
2. Chromogenic Peptide Substrate Assays: These 8. Tests should be fully completed within two hours
are qualitative assays. In these assays, the activated of the collection of the sample. If the samples are to
factor is allowed to act on a synthetic peptide to be stored, this should be done at -40°C.
which a dye is attached. The reaction releases the 9. The temperature of the water bath must be
dye, which is then measured photometrically. accurately maintained at 37±0.5°C during the test
However this activity is not the same as is the procedure.
physiological activity of the activated factor. 10. Before starting the tests, stop watches and timers
Therefore, the results of these assays also may not should be tested (if they are not electronic).
reveal the physiological defect. 11. The table lamp should be adjusted appropriately so
3. Coagulation Assays: These are qualitative that clot detection is easy and quick. Opaqueness of
assays. In these, the coagulation factor is the clot is inversely proportional to the length of
activated by means similar to those acting in time that it takes to form. Thus, in tests of a longer
vivo and is allowed to act on the natural time, the clot forms slowly. A uniform practice
substrate. Then, the action is also compared with should be adopted to read the end point.
a control or standard. These are the best assays 12. The trend for clotting to be prolonged with the
for clinical work. passage of time, due to a deterioration of reagents,
should be eliminated. If more than one sample is
GENERAL PRECAUTIONS: being tested in duplicate, the arrangement should be
something like A1 B1 B2 A2. The mean of two
1. Only venous blood should be used.
tests will take care of the difference in time.
2. Blood should be collected in a liquid
anticoagulant to allow a quick and thorough PLAN OF INVESTIGATIONS
mixing so that the process of coagulation
does not have time to progress. The If a patient with a suspected coagulation disorder is to
anticoagulant of choice is 31.3 g/L solution be investigated, the investigations should be pre-
of Trisodium Citrate Dihydrate or 38 g/L of planned. The most important, in this regard, are the
Trisodium Pentahydrate. history and clinical findings in the patient’s case.
3. The proportion of blood added to the These help in deciding whether the patient has a
anticoagulant must be exactly 9:1 otherwise vascular defect, a platelet defect or a defect in one or
the results will not be comparable. If the more of the coagulation factors. It also gives a clue as
PCV of the patient’s blood is less than 0.20 to whether the disorder is of a hereditary or an
l/L or more than 0.60 l/L, then the ratio of acquired nature and whether the inheritance is X-
blood to anticoagulant will have to be linked or autosomal (recessive or dominant). This
changed. The following formulae may be appreciably narrows down the number of tests to be
used to determine the amount of performed. The preliminary tests required are a
anticoagulant to be added to a volume of platelet count, Bleeding Time, PT, aPTT and
blood or the amount of blood to be added to Thrombin Time. A further line of action is decided
a fixed volume of anticoagulant. on the basis of these tests result.
319 | P a g e
Table 1: Plan for Investigations in a Patient with a Bleeding Disorder
N = Normal, ↑ = prolonged, ↓ = reduced
PLT BT PT PTTK TT CAUSES FURTHER
COUNT TESTS
N ↑ N N N Vascular abnormality, Plt function Hess’s test, Platelet function tests
defect
N ↑ N ↑ N Von Willebrand disease Platelet function tests, vWF assay`

N N N N N Factor XIII deficiency, Severe trauma, FXIII assay

Mild factor deficiency FVIII & FIX assay
N N ↑ N N Factor VII deficiency FVII assay

N N N ↑ N Intrinsic pathway factors deficiency Mixing studies

N N ↑ ↑ N Vit K deficiency, Oral anticoagulants, History, LFT, Mixing studies

Liver disease, FII, FV, FVII and FX
N N ↑ ↑ ↑ Heparin, Hypofibrinogenemia Thrombin time Mixing studies,
Dysfibrinogenemia, Systemic Reptilase time
? ↑ ↑ N Massive transfusion, Chronic liver History, LFT
disease
↓ ↑ ↑ ↑ ↑ DIC FDP, D-dimers

Table2: Plasma Preparations Required for Mixing Studies

PLASMA PREPARATION DEFICIENT FACTORS
Fresh normal plasma Nil
Plasma from patients on oral anticoagulants Factor VII
for 48-72 hrs
Plasma from patient on oral anticoagulants for a week or more Factors II, VII, IX, X

Aged plasma Factor V, VIIIC

Adsorbed plasma Factor II, VII, IX, X
Serum Factors I, V, VIIIC

Table 3: Correction of Prothrombin Time

Factor Prothrombin time corrected by mixing with
deficiency/
abnormality Normal Adsorbed Aged Coumarin
plasma plasma serum plasma
Factor I Yes Yes Yes Yes
Factor II Yes Partial Yes Yes
Factor V Yes Yes No Yes
Factor VII Yes No Yes No
Factor X Yes No Yes Yes
Anticoagulants No No No No

Table4: Correction of aPTT

PTTK corrected by mixing with

Factor deficiency/
abnormality Normal Adsorbed
Aged serum
plasma plasma

Factor VIIIC Yes Yes No

Factor IX Yes No Yes

Factor XI Yes Yes Yes

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MIXING STUDIES 4. To monitor replacement therapy
The basic test used for an assay depends upon the
These experiments are carried out on mixtures of test deficiency detected or suspected in screening tests
plasma with either normal plasma or plasma of (described previously). Prothrombin Time is used to
known factor(s) deficiency. The purpose of these assay factors II, V, VII and X. aPTT is used to assay
tests is to determine the cause of prolongation of factors VIII, IX, XI and XII. Factor-deficient plasmas
either PT or aPTT or sometimes of Thrombin Time. are required for the assays as these are used as
Factor-deficient plasma is commercially available but substrates. Serial dilutions of ‘test’ and ‘normal’
very expensive. Plasma with known factor plasma are mixed with the substrate plasma and on
deficiencies can be prepared in the laboratory and can each dilution, the corresponding clotting time is
then be used for mixing experiments. Once prepared, tested (i.e. Prothrombin Time or aPTT). The time
this plasma can be stored in small aliquots at -20°C obtained is plotted against the dilution or percentage
for future use. on suitable graph paper and activity of the factor in
THE PREPARATION OF ADSORBED ‘test’ plasma is estimated.
PLASMA FIBRINOGEN ASSAYS
Adsorbed plasma can be prepared by adsorption with Fibrinogen deficiency is indicated by a prolonged
Barium Sulphate. The procedure is as follows: Thrombin Time along with other abnormalities. It
1. To one ml normal plasma add 100 mg of Barium can be assayed by determining the clotting time after
Sulphate. the addition of Thrombin and comparing it with the
2. Place the tube at 37°C and continue stirring for 3 time obtained on known dilutions of fibrinogen.
minutes with a glass rod. Reagents are available commercially in kit form. The
3. Centrifuge at 1200-1500 g for 10 minutes and instructions and procedure supplied with the kit
collect the supernatant. should be strictly followed.
4. Test the Prothrombin Time--it should be more
than 60 seconds. Otherwise, carry out the FACTOR XIII DEFICIENCY
adsorption again.
THE UREA SOLUBILITY TEST
THE PREPARATION OF AGED PLASMA Principle
1. Collect blood in Oxalate. Factor XIII is activated during clotting. Thrombin
2. Centrifuge and separate the platelet-poor plasma. and Calcium ions are necessary for its activation.
3. Incubate at 37°C for 48 hours. Activated factor XIII stabilises the fibrin clot, which
4. The Prothrombin Time of the aged plasma is not soluble in 5 mol/L urea solution for at least 1
should be more than 90 seconds. hour, whereas clots formed in the absence of factor
5. Plasma is then dispensed in plastic containers XIII dissolve rapidly.
and stored at –20°C or lower. Reagents:
Procedure: 1. Patient’s citrated plasma
For mixing experiments the same test is used which 2. Normal citrated plasma
was abnormal. If the Prothrombin Time was 3. Urea 5 mol/L (300 g/L)
prolonged, then it is repeated after mixing with the 4. A Positive Control prepared by mixing 0.2 ml
appropriate reagents. If the aPTT was prolonged, then EDTA plasma with 0.2 ml Thrombin (20 NIH
it is repeated after mixing with the appropriate u/ml)
reagents. The correction of time is noted. To perform Procedure:
the test, one volume of ‘test’ plasma is mixed with 1. Place 0.2 ml of ‘test’ plasma in a 75X12 mm
one volume of the ‘reagent’ plasma or serum. The glass tube.
details of the test are the same as described earlier. 2. Place 0.2 ml ‘control’ plasma in another tube.
Significance: 3. To each, add 0.2 ml 10 NIH u/ml Thrombin
See Tables 3 and 4 for details. Solution and incubate at 37°C for 20 minutes.
4. Treat the positive control in the same way.
FACTOR ASSAYS 5. Add 3 ml Urea Solution to each tube and shake.
6. Leave overnight at room temperature
The precise activity of coagulation factors is assayed
undisturbed at 37°C.
to:
7. Inspect the next morning. A positive result is a
1. Diagnose a bleeding disorder
clot, which dissolves in the Urea Solution.
2. To assess the severity of a disorder
8. EDTA plasma can be used as a negative control.
3. To detect carriers
321 | P a g e

THE MEASUREMENT OF FDPS

Disorder ADP Collagen Adrenaline Ristocetin Arachidonic
Principle acid
The latex particles are coated with antibodies to FDP Glanzmann’s Abn Abn Abn N Abn
fragments D&E. If FDPs are present in the serum, Thrombasthenia
they will agglutinate with the latex particles. Serial Bernard Soulier N N N Abn N
dilutions of the serum are used and the agglutination syndrome
with the highest dilution of serum is noted. This gives von Willebrand N N Abn N
disease
a semi-quantitative estimation of the FDPs in the
Storage pool N Abn Abn N N
blood. disease
The test is available in kit form commercially. (the Aspirin defect N Abn Abn N Abn
procedure is given with the kit). It is important that Ehlers-Danlos N Abn N N N
samples should be collected in an agent that stops the Syndrome
fibrin breakdown, otherwise the results will be falsely
high. One such agent is ε-aminocaproic acid and the THROMBOPHILIA
tubes containing this reagent, for the collection of the
Thrombophilia are group of conditions associated
specimens, are provided with kits.
with an increased risk of thrombosis. These can be
PLATELET FUNCTION STUDIES hereditary or acquired. The common causes of
hereditary thrombophilia include Factor V Leiden,
Platelet function studiers are indicated in cases of Protein C Deficiency, Protein S Deficiency and
overt bleeding manifestations in which the Antithrombin III deficiency. The most commonly
Bleeding Time is prolonged in the absence of acquired cause is Lupus Anticoagulant.
significant thrombocytopenia and there are no
abnormalities of the coagulation pathway (except LUPUS ANTICOAGULANT SCREENING
in von Willebrand Disease).
The lupus anticoagulant is most commonly an IgG
Bleeding time should be performed prior to platelet
immunoglobulin. It is an immediate-acting coagulant
function studies.
inhibitor, which is characterised by a prolonged
A quantitative method has been devised to follow
Activated Partial Thromboplastin Time (aPTT). In
platelet aggregation by means of changes in light
mixing tests, the aPTT is not corrected by normal
transmissions of a sample of platelet-rich plasma
plasma. Although, aPTT is prolonged, it is rarely
(PRP). A known quantity of an aggregating agent
associated with bleeding problems. It is usually
is added to citrated PRP, which is contained in a
associated with thrombosis.
cuvette in a light- recording machine under
conditions of constant temperature and with Principle
continuous agitation. The changes in absorbance When aPTT is performed in the absence of a platelet
resulting from aggregation are measured directly substitute, it is particularly sensitive to the lupus
or graphically. The result is dependent on the anticoagulant.
platelet count.
Requirements:
If the platelet count of the patient is low, platelet
1. Kaolin 20 mg/ml
function studies can be performed by comparing it
2. Calcium Chloride 0.025 mol/L
with a sample from a healthy individual whose
3. Platelet-poor patient’s plasma (depleted of
sample is diluted to reduced platelet count. This
platelets by a second centrifugation, platelet
technique is not suitable with lipaemic samples. It
count <10x109/L)
is essential to obtain PRP from citrated venous
4. Normal platelet-poor plasma
blood collected into plastic tubes, with the tubes
5. Plastic Test Tubes with a Test Tube Stand
then capped to prevent loss of CO2 from the blood
6. Glass Test Tubes
to avoid a change in the pH. All handling of the
7. Water Bath
blood must be at room temperature, as any prior
8. Table Lamp
cooling inhibits the platelet-aggregating response.
9. Automated Micro-pipettes and tips
In screening studies, PRP is generally challenged
with a number of different aggregating agents, i.e., Procedure:
ADP, collagen, thrombin, adrenaline, and 1. Blood samples are collected and the plasma is
ristocetin. separated as for clotting tests.
2. Arrange 6 plastic tubes in the stand and prepare
Interpretations:
mixtures of ‘normal’ plasma and ‘patient’ plasma.
See Table 5
Table 5: Interpretation of Platelet Aggregation Studies. N = 3. Pipette 0.2 ml of each mixture into a glass tube
Normal Aggregation, that was previously placed in the water bath at
Abn =Impaired Aggregation. 37°C.
322 | P a g e
Tube 1 2 3 4 5 6 C, in its native form, is inactive. It is activated by
Normal plasma ml 1 0.9 .8 0.5 0.2 0 thrombin and thrombomodulin. It regulates blood
Test plasma ml 0 0.1 0.2 0.5 0.8 1 coagulation by inhibiting factors Va and VIIIa.
Protein C cleaves to activated V and VIII. Protein C
4. Add 0.1 ml Kaolin & incubate for 3 minutes. may be measured in three ways:
5. Add 0.2 ml CaCl2 and start the stop watch. 1. Clotting assay: generally measures function
Record the clotting time. 2. Antigenic assay: this measures total protein
6. Plot the clotting time (in seconds) against the 3. Chromogenic assay: this measures the binding site
dilution NP/TP.
Protein C deficiency may be acquired as a result of
Interpretations:
liver disease, Warfarin treatment and DIC or it may
1. Pattern-1: Curve convex near the y-
be hereditary. Protein S is also a Vitamin K-
axis=Classical lupus anticoagulant
dependant protein and acts as a co-factor of activated
2. Pattern-2: Sigmoid Curve=coexistent factor
Protein C. Its deficiency may also be acquired or
deficiency and lupus anticoagulant
hereditary as in Protein C and it is measured by the
3. Pattern-3: Curve with peak near the y-
same methods.
axis=coexisting deficiency of the lupus
anticoagulant and inhibitory co-factor ACTIVATED PROTEIN C
4. Pattern-4: Rather straight line=no lupus RESISTANCE (APCR)
anticoagulant
Activated factor V is a stimulus for the generation of
PLATELET-NEUTRALISATION TEST thrombin. Activated factor V, produced during the
Platelets absorb the lupus anticoagulant. Therefore, course of the coagulation process, is inactivated by
when platelets are used instead of Phospholipid in the activated Protein C (APC). When it cannot be
test system, the effect of the lupus anticoagulant is inactivated, it is called APC resistance. The stimulus for
neutralised. The platelets must be washed to remove the generation of thrombin continues, resulting in
contaminating plasma proteins and antibodies to thrombosis. The most common (>90%) cause of APC
expose their collagen factor-binding site. resistance is an abnormal factor V protein called factor
V Leiden resulting from a mutation (Arg506Glu) in the
factor V gene. This mutation removes the cleavage site
DILUTE RUSSELL VIPER VENOM for APC, hence greatly slowing the inactivation of
(DRVVT) TIME factor Va. It is the most common cause of Hereditary
Thrombophilia in the white population. Its prevalence in
Russell Viper Venom (RVV) activates factor X in the Pakistan is low (~1%)
presence of Phospholipids and Calcium ions. The
lupus anticoagulant prolongs the clotting time by COAGULATION ANALYZERS
binding to the phospholipids, thus preventing the
Chrono log, model 700 2– or 4 channel whole
action of RVV. In the case of factor deficiency, the
blood optical lumi-aggregation system.
time is not prolonged. Since RVV activates factor X The chrono-log uses three types of aggregation tests.
directly, defects of the contact system and factors
1. Optical aggregation in platelet rich plasma.
VIII, IX or XI deficiencies will not influence the test.
2. Impedance aggregation in whole blood.
ANTITHROMBIN 3. Measurement of ATP release (luminescence)

Antithrombin (AT), previously called Antithrombin- Platelet aggregation procedure:-

III is the major physiological inhibitor of thrombosis Aggregation can be measured with platelet
and factors IXa, Xa, XIa and XIIa. AT Deficiency is aggregometer either photometrically using (PRP) or
not uncommon and may be hereditary or acquired. In by measuring change in electrical impendence in
the presence of Heparin, AT reacts rapidly to whole blood.
inactivate the thrombin by forming a 1:1 complex. Impedence method:-
When serum is incubated with an excess of thrombin, The impedence (or electrical resistance) method of
the residual amount of thrombin left at the end of the aggregation is non-optical. The impedence method
incubation is proportional to the AT activity. Normal for measuring platelet aggregation allows the study of
levels of AT are usually in the range of 80-120 U/dl. platelets in the more physiological whole blood
An individual with congenital AT deficiency will environment. Sample preparation is greatly reduced.
have a level of around 50 U/dl. Newborns have a Preserving and thromboxane A2 resulting in a testing
lower AT concentration than adults. A low level of environment proven to be more sensitive to the effect
AT may be acquired during active thrombosis, liver of the many antiplatelet drugs (eg, Aspirin,
diseases or heparin therapy. Dipyridamole)
PROTEINS C AND S Luminescence Method:-
Protein C is a Vitamin K-dependant protein. Protein The photomultiplier is sensitive to light created by
323 | P a g e
CHRONO-LUME. Regent reacting with ATP 8. ATIII level
secreted by dense granules in platelet rich plasma. 9. The Protein C & S level
Luminescent measurement of ATP secretion provides 10. The D-dimer level (µg/m)
unequivocal evidence of normal or impaired dense 11. PTT LA ( Lupus anticoagulant)
granule release.
The % of the plasma being tested is displayed in the
‘’Test panel test status’’ screen of the STA
compact(R). The result is to be interpreted according
to the patient’s clinical and biological states.
In case of an evolution of the control values, it is
recommended to recalibrate the test or to adjust the
offset.
If the STA compact indicates that the control values
are outside the stated ranges check all compacts of
the test system to ensure that all are functioning
correctly i.e. assay conditions, reagents, Calibration,
Figure 1: Chrono log, model 700, optical lumi- integrity of the plasmas being tested etc. If necessary
aggregation system. repeat the assays.
Sysmex CA-1500:-
The sysmex CA-1500 is a fully automated blood
coagulation analyser for invitro diagnostic use to
perform blood coagulation tests.

Figure 3: Automated Coagulation Analyzer

STA Compact (Stago)

Bibliography:
Figure 2: Sysmex CA 1500 1. Dacie and Lewis. Practical Hematology 11th
Edition. S Mitchell Lewis, Barbra J Bain, Imelda
Automation (Coagulation) Bates eds. Churchill Livingstone London 2013
STA Compact:- 2. Wintrobe’s Clinical Hematology 12th edition.
Following tests are being done with automated John P. Greer, John Forester, Gerge M. Rogers
coagulation analyser STA Compact (Stago). ,Friox Paraskevas, Bertil Glader, Danial A
1. vWF : Ag Arber, Robert T. Means, Jr. Wolters Kluver,
Functional activity vWF. Lippincott Williams & Wilkins London 2009
vWF: Ag level (%) 3. Post Graduate Haematology 7th Edition . A.
2. PT (Sec) Victor Hoffbrand, Daniel Catovsky, Edward G.
3. aPTT (sec) D. Tuddenham eds. Blackwell Publishing
4. Fibrinogen (mg/dL or g/L) London 2016
5. Factor II level (%) 4. Glazier J: Measurement of Platelet aggregation
6. Factor V level (%) in whole blood. ACPR (26 - 30) April 1987.
7. Factor VII, VIII, IX, X, XI, XII
324 | P a g e

45. CLINICAL GENETICS

Pathology has, traditionally, been a descriptive science. visualised after Giemsa-staining. In order to identify
In describing the particular features of a morbid process, individual chromosomes, a special procedure of banding
pathologists have confronted only the consequences of is used in which the unstained chromosome slides are
biological processes and not the causative forces behind treated with trypsin. Subsequent Giemsa-staining imparts
them. Our ability to observe was greatly enhanced by each chromosome with a unique banded appearance that
the Light Microscope and then the Electron Microscope. can be seen under a Light Microscope. This type of
But, at the sub-microscopic level, explanations clearly banding is called G-Banding. Many different types of
lie with elements even smaller than the cellular banding techniques like C-Banding, Q-Banding, and R-
components. The centre point of all cellular activities at Banding, etc. are also used for the identification of
the sub-microscopic level is DNA. It carries within its chromosomes in special circumstances. Recently, it has
structure the hereditary information that determines the also become possible to visualise individual
structure of proteins, which are the prime molecules of chromosomes by using the technique of Fluorescent In
life. Our understanding of inherited and acquired Situ Hybridisation (FISH).
haematological disorder is of considerable
Common Indications for Cytogenetics
importance.New techniques are now available for the
Abnormalities in the number of chromosomes
study of normal, as well as abnormal, genes. These
(aneuploidy) involve either the loss or gain of one or
disorders include haemoglobinopathies allowing
more chromosomes. The structural abnormalities of
prenatal diagnosis, genetic risk factors in thrombophilia,
chromosomes involve the translocation of material from
monitoring of minimal residual disease and diagnosis
one chromosome to another, and the deletion or
and characterization of leukemia etc. This has opened
inversion of material from individual chromosomes.
new avenues for looking at things that are far beyond
The list of chromosomal disorders is very long; their
the reach of conventional diagnostic tools. For practical
description is beyond the scope of this discussion. The
purposes, ‘Genetics’ can be sub-divided into
common indications where cytogenetics may be
Cytogenetics and Molecular Genetics. Cytogenetics
required are either constitutional disorders or
deals with the study of whole chromosomes, whereas
malignancies and other acquired disorders. In both of
Molecular Genetics involves the study of genes at the
the categories, the abnormality can be either in the
molecular level.
number of chromosomes or in the structure of
CYTOGENETICS chromosomes. Table gives a list of the common
disorders and the usual chromosomal abnormalities
Chromosomes are thread-like structures that lie coiled found in them.
up in the nucleus of a non-dividing cell. At the time of
cell division (metaphase stage). A normal human cell Table1: Common Cytogenetic Disorders and Their
contains 46 chromosomes, including 22 pairs of Abnormalities
autosomes and one pair of sex chromosomes (XX in Disorder: Chromosomal abnormality:
females and XY in males). Each chromosome consists Constitutional disorders
of a pair of thread- like structures united together at a Down’s syndrome Trisomy 21 (47, XX or XY +21)
constriction called a ‘centromere’. Depending on the Patau’s syndrome Trisomy 13 (47, XX or XY +13)
Edward’s syndrome Trisomy 18 (47, XX or XY +18)
size of the chromosome and the position of the
Klinefelter’s syndrome 47, XXY
centromere, the chromosomes can be divided into seven Turner’s syndrome 45, X
groups (A-G). Karyotype refers to the number, size and Fragile X syndrome Fragile sites on X chromosome
shape of the total chromosomal content of an individual.
A normal male karyotype is written as 46, XY and that
of a female as 46, XX. Karyogram is the term that is
used for the photograph of an individual’s Malignancies
chromosomes, arranged in a standard manner. Acute lymphoblastic Hyperploidy, t(9;22).
leukaemia t(15;17); t(8;21)
Method of Chromosomal Analysis Acute myeloid leukaemia t(9;22)
Chronic myeloid leukaemia t(14;18)
The first step in study of chromosomes involves a culture Non Hodgkin’s lymphoma
of the cells. Most commonly, the lymphocytes of
peripheral blood are used. The lymphocytes, in presence
of Phytohaemagglutinin (PHA), are cultured in a suitable
medium like RPMI 1640. After 72 hours, cell division is
How to Refer a Patient for Cytogenetics
arrested at metaphase with Colchicin. These cells are first
The patients who require cytogenetic testing may be
suspended in a hypotonic KCl solution that causes them
referred to the Department of Genetics, AFIP. A
to swell and then fixed in acetic acid. A few drops of the
detailed clinical history of the patient and
fixed cell suspension are dropped onto a glass slide that
haematological information is recorded and 5 ml of
spreads the chromosomes. The chromosomes are then
peripheral blood is collected in a sterile tube with Na-
325 | P a g e
heparin (lithium-free) as an anticoagulant. In selected Several DNA-extraction kits are also available from
patients, particularly those with haematological commercial sources.
malignancies, cytogenetics is done on bone-marrow
The Analysis of the DNA
aspirates. Cytogenetics can also be done on
DNA analysis mostly involves amplification by the
Chorionic Villus Samples (CVS) and other solid
Polymerase Chain Reaction (PCR). The technique
tissues. The usual reporting time for cytogenetics is
involves use of a pair of 20-30 bp -long pieces of
one month.
DNA (primers) complementary to the DNA sequence
Flourescence in situ hybridization (FISH): of interest. The primers amplify the target sequence
Interphase FISH studies are performed on blood or by means of repeated cycles of de-naturation through
bone marrow specimens processed by standard heating the DNA, annealing the primers to the single-
methods for cultured samples. 3ml blood is collected stranded DNA & the extension of the primer DNA in
in sodium heparin. It is cultured at 37oC without PHA the presence of four nucleotides (G, A, T, C), heat-
for 24 hrs and then after adding 200ul colchicine for stable DNA Polymerase (Taq Polymerase) and a
1 hr. It is then be centrifuged at 1500rpm for 8 min. suitable reaction buffer. At end of each cycle, one
Supernatant will be discarded and KCl will be added molecule of DNA will yield two molecules. If cycles
followed by centrifugation. Three washings are given are repeated successively, 25-30 times for example,
with glacial acetic acid and methanol (3:1 ratio). The the target DNA can be amplified to over several
clear pellet will be placed on the slide. Slide is million-fold. .This is then directly visualised (after
prepared for FISH by treatment with ascending electrophoresis) on agarose or polyacrylamide gels..
concentrations of alcohol followed by PCR is done on automated equipment,
Salisodiumcitrate(SSC solution). The slide is air- THERMOCYCLER, having a computer controlled
dried in a dark place. 10ul of Metasystems XL heating block with the capacity to hold 24-96
different probes are applied to the target on the slide. reaction tubes.
Cover slip is applied and sealed with rubber cement. The Use of PCR in a Diagnostic Laboratory
Denaturation at 74oC for 05 min, hybridization at Areas of PCR use can be grouped as: inherited
370Cfor 18hrs and co-denaturation at 740C for 5 min disorders, malignant disorders, infectious disorders,
is done. The slide is then washed and air-dried. 10- forensic medicine, tissue-typing, etc.
20ul of DAPI-2 counterstain is added and then frozen 1. Inherited Disorders: Most of the inherited
at -20oC for 1 day. A total of 500 nuclei are analyzed disorders are with a single gene defect following
per probe set by using a fluorescent microscope using mandelian pattern of inheritance. Haemoglobin
an orange green spectrum filter. The specific probe is disorders, including thalassaemia, have been
labeled in orange while a green labeled probe which studied most extensively at the DNA level.
hybridizes to the centromere of target chromosome. Information is also rapidly emerging in mutation
disorders of the coagulation cascade, inborn
MOLECULAR GENETICS
errors of metabolism, endocrine disorders,
This deals with genetic analysis at sub-cellular level. lysosomal-storage disorders, premature
The genetic material of a cell consists of atherosclerosis, diabetes mellitus (insulin gene
Deoxyribonucleic Acid (DNA) and Ribonucleic Acid mutation), cystic fibrosis, muscular dystrophies,
(RNA). Most ofcellular DNA is present in nucleus congenital renal diseases and hereditary
with some traces in mitochondria. RNA, on the other enzymopathies. The two main types of molecular
hand, is present in the nucleus as well as cytoplasm. lesions (mutations) have been observed to be the
causes of these disorders are: gross abnormalities
The Extraction of DNA
(deletions, insertions or rearrangements) of genes
The first step in molecular genetics is extraction of
and, a single nucleotide abnormality (point
DNA from the test samples. DNA can be extracted
mutation) in a critical region of the genes.. The
from any dead or alive tissue that contains nucleated
most commonly used PCR-based technique is
cells. In routine practice 5-10 ml of peripheral blood
called the Amplification Refractory Mutation
collected in EDTA... The sample’s red cells are lysed
System (ARMS). In disorders where the genetic
by a buffered solution containing 2% Triton-X 100.
lesion is not well-characterised, an indirect
The white cells are sedimented by centrifugation at
approach of Restriction Fragment Length
3000 g for 5 min. The white cell pellet is lysed by
Polymorphism (RFLP) can be used. An exciting
overnight incubation at 37°C in 2% buffered solution
application is the diagnosis of inherited disorders
of SDS and Proteinase-K. The sample’s proteins are
during pregnancy (prenatal diagnosis). Prenatal
precipitated by phenol chloroform extraction. DNA,
diagnosis of a large number of inherited
in the final solution, is precipitated by 70% Ethanol.
syndromes is now possible with the use of the
The final DNA precipitate is re-dissolved in sterile
PCR. In practice, Chorionic Villous Sampling
distilled water. DNA can also be extracted from
(CVS) is done under ultrasound guidance
biological fluids containing nucleated cells, chorionic
between 10-12 weeks of gestation. The sample is
villus samples (CVS), archival bone- marrow smears,
dissected under the microscope and clean foetal
paraffin-embedded tissues, and other solid tissues.
tissue is separated. DNA is extracted from the
326 | P a g e
foetal tissue and the diagnosis of a genetic DNA or RNA can be detected in a clinical
abnormality can be made. .Prenatal diagnosis sample. An interesting application in viral
coupled with a therapeutic abortion in positive diseases is the in-situ PCR technique. The virus
cases, has proved to be very effective in particles, for example, Hepatitis B virus in the
eliminating the genetic disorder from a liver cells, CMV in the lung, and EBV in
community. With the PCR, it is also possible to association with lymphoma, can be demonstrated
diagnose an inherited disorder in an in-vitro in a tissue specimen.
fertilised embryo prior to its implantation (pre- 4. Miscellaneous Applications: PCR has
implantation diagnosis). tremendous potential for applications in forensic
pathology. It is based on fact that chance of
2. Neoplastic Disorders: The diagnosis of cancer, DNA being similar from two different
through the analysis of DNA, is based on the individuals is one in several million. Other useful
recognition that, at the cellular level, neoplasia is applications include HLA-typing for organ
almost certainly a genetic disorder. The genetic transplantation and the identification of auto-
alterations responsible for the neoplastic immune-linked HLA alleles.
proliferation of cells are usually acquired
5. Forensic DNA Testing:
somatically only in the neoplastic tissues of the
body. The genetics of cancer is intimately 1. The Collection/Storage of Samples -
associated with two topics that have received a. DNA can be extracted from any
considerable attention in recent years: oncogenes biological material that contains a
and chromosomal rearrangements. More sufficient number of living or dead
recently, the role of tumour-suppressor genes in nucleated cells. Since DNA is susceptible
the causation of cancer is also being recognised. to degradation by mishandling and
The activation of or aberrant expression of digestion by the enzymes derived from
oncogenes in some way lead to an excessive or bacterial contamination of the sample, try
uncontrolled cellular proliferation and seems to to obtain a sample that is clean and free of
involve at least three different mechanisms: point degradation as possible.
mutation, gene amplification, and a proximity to b. Depending on the indication, a wide
sites of chromosomal rearrangement. Each variety of samples may be used for
mechanism of oncogene activation carries a obtaining DNA. The usual samples are
potential for diagnosis through the analysis of whole or dried blood (stains), a buccal
DNA. Apart from oncogene analysis, the smear, fluid or dried semen and other
malignancies of lymphoid tissue can be body secretions, hair with the roots, soft
diagnosed by demonstrating clonal tissues, fresh and dried bones.
rearrangements of Immunoglobulin genes or T- c. Fresh blood: 2-3 ml of venous blood
cell receptor genes. The approach is also useful should be collected in EDTA (CP bottle).
for assigning the lineage commitment to The sample can be stored before dispatch
lymphoid malignancies. In addition, an abnormal at 4oC for 48 hours.
clone of lymphoid cells can be detected at a very d. Blood stains: these may be present on
early stage, or can be differentiated from a clothes or other objects and they can be an
benign polyclonal lymphocytic proliferation. The important source of DNA. If the stain is
PCR can be used to detect the minimal residual wet, it should be dried at room
disease in patients undergoing treatment for the temperature before dispatch. The dried
malignancy disorder. The PCR can also be used sample may be stored at -20oC, after
to demonstrate the association of some wrapping it in a polythene bag.
malignancies and viruses, e.g. Human e. Buccal smears: such smears obtained
Papillomavirus, Cervical Cancer, HTLV-1 with a clean throat swab contain a
Infection and leukaemia. sufficient number of mucosal cells to
yield enough and good-quality DNA. A
3. Infectious Disorders: The PCR is also disposable throat swab can be used to
becoming popular in the diagnosis of infectious obtain DNA from a dead body or its
disorders. DNA-based methods are very remains. Rub the swab several times over
sensitive for the detection of pathogens. PCR is a clean part of the buccal mucosa and then
particularly useful in tuberculosis where a place it back in its cover. The swab may
culture takes long time, or leprosy, where a be stored at 4oC for 48 hours before
culture may not even be possible. PCR is also dispatch.
being used for many fungal, parasitic and viral f. Semen and other body fluids: as these
infections like Hepatitis B and C, EBV, HIV, etc. contain nucleated cells, they are a good
PCR-based detection of viral genomes is an source of DNA. Any clothes or objects
extremely sensitive and specific method. that are so-stained should be air-dried and
Genomes as small as a single target molecule of treated as blood stains.
327 | P a g e
g. Hair: hair shafts themselves do not c. The Pathologist/ Medical Officer who is
contain nuclear DNA. However, hair that collecting the samples will verbally
has been plucked from the head/body and inform about the samples to the AFIP.
comes out with its roots can be used to d. The Request Form and the samples
extract the DNA. Sufficient DNA can be should be handed over to the
extracted from the roots of even just four investigation agency involved and those
pieces of hair. The hair may be stored in a personnel would be responsible for the
suitable container at -20oC for several transport and delivery of the samples to
days before dispatch. the AFIP.
h. Soft Tissues: these can be a very rich e. The investigation agency will dispatch
source of DNA. Skin is an easily the samples without delay to the
accessible tissue that can be used to Commandant, the AFIP under a
collect DNA from a dead body. A full covering letter from its OC.
thickness piece of skin that measures f. All correspondence pertaining to the
about 2X2 cm (from a clean part of the case should be classified as top
body or its remains) should be taken. If confidential.
skin is not available, then any other
3. The Transporting of Samples -
available soft tissue should be collected.
a. The samples should be transported as
Soft-tissue samples provide an excellent
quickly as possible. Any delay (unnecessary or
medium for bacterial growth. In a
otherwise) in transporting them can adversely
putrefied or heavily contaminated soft-
affect the quality of the sample(s).
tissue sample, the yield, as well as the
b. There is no special requirement of
quality of the DNA therein, can be very
transporting the samples in ice, etc. However,
poor. The soft-tissue samples can be
avoid exposing the sample(s) to extreme heat or
stored as such in a suitable container at -
direct sunlight.
20oC for several days. For transportation,
put the sample in normal saline. Note: do 4. The Chain of Custody -
not put samples for DNA testing in a. Forensic DNA testing is done for medico-
formalin at any stage of their handling. legal purposes and, therefore, it is essential to
i. Bones: bones can also be used as a source maintain the chain of custody.
of DNA, but the extraction of DNA from b. A record of the individuals receiving and
a bone is difficult and, therefore, the yield handing over the samples must be maintained at
and quality of the DNA that is extracted all steps, as they may be called by the court as
from such a material is also variable. witnesses.
Depending on its availability, a bone with
a compact structure like a humerus or
femur bone, complete or in part, should be Bibliography:
collected. If these are not available, then 1. Dacie and Lewis. Practical Hematology 11th
any other compact bone may be collected Edition. S Mitchell Lewis, Barbra J Bain, Imelda
and used. Bates eds. Churchill Livingstone London 2013.
2. Wintrobe’s Clinical Hematology 12th edition.
2. The Dispatch of Samples - John P. Greer, John Forester, Gerge M. Rogers
a. All samples should be properly labelled ,Friox Paraskevas, Bertil Glader, Danial A
and sealed. Arber, Robert T. Means, Jr. Wolters Kluver,
b. The Request Form should contain all Lippincott Williams & Wilkins London 2009
available details of the individual(s) to 3. Post Graduate Haematology 7th Edition . A.
be tested, along with a brief summary of Victor Hoffbrand, Daniel Catovsky, Edward G.
the incident and what exactly is D. Tuddenham eds. Blackwell Publishing
required to be solved by the DNA test. London 2016
328 | P a g e

46. TRANSFUSION MEDICINE

Transfusion medicine integrates the field of blood Antibodies produced as a result of an antigenic stimulus
banking and clinical medicine in an effort to serve the are known as immune, acquired or ‘warm’ antibodies.
ailing members of humanity with the best possible These are usually IgG e.g., Rh, Kell, Kidd, Duffy, etc.
outcome. Assurance of safety in transfusion medicine Those antibodies that appear without any apparent
depends upon under- standing the subject and antigenic stimulation as in the cases of transfusion,
applying the knowledge in different clinical pregnancy or vaccination, are known as natural or ‘cold’
situations. One must be fully aware of the meaning of antibodies. The latter are mostly IgM antibodies and are
‘safe’ blood, which includes knowledge of the commonly found in the ABO, M, N, Lewis, P and Ii
transmission of viral diseases like Hepatitis and systems of blood groups.
AIDS. The field of transfusion medicine has acquired Antibodies involved in blood group systems are IgG,
its present status primarily due to a better IgM and, occasionally, IgA. Table 2 clarifies the
understanding of immunology in general and a grasp properties of these immuno-globulins.
of immunohaematology in particular.
Table 2: Immunoglobulins in Transfusion Medicine
ANTIGENS PROPERTIES IgG IgM IgA
Structure Monomer Pentamer Monomer
An antigen is a substance which, when introduced Molecular weight 150,000 900,000 160,000
into an immuno-competent host, causes a production Carbohydrate 3 12 8
of antibodies with which it reacts specifically. percentage
Considerable structural diversity exists among Serum concentration 150,00 200 350
antigens. Blood-group antigens are chemical (mg/dl)
Serum half-life(days) 23 5 6
structures embedded in or protruding from red blood Present in secretions No No Yes
cells, white blood cells and platelet membranes. The Antibody activity Yes Yes Yes
three most common forms of blood-group antigens Antigen binding sites 2 5-10 2
are glycoproteins (HLA system), glycolipids (ABO, per molecule
Lewis, Ii, and P blood-group systems) and proteins Complement fixation Occasional Yes No
Cross placenta Yes No No
(Rh, M, and N blood-group systems). Broadly Serological behaviour Non Agglutinati Non
speaking, antigens may be classified into two major Agglutinati ng Agglutinati
types: exogenous & endogenous. In blood transfusion ng ng
services, we are concerned primarily with antigens
defined as allogenic (from another human) and
autologous (self). These antigens are important in ANTIGEN ANTIBODY REACTIONS
relation to pregnancy, transfusion & organ A wide variety of antigen-antibody reactions are
transplantation. known but only those which are important from a
transfusion point of view, are described.
IMMUNE RESPONSE
When a foreign antigen is introduced into the body Agglutination:
for the first time, a primary antibody response Agglutination is the formation of aggregates of particles,
(characterised by a slow production of IgM e.g. red blood cells that bear antigenic determinants on
antibodies) occurs. When the same antigen is their surface and combine with the antibodies present in
introduced for the second time, a secondary immune the test serum. The mechanism used is the formation of
response occurs with the production of larger amount antibody bridges that connect these with the antigenic
of antibodies, mainly of IgG type. This is called determinants of adjacent cells. Agglutination can be
humoral immunity. observed both through direct (ABO grouping) and
indirect techniques (antiglobulin procedures).
ANTIBODIES Agglutination occurs in two stages:
Antibodies are immunoglobulins produced by B- 1. The antibody attaches itself specifically to the
lymphocytes and plasma cells in response to polysaccharide/lipid/protein complexes that form
antigenic stimuli. Upon exposure to a particular the antigen sites on the red cell. This process is
antigen, plasma cells proliferate and synthesize known as sensitisation; it requires proper
immunoglobulin’s that are capable of specifically temperature, pH and ionic strength of the
combining with the original antigen, a functional medium and antigen-antibody ratio.
characteristic referred to as ‘antibody specificity.’ 2. The second phase is the physical process of
In humans, an antibody is associated with five major agglutination, in which cells come together to
classes of proteins, known as the immunoglobulins. form clumps. This depends upon the type of
These can be differentiated from one another on the antibody involved, the antigen sites available and
basis of size, biological function, bio-chemical the ‘zeta’ potential of the medium in which the
properties and serological activity. cells are suspended.
329 | P a g e
Zeta Potential: Red cells, when in a suspension, occurs in two stages:
carry a negative charge on their surface in the form of 1. The antibody combines with the antigen, thus
sialic acid residues. These charges serve to repel the sensitising the red cell.
adjacent cells to avoid sledging and achieve 2. Sensitised red cells lyse with the help of
satisfactory oxygen carriage. When the cells are activated complement components.
suspended in an electrolyte solution, electro-positive
Table 2: Reading & Interpreting Agglutination
charges are attracted towards the cells, thus carrying
Reactions and Haemolysis
a double ionic cloud that moves along with each cell.
The farther end of this edge is known as the surface Symbol Agglutinatio Description
of shear or the slipping plane. This determines the n score
effective charge of the red blood cells and is 4+ or C 12 Cell button remains in one clump,
(complete macroscopically visible
designated as the zeta potential. In order to bring an )
antibody close to the surface of the cell, this potential 3+ 10 Cell button dislodges into several
large clumps, macroscopically
has to be reduced. Even those antibodies that do not visible
2+ 8 Cell button dislodges into many
exhibit agglutination normally can do so if this small clumps, macroscopically
potential is reduced. This can be brought about by the visible
1+ 5 Cell button dislodges into finely
following procedures: granular clumps, macroscopically
(+) or w 3 just visible
Cell button dislodges in fine
1. Proteolytic Enzymes: The enzymes that are
(weak) granules, only visible
used to enhance antigen-antibody reactions microscopically
include papain, ficin and bromelin. They also - 0 Negative result-all cells free and
evenly distributed
remove structures on the red cells membranes, so
as to facilitate the interaction of the antibody REQUIREMENTS OF A STANDARD
with the corresponding antigen. The red cell
antigens that are enhanced by enzymes include BLOOD BANK
Rh, Kell, Kidd & Lewis blood groups. Certain AREAS WITHIN THE BLOOD BANK’S PREMISES:
red cell antigens, including Duffy (Fy) and M, N,
S and s, are actually destroyed by enzymes. A general work area required varies greatly with the
number of expected blood donations and the
2. Albumin: It is prepared from bovine sources and workload. However, it should be divided into the
is commercially available as a 22% preparation. following sections:
Over time, the use of albumin has been • Reception and donation sections comprising of a
abandoned by many blood banks, due to the waiting area, donor’s assessment room (where
availability of better potentiators like LISS and donor is weighed & haemoglobin is tested), a
Polyethylene Glycol. donation room and an area marked for the
3. LISS: Low-Ionic Strength Saline is widely used refreshment of donors.
in blood banks as an enhancing medium. The • a screening section
incubation time is shortened to 10 minutes and • blood banks for the storage of blood
most antibodies are well- detected by this • A Laboratory that includes cross-match & issue
technique. There are reports of a diminished sections
reactivity of anti-K in the low ionic strength • Stores
medium. • Offices
• Components section (if the facilities are available)
4. PEG (Polyethylene Glycol): It potentiates
agglutination by taking out water in hydration, STAFF:
thus binding the cells together and enhancing
second-stage agglutination. • Pathologists (Haematologists) with experience in
transfusion medicine
Haemolysis: • Preferably, a Microbiologist for screening the tests
This is an important antigen-antibody reaction that • Laboratory Technicians who are trained in
results in the lysis of cells. The red colour of free transfusion medicine
haemoglobin (released during the immune • Phlebotomy Nurses
destruction of cells) is an important end point of an
• Auxiliary Staff members
antigen-antibody reaction. Haemolysis represents
destruction of the red blood cell membrane through EQUIPMENT:
the action of complement proteins that are activated
by the attachment of a specific antibody to a surface The following equipment is required for a routine blood
antigen. Haemolysis is a positive result that indicates bank. In cases where more specialised services are
the presence of a complement-activating antibody. required, then additional equipment may also be needed.
Antibody-mediated haemolysis does not occur in the • Donation beds
absence of complement or in plasma when a calcium- • Height and weight scales
chelating agent is present. A haemolytic reaction • Mixing and weighing equipment (haemoscale)
330 | P a g e
for blood units during donation or at least • Ether
hanging-weighing scales.
• Sphygmomanometers THE PREPARATION OF BASIC
• Stethoscopes REAGENTS
• Oxygen-inhalation equipment
BUFFERED NORMAL SALINE:
• Suction machines
• Air-ways for emergency use 1. Phosphate Buffer pH 7.0
• Normal Saline infusion with IV set (500/1000 ml). a. Solution A: NaH2PO4.2H2O 23.4 g/L
• Crape Bandages b. Solution B: Na2HPO4 (anhydrous): 21.3 g/L
• Blood-Bank refrigerators with continuous c. Mix: 32 ml Solution A & 68 ml Solution B
temperature recorders and alarms. 2. Normal Saline 9.0 g/L
• Blood bag centrifuges--preferably refrigerated. 3. Buffered Saline: Mix equal volumes of Solutions
• a deep freezer (-30 to -80°C) for freezing plasma 1&2
and cryoprecipitate LOW IONIC STRENGTH SOLUTION
• Platelet Incubators
(LISS)
• a Laboratory Incubator (37°C)
• Refrigerators Low Ionic Strength Solution (LISS) reduces the zeta
• Water Baths with temperature controls potential and thus enhances the association of an
• Laboratory Centrifuges antibody with an antigen. The major advantage is that
• a high-speed Centrifuge for 75x10 mm test tubes the incubation period in the Indirect Antiglobulin
• Glass Test Tubes 75x10 mm Test can be reduced while maintaining or increasing
the sensitivity of detecting a majority of the red cells’
• Metallic Test Tube Racks (with 12 holes in each
antibodies. The red cells should be washed in saline
row) to hold 75x10 mm test tubes
and suspended in LISS. One volume of cell
• Glass Pasteur Pipettes
suspension & two volumes of serum should be used.
• Grouping Tiles The incubation period can be reduced to 10 minutes,
• Glass Slides with its pH at 6.6-6.8, osmolality 270-285 and
• a Microscope conductivity of 3.5-3.8 mS/cm.
• Automatic ELISA equipment (for screening
workloads that are > 50 samples per day) PREPARATION:
• Tube Sealers 1. Stock Solution: Dissolve 42.9 g Na2HPO4: and
• Stationery and rubber stamps marked with the 10.2 g KH2PO4: separately in 500 ml water.
labels of blood groups, components and the 2. Working Solution: Dissolve 1.75 g NaCl, 18.0 g
names of tests Glycine1 in water. Add 8.7 ml Na2HPO4, 11.3 ml
• Computers KH2PO4 and make the volume up to 1 litre with
REAGENTS: distilled water’
3. Adjust the pH to 6.7 with NaOH
• Full range of blood-grouping sera (anti-A, anti- 4. Add 0.5 g of Sodium Azide, as preservative.
B, anti-AB, anti-D) 5. The LISS should have the following
• Bovine Albumin 22% / LISS characteristics:
• Polyspecific Coombs Reagent a. pH: 6.6.-6.8
• Three-cell panel for antibody screening b. Osmolality: 270-285 mmol
• Eleven-cell panel for antibody identification c. Conductivity: 3.5-3.8 ms/cm at 23oC
• Phosphate-Buffered saline (can be made in the Note:Glycine prevents the non-specific uptake of complement on the red
laboratory) cell’s surface .
• Low Ionic-Strength Saline, LISS (can be made in
the laboratory)
331 | P a g e

BLOOD DONATION

THE RECEPTION OF DONORS: Sulphate Specific Gravity Method, with Hemacue

Strips, by the Spectrophotometric Method or with a
haematology analyser. The acceptable level is 12.0
A blood donor is not an ordinary person, particularly g/dl for females and 13.0 g/dl for male donors.
in our community where baseless prejudice and fears Medical History: A thorough medical history should
against blood donation still persist. She/he deserves be taken to ensure that the donor is free of all
our special attention and care. It must be ensured diseases. Special emphasis should be given to ask
that: about the history of viral hepatitis within the past 1
1. The environment of the donation centre is clean, year, venereal diseases, AIDS, cardiovascular
comfortable and quiet. It should be well-lit, well- diseases, hypertension, renal diseases, diabetes
furnished, well-ventilated and, preferably, air- mellitus, tuberculosis, bleeding disorders, central
conditioned/ heated. nervous system disorders, gastrointestinal disorders,
2. All of the personnel on duty should exhibit an respiratory diseases and malignancies. Potential
attitude of professional competence and good donors who have any of the above-mentioned
manners. diseases are excluded from making blood donations.
3. Every donor is a VIP and should be treated
accordingly. Unnecessary and non-professional Current Conditions: Pregnancy, lactation,
arguments with a donor should be avoided at all infections, and a blood donation given less than 12
times. weeks prior to the present time are reasons for a
4. Introduce yourself by name to the donor, offer temporary rejection of accepting a blood donation.
her/him a seat and then ask about the recipient Donor Registration Card: the Blood Donor
for whom she/he wants to donate blood, or if Registration Card is filled in the presence of the
she/he is a voluntary donor. donor. The details of the physical examination and
5. Explain the procedure to the donor, reassure the medical history are printed on the card. Note: all
her/him that the procedure is safe, and will entail questions must be asked in the language that the
only a single prick of a needle. donor fully understands.
6. Give her/him the chance to ask questions. Copper Sulphate Method for the Screening of the
7. First-time donors must be handled very carefully Haemoglobin’s Concentration:
and completely reassured. Aqueous Copper Sulphate, coloured blue, with a
DONOR SELECTION / REGISTRATION: specific gravity of 1.053, equivalent to 12.5 g/dL
haemoglobin is normally used to test female donors.
Considerable care must be exercised in selecting Copper Sulphate, coloured green with a specific
potential blood donors, for the protection of both donor gravity of 1.055 equivalent to 13.5 g/dL is used to
and the recipient. Most donors may be accepted on the test male donors. The donor's fingertip is cleaned
basis of medical history, general appearance and with a swab of methylated spirit and punctured by a
haemoglobin estimation, although it is advisable to lancet. The first drop of blood is wiped off by a piece
examine all of a potential donor’s vital signs. of sterile gauze. The second drop is allowed to reach
General Appearance: The donor should appear to as big a size as possible and allowed to drop by itself
have good health. Signs of poor physique, from a height of 10 mm into the appropriate Copper
debilitation, poor nutrition, plethora, jaundice, Sulphate Solution. The drop is observed for 15
cyanosis, dyspnoea and mental instability should be seconds. If the drop of blood has a higher specific
noted. A clinical examination that suggests gravity than the Copper Sulphate Solution, then it
intoxication either by alcohol or narcotic drugs will sink within 15 seconds. If not, then it either takes
should be a reason to exclude that donor. The skin at a longer time to sink or it remains suspended or even
the venepuncture site should be free from lesions. may rise to the top of the solution. The results are
Weight: The donor must weigh more than 50 kg to interpreted as “Pass” or “Fail” accordingly.
donate a full 450 ml quantity of blood. Those who THE COLLECTION OF BLOOD:
weigh less, but are otherwise healthy, may donate
250 ml of blood for which either a reduced volume of After taking the medical history, physical
anticoagulant or a special donation bag is used. examination and checking the haemoglobin level, the
Age: First-time donors should be 18-60 years of age. donor is guided to the Blood Donation Room. The
Donors may continue to donate regularly until they name and other particulars of the donor are counter
are 65 years of age. checked and the following procedure is adopted:
1. Blood should be drawn from a suitable vein in
Haemoglobin: The haemoglobin concentration the antecubital fossa in an area that is free of
should be determined before every donation. any skin lesions.
Haemoglobin screening can be done by the Copper
332 | P a g e
2. Clean it thoroughly with iodine and methylated 4. Before the donor leaves the blood donation
spirit. centre, it is confirmed that she/he is perfectly all
3. A sphygmomanometer cuff is wrapped around the right and that there is no more bleeding from site
upper arm. of the phlebotomy.
4. Inspect the bag that contains the anticoagulant
(CPDA-1, shelf life 35 days). It should be clear and THE SCREENING OF BLOOD:
colourless. Once the blood has been donated, pilot tubes are sent
5. Label the bag and two plain, glass test tubes/screw- to the Screening Department. The following tests
capped bottles (to be used later as pilot tubes). should be performed routinely on the donor's blood:
6. Now raise the pressure in the sphygmomanometer 1. ABO and Rh typing
cuff to 50-80 mm of Hg. The veins will become 2. Screening for antibodies other than the ABO
prominent. group antibodies
7. Perform the phlebotomy. The blood will start 3. Hepatitis B and Hepatitis C screening (HBsAg,
flowing into the bag. Anti-HCV antibody)
8. The attendant should observe and ensure that the 4. Screening test for AIDS (Anti-HIV antibody)
blood is flowing at a steady speed and she/he should 5. Screening test for Syphilis (VDRL)
gently mix the blood in the bag (or automatic 6. Screening for malarial parasites in high-risk
mixing/weighing equipment should be used). areas
9. The attendant should also look for the condition of Any positive reaction observed in ANY of the
the donor. If she/he manifests signs of fainting, screening tests makes the blood unit unfit for
sweating or palpitation, then the process should be transfusion and it should be discarded.
stopped at once.
10. When the required quantity of blood has been BLOOD GROUP SYSTEMS
collected (this takes less than 10 minutes) the
There are many blood group systems including ABO,
pressure in the sphygmomanometer cuff is released.
Rh, Kell, Kidd, Duffy, Lutheran, Lewis, MNS.
Two clamps are applied as close to the needle as
However, in routine practice only the ABO and Rh
possible. The tubing is then cut between the clamps
systems are considered important.
with small scissors. Take a sample of blood in pilot
tubes by releasing the clamp near the needle and ABO AND Rh-D GROUPING
then apply it again.
11. Sterile gauze is placed over the puncture site, the It is the most important part of blood screening. It can be
needle is withdrawn and the puncture site is sealed performed on the cells as well as on the serum. When it
aseptically with adhesive dressing. is performed on the cells it is called direct grouping
12. The arm and the general well-being of the donor (forward typing) in which unknown red cells (test cells)
should be checked. are tested against known antisera. When it is performed
on the serum it is called indirect grouping (reverse
THE STORAGE OF BLOOD: typing) in which the unknown serum (test serum) is
tested against known red cells. Ideally both should be
Blood must be stored in a blood bank refrigerator that
performed on each specimen.
operates between 2-6°C. It should be well-lit and should
be equipped with alarm and temperature-recording DIRECT GROUPING (FORWARD
devices. The red cell concentrates/whole blood can be TYPING)
stored for 35 days from the date of collection, in blood
bags containing CPD-A1, as the solution. Citrate is a This can be performed either by the Tile Method or
calcium-chelating agent that prevents the blood from by the Test Tube Method.
clotting. Dextrose is provided as a nutrient for the red
The Tile Method:
cells to support the generation of ATP by glycolysis,
1. Allow the blood to clot. Clear supernatant serum
thus increasing the red cells’ viability. The addition of
should be aspirated carefully with the help of a
adenine is also associated with the improved synthesis
Pasteur Pipette into another clean tube.
of ATP in stored blood.
2. Prepare 5% cell suspension from the cells by
AFTER-PROCEDURE CARE OF THE mixing one drop of packed cells with 19 drops of
buffered normal saline.
DONOR:
3. Divide the tile (with a grease pencil) into A, A1, B,
1. Make sure that the bleeding has stopped from the AB and Rh D squares.
site of the phlebotomy. 4. Place one drop of the corresponding antiserum in
2. Let the donor remain lying on the couch for at each square.
least 10 minutes, so that her/his circulation can 5. Add a drop of test cell suspension into each of the
re-adjust itself. squares that contain antiserum.
3. The donor is provided with light refreshment 6. Mix with a glass rod, cleaning its tip thoroughly
(particularly tea/coffee) and advised to refrain after mixing in each square.
from smoking for at least one hour. 7. Gently tilt the slide backwards and forwards at
333 | P a g e
room temperature for a maximum of two minutes. O and auto-control squares.
8. Macroscopically read for agglutination and record 2. Place one drop of the patient’s serum in all of the
the result. squares.
9. Rh D-negative persons may be tested for Du 3. Add one drop of the corresponding 2-3%
(variant). suspension of red cells to the labelled squares.
4. Add one drop of the 2-3% suspension of the
The Test Tube Method:
patient’s red cells to the square labelled ‘auto-
1. Prepare a 2-3% suspension of red cells in isotonic
buffered saline. control’.
2. Arrange test tubes in the rack, marked anti-A, anti- 5. Mix well with a glass rod, cleaning its tip after each
A1, anti-B, anti-AB and anti-Rh D. application. Gently tilt the slide backwards and
3. Add one drop of the corresponding antiserum to forwards at room temperature for a maximum of
each of the test tubes. two minutes.
4. Using a small pipette, add one drop of 2-3% cell 6. Macroscopically read for agglutination.
suspension to all the test tubes.
5. Mix well and centrifuge at 3400 RPM (900-1000g) The Test Tube Method:
for 30 seconds. 1. Place 2 drops of the serum to be tested into test
6. Try to re-suspend the cells by gentle agitation and tubes labelled A, B, O and auto-control.
read macroscopically for agglutination and/or 2. Using a Pasteur Pipette, add one drop of 2-3%
haemolysis.
7. Confirm a negative result by microscopy. suspension of corresponding cells into each tube.
8. Rh D-negative persons may be tested for Du 3. Add one drop of test red cells (patient’s red cells)
(variant). to the test tube labelled ‘auto-control’.
4. Mix well and centrifuge at 3400 RPM (900-
Quality Control: The antisera should be tested with 1000g) for 30 seconds.
known A, B and O red cells with each batch on (a tile 5. Try to re-suspend the cells by gentle agitation
or in the test tubes) to determine their effectiveness. and read macroscopically for agglutination or
haemolysis.
INDIRECT GROUPING (REVERSE 6. Confirm a negative result by microscopy.
TYPING):
Quality Control: The reagent red cells used for
This can also be performed by the tile or the test- reverse grouping should be cross-checked against
tube method. known antisera with each batch. It is better to adopt a
The Tile Method: standard procedure for recording the results on a
1. Divide the tile (with a grease pencil) into the A, B, work sheet. A sample work sheet is shown in table.

.Table3: Work Sheet for Blood-Grouping Results

Donor/
Anti-A Anti-A1 Anti-B Anti-AB Anti-D A cells B cells Auto control Result
Patient ID
1 +++ +++ - +++ +++ - +++ - A, ’D’ Pos
2 +++ - - ++ +++ - +++ - A2 ’D’ Pos
3 - - +++ +++ - +++ - - B, ‘D’ Pos
4 +++ ++ +++ +++ ++ - - - AB, ‘D’ Pos
5 - - - - + +++ ++ - O, ‘D’ Pos
6 - - - - - +++ ++ - O, ‘D’ Neg

Table 4: Causes of Discrepancies in ABO and Rh Grouping

False positive result False negative results
Rouleaux formation Impotent sera
Auto/allo antibodies Failure to add grouping sera
Cell lysis in reverse grouping

Table 5: ABO Blood Groups, Sub-groups, Antigens & Antibodies

Blood group subgroup Antigens on cells Antibodies in plasma
A A1 A + A1 Anti B
A2 A Anti A1#
B - B Anti A
AB A1B A + A1 + B None
A2B A+B Anti A1#
O - - Anti A
- - Anti B

#
In 2% of A2 subjects and 25-30% A2B subjects.
334 | P a g e
4. Mix and centrifuge for 15 seconds.
Du TESTING: 5. Re-mix the cells gently and observe for
1. Place one drop of anti-D serum in a test tube. agglutination.
2. Add one drop of the patient’s cell suspension to it. 6. Confirm microscopically, for the presence of
3. Incubate the test tube at 37°C for 30-60 min. agglutinates or otherwise.
4. Wash the cells five times with saline. Interpretations:
5. Add 2 drops of antiglobulin (Coombs) serum, mix Agglutination indicates a positive test which means that
and centrifuge at 3400 RPM for 15 seconds. the red cells have been sensitised in vivo either with an
6. Read for agglutination. Agglutination in the test antibody alone or with complement components. A valid
indicates a Du variant. negative test indicates a lack of in vivo sensitisation or
7. If there is no agglutination, add one drop of check insufficient globulin or complement molecules on the red
cells to the test tube. Centrifuge at 3400 RPM for cell surface to allow detection.
10-15 seconds and read for agglutination. If the
check cells also show no agglutination, then the INDIRECT ANTIGLOBULIN TEST
antiglobulin (Coombs) test is invalid and must be (IAT)
repeated.
The Indirect Coombs Test is used to demonstrate
Quality Control: Anti-D serum should be tested circulating antibodies in the serum, which do not
against known Rh-positive and Rh-negative red cells agglutinate cells suspended in saline. This
with each batch of tests. depends on the combination in vitro of an
antibody with its specific antigen. In the indirect
ANTIGLOBULIN TEST (COOMBS test, normal O + Group red cells are exposed to a
TEST) serum suspected of containing an antibody and
are subsequently tested after washing to see
In some cases, a small antibody molecule such as IgG whether they have been sensitised or otherwise.
can sensitise red blood cells but cannot produce Two steps are required. The first step involves
agglutination. The small size of the antibody’s the incubation of the serum with known O Group
molecules makes them unable to overcome the forces red cells to allow them to become coated with the
that cause red blood cells to repel one another and antibody (if it is present in the serum). The
hence fail to form cross-linked bridges that connect second step involves testing for sensitised cells
cells. In 1945, Coomb et al described a test for as in a Direct Coombs Test.
detecting these non-agglutinating, coating (sensitising)
antibodies. Later, the same test was used to Indications:
demonstrate the coating of red blood cells with 1. Compatibility testing (cross match).
complement components as well. This test is known as 2. Detection and identification of irregular
the Antiglobulin Test or Coombs Test. The antibodies.
antiglobulin test is performed in two ways: The Direct 3. Detection of antibodies, e.g. Kell, Duffy and Kidd,
Antiglobulin Test (DAT) and indirect Antiglobulin etc.
Test (IAT). 4. Investigation of Immune Haemolytic Anaemias.

THE DIRECT ANTIGLOBULIN TEST Procedure:

1. Two volumes (drops) of serum are placed in a test
(DAT) tube.
The Direct Antiglobulin Test initiates the agglutination 2. One volume (drop) of 3% red cell suspension is
of human red blood cells that have already been added to it.
sensitised in vivo by antibodies or complement 3. Mix thoroughly.
components. Coombs serum, containing both anti- 4. Incubate at 37°C for 50 minutes.
human globulin and anti-complement antibodies, can 5. Wash these cells three times with normal saline.
detect both of these sensitised cells by inducing visible 6. After the removal of the saline of the last wash,
agglutination. add 2 drops of Coombs Reagent.
7. Mix and centrifuge for 15 seconds.
Indications: 8. Re-mix the cells gently and observe for
It is indicated for the determination of antibody-coated agglutination. Confirm microscopically.
red cells in the haemolytic disease of newborns, auto- 9. If the test is negative, add 1 drop of check cells to
immune haemolytic anaemia and following haemolytic confirm the validity of the Coombs serum.
transfusion reactions. 10. Reduce the incubation time to 10 minutes if an
Procedure: equal volume of LISS is added to the patient’s
1. Wash the patient’s red cells three times with serum.
normal saline. Interpretations:
2. Add a volume (drop) of 3% washed red cell
suspension in a test tube. The presence of agglutination indicates the presence of
3. Add 2 drops of Coombs Reagent. antibodies in the test serum that are capable of reacting
335 | P a g e
with the test cells. If a known antiserum is used, the when transfused, will have acceptable survival
test will indicate the presence of the corresponding and to prevent transfusion of incompatible donor
antigen. RBC’s that may result in immune mediated
haemolytic transfusion reaction.
Quality Control: The antiglobulin serum should be
checked against known, sensitised cells. The sensitised
red cells may either be commercially purchased or Steps of pre-transfusion testing
prepared in the laboratory. 1. Transfusion Request Form
Oral, electronic or written
Preparing Check Cells: take 1 ml serum from a
2. Positive identification of recipient and recipient’s
D-negative patient who has already been
blood sample
sensitised by exposure to D-positive fetal cells
Positive patient identifies include first and last
during pregnancy/delivery. The titre of anti-D
name, identity card, identification mark or unique
antibodies should be at least 1/16. Mix this
identification number, bed number diagnosis
serum (containing anti-D IgG) with 1 ml washed
3. ABO and D typing of recipient’s blood
O positive cells and incubate at 37°C for 30
4. Antibody detection tests using the recipient’s
minutes. Wash the cells and make a 3%
serum of plasma
suspension with saline. These IgG-coated check
5. Selection of blood components of the appropriate
cells may be used to check the validity of the
ABO and D types (Tubes)
Coombs Test.
Table 6: Red cell concentrate (RCC) transfusion
SOURCES OF ERROR IN policy
ANTIGLOBULIN TESTS
Recipient 1st 2nd 3rd 4th
False ‘Negative’ Tests: Group Choice Choice Choice Choice
1. The test tubes or pipettes may be dirty. OO OO NIL NIL
Positive Positive
2. The red cells may have been inadequately washed.
OO OO OO -
3. Proteins on the fingertips may neutralise AHG and Negative Negative Positive*
thus a false negative result may be obtained. AYE AYE OO NIL NIL
4. The incubation time was too short /too long. Positive Positive Positive
5. The incubation was at a temperature that did not AYE AYE OO AYE OO
Negative Negative Negative Positive* Positive
activate the antibody. BEE BEE OO Nil Nil
6. There was a delay in reading the test or in Positive Positive Positive
performing the test, thus allowing the antibody to BEE BEE OO Bee OO
be eluted off the red cells. Negative Negative Negative Positive Positive
7. The test cells were stored improperly, causing AB AB AYE BEE OO
Positive Positive Positive Positive Positive
them to lose activity. AB AB AYE BEE OO
8. The antiglobulin serum is inactive (improper Negative Negative Negative Negative Negative
storage) or it was not added at all.
9. A change in the optimal ratio of antibody to *Only for dire emergencies and non-availability of OO
antigen. negative. This is appropriate for male patients and
10. If plasma, rather than serum, was used. elderly females. Clinician in charge of the case and
11. Under-centrifugation. pathologist should approve the use of such blood.
False ‘Positive’ Tests:
1. A presence of heavy metal ions and colloidal silica Table7: FFPs & Platelet transfusion policy
in the saline solution can cause non-specific
agglutination. Recipient 1st 2nd 3rd 4th
2. Bacterial contamination of the test cells due to Group Choice Choice Choice Choice
improper storage.
O O* A B AB**
3. Refrigerated, clotted blood results in a non- A A AB** B
specific binding of C4, which can react with the B B AB** A
anti-complement of the antiglobulin serum. AB AB** A** B
4. Over centrifugation will result in a false positive
test. *Group O FFP must only be given to group O patients.
Tested and found to be negative for high-titer ABO
antibodies
PRE-TRANSFUSION COMPATIBILITY **Efforts must be made to maintain a sufficient of AB
TESTING plasma in the inventory.
Purpose Note: Positive or negative for Rh D group while
The purpose of pre-transfusion testing is to issuing FFP is not very important because there are no
select, for each recipient, blood components that, red cells in the plasma and so almost no chance of
336 | P a g e
alloimmunization of the recipient. is being used or 50 min if albumin is used.
7. Enhancement media use is not recommended
6. Compatibility Testing because it is difficult to maintain ratios.
Type and screen protocol: Has emerged as an 8. Wash the cells three times with normal saline.
acceptable alternative to cross-matching blood. The 9. After the last wash, add 2 drops of Coombs serum
protocol consists of performing an ABO, D and in all of the test tubes.
antibody screen for the presence of alloantibodies. 10. Centrifuge for 15 seconds at 3400 rpm and read
Cross-Match: Testing the recipient’s serum or plasma the results.
and donor’s RBC’s 11. The results are read macroscopically and
microscopically.
The Coombs Cross-match (Major Cross-match):
Patient’s serum is added to each potential donor cell Interpretations:
and read after 3 phases: IS (Immediate spin), 37oC and The highest dilution showing agglutination
AHG. Reactivity at any phase was evaluated. microscopically indicates the titre of the antibody in
the serum. While reporting the titre, the dilution prior
The Immediate Spin Cross-match (Abbreviated to the highest one showing agglutination is reported.
Cross-match): When no clinically significant
antibodies are detected in the antibody screen, and Caution:
there is no history of such antibodies, the antiglobulin Store the previous sample (frozen in the lab) and re-test
phase of the cross-match is not required. It can also be it along with the next sample of the same patient for
done for emergency release of blood, when there is no antibody titration. The results should always be
time for major cross-match. compared with those that were previously reported.
Table 8: Work Sheet for Recording Compatibility Test Results
Computerized Cross-match
Patient ID: Patient Blood Group:
If a computer has been validated on site to
Donor ID/ Donor Saline Saline
present the release of ABO – incompatible blood Bag blood phase phase
Coombs
Result
components, it may be used when the antibody screen phase
number group Room temp 37°C
37°C
is negative. It is important that there have been 2
determinations of the recipient’s ABO group. The
computer system contains the donor unit number, the
component name, ABO and D types. There should be a
method to ensure correct entry. ANTIBODY SCREENING
The testing of donor’s serum for unexpected blood-
7. Labelling of the components with the recipient’s group antibodies is required because these antibodies
information. adversely affect the red cells of recipients. Cell panels
of known antigen specificity are available
Rh D ANTIBODY TITRATION commercially. The range varies with each size of the
panel. The important antibodies are covered in a three-
Antibody titration is a semi-quantitative means of cell panel.
assessing the amount of antibody in the serum. This is
usually done in Rh-incompatible mothers with a view Procedure:
to induce labour if the titre progressively rises. It 1. Place three test tubes in a rack and label them as I,
should be done after detection of the antibody by IAT II and III.
and identification by the cell panels. 2. Add 2 drops of patient’s serum in each tube.
3. Add 1 drop of commercial, phenotyped red cells
Procedure: from each vial to the corresponding test tube.
1. Set up 10 test tubes in a rack. Label them as 1/1, 4. Add 2 drops of LISS. Incubate at 37°C for 10 minutes.
1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512 5. Wash three times with normal saline.
and 1/1024. 6. Add 2 drops of Coombs serum and centrifuge for
2. Add 2 drops of saline in each starting from the 15 seconds at 3400 RPM.
second (1/2) tube. 7. Look for agglutination and record.
3. Add 2 drops of the patient's serum in the first and
second tubes. Interpretations:
4. Mix well and transfer 2 drops from the 2nd test Agglutination indicates the presence of an antibody.
tube to the third. Mix well and transfer 2 drops to The results are interpreted according to the sheets that
the next tube and so on until the last tube is are provided with the commercially-prepared red cell
reached. Discard 2 drops from the last tube. panels.
5. Add one drop of 2-5% known O Rh D- positive
cell suspension in saline in each tube and ANTIBODY SPECIFICITY
centrifuge for 15 seconds at the rate of 3400 RPM. For the purpose of identifying the specificity of the
Look for agglutination. antibody detected in the screening, larger cell panels with
6. Incubate the test tubes at 37°C for 15 min if LISS known specificity are required. The most commonly used
337 | P a g e
is the commercially available 11-cell panel. 4. Incubate for 30 minutes.
5. Centrifuge at the rate of 3400 RPM for 10 minutes
Procedure:
(the centrifuge cups should be pre-chilled to 4°C
1. Place 11 test tubes in a rack and label them from 1
or warmed to 37°C, depending upon the antibody
to 11.
involved).
2. Add 2 drops of patient’s serum in each tube.
6. Remove the supernatant (serum) and test it for
3. Add 1 drop of commercial cells from each vial to
complete adsorption of the antibody with cells
the corresponding test tube.
carrying the antigen.
4. Add 2 drops of LISS. Incubate at 37°C for 10 minutes.
7. Further adsorptions may be required, if the
5. Wash four times with normal saline.
antibody is not completely removed.
6. Add 2 drops of Coombs serum and centrifuge for
15 seconds at the rate of 3400 rpm. ELUTION
7. Look for agglutination and record.
Elution is the process by which an adsorbed antibody is
Interpretations: broken down from the antigen antibody complex with
Read the antibody specificity from the manufacturer's the help of heat, alcohol, ether or acid so that the
chart (provided with the panel). antibody is liberated. This technique is used for the
identification of certain antibodies. The elution should
SCREENING FOR HAEMOLYSINS be tested immediately. If it is to be stored, then bovine
Blood Group O contains anti-A and anti-B antibodies, albumin (to a final concentration of 10 mg/100 ml)
which may be haemolysins. When such blood having a should be added to protect the antibodies.
high titre of these antibodies is transfused to persons of
blood groups A, B, or AB it may induce haemolysis of HEAT ELUTION:
the recipient’s red cells. Such Group O blood units are Heat elution is best suited for the investigation of ABO
designated as dangerous universal donor. This blood haemolytic disease of the newborn and the elution of
must be identified and used only for O-Group IgM antibodies from the red cells.
recipients. A label reading ‘For O-Group Recipients 1. Wash the red cells in saline (at least four
Only’ must be applied to such a bag to avoid using it washings).
for non-group O recipients. 2. Centrifuge at the rate of 3400 rpm for 5 minutes.
Procedure: 3. To the washed, packed cells, add an equal amount
1. Take two test tubes and label them as A and B. of saline.
2. Add 2 drops of the donor’s serum in each test 4. Mix and agitate continuously in a water bath at
tube. 56°C for 10 minutes.
3. In Test Tube A, add one drop of known A cell 5. Centrifuge rapidly while still hot and remove the
suspension. cherry-red supernatant. This is the elution.
4. In Test Tube B, add one drop of known B cell ETHER ELUTION:
suspension.
5. Keep both test tubes at 37°C for 2 hours. Ether elution is suitable for investigating a positive
6. Centrifuge and examine for evidence of Direct Antiglobulin Test associated with warm reactive
haemolysis (a pink-coloured supernatant). (IgG) auto or allo antibodies.
1. Wash the red cells in saline (at least four washings).
Interpretations: 2. Centrifuge at the rate of 3400 RPM for five
If there is haemolysis, it means that the blood is not minutes.
safe and should not be given to any other bood group, 3. To the washed packed cells, add an equal amount
only to Group O recipients. of saline.
ANTIBODY ADSORPTION 4. Add a volume of ether twice that of the packed red
cells.
Adsorption is a process by which an antibody is 5. Shake the tube vigorously for one minute by
allowed to react with an antigen of the cell’s membrane keeping the thumb on it and frequently allowing
to isolate it from the serum. The process is used for the release of vapours.
removing the unwanted antibodies from the serum for 6. Place at 37°C for 30 minutes, mixing frequently.
various purposes. It is also used for antibody 7. Centrifuge at the rate of 3400 RPM for five minutes.
identification after elution and for the detection of Three layers will be formed: a top layer of clear ether,
weakly- expressed antigens on the red cells. a middle layer of de-natured red cell stroma and a
Procedure: bottom layer of haemoglobin-stained elution.
1. Wash the cells in normal saline six to eight times. 8. Remove the top two layers by aspiration and
2. Mix one volume packed cells with one volume discard.
serum. 9. Centrifuge the elution at high speed and transfer it
3. Place the mixture in a water bath at 37°C for warm into another tube.
antibody adsorption or in the refrigerator (at 4°C) 10. The elution can be tested immediately or stored
for cold antibodies. frozen at -20°C.
338 | P a g e
THE TEST FOR COLD AGGLUTININS ii) Protozoal
(1) Malaria (Plasmodium spp.)
Cold agglutinins are antibodies that react best at cold (2) Chaga’s disease (T. cruzi)
temperatures. The following procedure is used for their iii) Bacterial
detection/ titration: (1) Syphilis (Treponema pallidum)
1. Place 11 test tubes in a rack and label them as 1/1, (2) Yersinia enterocolitica
1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512 (3) Pseudomonas spp.
and 1/1024. (4) Citrobacter, E. coli
2. Add 2 drops of saline into each tube. (5) Others
3. Add 2 drops of the patient's serum in the first tube. iv) Prions
4. Mix well and transfer 2 drops from the 1st tube to (1) CJD (Creutzfeld-Jakob disease)
the 2nd tube. Repeat this transfer to the last tube b. Circulatory overload
from which 2 drops are discarded. c. Citrate toxicity
5. Add 2 drops of a 5% suspension of pooled Group d. Potassium toxicity
O cells to each tube. e. Acid overload
6. Mix gently and place the rack at 4°C overnight f. Thrombophlebitis
(not less than 6 hours). g. Air embolism
7. Remove the rack, centrifuge, examine for h. Transfusion haemosiderosis
agglutination and record the findings. i. Complications of massive transfusions -
Interpretation: i) Dilution of coagulation factors
The highest dilution that shows agglutination indicates ii) Dilutional thrombocytopenia
the titre of the ‘cold’ antibodies. iii) Hyperkalaemia
iv) Hypocalcaemia
THE COMPLICATIONS OF BLOOD
TRANSFUSIONS THE INVESTIGATION OF HAEMOLYTIC
TRANSFUSION REACTIONS
The transfusion of blood and blood products are
associated with certain risks and unfavourable effects. The following tests should be carried out in cases of
Due to this, blood products should only be any untoward reaction following a blood transfusion.
administered when alternate forms of therapy do not 1. Bedside check: Immediately check all of the issue
exist or are less effective. The side effects can be forms, the blood bag and the patient’s identification.
classified as follows: Record and inform if any discrepancy is found.
1. IMMUNOLOGICAL: 2. Check for haemolysis:
a. Examine the patient’s plasma and urine for
a. Due to red cell antibodies -
haemoglobin.
i) Sensitisation to red cell antigens
b. The blood film may show spherocytosis or
ii) Haemolytic transfusion reactions
agglutination.
(Immediate and delayed)
c. Bio-chemical evidence – including the
b. Due to white cell antibodies -
bilirubin and haptoglobin levels
i) Febrile transfusion reactions
ii) Transfusion-related acute lung injury
(TRALI) 3. Check for incompatibility:
a. Clerical errors: An identification error will
iii) Transfusion-associated graft versus host
indicate the type of incompatibility. Re-check
disease (TA GVHD)
the patient’s particulars on the Requisition
c. Due to platelet antibodies -
Form, pre-transfusion cross- match sample
i) Platelet refractoriness
and post-transfusion sample.
ii) Post-transfusion purpura
b. Serological workup:
d. Due to plasma protein antibodies -
i) Repeat the ABO and Rh D group of the
i) Urticaria
patient (pre and post-transfusion samples)
ii) Anaphylaxis
and the donor’s unit.
2. NON IMMUNOLOGICAL: ii) Screen the patient’s serum (pre and post-
transfusion) for red cell antibodies.
a. Transfusion-transmitted infections - iii) Repeat a cross-match with pre and post-
i) Viral transfusion serum.
(1) Hepatitis B Virus(HBV) iv) Direct Antiglobulin Test (pre and post-
(2) Hepatitis C Virus(HCV) transfusion samples).
(3) HIV 1 and 2 v) When the Direct Antiglobulin Test is
(4) Cytomegalovirus (CMV) positive, elute the antibody from the cells.
(5) HTLV I and II 4. Check for Disseminated Intravascular Coagulation
(6) Parvovirus B19 (DIC) -
(7) Epstein Barr (EB) virus
339 | P a g e
a. Blood film (red-cell fragmentation) d. If the mother’s serum is not available, use the
b. Platelet count infant’s serum/eluate from the red cells
c. Coagulation screen
In ABO haemolytic disease of the newborn, always
5. Check for bacterial infection -
use group ‘O’ blood preferably suspended in AB
a. Gram stain and culture both the donor’s and
plasma. This is because the corresponding maternal
the recipient’s blood.
antibody is going to cause rapid haemolysis, if adult
6. Check the baseline renal function (urea, creatinine,
A or B cells are used for the exchange transfusion.
electrolytes).
The appropriate blood required in Haemolytic
SPECIAL TRANSFUSION SITUATIONS Disease of the Newborn (other than ABO haemolytic
disease) is shown in Table 33.7)
There are some situations where the provision of
AB B B
compatible blood requires special consideration. A AB A
B AB B
Compatibility Tests in Newborn Infants: O A O
For infants under 4 months of age, both the baby’s and A O O
maternal blood samples should be ABO and Rh D O B O
B O O
grouped, with the maternal serum screened for atypical A B O
antibodies and a DAT done on the baby’s cells. If a B A O
maternal antibody screen is negative and the baby’s AB AB AB
AB A A
DAT is negative, blood of the same ABO and D group
as the infant may be issued without cross matching, Table 9: Haemolytic Disease of Newborns (Cross-
even when repeated small-volume transfusions are Match Policy
required. Infants under the age of 4 months do not
Selection of bloodfor Intra-UterineTransfusions:
make red cell allo-antibodies even after multiple small-
Blood for an intra-uterine transfusion should be tested
volume transfusions. Haemolytic disease of
for compatibility with the mother’s serum. It should be
newborns: Haemolytic disease of newborns is defined
group O, Rh D-negative and K- negative. It is essential
as a decrease in red-cell survival of the infant due to
to repeat the antibody identification on each fresh
the presence of antibodies derived from the mother.
sample of the mother’s serum to identify any new allo-
These antibodies are IgG antibodies that cross the
antibodies that may have formed. Blood for intra-
placenta and enter the foetal circulation. They are
uterine transfusion should be less than 5 days old. Also,
produced in response to a transplacental haemorrhage
it should be CMV-negative, have a PCV <0.6 L/L, be
during pregnancy, in which the foetal red cells carrying
irradiated to a minimum of 25 Gy (to avoid graft versus
antigens that are not present in the mother stimulate the
host disease) and be transfused within 24 hours of
maternal immune system. The most common antibody
irradiation. Plasma-reduced blood or washed red cells
detected in haemolytic disease of newborns is anti D,
suspended in saline should be used.
followed by anti C and, rarely, anti K. Anti A or anti B
in group ‘O’ mothers may have an IgG component that
may result in ABO haemolytic disease of the newborn. AUTOMATION IN BLOOD BANKING
With advanced diagnostic methods, it is possible to
detect haemolytic disease of the newborn during the The increase in workloads and the requirement of
pregnancy and fetal exchange transfusions reliability of tests has resulted in the introduction of
carried out using O Rh D-negative, fresh blood (Hct various automated serological procedures in the blood
>70%), which is leuco-depleted and irradiated prior to bank. These include automation in blood grouping,
the transfusion. The following serological procedures antibody screening, anti-D quantitation and viral
are carried out in the laboratory in order to select the screening of blood donations by ELISA systems.
appropriate blood for transfusion: Various machines used for this purpose are designed
for large workloads and are not suitable for a normal,
1. Mother’s sample - hospital-based blood bank. Some of the techniques and
a. ABO and Rh ‘D’ grouping methods used in automated systems for blood grouping
b. Antibody screening & identification and red cell serology are as follows:
2. Infant’s sample -
a. ABO and Rh ‘D’ grouping 1. Individual Reaction Wells: In this antisera technique,
b. Direct Antiglobulin Test the serum and the red cells are mixed in reaction
c. Identification of an antibody from the elution wells, centrifuged and remixed. The results are read
(if required) by photometric method. Examples include Kontron
3. Cross-Match - Groupamatic Systems.
a. Maternal serum is to be used 2. Microplate method: Several systems are available, in
b. Donor blood unit that is compatible with which the serological reactions are carried out in
mother’s & infant’s blood group(s) microplates.
c. If in doubt, select O negative blood suspended 3. Continuous Flow Systems: In this the antisera react
in AB plasma with the red cells in a continuous system of coils.
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Technicon Autogrouper utilises this technique, which is equipments are interfaced Flow chart for Blood
then interfaced with computer for recording of results. component preparation with computers and printers
4. Gel microcolumns: This includes interaction antisera for recording of the results. It should be emphasized
and red cells in solid phase Sephadex columns. that introduction of automation in the laboratory will
Special centrifuge is required for the centrifugation of require more stringent quality control procedures and
cards holding specific number of columns. This closer monitoring and maintenance. Hence, each
technique has the advantage that it is more laboratory should have critical analysis of costs and
reproducible and does not require any washing step. benefits of any such system, before introducing them
Examples include DiaMed and all the automated as a routine.

FIGURE1:BLOOD COMPONENT PREPARATION

Whole blood
Soft spin3500rpm
4 minutes

Platelet rich plasma Red

Hard spin 4000rpm Cells
7 minutes

Plasma Platelets
Frozen at -30°C

FFP
Controlled
thawing 4°C

Cryosupernatent Cryoprecipitate
341 | P a g e

Table 10: Storage requirements and other information of various blood components.

Fresh Frozen Plasma Cryoprecipitate Red cell concentrate Platelet concentrate

Preparation Fresh plasma rapidly frozen to Thawing FFP unit at 4±2°C Whole blood, centrifugation Whole blood <8 hours
-30°C
Volume 150-275 ml 20±5 ml 280±60 ml 50±10 ml
Contents All coagulation factors, FVIII FVIII, vWF, Fibrinogen, Hct: 0.55-0.75 l/l Platelet count
200 units, Fibrinogen 250-400 FXIII 5.5x1010/unit, erythrocyte
mg/ unit count <1x109/unit, Stable
factors, FV,FVIII
Storage ≤-30°C ≤-30°C 4±2°C 22±2°C
Shelf life >12 months >12 months CPDA-1: 35 days, RCC in 5 days, pooled platelets
AS-1 42 days must be used within 4
hours
Thawing At 37°C water bath within 15- At 37°C water bath for 15 - -
30 minutes minutes, do not warm.
Administration Through filter, without cross Through filter, within 2-6 Through 170 µm filter, ≥19 gauge needle, 170
match hours, pooled precipitate µm filter
within 4 hours
Dosage 10 ml/kg body weight One conc/5kg body weight - Increment 5000/unit
concentrate
Rate of Within 4 hours 10 ml/minute as loading dose Within 2-4 hours 10 minutes/unit
infusion
Required level - Minor bleed: 10-50% of - Corrected count
factor, Major surgery: 80- increment (CCI) >7.5
100%,
Post-op: >50% for 10-14 days
Turnaround - - 30-45 minutes -
time
Holding time - - Normally: 24 hours, -
Exceptionally: 72 hours
Caution May transmit disease May transmit disease Avoid if signs of May transmit disease
deterioration obvious
May transmit disease
Demand type - - Routine: ABO-Rh Single unit platelets
compatible Pooled platelets
Emergency: ABO type
specific without cross match
Disparate situation: O Rh
negative without cross mach
Avoid - - Glucose solutions, lactate -
simultaneous ringer, dextrose, dextrose
administration saline, any other hypotonic
of solution, calcium, etc
Any medication
QC Totoal protein >50 g/L Factor VIII > 70 IU/unit Hct 0.65 – 0.75 Random donor
1% of total Platelet < 50 x 109/L Fibrinogen > 140 mg/dl Haemolysis < 0.8% of red Plt 5.5 x 1010/unit
prepared are Red cell < 6 x 109/unit cells Hb content > 40 g/unit Leucocytes < 0.2 x
tested F VIII 0.7 IU/ml 109/unit
At least 75% or pH <6.4 at the end of
of units tested 70 IU/100ml shelf life
meet Leukocytes < 5 x 106/unit Single donorL
specification (if leukoreduced) Plt 300 x 109 /unit
Leukcocytes < 0.3 x
109/unit
342 | P a g e

Suspected haemolytic transfusion

reaction

Immediate laboratory tests

Evidence of Haemolysis

YES NO

Check for errors

Serological tests
Search for infections and other
Repeat grouping, cross match, causes
antibody screening
Evidence of incompatibility

YES NO

Search for non immune causes of

Provision of compatible blood
haemolysis

Transfusion of
•Cold blood
•Lysed blood
•Osmotic lysis

Figure2: Flow diagram for investigation of suspected transfusion reaction.