2. Blood
• Hematology (Greek Haema = Blood; logy = Study of). Hematology is the branch of
medical science that deals with the study of blood. Blood, along with the
cardiovascular system constitutes the Circulatory system and performs the
following functions:
• 1. Transport. Blood provides a pickup and delivery system for the transport of
gases, nutrients, hormones, waste products, etc. over a route of some 1,12,000 km
of blood vessels, with 60–70 trillion customers (cells).
• 2. Regulation. It regulates the body temperature by transporting heat from the
tissues (mainly liver and muscles) to the skin from where it can be lost. Its buffers
regulate pH of the body fluids, while its osmotic pressure regulates water content
of cells through the actions of its dissolved proteins and ions.
• 3. Protection. The blood protects the body against diseases caused by harmful
organisms by transporting leukocytes and antibodies against more than a million
foreign invaders. It also protects the body against loss of blood after injury by the
process of blood clotting.
3. • Physical features. The blood is denser and more viscous than water, slightly alkaline, sticky to
touch, and salty in taste. It clots on standing, leaving behind serum. The normal total circulating
blood volume amounts to 8% of the body weight, i.e. 5–6 liters in an average adult male weighing
70kg, and 4–5 liters in a female. The interplay of various hormones that control salt and water
excretion in the urine keep the blood volume remarkably constant.
• Composition. Blood consists of 55% of watery liquid plasma that contains various proteins and
other solutes dissolved in it. The rest 45% is the formed elements— mainly the red blood cells
(RBCs) but also white blood cells (WBCs), and platelets (cell fragments). The RBCs are the most
numerous (4.5–5.5 million/mm3) and are medium sized (7–8 μm). Next in number are platelets
(2.5–4.5 lacs/mm3) and are the smallest (2–4 μm) in size. The WBCs number 4000–11000/mm3
and vary in size from 8 to 20 μm. The percentage of whole blood that is red cells is called
hematocrit, its value being 45.
4. Collection of Blood Samples
• Since blood is confined within the cardiovascular system,
the skin has to be punctured before blood can be obtained.
There are two common sources of blood for routine
laboratory tests: blood from a superficial vein by
puncturing it with a needle and syringe, or from skin
capillaries by skin-prick. Arterial blood and blood from
cardiac chambers may be required for special tests.
• None of these samples can be called a representative
sample because there are minor variations in their
composition. But for routine hematological tests, however,
these differences can safely be ignored.
5. THE BLOOD SAMPLE
• The term “blood sample” refers to the small
amount of blood—a few drops or a few
milliliters—obtained from a person for the
purpose of testing or investigations.
• These tests are carried out for aiding in
diagnosis and/or prognosis of the disease or
disorder.
6. Sources and Amount of Blood Sample
i. Capillary blood. The skin and other tissues are richly supplied with
capillaries, so when a drop or a few drops of blood are required, as for
estimation of Hb, cell counts, BT and CT, blood films, micro chemical tests,
etc, blood from a skin puncture (skin-prick) with a lancet or needle is
adequate.
• ii. Venous blood. When larger amounts (say, a few ml that cannot be
obtained from a skin puncture) are needed as for complete hematological
and biochemical investigations, venous blood is obtained with a syringe
and needle by puncturing a superficial vein.
• iiii. Arterial blood. When arterial blood is needed for special tests such as
blood pH, gas levels, etc, an artery such as radial or femoral is punctured
with a syringe and needle. This, however, is not a routine procedure.
• iv. Cardiac catheterization. Blood from a heart chamber, taken through a
cardiac catheter, may be required for special tests.
7. Containers for Blood Sample
• A container is a receptacle into which blood is transferred from the
syringe before sending it to the laboratory. Clean and dry 10 ml
glass test tubes, collection bottles such as clean and dry 10 ml
discarded medicine vials, glass bulbs, etc are the usual ones in use.
• A container may or may not contain an anticoagulant depending on
whether a sample of blood/plasma, or serum is required.
• For a sample of whole blood or plasma. The blood is transferred to
a container containing a suitable anticoagulant. This is to prevent
clotting of blood.
• For a sample of serum. No anticoagulant is used. The blood is
allowed to clot in the container and serum is collected as described
later.
• Obviously, capillary blood does not require a container or
anticoagulant
8.
9. Determination of Hematocrit (Hct)
• Measurement of hematocrit (Hct) or packed cell
volume (PCV) is the most accurate and simplest
of all tests in clinical hematology for detecting the
presence and degree of anemia or polycythemia.
Also, if Hb, RBC count, and PCV are determined at
the same time, various absolute corpuscular
values (e.g., volume and Hb content of a single
red cell) of a person can be determined. These
values help in the laboratory diagnosis of the
type of anemia in a person
10. (Hct)
• The hematocrit represents the packed cell volume; plasma accounts for the rest of the volume.
• Blood represents about 8% of total body weight and has an average volume of 5 liters in women and 5.5 liters in men. It
consists of three types of specialized cellular elements, erythrocytes (red blood cells), leukocytes (white blood cells), and
platelets (thrombocytes), suspended in the complex liquid plasma
• Erythrocytes and leukocytes are both whole cells, whereas platelets are cell fragments.
• The constant movement of blood as it flows through the blood vessels keeps the cellular elements rather evenly dispersed
within the plasma. However, if you put a sample of whole blood in a test tube and treat it to prevent clotting, the heavier
cells slowly settle to the bottom and the lighter plasma rises to the top. Th is process can be speeded up by centrifuging,
which quickly packs the cells in the bottom of the tube
11. (Hct)
• Because more than 99% of the cells are erythrocytes, the hematocrit,
• or packed cell volume, essentially represents the percentage of erythrocytes in the total blood
volume.
• The hematocrit averages about 42% for women and slightly higher,
45%, for men. Plasma accounts for the remaining volume. Accordingly, the average
• volume of plasma in the blood is about 58% for women and 55% for men. White blood
• cells and platelets, which are colorless and less dense than red cells, are packed in a
• thin, cream-colored layer, the buff y coat
12. Plasma water is a transport medium for many inorganic
and organic substances
• Plasma, being a liquid, consists of 90% water. Plasma
water is a medium for material being carried in the
blood. Many inorganic and organic substances are
dissolved in the plasma. Plasma also carries heat
generated metabolically within tissues to the surface of
the skin, where heat energy not needed to maintain
body temperature is eliminated to the environment.
Inorganic constituents account for about 1% of plasma
weight. Th e most abundant electrolytes (ions) in the
plasma are Na+ and Cl–, the components of common
salt. Smaller amounts of HCO3–, K+, Ca2+, and other
ions are present. The most notable functions of these
ions are their roles in membrane excitability, osmotic
distribution of fl uid between the extracellular fl uid
(ECF) and the cells, and buff ering of pHchanges; these
functions are discussed elsewhere.
• The most plentiful organic constituents by weight are
the plasma proteins, which make up 6% to 8% of plasma
weight. We examine them more thoroughly in the
nextsection. Th e remaining small percentage of plasma
consists of other organic substances,including nutrients
(such as glucose, amino acids, lipids, and vitamins),
waste products(creatinine, bilirubin, and nitrogenous
substances such as urea), dissolved gases (O2 andCO2),
and hormones. Most of these substances are merely
being transported in the plasma. For example, endocrine
glands secrete hormones into the plasma, which
transports these chemical messengers to their sites of
action.
13.
14. Blood Constituents and Their
Functions
• Constituent Functions
• Plasma
• Water Acts as transport medium; carries heat
• Electrolytes Play a role in membrane excitability; distribute fl uid osmotically between ECF and ICF; buff er pH changes
• Nutrients, wastes,
• gases, and hormones
• Are transported in blood; blood CO2 plays a role in acid–base balance
• Plasma proteins Exert an osmotic eff ect important in the distribution of ECF between the vascular and the interstitial compartments;
• buff er pH changes; transport many substances; include clotting factors, inactive precursor molecules,
• and antibodies
• Cellular Elements
• Erythrocytes Transport O2 and CO2 (mainly O2)
• Leukocytes
• Neutrophils Engulf bacteria and debris
• Eosinophils Attack parasitic worms; play a role in allergic reactions
• Basophils Release histamine, which is important in allergic reactions, and heparin, which helps clear fat from the
• blood
• Monocytes Are in transit to become tissue macrophages
• Lymphocytes
• B lymphocytes Produce antibodies
• T lymphocytes Produce cell-mediated immune responses
• Platelets Contribute to hemostasis
15. Plasma proteins
MAIN FUNCTIONS
• Plasma proteins are the one group of plasma constituents that are not
along just for the ride. Th ese important components normally stay in the
plasma, where they performmany valuable functions. Here are the most
important of these functions, which are elaborated on elsewhere in the
text:
• 1. Unlike other plasma constituents that are dissolved in the plasma water,
plasma proteins are dispersed as a colloid Furthermore, because they are
the largest of the plasma constituents, plasma proteins usually do not exit
through the narrow pores in the capillary walls to enter the interstitial fl
uid. By their presence as a colloidal dispersion in the plasma and their
absence in the interstitial fl uid, plasma proteins establish an osmotic
gradient between the blood and the interstitial fluid.Th is colloid osmotic
pressure is the primary force preventing excessive loss of plasma from the
capillaries into the interstitial fluid and thus helps maintain plasma volume
• 2. Plasma proteins are partially responsible for plasma’s capacity to buff er
changes in pH
• 3. Some plasma proteins bind substances that are poorly soluble in plasma
for transport in the plasma. Examples of substances
• carried by plasma proteins include cholesterol and thyroid hormone .
• 4. Many of the factors involved in the blood-clotting process are plasma
proteins .
• 5. Some plasma proteins are inactive, circulating precursor molecules,
which are activated as needed by specifi c regulatory inputs. For example,
the plasma protein angiotensinogen is activated to angiotensin, which
plays an important role in regulating salt balance in the body .
• 6. One specifi c group of plasma proteins are antibodies, or
immunoglobulins, which are crucial to the body’s defense mechanism
• Plasma proteins are synthesized by the liver, with the exception of
antibodies, which are produced by lymphocytes, one of the types of white
blood cells.
16. Erythrocyte Sedimentation Rate (ESR)
Draw 2.0 ml of venous blood and transfer it into a vial containing 0.5 ml of 3.8%
sodium citrate solution. This will give a blood: citrate ratio of 4:1. Mix the contents by
inverting or swirling the vial. Do not shake, as it will cause frothing.
2. Fill the Westergren’s pipette with blood-citrate mixture by sucking, after placing the
tip of your finger over the top of the pipette to control the flow of blood into and out
of it, or with a rubber bulb. Bring the blood column to exact zero mark. (If there is a
difference of 1–2 mm, it should be noted and taken into account before giving the
final report at the end of one hour).
3. Keeping your finger (or the rubber bulb) over the pipette, transfer it to the
Westergren stand by firmly pressing its lower end into the rubber cushion. Now slip
the upper end of the pipette under the screw cap. Confirm that there is no leakage of
blood and that the pipette will remain vertical.
4. Leave the pipette undisturbed for one hour at the end of which read the mm of
clear plasma above the red cells.
Express your results as:………………mm 1st hour (Westergren).
Normal values
Males : 3–9 mm 1st hour
Females : 5–12 mm 1st hour
18. Factors Affecting ESR
• The factors that affect the ESR include the following:
• 1. Technical and Mechanical Factors. Factors like the
length of the tube, diameter of the bore (if less than 2
mm), and the anticoagulant used (liquid anticoagulant
in Westergren’s method gives somewhat higher values.
• 2. Physiological Factors. The red cell count, their size
and their shape, raised body temperature, viscosity of
plasma, and tendency to rouleaux formation, all affect
the ESR.
19. Erythrocytes
• Each milliliter of blood on average contains about 5 billion
erythrocytes (red blood cells, or RBCs), commonly reported
clinically in a red blood cell count as 5 million cells per cubic
millimeter (mm3).
• The structure of erythrocytes is well suited to their main
function of O2 transport in the blood.
• The shape and content of erythrocytes are ideally suited to
carry out their primary function, namely, transporting O2.
• Erythrocyte Structure Two anatomic features of erythrocytes
contribute to the efficiency with which they transport O2.
• First, erythrocytes are flat, disc-shaped cells indented in the
middle on both sides, like a doughnut with a fl attened center
instead of a hole (that is, they are biconcave discs) . This
unique shape provides a larger surface area for diffusion of O2
from the plasma across the membrane into the erythrocyte
than a spherical cell of the same volume woul
20. • Second, the most important anatomic feature that enables
• RBCs to transport O2 is the hemoglobin they contain.
• Hemoglobin’s Transport Capacity Hemoglobin is found
• only in red blood cells. A hemoglobin molecule has two parts:
• (1) the globin portion, a protein made up of four highly folded
• polypeptide chains, and (2) four iron-containing, nonprotein
• groups known as heme groups, each of which is bound to one
• of the polypeptides . Each of the four iron atoms can combine reversibly with one molecule of O2;
• thus, eachhemoglobin molecule can pick up four O2 passengers in the
• lungs. Because O2 is poorly soluble in the plasma, 98.5% of the
• O2 carried in the blood is bound to hemoglobin .
• Hemoglobin is a pigment (that is, it is naturally colored).
• Because of its iron content, it appears reddish when combined
• with O2 and bluish when deoxygenated. Thus, fully oxygenated
• arterial blood is red, and venous blood, which has lost some of
• its O2 load at the tissue level, has a bluish cast.
• In addition to carrying O2, hemoglobin can combine with
• the following:
1. Carbon dioxide (CO2). Hemoglobin helps transport this gas
• from the tissue cells back to the lungs .
• 2. The acidic hydrogen-ion portion (H+) of ionized carbonic
• acid, which is generated at the tissue level from CO2. Hemoglobin
• buffers this acid so that it minimally alters the pH of
• the blood .
• 3. Carbon monoxide (CO). This gas is not normally in the blood,
• but if inhaled, it preferentially occupies the O2-binding sites
• on hemoglobin, causing CO poisoning (see p. 369).
• 4. Nitric oxide (NO). In the lungs, the vasodilator nitric oxide
• binds to hemoglobin. This NO is released at the tissues,
• where it relaxes and dilates the local arterioles .
• Vasodilation helps ensure that the O2-rich blood can make
• its vital rounds and helps stabilize blood pressure.
• Therefore, hemoglobin plays the key role in O2 transport
• while contributing signifiantly to CO2 transport and the
• pH-bufffering capacity of blood. Furthermore, by toting along
• its own vasodilator, hemoglobin helps deliver the O2 it is
• carrying.
22. • Hayem’s fluid (RBC diluting fluid): The ideal fluid for
diluting the blood should be isotonic and neither cause
hemolysis nor crenation of red cells. It should have a
fixative to preserve the shape of RBCs and also prevent
their autolysis so that they could be counted even several
hours after diluting the blood if necessary. It should prevent
agglutination and not get spoiled on keeping. All these
properties are found in Hayem’s fluid.
• Composition of Hayem’s fluid.
• Sodium chloride (NaCl) 0.50 g
• Sodium sulfate (Na2SO4) 2.50 g
• Mercuric chloride (Hg Cl2) 0.25 g
• Distilled water 100 ml
23.
24. The Red Cell Count
Compound microscope: (1) Base,
(2) Pillars, (3) Handle, (4) Body
tube, (5) Coarse adjustment screw,
(6) Fine adjustment screw, (7) Fixed
stage, (8) Mechanical stage, (9)
Fixed and revolving nose pieces,
(10) Objective lenses, (11) Mirror,
(12) Condenser, and (13) Eye-piece
25. The Red Cell Count
The Counting Grid
• The ruled area on each floor piece, the counting grid, has the following dimensions:
• • The central densely etched large square (1 mm × 1 mm), called the RBC square, is divided into 25
medium-sized squares, each of which has a side of 1/5 mm.
• • Each of these medium squares is set off (separated) from its neighbors by very closely placed double
lines (tram lines) or triple lines. These double or triple lines extend in all directions
27. The Red Cell Count
Formula: N/80*S*H*D
N=nr of rbc
80=nr of small square
S=area of a small sguare=1/400
H=hight of chamber=1/10
D=dilution=100 or 200
Normal Red Cell Count
• Express your result as ..... million/mm3
Males = 5.0 million/mm3 (4.75 – 6.0 million/mm3)
Females = 4.5 million/mm3 (4.0 – 5.5 million/mm3).
28. Estimation of Hemoglobin
• The hemoglobin concentration is always estimated as part of routine tests in
outpatients department and also as a bedside test in indoor patients. It is
indicated as part of complete hematological studies in all diseases of blood,
especially all types of anemias, leukemias, and in chronic diseases such as
tuberculosis, Hemoglobinometry
• The term refers to measurement of the concentration
• (amount) of Hb in the blood. For this purpose,
• Hematology 35
• advantage is taken of the following characteristics
• of Hb:
• 1. Ability to combine with oxygen.
• 2. Presence of known amount of iron in each gram
• of Hb.
• 3. Ability of a solution of a derivative of Hb to refract
• specific wavelengths of light, thus giving typical
• absorption bands.
29. • PRINCIPLE
• The Hb present in a measured amount of blood
• is converted by dilute hydrochloric acid into acid
• hematin, which in dilution is golden brown in color. The
• intensity of color depends on the concentration of acid
• hematin which, in turn, depends on the concentration
• of Hb. The color of the solution (i.e. its hue and depth),
• after dilution with water, is matched against
goldenbrown
• tinted glass rods by direct vision. The readings
• are obtained in g%.
30. • Sahli (Sahli-Adams) Hemoglobinometer (Hemometer). The set consists of:
• 1. Comparator. It is a rectangular plastic box with a slot in the middle which accommodates the calibrated Hb tube.
Non-fading, standardized, golden-brown glass rods are fitted on each side of the slot for matching the color. An opaque
white glass (or plastic) is fitted behind the slot to provide uniform illumination during direct visual color matching.
• 2. Hemoglobin tube. The square or round glass tube is calibrated in g Hb % (2–24 g%) in yellow color on one side,
and in percentage Hb (20–160%) in red color on the other side. There is a brush to clean the tube (Figure 1-10A).
• 3. Hemoglobin pipette. It is a glass capillary pipette with only a single calibration mark-0.02 ml (20 cmm, cubic
millimeters; or 20 ml, micro liters). There is no bulb in this pipette (as compared to cell pipettes) as no dilution of
blood is done. Figure 1-10B shows the Hb pipette.
• Note The calibration mark 20 cmm indicates a definite, measured volume and not an arbitrary volume, as is the case
with diluting pipettes.
• 4. Stirrer. It is a thin glass rod with a flattened end which is used for stirring and mixing the blood and dilute acid.
• 5. Pasteur pipette. It is a 8–10 inch glass tube drawn to a long thin nozzle, and has a rubber teat. Ordinary glass
dropper with a rubber teat also serves the purpose.
• 6. Distilled water.
• B. Decinormal (N/10) hydrochloric acid (0.1 N HCl) solution. Mixing 36 g HCl in distilled water to 1 liter gives
‘Normal’ HCl; and diluting it 10 times will give N/10 HCl solution.
• C. Materials for skin prick.
• • Sterile lancet/needle
• • Sterile gauze and cotton swabs
• • Methylated spirit/70% alcohol
31. • Using a dropper, place 8–10 drops of N/10 HCl in the Hb tube,
or up to the mark 20% or 3 g, or a little more till the tip of the
pipette will submerge, and set it aside.
• 2. Get a finger prick under aseptic conditions, wipe away the
first 2 drops of blood. When a large drop of free-flowing blood
has formed again, draw blood up to the 20 cmm mark (0.02
ml). Carefully wipe the blood sticking to the tip of the pipette
with a cotton swab, but avoid touching the bore or else blood
will be drawn out by capillarity.
• Note
• If any blood remains sticking to the outside of the pipette, it
will be that much extra blood in addition to 20 cmm.
32. • Without any waiting, immerse the tip of the pipette to the bottom of the acid
solution and expel the blood gently. Rinse the pipette 3–4 times by drawing up and blowing out the clear upper part of
the acid solution till all the blood has been washed out from it. Avoid frothing of the mixture. Note the time.
• 4. Withdraw the pipette from the tube, touching it to the side of the tube, thus ensuring that no mixture is carried out
of the tube. Mix the blood with the acid solution with the flat end of the stirrer by rotating and gently moving it up and
down.
• 5. Put the Hb tube back in the comparator and let it stand for 6–8 minutes (or as advised by the manufacturer). During
this time, the acid ruptures
• the red cells, releasing their Hb into the solution (hemolysis). The acid acts on the Hb and converts it into acid hematin
which is deep golden brown in color.
• • The color of acid hematin does not develop fully immediately, but its intensity increases with time, reaching a
maximum, after which it starts to decrease. An adequate time, usually 6–8 minutes, must be allowed before its dilution
is started. Too little time and all Hb may not be converted into acid hematin. And, waiting too long, may result in
fading of color. In either case, the result will be falsely low.
• 6. Diluting and matching the color. The next step is to dilute the acid hematin solution with distilled water
(preferably buffered water, if available) till its color matches the color of the standard tinted glass rods in the
comparator.
• Take the Hb tube out of the comparator and add distilled water drop by drop (or
larger amounts depending on the experience), stirring the mixture each time and comparing the color with the
standard.
• 8. Hold the comparator at eye level, away from your face, against bright but diffused light. Read the lower meniscus
(lower meniscus is read in colored transparent solutions).
33. • The average levels and their
ranges are as follows:
• Males: 14.5 g/dl (13.5–18 g/dl).
• Females: 12.5 g/dl (11.5–16 g/dl
• https://www.youtube.com/watch?v=mWAEIv
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34. Normal Blood Standards
Absolute Corpuscular Values and Indices
• The basic values of Hb, RBC count, and PCV (Hct) do
not give any information about the condition of an
average red cell, such as its volume, Hb content, or
its percentage saturation with Hb. Neither can this
information, which is important in diagnosing the type
of anemia in a patient, be obtained directly from any
experimental method. However, this information, in
the form of absolute corpuscular values, especially if
these are done electronically, can be calculated from 3
basic values of Hb, RBC count, and PCV.
• Further, the basic values found in a patient/subject
can be compared with arbitrarily set “normal” values.
This information, the red cell indices, have been
discarded in favor of absolute corpuscular values.
35. I. Mean Corpuscular Volume (MCV)
• The MCV is the average or mean
volume of a single red blood cell
expressed in cubic micrometers (μm3 or
femtoliters). It is calculated from the
following two basic values:
• i. Red cell count in million/mm3
• ii. Packed cell volume (PCV) in 100 ml
blood.
36. II. Mean Corpuscular Hemoglobin (MCH)
The MCH, which is also determined indirectly, is the
average hemoglobin content (weight of Hb) in a single red
blood cell expressed in picograms (micro-microgram, μμg).
It is calculated from the following basic values:
• i. RBC count in million/mm3.
• ii. Hb in g percent.
Formula
37. III. Mean Corpuscular Hemoglobin Concentration (MCHC)
• The MCHC represents the relationship between the red cell
volume and its degree or percentage saturation with
hemoglobin, that is, how many parts or volumes of a red
cell are occupied by Hb. The MCHC does not take into
consideration the RBC count, but represents the actual Hb
concentration in red cells only, (i.e., not in whole blood)—
expressed as saturation of these cells with Hb.
• The Hb synthesizing machinery of red cells does not have
the Hb concentrating capacity beyond a 59 Hematology
• certain limit, i.e., RBCs cannot be, say 70% “filled” with Hb;
this upper limit is only 36%.
