Hematology Quick study

http://www.irvingcrowley.com/cls/maturation.htm

Identify the sites of haemopoeitic tissue in fetal, neonate and adult life

Sites of haemopoiesis.
Fetus
02 months (yolk sac)
27 months (liver, spleen)
59 months (bone marrow)
Infant
Bone marrow (practically all bones)
Adults
Vertebrae, ribs, sternum, skull, sacrum and pelvis, proximal
ends of femur

List the cell lines in the bone marrow that are derived from the pluripotential

stem cells

Outline the development of red cells, granulocytes and platelets from the
pluripotential stem cell to the end cell.

Identify the growth factors (including cytokines) which infl uence the
development of various cell lines
Haemopoietic tissue growth factors

This is a diagram of the role of growth factors in normal haemopoiesis. Multiple growth factors
act on the earlier marrow stem and progenitor cells. EPO, erythropoietin; PSC, pluripotential stem
cell; SCF, stem cell factor; TPO, thrombopoietin.

Outline the role of connective tissues elements in Haemopoietic function

FUNCTIONS OF CONNECTIVE TISSUE

Structural support

The connective tissues serve several functions, of which the most prominent function is structural
support to enable maintenance of anatomical form of organs and organ systems. Examples include
the connective tissue capsules surrounding organs (such as the kidney, lymph nodes). The loose
connective tissue acts to fill the spaces between organs. The tendons (connecting muscles to bone)
and the elastic ligaments (connecting bones to bones) are examples of specialized orderly forms of
connective tissue. The skeletal tissues (cartilage and bone) are special forms of connective tissue.

Metabolic functions

The connective tissues serve a nutritive role. All the metabolites from the blood pass from
capillary beds and diffuse through the adjacent connective tissue to cells and tissues. Similarly
waste metabolites from the cells and tissues diffuse through the loose connective tissue before
returning to the blood capillaries.
The adipose tissue (especially that of the hypodermis) serves as an energy store and also provides
thermal insulation. Surplus calories can be converted into lipid and stored in adipocytes.

Blood components and blood vessels

The hematopoietic tissues (blood-forming tissues) are a further specialized form of connective
tissue. These include the myeloid tissue (bone marrow) and the lymphoid (lymphatic) tissue. The
lining of the blood and lymphatic vessels (endothelial cells) as well as the peripheral blood, are
also specialized forms of connective tissue.

Defensive functions

Various components of the connective tissue play roles in the defense or protection of the body
including many of the components of the vascular and immune systems (plasma cells,
lymphocytes, neutrophils, eosinophils, basophils, mast cells). The various macrophages of the body
are also categorized as connective tissue cells. These all develop from monocytes and are grouped
as part of the Mononuclear Phagocyte System of the body. Macrophages are important in tissue
repair as well as defense against bacterial invasion. The fibroblasts of connective tissue proliferate
in response to injury of organs and migrate to and deposit abundant new collagen fibers, resulting
in the formation of fibrous scar tissue.
Cell type

Chief function

Mesenchyme

Embryonic source of all connective tissue cells

Fibroblasts
Chondroblasts
Osteoblasts

Structural support

Plasma cells
Lymphocytes
Neutrophils
Eosinophils
Basophils
Mast cells
Macrophages

Defense and immune

Adipocytes

Metabolic
Energy storage
Thermal insulation

The formed elements of the blood. List the cellular components in peripheral
blood.

Identify site of action of erythropoietin, GCSF and GMCSF

This is a
diagrammatic representation of the bone marrow pluripotent stem cell and the cell lines that
arise from it.
Various progenitor cells can be identified by culture in semi-solid medium by the type of colony
they form.
Baso, basophil; BFU, burst-forming unit; CFU, colony-forming unit; E, erythroid; Eo, eosinophil;
GEMM, granulocyte, erythroid, monocyte and megakaryocyte; GM, granulocyte, monocyte; Meg,
megakaryocyte; NK, natural killer.

Outline factors which lead to increased production of erythropoietin

List the parameters in the CBC/FBC. Discuss the importance of the CBC/FBC.
To determine general health status and to screen for a variety of disorders, such as anaemia and infection,
inflammation nutritional status and exposure to toxic substances

A CBC test usually includes:

White blood cell (WBC, leukocyte) count. White blood cells protect the body against infection. If an infection
develops, white blood cells attack and destroy the bacteria, virus, or other organism causing it. White blood cells are
bigger than red blood cells but fewer in number. When a person has a bacterial infection, the number of white cells rises
very quickly. The number of white blood cells is sometimes used to find an infection or to see how the body is dealing with
cancer treatment.

White blood cell types (WBC differential). The major types of white blood cells are neutrophils, lymphocytes,
monocytes, eosinophils, and basophils. Immature neutrophils, called band neutrophils, are also part of this test. Each type
of cell plays a different role in protecting the body. The numbers of each one of these types of white blood cells give
important information about the immune system. Too many or too few of the different types of white blood cells can help
find an infection, an allergic or toxic reaction to medicines or chemicals, and many conditions, such as leukemia.

Red blood cell (RBC) count. Red blood cells carry oxygen from the lungs to the rest of the body. They also carry
carbon dioxide back to the lungs so it can be exhaled. If the RBC count is low (anemia), the body may not be getting the
oxygen it needs. If the count is too high (a condition called polycythemia), there is a chance that the red blood cells will
clump together and block tiny blood vessels (capillaries). This also makes it hard for your red blood cells to carry oxygen.

Hematocrit (HCT, packed cell volume, PCV). This test measures the amount of space (volume) red blood cells
take up in the blood. The value is given as a percentage of red blood cells in a volume of blood. For example, a hematocrit
of 38 means that 38% of the blood's volume is made of red blood cells. Hematocrit and hemoglobin values are the two
major tests that show if anemia or polycythemia is present.

Hemoglobin (Hgb). The hemoglobin molecule fills up the red blood cells. It carries oxygen and gives the blood
cell its red color. The hemoglobin test measures the amount of hemoglobin in blood and is a good measure of the blood's
ability to carry oxygen throughout the body.

Red blood cell indices. There are three red blood cell indices: mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). They are measured by a
machine and their values come from other measurements in a CBC. The MCV shows the size of the red blood cells. The
MCH value is the amount of hemoglobin in an average red blood cell. The MCHC measures the concentration of
hemoglobin in an average red blood cell. These numbers help in the diagnosis of different types of anemia. Red cell
distribution width (RDW) can also be measured which shows if the cells are all the same or different sizes or shapes.

Platelet (thrombocyte) count. Platelets (thrombocytes) are the smallest type of blood cell. They are important in
blood clotting. When bleeding occurs, the platelets swell, clump together, and form a sticky plug that helps stop the
bleeding. If there are too few platelets, uncontrolled bleeding may be a problem. If there are too many platelets, there is a
chance of a blood clot forming in a blood vessel. Also, platelets may be involved in hardening of the arteries
(atherosclerosis).

Mean platelet volume (MPV). Mean platelet volume measures the average amount (volume) of platelets. Mean
platelet volume is used along with platelet count to diagnose some diseases. If the platelet count is normal, the mean
platelet volume can still be too high or too low

Give the specimen tube/ anticoagulant used to collect a specimen for a

complete blood count / full blood count (CBC/FBC).

Blood Collection Tubes

Complete Blood Count (CBC):

EDTA tubes (purple top):

Purple top tubes should be 50-60% full. Do not overfill tubes.

Gently mix specimen by inverting 5-10 times and place it on a rocker for up to 30 minutes, then refrigerate at 28C. When a differential is required as part of a CBC, slides must be prepared within 12 hours of blood
collection.

Refrigerated EDTA blood is stable for CBC for up to 24 hours. Clotted or hemolyzed specimens are
unacceptable. Check for clots by using a clean wooden applicator stick and gently swirling blood in tube.

EDTA microtainers must be shaken 10-15 times to overcome the surface tension within the tube

Serum Chemistry:

Plain or serum separator tube (red top or SST, red/camouflage top):

SST tubes should be at least 50-80% full and should be mixed.

No mixing is necessary with plain tubes.

Both plain and SST tubes must be allowed to clot at 4C for 30 minutes - 60 minutes. Then the samples must
be centrifuged at 7000rpm for 10 minutes and the serum decanted and frozen at -80C for clinical chemistry
analysis.

Heparin tube (green top chemistry/blood gas):

Tubes should be at least 50% full.

Gently mix specimen by inverting 8-10 times and place immediately on at 4C for 30 minutes - 60 minutes
before spinning in a centrifuge, although the exact time allowable may be protocol specific.

Coagulation:

Sodium citrate tubes (blue top):

Citrate tubes should be filled to the top.

Gently mix specimen by inverting at least 5-6 times and place immediately at 4C for up to 30 minutes before
spinning in a centrifuge.

Blood Glucose:

Sodium fluoride tubes (gray top):

Tubes should be at least 65-80% full.

Gently mix specimen by inverting 5-6 times and place immediately at 4C for up to 30 minutes before spinning
in a centrifuge

If blood is placed into tubes after removing the tube tops, care must be taken not to cross contaminate tubes
containing anticoagulant. It is best to remove the needle before filling the tubes.

Capillary tubes:

Take capillary tube and place finger on the end of tube that has the blue or red fill line mark on it.

Place other end of the capillary tube into the tip of a blood filled syringe, in a drop of blood on Parafilm, or along
the edge of a tube of blood (being careful not to spill it!).

Release finger and allow blood to fill the capillary tube (If possible to the blue or red line) by capillary action. If
less blood is available, fill as full as is possible.

Green-Top Tube (Sodium Heparin): This tube contains sodium heparin -- used for collection of heparinized plasma or whole blood
for special tests. Note: After tube has been filled with blood, immediately invert tube several times to prevent coagulation.
Grey-Top Tube (Potassium Oxalate/Sodium Fluoride): This tube contains potassium oxalate as an anticoagulant and sodium
fluoride as a preservative -- used to preserve glucose in whole blood and for some special chemistry tests. Note: After tube has been
filled with blood, immediately invert tube several times to prevent coagulation.
Lavender-Top Tube (EDTA): This tube contains EDTA as an anticoagulant -- used for most hematological procedures. Note: After
tube has been filled with blood, immediately invert tube several times to prevent coagulation.

Light Blue-Top Tube (Sodium Citrate): This tube contains sodium citrate as an anticoagulant -- used for drawing blood for
coagulation studies. Note: It is imperative that the tube be completely filled. The ratio of blood to anticoagulant is critical for valid
prothrombin time results. Immediately after draw, invert tube 6 to 10 times to activate the anticoagulant.
Red-Top Tube: This tube is a plain VACUTAINER containing no anticoagulant -- used for collection of serum for selected
chemistry tests as well as clotted blood for immunohematology.
Royal Blue-Top Tube: There are two types of royal bluetop Monoject tubes -- one with the anticoagulant EDTA and the other plain.
These are used for collection of whole blood or serum for trace element analysis. To determine tube type necessary, refer to individual
metals in individual test listings.
Serum Gel Tube: This tube contains a clot activator and serum gel separator -- used for various laboratory tests. Note: Invert tube to
activate clotting; let stand for 20 to 30 minutes before centrifuging for 10 minutes. If frozen serum is required, pour off serum into
plastic vial and freeze. Do not freeze VACUTAINER(S).
Special Collection Tubes: Some tests require specific tubes for proper analysis. To obtain correct tubes for metal analysis or other
tests as identified in individual test listings. Yellow-Top Tube (ACD): This tube contains ACD -- used for drawing whole blood for
special tests

List the functions of the erythrocytes in relation to its structure

The majority of normal red cells or erythrocytes are disciform in shape (Fig. 3.12) [4]; a minority are bowl-shaped. On a
stained peripheral blood film they are approximately circular in outline and show only minor variations in shape and
moderate variations in size (Fig. 3.13). The average diameter is about 7.5 m. In the area of a film where cells form a
monolayer, a paler central area occupies approximately the middle third of the cell.
The normal shape and flexibility of a red cell are dependent on the integrity of the cytoskeleton to which the lipid
membrane is bound. An abnormal shape can be caused by a primary defect of the cytoskeleton or membrane or be
secondary to red cell fragmentation or to polymerization, crystallization or precipitation of haemoglobin.

Fig. 3.12 Scanning electron micrograph of a normal red

cell (discocyte). Courtesy of Professor A. Polliack,
Jerusalem, from Hoffbrand and Pettit [4].

Fig. 3.13 Peripheral blood film of a healthy subject

showing normal red cells and platelets. The red cells show
little variation in size and shape. Some of the platelets
show granules dispersed through the cytoplasm while
others have a granulomere and a hyalomere.

Functions of Red Blood Cells

Apart from carrying oxygen, which is the main function of red blood cell, it can also conduct the
following functions.
1. Release the enzyme carbonic anhydrase which allows water in the blood to carry carbon
dioxide to the lungs where it is expelled.
2. Control the pH of the blood by acting as an acid-base buffer.

Shape and Size of a Red Blood Cell

A red blood cell is a biconcave disc. Simply it is a round ball that is squeezed from two opposite
ends to appear, widest at the sides and narrowest in the middle.
A red blood cell measures about 6 to 8 micrometers in diameter (average = 7.8 um) with an
average thickness of 2 micrometers (2.5 um at the thickest point and less than 1um at the
center). Although a red blood cell is wider than some capillaries, its flexibility allows it to
become distorted as it squeezes through narrow passages and then restores to its original shape.
There shape confers them a degree of flexibility as some capillaries are smaller than the
diameter of a RBC. Secondly, the biconcave shape of the RBC gives it an ideal surface area to
volume ratio for maximum gas exchange.

List the major blood group antigens (e.g. ABO), discuss rhesus blood group
systems in humans and just make note of other systems

RHESUS BLOOD GROUPING SYSTEM

The Rh system was named after rhesus monkeys, since they were initially used in the research to make the
antiserum for typing blood samples. If the antiserum agglutinates your red cells, you are Rh+ . If it doesn't, you
are Rh- . Despite its actual genetic complexity, the inheritance of this trait usually can be predicted by a simple
conceptual model in which there are two alleles, D and d. Individuals who are homozygous dominant (DD) or
heterozygous (Dd) are Rh+. Those who are homozygous recessive (dd) are Rh- (i.e., they do not have the key
Rh antigens).
Clinically, the Rh factor, like ABO factors, can lead to serious medical complications. The greatest problem with
the Rh group is not so much incompatibilities following transfusions (though they can occur) as those between
a mother and her developing fetus. Mother-fetus incompatibility occurs when the mother is Rh- (dd) and her
fetus is Rh+ (DD or Dd). Maternal antibodies can cross the placenta and destroy fetal red blood cells. The risk
increases with each pregnancy. Europeans are the most likely to have this problem--13% of their newborn
babies are at risk. Actually only about of these babies (6% of all European births) have complications. With
preventive treatment, this number can be cut down even further. Less than 1% of those treated have trouble.
However, Rh blood type incompatibility is still the leading cause of potentially fatal blood related problems of
the newborn. In the United States, 1 out of 1000 babies are born with this condition.
Rh type mother-fetus incompatibility occurs only when an Rh+ man fathers a child with an Rh- mother. Since
an Rh+ father can have either a DD or Dd genotype, there are 2 mating combinations possible with differing
risks as shown below. Regardless of the father's genotype, if he is Rh+ and the mother is Rh-, doctors assume
that there will be an incompatibility problem and act accordingly.

Human fetus in a mother's uterus

(the umbilical cord and placenta
connect the fetus to its mother)

Keep in mind that only the Rh+ children (Dd) are likely to have medical complications. When both the mother
and her fetus are Rh- (dd), the birth will be normal.
The first time an Rh- woman becomes pregnant, there usually are not incompatibility difficulties for her Rh+
fetus. However, the second and subsequent births are likely to have life-threatening problems for Rh+ fetuses.
The risk increases with each birth. In order to understand why first born are normally safe and later children are
not, it is necessary to understand some of the placenta's functions. It is an organ that connects the fetus to the
wall of the uterus via an umbilical cord. Nutrients and the mother's antibodies regularly transfer across the
placental boundary into the fetus, but her red blood cells usually do not (except in the case of an accidental
rupture). Normally, anti-Rh+ antibodies do not exist in the first-time mother unless she has previously come in
contact with Rh+ blood. Therefore, her antibodies are not likely to agglutinate the red blood cells of her Rh+
fetus. Placental ruptures do occur normally at birth so that some fetal blood gets into the mother's system,
stimulating the development of antibodies to Rh+ blood antigens. As little as one drop of fetal blood stimulates
the production of large amounts of antibodies. When the next pregnancy occurs, a transfer of antibodies from
the mother's system once again takes place across the placental boundary into the fetus. The anti-Rh+
antibodies that she now produces react with the fetal blood, causing many of its red cells to burst or
agglutinate. As a result, the newborn baby may have a life-threatening anemia because of a lack of oxygen in
the blood. The baby also usually is jaundiced, fevered, quite swollen, and has an enlarged liver and spleen.
This condition is called erythroblastosis fetalis . The standard treatment in severe cases is immediate
massive transfusions of Rh- blood into the baby with the simultaneous draining of the existing blood to flush
out Rh+ antibodies from the mother. This is usually done immediately following birth, but it can be done to a

fetus prior to birth. Later, the Rh- blood will be replaced naturally as the baby gradually produces its own Rh+
blood. Any residual anti-Rh+ antibodies from the mother will leave gradually as well because the baby does not
produce them.
Erythroblastosis fetalis can be prevented for women at high risk (i.e., Rh- women with Rh+ mates or mates whose blood
type is unknown) by administering a serum (Rho-GAM) containing anti-Rh+ antibodies into the mother around the 28th
week of pregnancy and again within 72 hours after the delivery of an Rh+ baby. This must be done for the first and all
subsequent pregnancies. The injected antibodies quickly agglutinate any fetal red cells as they enter the mother's blood,
thereby preventing her from forming her own antibodies. The serum provides only a passive form of immunization and will
shortly leave her blood stream. Therefore, she does not produce any long-lasting antibodies. This treatment can be 99%
effective in preventing erythroblastosis fetalis. Rho-GAM is also routinely given to Rh- women after a miscarriage, an
ectopic pregnancy, or an induced abortion. Without the use of Rho-GAM, an Rh- woman is likely to produce larger
amounts of Rh+ antibodies every time she becomes pregnant with an Rh+ baby because she is liable to come in contact
with more Rh+ blood. Therefore, the risk of life-threatening erythroblastosis fetalis increases with each subsequent
pregnancy.
Anti-Rh+ antibodies may be produced in an individual with Rh- blood as a result of receiving a mismatched blood
transfusion. When this occurs, there is likely to be production of the antibodies throughout life. Once again, Rho-GAM can
prevent this from happening.
Mother-fetus incompatibility problems can result with the ABO system also. However, they are very rare--less than .1% of
births are affected and usually the symptoms are not as severe. It most commonly occurs when the mother is type O and
her fetus is A, B, or AB. The symptoms in newborn babies are usually jaundice, mild anemia, and elevated bilirubin levels.
These problems in a baby are usually treated successfully without blood transfusions.
NOTE: Identifying someone as being Rh+ or Rh- is a simplification. There are many variations of Rh blood types
depending on which of the 45 Rh antigens are present. The most important of these antigens for mother-fetus
incompatibility and transfusion problems apparently are D, C, c, E, and e. When an individual is identified as being Rh+ or
Rh-, it is usually is in reference to the D antigen. In other words, the individual is RhD+ or RhD-.
Other blood grouping systems
http://faculty.matcmadison.edu/mljensen/BloodBank/lectures/other_blood_group_systems.htm

Explain the importance of blood group antigens in blood transfusion

Blood Transfusions
Blood that has antibodies on it that is not recognized by the
body will be attacked by your immune system
O is the Universal Donor because a person with this type of
blood does not have antigens on the surface of the blood cells
- hence will not cause an immune reaction in the patient.
AB is the universal Acceptor because this person will not have
an immune reaction to A, B, AB, or O
*Just remember, the antigens on the surface of your cells (or
donated cells) will cause a reaction if your immune system does not recognize them as being part of

you. Hence, if you are Type A, and transfused with Type B, your body will mobilize a massive immune
response against the "invading" blood. This will cause coagulation of blood and death.
----- AGGLUTINATION (the clumping of red blood cells following a transfusion reaction; likely fatal)

Blood Safety
Blood can carry diseases and health care professionals must be careful when working with blood. A
bloodborne pathogen is any disease causing agent that is present in the blood and can be transferred
from one person to another.
HEPATITIS B (HBV)
HEPATITIS C (HCV)
HUMAN IMMUNODEFICIENCY VIRUS (HIV)
MALARIA

Testing Your Blood

A test kit can be used to test your blood type. It involves pricking your finger and placing a drop of
blood on a card that will react to a serum on the card that contains antibodies. You will be given the
opportunity to test your blood type using this technique.

List the white blood cells present in peripheral blood giving their functions and
identify them on a blood fi lm
Normal peripheral blood
The usual diagnostic approach to blood disorders is blood counting and blood film examination. Blood films on glass
slides are stained with a Romanowsky stain (usually Wright's, Giemsa, or May-Grnwald). Red cells in normal
peripheral blood are circular and fairly uniform in size. Mild variation in shape (poikilocytosis) and size (anisocytosis) is
seen. Platelets appear as small bluish-purple discs. During blood film examination, the individual types of white blood
cells are enumerated; this is referred to as the differential count.

Band neutrophils

Basophil

Eosinophil

Erythrocyte (red blood cell)

Lymphocyte

Monocytes

Platelets

Segmented neutrophils

Band neutrophil
Band neutrophils comprise approximately 1 to 3% of the peripheral leukocytes. They are usually 9 to 15 m in
diameter. The nucleus forms a "U" or curled rod prior to segmentation. The chromatin pattern is coarse and clumped.
The cytoplasm is moderate to abundant with a few nonspecific granules and many specific granules.

Basophil
Basophils are granulocytes that contain purple-blue granules that contain heparin and vasoactive compounds. They
comprise approximately 0.5% of the total leukocyte count. Basophils participate in immediate hypersensitivity
reactions, such as allergic reactions to wasp stings, and are also involved in some delayed hypersensitivity reactions.
Basophils are the smallest circulating granulocytes, averaging 10 to 15 m in diameter. The nucleus to cytoplasm ratio
is about 1:1, and the nucleus is often unsegmented or bilobed, rarely with three or four lobes. The chromatin pattern
is coarse and patchy, staining a deep blue to reddish-purple. The cytoplasm is a homogenous pale blue, but this is
often obscured by the large dark granules.

Eosinophil
Eosinophils are the mature granulocytes that respond to parasitic infections and allergic conditions. Eosinophils
comprise about 1 to 4% of the peripheral leukocytes. They are usually 9 to 15 m in diameter. Granules stain a bright
reddish-orange with Wright's or Giemsa stains. The nucleus contains one to three lobes. The chromatin pattern is
coarse and clumped. The cytoplasm is abundant with a full complement of bright reddish-orange specific granules.

Erythrocytes (red blood cells)

The mature red blood cell (rbc) consists primarily of hemoglobin (about 90%). The membrane is composed of lipids
and proteins. In addition, there are numerous enzymes present which are necessary for oxygen transport and cell
viability. The main function of the red cell is to carry oxygen to the tissues and return carbon dioxide from the tissues

to the lungs. The protein hemoglobin is responsible for most of this exchange. Normal red blood cells are round, have
a small area of central pallor, and show only a slight variation in size. A normal red cell is 6-8 m in diameter. As the
relative amount of hemoglobin in the red cell decreases or increases, the area of central pallor will decrease or
increase accordingly.

Lymphocyte
Lymphocytes in the peripheral blood have been described on the basis of size and cytoplasmic granularity. Small
lymphocytes are the most common, ranging in size from 6 to 10 m. The nucleus is usually round or slightly oval,
occasionally showing a small indentation due to the adjacent centrosome. Except in the smallest cells, the nucleus is
about 7 m in diameter, a size that has been convenient for estimating the size of the surrounding erythrocytes.
Nuclear chromatin stains a dark reddish-purple to blue with large dark patches of condensed chromatin. The nuclear
cytoplasm ratio is 5:1 to 3:1, and the cytoplasm is often seen only as a peripheral ring around part of the nucleus.

Platelets
Platelets are the cytoplasmic fragments of megakaryocytes, circulating as small discs in the peripheral blood. They are
responsible for hemostasis (the stoppage of bleeding) and maintaining the endothelial lining of the blood vessels.
During hemostasis, platelets clump together and adhere to the injured vessel in this area to form a plug and further
inhibit bleeding. Platelets average 1 to 4 m in diameter. The cytoplasm stains light blue to purple, and is very
granular. There is no nucleus present. Normal blood concentrations range from 130,000 to 450,000/L.

Segmented neutrophil (seg)

Segmented neutrophils (polymorphonuclear leukocytes, or segs) are the mature phagocytes that migrate through
tissues to destroy microbes and respond to inflammatory stimuli. Segmented neutrophils comprise 40-75 % of the
peripheral leukocytes. They are usually 9 to 16 m in diameter. The nuclear lobes, normally numbering from 2 to 5,
may be spread out so that the connecting filaments are clearly visible, or the lobes may overlap or twist. The
chromatin pattern is coarse and clumped. The cytoplasm is abundant with a few nonspecific granules and a full
complement of rose-violet specific granules.

List the features of the blood count and blood fi lm that are associated with:
acute bacterial infection, viral infection, parasitic infestations and chronic
infection.
White cells
Acute Bacterial infection: increase neutrophils
Parasitic infestation: increase eosinophils
Acute Viral infection: increase lymphocytes

Normal values vary with age. White counts are highest in children under one year of age and then decrease
somewhat until adulthood. The increase is largely in the lymphocyte population. Adult normal values are shown
below.

WBC count: 4,500-11,000/L

polymorphonuclear neutrophils: 1800-7800/L; (50-70%)

band neutrophils: 0-700/L; (0-10%)

lymphocytes: 1000-4800/L; (15-45%)

monocytes: 0-800/L; (0-10%)

eosinophils: 0-450/L; (0-6%)

basophils: 0-200/L; (0-2%)

Acute lymphocytic leukemia (ALL)

Acute
ages of

lymphocytic leukemia (ALL) usually strikes children between the

2 to 10. A second peak in incidence is seen in elderly patients.
Only half of all patients with ALL have increased leukocytes and may
not have lymphoblasts in their peripheral blood. Neutropenia,
thrombocytopenia, and anemia are usually present. Patients have
symptoms of fatigue, fever and bleeding. There is often lymph
node enlargement. Enlargement of the spleen and of the liver may be
seen. The cells depicted in the image below are two lymphoblasts
and a neutrophil.

Acute myelocytic leukemia (AML)

Acute myelocytic leukemia (AML), is the most common leukemia in
children less than 1 year of age. It is rare in older children and
adolescents, but a second peak of incidence occurs among adults 40
years of age. The patient usually has an elevated white blood cell
count, and myeloblasts are present.
Anemia, thrombocytopenia, and neutropenia give rise to the clinical
findings of pallor, bruising and bleeding, fever with infections, and
fatigue. Bone pain and joint pain are seen as the first symptoms in 25%
of patients. Enlarged spleen is seen in 50% of all AML patients, but
lymph node enlargement is rare. The cell depicted in the image below
is a myeloblast.

Chronic granulocytic leukemia (CGL)

Chronic granulocytic leukemia or chronic myelogenous leukemia can occur at any
age, but is most common after the age of 45 years. Weight loss and fatigue are
often the initial symptoms. There is usually massive spleen enlargement, which
may cause left upper abdominal pain. There is anemia, markedly elevated levels of
leukocytes, thrombocytosis, eosinophilia, basophilia, and a predominance of
myelocytes in the peripheral blood. Myeloblasts constitute fewer than 10% of
circulating leukocytes. Occasional nucleated red blood cells are seen. The cells
depicted in the image below are two myeloblasts and a hypersegmented neutrophil.

Chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia is the most common type of leukemia and usually
occurs in older patients; it is rare in patients less than 40 years of age. The disease
is usually discovered when other medical problems are present. Weakness, fatigue,
and weight loss are usually seen. The malignant cell in CLL is usually a small,
mature-appearing lymphocyte.

Hairy cell leukemia

Hairy cell leukemia, or leukemic reticuloendotheliosis, is a rare malignant disorder.
It usually occurs in middle-aged patients over 50. The first symptoms of disease
include weakness and lethargy. Enlarged spleen occurs in 80% of patients.
Hairy cells are characterized by their fine, irregular pseudopods and immature nuclear
features. Bone marrow aspiration is often unsuccessful because of complete
infiltration by hairy cells, resulting in a dispersed spongy web of cells in an increased
meshwork of reticulin fiber.

Infectious mononucleosis
Infectious mononucleosis is caused by the Epstein-Barr virus, a DNA herpes-type
virus that infects B lymphocytes. Patients present with mild to severe
adenopathy, hepatosplenomegaly, fever, malaise, pharyngitis, and a
characteristic peripheral blood smear demonstrating reactive lymphocytes.

Malaria
Malaria is a disease caused by the parasite Plasmodium. The four species most
commonly found in man are vivax, malariae, falciparum, and ovale.
"Malaria

is mainly transmitted from person to person through the bite of the female
Anopheles mosquito. Other means of transmission are through the use of
contaminated needles, by congenital means, and through blood transfusions.

When the infected Anopheles mosquito bites a human, sporozoites are injected
the peripheral blood of the individual. The sporozoites then invade the liver.
When the red blood cell has been penetrated by the merozoite, the parasite
develops into the trophozoite ring form and thence to a mature schizont. The merozoites rupture from the mature
schizonts and penetrate other red blood cells. Fever and chills are associated with the rupture of the red blood cells.
into

Pelger-Hut Anomaly
Pelger-Hut anomaly is a benign hereditary condition characterized by
decreased segmentation in the neutrophils. These neutrophils usually
contain two lobes, but appear to function normally.

Describe the role of platelets in haematostasis

The platelet Role in Hemostasis

In hemostasis, platelets play three main roles:

1. They maintain the endothelial surface. Loss of circulating platelets quickly results in morphologic changes in the
endothelial cells of the capillaries. These changes cause intravascular material to leak into the capillary bed.
2. They initially arrest bleeding in severed blood vessels.
3. They provide phospholipid, which acts as the catalytic surface for initiation of the coagulation process.
When platelets encounter a break in the endothelial surface, several important actions cause the bleeding to cease.
A. Adhesion occurs when they encounter collagen, membranes or non-collagenous microfibrils beneath the basement
membranes. Adhesion is mediated by GP-Ib and von Willebrand Factor (vWF).
a. Receptors (especially for glycoprotein-Ib) bind vWF and facilitate adhesion.
b. vWF is an extremely large molecule (a multimer) that bridges the gap between different cells, the platelet, and
subendothelial surfaces.
c. vWF is a very sticky protein and binds readily.
d. Besides binding GP-Ib, vWF also binds to GP-IIb-IIIa to facilitate adhesion.
e. Fibrinogen, binding to the GP IIb-IIIa complex on two separate platelets, can bridge the gap between those two
platelets (this is important to a subsequent functionaggregation).
f. In the absence of these factors, (or their receptors) both adhesion and aggregation are abnormal and certain types of
bleeding problems can occur.
B. Release reaction: Platelets then undergo a "release reaction". This process involves change from a disc shape to a
spherical shape, constriction of the microtubules toward the center of the platelet, and the release of contents of the
granules (primarily ADP, catecholamine, and serotonin) into the open canalicular system. These platelets thus become
activated.
a. During the release reaction, the granules within the platelets release their contents into the canalicular system.
b. These granule components then leak into the plasma around the platelet.

c. The released ADP binds other circulating platelets in close proximity to activated platelets and this binding to surface
receptors initiates the release reaction in these recruited platelets.
d. The release reaction is mediated by means of Thromboxane A2. Arachidonate is integrated in the phospholipids in the
cell wall and is freed by a phospholipase, activated during the process of adhesion or by binding of certain ligands to
receptors on the platelet surface.
e. Cyclo-oxygenase converts arachidonate to an intermediate, prostaglandin H2.
f. In the platelets, PGH2 is acted upon by thromboxane synthetase to form thromboxane A2. Thromboxane A2 promotes
the release reaction, change in shape, and aggregation. In the endothelial cell, the pathway is different from that of the
platelet.
g. Following the formation of PGH2, prostacyclin synthetase produces PGI2, which inhibits adhesion, aggregation and the
release reaction, forces that oppose those of Thromboxane A2.
h. Aspirin blocks cyclo-oxygenase and therefore the pathway that leads to both Thromboxane A2 and PGI2. In the platelet,
the block is permanent for the life of the platelet, because the platelet does not have a nucleus to direct the formation of
more cyclo-oxygenase. This yields a platelet that cannot function. Since the life of the platelet is about 7-10 days, the
effect of the aspirin on bleeding will gradually decrease over a week, as new platelets replace those that were exposed to
aspirin. In the endothelial cell, however, cyclo-oxygenase is regenerated.
C. Platelet aggregation occurs as platelets are "recruited" from the immediate area by the released contents, for
example, ADP. This process is accomplished by fibrinogen, binding to the GP IIb-IIIa complex on separate platelets, and
bridging the gap between platelets When the release of ADP, or other aggregating agents, is minimal, the local
concentration of these agents do not reach a high level, this aggregation may be reversible; with higher concentrations,
aggregation is irreversible.
Associated with the change of shape of the platelet and the release reaction, is the appearance of clotting promoting sites
(historically referred to as platelet factor 3) on the platelet membranes. The receptor sites for the coagulation proteins
serve as a catalytic site for the clotting proteins and assists in initiating the clotting mechanism. Important coagulation
proteins that are bound to the surface include factors V and VIII among others.
E. Clot retraction occurs when platelets are trapped within the enlarging blood clot. During the release reaction,
pseudopodia like structures extend some distance from the surface of the platelet, and attach to similar structures on
adjacent platelets. With time, these structures retract, pulling the body of the clot together, and sealing the vessel wall at
the site of injury.

Identify the diff erence between plasma and serum. List the components of
plasma. Describe the functions of the diff erent plasma proteins
Blood serum; the clear liquid that separates from blood when it is allowed to clot completely, and is therefore blood
plasma from which fibrogen has been removed during clotting.
Blood plasma; the fluid portion of the blood in which the particulate components are suspended.

BLOOD PLASMA COMPONENTS

Water
The primary component of blood plasma is water. The American Association of Blood Banks (AABB) estimates
that water makes up 90 percent of blood plasma. Blood plasma can provide water to body tissues that are in
need of additional fluid or it can absorb excess water and pass it along to the kidneys to be excreted as urine.
Albumin
Albumin is the predominate protein present in blood plasma. This protein prevents blood fluid from
inadvertently leaking out of blood vessels, explains the Merck Manual: Home Edition, a medical reference
guide for patients and caregivers. Albumin also helps carry certain substances, such as hormones and
ingested drug products, through the body to the appropriate cells and tissues.
Antibodies
Blood plasma contains a number of additional proteins called immunoglobulins or antibodies. Antibodies are
part of your body's immune system and are produced by the white blood cells, explain health professionals at
the National Blood Service in the United Kingdom. If bacteria or viruses invade your body, antibodies within
your blood plasma help to identify and fight off infection.
Blood Clotting Factors
If you sustain an injury to your body, you can begin to bleed. The body uses blood clotting factors---which are
major components of blood plasma---to prevent you from losing excessive amounts of this vital body fluid.
Blood clotting factors, such as fibrinogen or Factor VIII, help the body seal damaged blood vessels and stop
bleeding.

BLOOD PLASMA PROTEINS

Draw a diagram of the intrinsic, extrinsic and fi nal common pathways of

coagulation and complement systems

The coagulation mechanism

The blood clothing system or coagulation pathway,
like the complement system, is a proteolytic
cascade. Each enzyme of the pathway is present in
the plasma as a zymogen, in other words in an
inactive form, which on activation undergoes
proteolytic cleavage to release the active factor from
the precursor molecule. The coagulation pathway
functions as a series of positive and negative
feedback loops which control the activation process.
The ultimate goal of the pathway is to produce
thrombin, which can then convert soluble fibrinogen
into fibrin, which forms a clot. The generation of
thrombin can be divided into three phases, the
intrinsic and extrinsic pathways that provide
alternative routes for the generation of factor X, and
the final common pathway which results in thrombin
formation (Figure 1.3).

Figure 1.3: The intrinsic, extrinsic, and common

pathways of the coagulation (clotting) cascade
The intrinsic pathway is activated when blood
comes into contact with sub-endothelial connective
tissues or with negatively charged surface that are
exposed as a result of tissue damage. Quantitatively
it is the most important of the two pathways, but is
slower to cleave fibrin than the extrinsic pathway.
The Hageman factor (factor XII), factor XI,
prekallikrein, and high molecular weight kininogen
(HMWK) are involved in this pathway of activation.
Thus this pathway provides a further of the
interrelationship between the various enzyme
cascade systems in plasma. The first step is the
binding of Hageman factor to a sub-endothelial surface exposed by an injury. A complex of prekallikrein and HMWK also
interacts with the exposed surface in close proximity to the bound factor XII, which becomes activated. During activation, the
single chain protein of the native Hageman factor is cleaved into two chains (50 and 28 kDa), that remain linked by a disulphide
bond. The light chain (28kDa) contains the active site and the molecule is referred to as activated Hageman factor (factor XIIa).
There is evidence that the Hageman factor can autoactivate, thus the pathway is self-amplifying once triggered (compare with the
alternative pathway of complement).
Activated Hageman factor in turn activates prekallikrein. The kallikrein produced can then also cleave factor XII, and a further
amplification mechanism is triggered. The activated factor XII remains in close contact with the activating surface, such that it
can activate factor XI, the next step in the intrinsic pathway which, to proceed efficiently, requires Ca2+ . Also involved at this
stage is HMWK, which binds to factor XI and facilitates the activation process. Activated factors XIa, XIIa, and kallikrein are all
serine proteases, like many of the enzymes of the complement system.
Eventually the intrinsic pathway activates factor X, a process that can also be brought about by the extrinsic pathway. Factor X is
the first molecule of the common pathway and is activated by a complex of molecules containing activated factor IX, factor VIII,

calcium, and phospholipid which is provided by the platelet surface, where this reaction usually takes place. The precise role of
factor VIII in this reaction is not clearly understood. Its presence in the complex is obviously essential, as evidenced by the
serious consequences of factor VIII deficiency experienced by haemophiliacs. Factor VIII is modified by thrombin, a reaction
that results in greatly enhanced factor VIII activity, promoting the activation of factor X.
The extrinsic pathway is an alternative route for the activation of the clothing cascade. It provides a very rapid response to tissue
injury, generating activated factor X almost instantaneously, compared to the seconds or even minutes required for the intrinsic
pathway to activate factor X. The main function of the extrinsic pathway is to augment the activity of the intrinsic pathway.
There are two components unique to the extrinsic pathway, tissue factor or factor III, and factor VII. Tissue factor is present in
most human cells bound to the cell membrane. The activation process for tissue factor is not entirely clear. Once activated, tissue
factor binds rapidly to factor VII which is then activated to form a complex of tissue factor, activated factor VII, calcium, and a
phospholipid, and this complex then rapidly activates factor X.
The intrinsic and extrinsic systems converge at factor X to a single common pathway which is ultimately responsible for the
production of thrombin (factor IIa).
Clot formation. The end result of the clotting pathway is the production of thrombin for the conversion of fibrinogen to fibrin.
Fibrinogen is a dimer soluble in plasma. Exposure of fibrinogen to thrombin results in rapid proteolysis of fibrinogen and the
release of fibrinopeptide A. The loss of small peptide A is not sufficient to render the resulting fibrin molecule insoluble, a proces
that is required for clot formation, but it tends to form complexes with adjacent fibrin and fibrinogen molecules. A second
peptide, fibrinopeptide B, is then cleaved by thrombin, and the fibrin monomers formed by this second proteolytic cleavage
polymerize spontaneously to form an insoluble gel. The polymerized fibrin, held together by noncovalent and electrostatic forces,
is stabilized by the transamidating enzyme factor XIIIa, produced by the action of thrombin on factor XIII. These insoluble fibrin
aggregates (clots), together with aggregated platelets ( thrombi), block the damaged blood vessel and prevent further bleeding.
There is an interrelationships between the coagulation pathway and other plasma enzyme systems. Contact activation of the
coagulation pathway, in addition to promoting blood clotting, results in the generation of plasminogen activator activity, which is
involved in fibrinolysis or clot removal. Activated Hageman factor and its peptides can also initiate the formation of kallikrein
from plasma prekallikrein, and this triggers the release of bradykinin from kininogens in the plasma. Kinins are responsible for
dilating small blood vessels, inducing a fall in blood presssure, triggering smooth muscle contraction, and increasing the
permeability of vessel walls. In addition, activation of the coagulation pathway produces a vascular permeability factor, as well as
chemotactic peptides for professional phagocytes.

Describe the vascular epithelium and its role in haematostasis as a procoagulant and anti-coagulant surface

Platelet pro-coagulant activity

After platelet aggregation and release, the exposed membrane phospholipid (platelet factor 3) is available for two reactions in the coagulation
cascade. Both phospholipid-mediated reactions are calcium ion dependent. The first (tenase) involves factors IXa, VIIIa and X in the formation of
factor Xa(Fig. 22.7). The second (prothrombinase) results in the formation of thrombin from the interaction of factors Xa, Va and prothrombin (II).
The phospholipid surface forms an ideal template for the crucial concentration and orientation of these proteins.

Growth factor

PDGF found in the specific granules of platelets stimulates vascular smooth muscle cells to multiply and this may hasten vascular healing following
injury.
Natural inhibitors of platelet function
Nitric oxide (NO) is constitutively released from endothelial cells and also from macrophages and platelets. It has a short half-life of 3-5 s. It inhibits

platelet activation and promotes vasodilatation. Prostacyclin synthesized by endothelial cells also inhibits platelet function (Fig. 22.8) and causes
vasodilatation by raising cyclic guanosine monophosphate (GMP) levels. The transmembrane protein PECAM-1 is expressed also on endothelial
cells. It is its own ligand and inhibits platelet activation by collagen.

Defi ne bleeding time (BT), prothrombin time (PT) and partial thromboplastin
time (PTT). State the importance of each test and the relevant specimen tube/
anticoagulant.
Bleeding Time is a crude test of hemostasis (the arrest or stopping of bleeding). It indicates how well platelets interact
with blood vessel walls to form blood clots.
Bleeding time is used most often to detect qualitative defects of platelets, such as Von Willebrand's disease. The test
helps identify people who have defects in their platelet function. This is the ability of blood to clot following a wound or
trauma. Normally, platelets interact with the walls of blood vessels to cause a blood clot. There are many factors in the
clotting mechanism, and they are initiated by platelets. The bleeding time test is usually used on patients who have a
history of prolonged bleeding after cuts, or who have a family history of bleeding disorders. Also, the bleeding time test is
sometimes performed as a preoperative test to determine a patient's likely bleeding response during and after surgery.
However, in patients with no history of bleeding problems, or who are not taking anti-inflammatory drugs, the bleeding
time test is not usually necessary.

Prothrombin time (PT) The rate at which prothrombin is converted to thrombin in citrated blood with added calcium;
used to assess the extrinsic coagulation system of the blood
The PT test is used to monitor patients taking certain medications as well as to help diagnose clotting disorders.
Diagnosis
Patients who have problems with delayed blood clotting are given a number of tests to determine the cause of the
problem. The prothrombin test specifically evaluates the presence of factors VIIa, V, and X, prothrombin, and fibrinogen.
Prothrombin is a protein in the liquid part of blood (plasma) that is converted to thrombin as part of the clotting process.
Fibrinogen is a type of blood protein called a globulin; it is converted to fibrin during the clotting process. A drop in the
concentration of any of these factors will cause the blood to take longer to clot. The PT test is used in combination with the
partial thromboplastin time (PTT) test to screen for hemophilia and other hereditary clotting disorders.
Monitoring
The PT test is also used to monitor the condition of patients who are taking warfarin (Coumadin). Warfarin is a drug that is
given to prevent clots in the deep veins of the legs and to treat pulmonary embolism. It interferes with blood clotting by
lowering the liver's production of certain clotting factors.
Partial Thromboplastin Time
A test for detecting coagulation defects of the intrinsic system by adding activated partial thromboplastin to a sample of
test plasma and to a control sample of normal plasma. The time required for the formation of a clot is compared with the
normal plasma. It is also used to monitor the activity of heparin in patients who are being treated for a variety of
cardiovascular disorders.
The partial thromboplastin time (PTT) test is a blood test that is done to investigate bleeding disorders and to monitor
patients taking an anticlotting drug (heparin).
Diagnosis
Blood clotting (coagulation) depends on the action of substances in the blood called clotting factors. Measuring the partial
thromboplastin time helps to assess which specific clotting factors may be missing or defective.
Monitoring
Certain surgical procedures and diseases cause blood clots to form within blood vessels. Heparin is used to treat these
clots. The PTT test can be used to monitor the effect of heparin on a patient's coagulation system.

Light blue: A reversible anticoagulant Sodium citrate in measured amount is present. Used for
coagulation assays (Prothrombin time, Partial Thromboplastin Time). Full draw is essential since
dilution factor with liquid citrate should be maintained.

List the anticoagulants used in the laboratory and state their mechanisms of
action

Describe naturally occurring anticoagulants and outline their role in health and
disease

Describe the fi brinolytic system