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1. SEROLOGY

1.1 What does mean by serology?

Serology is the scientific study of serum and other bodily fluids. In practice, the term usually refers to

the diagnostic identification of antibodies in the serum. Such antibodies are typically formed in

response to an infection (against a given microorganism), against other foreign proteins (in response,

for example, to a mismatched blood transfusion), or to one's own proteins (in instances of

autoimmune disease).Serological tests may be performed for diagnostic purposes when an infection is

suspected, in rheumatic illnesses, and in many other situations, such as checking an individual's type.

Serology blood tests help to diagnose patients with certain immune deficiencies associated with the

lack of antibodies, such as X-linked gamma globulinemia. In such cases, tests for antibodies will be

consistently negative.

There are several serology techniques that can be used depending on the antibodies being

studied. These include:

ELISA

agglutination

precipitation

complement-fixation and

Fluorescent antibodies.

Some serological tests are not limited to blood serum, but can also be performed on other

bodily fluids such as semen and saliva, which have (roughly) similar properties to serum.

Serological tests may also be used forensically, specifically for a piece of evidence (e.g.,

linking a rapist to semen sample).

1.2 How can we get the sample of serum from the blood sample?

Blood is centrifuged to remove cellular components. Anti-coagulated blood yields plasma containing

fibrinogen and clotting factors. Coagulated blood (clotted blood) yields serum without fibrinogen,

although some clotting factors remain.

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1.3 For what types of tests we use serum samples? Please list all possible

test performed in the clinical laboratory used in the analysis of serum.

Don’t miss to specify the purpose (objectives) of each test

Any of several laboratory procedures carried out on a sample of blood serum, the clear liquid that

separates from the blood when it is allowed to clot. The purpose of such a test is to detect serum

antibodies or antibody-like substances that appear specifically in association with certain diseases.

The various types of serological tests include:

1) Flocculation tests:

The complement-fixation tests are the most common. They are based on the precipitation,

or flocculation, that takes place when antibody and specially prepared antigens are mixed

together.

2) Neutralization tests:

Depend on the capacity of antibody to neutralize the infectious properties of the

infectious organisms.

3) Hemagglutinin-inhibition tests:

Which make use of the finding that certain viruses will cause the red blood cells of certain

animal species to agglutinate (congeal, or clump together) and that this agglutination will be

prevented by antibody.

4) Enzyme linked immuono sorbent assay (ELISA)

Its objectives are:

To evaluate either the presence of antigen or the presence of antibody in a sample and

determining serum antibody concentrations.

ELISA test has a wide application in the detection of antigens and antibodies of different

diseases.

It is a helpful diagnostic tool to detect diseases and determining the presence of allergen in

food.

ELISA testing is a double antibody ―sandwich‖ immunoassay, which employs specific antibodies

against the disease to be tested: monoclonal antibody immobilized at the bottom of the microtiter

wells, and polyclonal antibodies coupled with horseradish peroxidase as the conjugate solution.

During the assay, existing antigens in the specimen will react with the antibodies to form an

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―antibody-antigen-antibody-HRP‖ immuno-complex. After the unbound material is washed off

during the assay procedure, substrate is applied to indicate the test result.

5) Immuno fluorescent antibody technique (IFAT):

To evaluate an immunofluorescence antibody test (IFAT) for diagnosis of

schistosomiasis in non-immune travellers and immigrants from endemic areas.

It used to detect serum antibodies and immune complexes in tissues and microorganisms

in specimens from patients with infectious diseases (IDFAT).

Utilizing a fluorochrome conjugated to an antibody, which is added directly to a tissue or

cell suspension for the detection of a specific antigen (DFAT) and

To examine tissue fluorescence microscope.

6) Radio immuno assay (RIA):

Immunoassay is the method of choice for measuring analyses normally present at very low

concentrations that cannot be determined accurately by other less expensive tests.

It uses for measurement of drugs, hormones, specific proteins, tumor markers and markers of

cardiac injury.

Qualitatively it used to detect antigens on infectious agents and antibodies that the body

produces to fight them. For example, immunoassays are used to detect antigens on

Hemophilus, Cryptococcus and Streptococcus organisms in the cerebrospinal fluid (CSF) of

meningitis patients.

To detect antigens associated with organisms that are difficult to culture, such as hepatitis B

virus and Chlamydia trichomatis.

7) Agglutination tests:

Determination of blood types or antibodies to blood group antigens and to assess bacterial

infections

Determination of blood types or antibodies to blood group antigens.

To assess bacterial infections. Example , typhoid fever

To detect the presence and relative amount of specific antibody in a patient’s serum.

8) Complement fixation tests (CFT):

Looking for evidence of infection tests for the presence of either specific antibody or specific

antigen in a patient's serum

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for the aid of diagnosis of infectious disease

9) Precipitation tests:

For the detection of immunoglobulin levels in the serum of a patient

10) Serum neutralization tests (SNT):

11) Toxin-antitoxin test:

12) Determination of the protective value of an ant serum in an animal:

2. HEMOCYTOMETER

2.1 What is hemocytometer?

Hemocytometer is a device used to count cells. It was originally designed for the counting of

blood cells. It consists of a thick glass microscope slide with a rectangular indentation that

creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. The

device is carefully crafted so that the area bounded by the lines is known, and the depth of the

chamber is also known. It is therefore possible to count the number of cells or particles in a

specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

To use the hemocytometer, first make sure that the special coverslip provided with the counting

chamber is properly positioned on the surface of the counting chamber. When the two glass

surfaces are in proper contact Newton's rings can be observed. If so, the cell suspension is

applied to the edge of the coverslip to be sucked into the void by capillary action which

completely fills the chamber with the sample. The number of cells in the chamber can be

determined by direct counting using a microscope, and visually distinguishable cells can be

differentially counted. The number of cells in the chamber is used to calculate the concentration

or density of the cells in the mixture the sample comes from. It is the number of cells in the

chamber divided by the chamber's volume, which is known from the start, taking account of any

dilutions and counting shortcuts:

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Fig.1. parts of hemocytometer

2.2 How many squares does hemocytometer have? Whether the squares

are used for counting or not? Consider the size.

hemocytometer is composed of nine equally sized bigger squares. The central one is different

from the other ones because it is divided into 25 smaller squares, while the ones in the

corners are divided into 16 smaller squares. The rest of squares are not used. In addition, the

smaller squares inside the central square are subdivided into 16 even smaller squares each.

This allows counting very tiny cells with the same precision level as larger ones (but with a

higher magnification).

Dimensions

Area

Volume at 0.1 mm depth

1 x 1 mm 1 mm2 100 nL

0.25 x 0.25 mm 0.0625 mm2 6.25 nL

0.25 x 0.20 mm 0.05 mm2 5 nL

0.20 x 0.20 mm 0.04 mm2 4 nL

0.05 x 0.05 mm 0.0025 mm2 0.25 nL

Table.1 dimension, areas and volume of hemocytometer.

The proportion of the cells counted applies if not all inner squares within a set square are

counted (i.e., if only 4 out of the 20 in a corner square are counted, then this term will equal

0.2).The parts of the hemocytometer (as viewed from the side) are identified. For most

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applications, the four large corner squares are only used. The cells that are on or touching the

top and left lines are counted, but the ones on or touching the right or bottom lines are

ignored.

2.3 How many squares used for RBC in one hemocytometer?

The central square, each of the 25 smaller squares will be 1 mm/5 = 0.2 mm in width and 0.2

mm x 0.2 mm = 0.04 mm2 in area (or 1 mm 2/25 = 0.04 mm2). In turn, each of the 25

smaller squares contains 16 even smaller squares which measure: 0.2 mm/4 = 0.05 mm in

width and 0.05 mm x 0.05 mm = 0.0025 mm2 = 2500 μm2 (or 0.04 mm2/16 = 0.0025 mm2).

Cells that are 10 μm or smaller should be counted in the central square – sometimes even in

one of the smaller squares inside the central square. Typically you would count red blood

cells, platelets, most types of yeast, and sperm cells.

Fig.2 centeral squares used to count RBC.

2.4 How many squares used for WBC counting in one hemocytometer?

The four corner squares are further divided into sixteen smaller squares and are used for

WBC counting. Four corner squares are meant for WBC counting. Total = 64 small squares

Counting procedures of WBCs on hemacytometers Count cells starting in the upper the left

top large corner square. Move to the upper right corner square, bottom right corner square,

and end in the bottom left corner square. Count cells touching the left and top side lines.

Count all cells that touch any of the right and bottom lines, do not count any cell that touches

the upper and left boundary lines.

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Each of the 16 smaller squares will be 1 mm/4 = 0.25 mm in width and 0.25 mm x 0.25 mm

= 0.0625 mm2 in area (or 1 mm2/16 = 0.0625 mm2). Therefore, cells that are 10 μm or more

should be counted in these corner squares (although it doesn’t hurt if you also include a count

from the central square). For example, white blood cells (leukocytes).

Fig.3 corner squares (labeled by ―W‖ used for WBC count.

2.5 Why not those squares used for WBC counting is also used for RBC

counting and vice versa?

White blood cells: because they’re bigger, we are going to count those in the four corner

squares. We should establish a rule for the cells that are touching the peripheral lines: we can

count the ones touching the top and left and skip the ones on the bottom and right, or any

other combinations of two consecutive lines that we want. We have to note down our counts

(discriminating between live and dead if we added a dye).

Red blood cells: zoom into the central square, where smaller squares have been drawn. Count

the cells in the four small corner squares and the small central square, and do as with the

counts of the WBC

3. DIFFERENTIAL CELL COUNTING

3.1Define differential cell counting

A blood differential test, also called a white blood cell count differential, measures the

number of each of the five types of white blood cells present in blood. The blood differential

test measures the percentage of each type of white blood cell (WBC) in blood. It also reveals

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if there are any abnormal or immature cells and can diagnose an infection, inflammation,

leukemia, or an immune system disorder. It helps to tell the difference between various types

of white blood cells. Five types of white blood cells, also called leukocytes, normally appear

in the blood: Neutrophils, Lymphocytes (B cells and T cells), Monocytes, eosinophil and

Basophils.

3.2 Which test is used for differential cell counting?

It helps reveal abnormal white blood cell populations (e.g., blasts, immature granulocytes, or

circulating lymphoma cells in the differential totals the number of each type and determines

if the cells are present in normal proportion to one another, if one cell type is increased or

decreased, or if immature cells are present. This information is useful in helping to diagnose

the specific cause of an illness, such as:

Infections caused by bacteria, viruses, fungi or parasites

Inflammation

Allergies, asthma

Immune disorders (e.g., autoimmune disorders, immune deficiency)

Leukemia (e.g., chronic myeloid leukemia, chronic lymphocytic leukemia)

Myelodysplastic syndrome

Myeloproliferative neoplasms (e.g., myelofibrosis) the peripheral blood).

Some diseases trigger a response by the immune system that causes an increase in certain

types of WBCs. A differential may give clues to the specific cause of that immune response.

For example, it may help determine whether an infection is caused by bacteria or by viruses.

Other conditions affect the production of certain WBCs by the bone marrow or their survival

in the circulation, resulting in either an increase or decrease in their number. A differential

informs the healthcare provider as to which type of WBC is low or high. An abnormal

differential result may be followed by other tests such as a blood smear, bone marrow biopsy,

chromosome analysis, or immunopheno typing (e.g., flow cytometry). These tests can reveal

the presence of abnormal and/or immature populations of WBCs.

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Type of WBC

Abbreviations

Examples of causes of

a high count

Examples of causes

of a low count

Neutrophils

(Absolute neutrophil

count, percent

neutrophils)

Neu, Polys, PMNs,

ANC, % Neu

Known as

neutrophilia

Acute bacterial

infections and also

some infections

caused by viruses and

fungi

Inflammation (e.g.,

inflammatory bowel

disease, rheumatoid

arthritis)

Tissue death

(necrosis) caused by

trauma, major

surgery, heart attack,

burns

Physiological

(stress, rigorous

exercise)

Pregnancy—last

trimester or during

labor

Chronic leukemia

(e.g., myelogenous

leukemia

Known as

neutropenia

Myelodysplastic

syndrome

Severe,

overwhelming

infection (e.g.,

sepsis--neutrophils

are used up)

Reaction to drugs

(e.g., penicillin,

ibuprofen, phenytoin,

etc.)

Autoimmune

disorder

Chemotherapy

Cancer that

spreads to the bone

marrow

Aplastic anemia

Lymphocytes

(Absolute

Lymphs, lym, ly,

ALC, % lymphs

Known as

lymphocytosis

Known as

lymphopenia or

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lymphocyte count,

percent lymphocytes

Acute viral

infections (e.g.,

hepatitis, chicken

pox, cytomegalovirus

(CMV), Epstein-Barr

virus (EBV), herpes,

rubella)

Certain bacterial

infections (e.g.,

pertussis (whooping

cough), tuberculosis

(TB))

Lymphocytic

leukemia

Lymphoma

lymphocytopenia

Autoimmune

disorders (e.g., lupus,

rheumatoid arthritis)

Infections (e.g.,

HIV, TB, hepatitis,

influenza)

Bone marrow

damage (e.g.,

chemotherapy,

radiation therapy)

Immune

deficiency

Eosinophils

(Absolute eosinophil

count, percent

eosinophils)

Eos, AEC, % eos Known as

eosinophilia

Asthma, allergies

such as hay fever

Drug reactions

Inflammation of

the skin (e.g., eczema,

dermatitis)

Parasitic infections

Inflammatory

disorders (e.g., celiac

disease, inflammatory

bowel disease)

Certain

Known as

eosinopenia

This is often difficult

to determine because

numbers are

normally low in the

blood. One or an

occasional low

number is usually not

medically significant

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malignancies/cancers

Hypereosinophilic

myeloid neoplasms

Basophils (Absolute

basophil count,

percent basophils

Baso, ABC, % baso Known as basophilia

Rare allergic

reactions (e.g., hives,

food allergy)

Inflammation

(rheumatoid arthritis,

ulcerative colitis)

Some leukemias

(e.g., chronic myeloid

leukemia

Known as basopenia

As with eosinophils,

numbers are

normally low in the

blood; usually not

medically significant

Table.2 Possible Causes of High and Low WBC Differential Results

3.3 Why differential cell count is preferred than total white blood cell

count?

A white blood cell (WBC) count determines the concentration of white blood cells in the

patient's blood. A differential determines the percentage of each of the five types of mature

white blood cells. The white blood cell differential is often used as part of a complete blood

count (CBC) as a general health check. It may be used to help diagnose the cause of a high or

low white blood cell (WBC) count, as determined with a CBC. It may also be used to help

diagnose and/or monitor other diseases and conditions that affect one or more different types

of WBCs.

4. WHY THE SHAPE OF RBC IS BICONCAVE? EXPLAIN BRIEFLY.

Because their biconcave shape gives blood cell a larger surface area which increases the

ability to absorb oxygen. They are tiny and flexible; therefore they can squeeze through the

narrowest of blood capillaries to deliver oxygen. Red blood cells or Erythrocytes are

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biconcave because they do not contain nucleus. They are a-nucleated. This provides the cell

to attain large surface area to accommodate and maximum space for hemoglobin. And also

use for the subsequent accumulation of non-coding DNA in the genome. Efficient gas

transport requires erythrocytes to pass through very narrow capillaries, and this constrains

their size. In the absence of nuclear elimination, the accumulation of repeat sequences is

constrained by the volume occupied by the nucleus, which increases with genome size.

Generally Red blood cells, or erythrocytes, are biconcave for two reasons.

First, erythrocytes have no nucleus or internal membranes.

Second, erythrocytes have an unusual cytoskeleton.