Immunology first practical work. Natural immunity.

1. IMMUNOLOGY
Laboratory methods for detection of complement
system and phagocytosis.
First Practice – Topic 1
Fadi Marroushi
UPJS
Dr. Sabol
2014 – 2015

2.  Natural Immunity
Also known as innate, native, non-specific and non-adaptive immunity.
It is present before birth and does not have to be learned through exposure to an
invader. It is present prior to exposure to antigen. It thus provides an immediate rapid
response to foreign invaders and reacts nonspecifically. However, its components treat
all foreign invaders in much the same way. They recognize only a limited number of
identifying substances (antigens) on foreign invaders. However, these antigens are
present on many different invaders. Natural immunity, unlike acquired immunity, has no
memory of the encounters, does not remember specific foreign antigens, and does not
provide any ongoing protection against future infection.
 Phagocytes
The two types of circulating phagocytes, neutrophils and monocytes, are blood cells that
are recruited to sites of infection, where they recognize and ingest microbes for
intracellular killing.
They are essential for fighting infections and for subsequent immunity.
Neutrophils are the most abundant leukocytes in the blood, numbering 4000 to 10,000
per μL. In response to infections, the production of neutrophils from the bone marrow
increases rapidly, and their number may rise to 20,000 per μL.
Neutrophils are the first cell type to respond to most infections, particularly bacterial and
fungal infections. They ingest microbes in the circulation, and they rapidly enter
extravascular tissues at sites of infection, where they also ingest microbes and die after a
few hours.
Monocytes are less abundant than neutrophils, numbering 500 to 1000 per μL of blood.
They too ingest microbes in the blood and in tissues. Unlike neutrophils, monocytes that
enter extravascular tissues survive in these sites for long periods; in tissues, these
monocytes differentiate into cells called macrophages.
Neutrophils and monocytes migrate to extravascular sites of infection by binding to
endothelial adhesion molecules and in response to chemoattractant that are produced
on encounter with microbes.
 Methods
In-vitro phagocytosis test- Blood smear assay:
- Principle: A drop of whole blood is mixed with a drop of a bacterial culture and
incubated at 37o
C to demonstrate engulfment of microbes by leukocytes.
- Procedure: 1) Pipete 1ml of the blood with heparin into test tube.
2) Add 50 ul of Candida albicans (yeast) culture to the tube with
blood.
3) Gently shake the tube to mix blood and yeasts.
4) Incubate tubes for 30 minutes at 37o
C
5) Prepare blood smears.

3. - How to make blood smear:
1) With a pipette, carefully place a small drop of blood
mixed with Candida at one end of a microscope slide.
2) Holding the spreader slide with the thumb and forefinger, place
the spreader slide slightly in front of the drop of blood on the other
slide, maintaining a 25-degree angle between the slides.
3) Move the spreader slide back toward the drop of blood. As soon
as the slide comes in contact with the drop of blood, the blood will
start to spread along the edge.
4) Keeping the spreader slide at a 25-degree angle, move it rapidly
over the length of the slide. There should be a feathered edge on the
end of the smear.
- May-Grünwald Giemsa-Romanowski staining:
1) Dry the smear with the help of hot air.
2) Place slides on staining try.
3) Cover the smear with May-Grünwald solution for 3 minutes.
4) Add 4ml water to dilute stain on the slides. Stain 1 minute and
drain.
5) Drop the Giemsa-Romanowski stain to cover smear for 15
minutes.
6) Drain, dry with hot air and observe with 100x objective.
- Calculation and evaluation:
* Phagocytic Activity:
* Phagocytic Index:
Killing activity of phagocytes- Fluorescent method:
- PMNL Monolayer:
1) Pipet 100l of freshly collected venous blood on coverslip.
2) Put the coverslip to wet chamber for 60 min. at 37°C.
3) After 60 min. of incubation, when coagulum was formed, take
the coverslip with a tweezers and wash gently in prewarmed
(37°C) Hanks solution to remove nonadherent cells. Only
monolayer of PMNL and monocytes will be present on coverslip.
- Suspension of Candida albicans:
1) Sterilize the bacteriological loop in the flame and let it cool to
room temperature.
PMNLcountedallofnumber
CandidaoneleastatingestedwhichPMNLofcount
PA
CandidaoneleastatingestedwhichPMNLallofcount
countedPMNLallinCandidaingestedallofcount
PI

4. 2) Fill ¼ of loop with yeasts mass and transfer to the tube with 2ml
of Hanks solution. Firstly smear the yeast on the wall of the tube
and then mix with saline.
3) Adjust the optical density with the spectrophotometer or
McFarland density standards.
- Osponization:
1) Transfer 100l of yeast suspension to 5ml plastic tube, change
the pipet tip and add 100l of fresh human serum.
2) Place the tubes at 37°C for 15-20 min.
3) Centrifuge the tubes 1 minute at 1500rpm.
4) Pour off the supernatant and add 1ml of Hanks solution and 5l
of diluted acridine orange to the pellet.
5) Mix gently.
- Test of phagocytosis:
1) Mix gently the suspension of opsonized yeasts (They tend to
settle down with prolonged time).
2) Pipet 40l of opsonized yeasts on coverslip with adhered
polymorphonuclear leukocytes.
3) Incubate chamber with coverslips in incubator at 37°C.
- Staining of Preparation:
1) Stain coverslips 45 seconds with freshly prepared solution of
acridine orange.
2) After staining touch the edge of coverslip to filter paper to
remove the abundant stain.
3) Put the coverslip on microscopic glass monolayer down and lace
the coverslip with mounting medium.
- Evaluation: 1) Preparation is evaluated in fluorescent microscope with 1000x
Magnification.
2) In neutrophils there are ingested yeasts, killed yeasts are red
colored, viable yeasts are green or orange.
 Complement System
The complement system consists of more than 30 proteins, present in blood and tissues,
as well as other proteins anchored on the surfaces of cells. The primary functions of the
complement system are to protect from infection, to remove particulate substances, (like
damaged or dying cells, microbes or immune complexes) and to help modulate adaptive
immune responses. As part of the innate immune system, complement acts immediately
to start the process of removal and resolution of the problem. Complement works with
the inflammatory cells of the innate immune system and those of adaptive or acquired
immunity. It also interacts with proteins of the coagulation and kinin generating systems
along with others.

5. Three biochemical pathways activate the complement system: the classical complement
pathway, the alternative complement pathway, and the lectin pathway. The following
are the basic functions of the complement: opsonization (enhancing phagocytosis of
antigens); chemotaxis (attracting macrophages and neutrophils); cell lysis (rupturing
membranes of foreign cells); and clumping (antigen-bearing agents).
 Methods
Hemolytic assay (Titration of complement 1/2):
- Introduction: Patient serum is used for estimation of complement.
In the titration method, several dilutions (titration) of serum sample
are used in order to determine haemolytic unit CH50.
- Principle:
1) Determination of complement concentration is based on the
haemolysis of erythrocytes covered by antibody (haemolytic
system).
Haemolysis is proportional to haemoglobin release and measured
with spectrophotometer. The number of erythrocytes as well as
other test conditions is strictly standardized.
2) Amount of the serum, which causes the lysis of 50 %
erythrocytes, is defined as one haemolytic unit - CH50.
Haemolytic unit is variable and depend on the concentration of
complement in patient serum.
The amount of complement in patient sample is expressed as the
number of haemolytic units per mL.
Normal concentration of complement in serum is 37.5 +/- 4
CH50/mL.
Hemolytic assay (Titration of complement 2/2):
- Procedure: 1) Titration of serum (serum, VFR buffer).
2) Drop the VFR buffer into the tubes as indicated in table below.
3) Drop the serum.
4) Mix tubes gently.
5) Drop haemolytic system (HS)
6) Incubate and read.