‫آزمايشگاه ايمونولوژي‬
‫‪‬‬
‫كارشناس مسئول برنامه خانم قارداشي‬
‫كاربرد ايمونولوژي‬
‫درمان‬
‫تشخيص‬
‫تحقيق‬
‫پيش گيري‬
‫چرا من بعنوان يك پزشك بايد ايمونولوژي عملي بدانم‬
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‫‪‬‬
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‫روشهاي مختلف آزمايشگاهي را كه مي توان آناليت ها را‬
‫اندازه گيري كرد بشناسيد‬
‫از محدوديت هاي كار در آزمايشگاه آگاه باشيد‬
‫پشت صحنه اعداد و ارقامي كه در مطب بدستتان مي‬
‫رسد با خبر باشيد‬
‫منابع خطا را بشناسيد‬
‫بدانيد تا چه ميزان بايد به روشها و جوابها اعتماد كنيد‬
Errors
Requesting appropriate tests
Writing prescription
Reading prescription
Sample collection
Sampling times
environmental factors
Drug interferences
Patient identification
Sample transfer
Technician errors
Instrumental errors
Method limitations
Data entry mistakes
Interpretation of results
How can we trust
Lab Equipment
Safety & Sterilization
Beakers hold and/or
heat solids or liquids
that will not release
gases when reacted,
or are unlikely to
splatter if stirred.
Beaker
Very poor item to
measure volume with
(+/- 5% error!)
Note the total size
capacity = 250 mL
(upper mark is 200 mL)
There are six sizes of beakers in
your lab table for you to use:
50, 100, 150, 250, 400, & 600 mL
Beaker Tongs
Beaker tongs
are used to hold
and move
beakers
containing hot
liquids.
Note the rubber
coating to
improve grip on
the glass beaker
- do not hold these
in a burner flame.
Erlenmeyer Flask
Erlenmeyer flasks
hold and/or heat
solids or liquids
that may release
gases during a
reaction, or that are
likely to splatter if
stirred.
Note the size = 125 mL
Florence Flask
Rarely used in first
year chemistry, it
is used for the
mixing of
chemicals. The
narrow neck
prevents splash
exposure.
Also called round-bottom flasks
Flask Tongs
Flask tongs are used only to handle
flasks – use beaker tongs for beakers.
A graduated cylinder
is used to measure
volumes of liquids;
probably your best
everyday measuring
tool, there are three
sizes in your desk:
10, 50 and 100 mL
*NOT to be used
for heating or
mixing chemicals
Note the rubber
“bumpers”.
Graduated Cylinder
Some graduated cylinders that are
smaller may not have “bumpers”, but
have reinforced glass rims.
The top
plastic
bumper
ALWAYS
stays at
the top, to
prevent
breakage if
it falls
over.
Reading the Volume
10 mL has approx 6.62 mL
100 mL 52.7 mL
25 mL has 11.5 mL
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Test Tube – we commonly use 2 sizes:
18 x 150 mm
Larger
tube
(25 x 200 mm)
sometimes
used
x 100the
mm
The size is determined by the diameter13
across
top and the
length of the test tube. Example: 13 mm x 100 mm
(diameter)
(length)
Test tubes are used to mix chemicals,
and also used to heat chemicals in.
Test Tube Holder
A test tube
holder is useful
for holding a test
tube which is too
hot to handle
with your hands.
Knowing where
to hold this piece
of equipment is Holding it here will keep your hand as far as
possible from the fire, and prevent you from
important.
squeezing the holder and dropping the tube.
Stopper
Rubber and cork
stoppers are used
to close test tubes
and flasks, thus
avoiding spillage
or contamination.
Containers should NEVER be heated when
there is a stopper in place – pressure will
build up, and an explosion could occur.
Test Tube Rack
Test tubes can be placed
upside down on these
pegs for drying.
Test tube racks are for holding, drying, and
organizing test tubes in a vertical position,
and are located in the side wall cabinets.
Test tube brushes
are used to clean
test tubes and
graduated
cylinders.
Test Tube Brush
Small test tube brush
Caution: Forcing a
large brush into a
small test tube will
often break the tube.
Don’t worry about
drying the inside of a
tube or cylinder - Let
them air dry instead of forcing a
paper towel down inside.
Large test tube brush
TYPES OF PIPETTES
•Volumetric
•Measuring
•Mohr
•Serological
Precision & Accuracy
VOLUMETRIC PIPETTES
Used to deliver a single specific volume of liquid, usually
between 1 and 100 ml.
Shaped like rolling pins with a large belly, one blunt end, the
neck, and one tapering end, the tip.
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MOHR AND SEROLOGICAL
PIPETTES
Measuring pipettes are
divided into:
MOHR PIPETTES 
the graduations on 
these always end
before the tip
SEROLOGICAL PIPETTES
the graduation marks 
continue to the tip

Examine pipettes A and B.
Which is the serological and which is the Mohr?
5 in 1/10 ml
TD 20oC
BLOW OUT PIPETTES
The frosted band should
not be confused with
thicker colored rings or
colored dots, which are a
manufacturer’s code for
the maximum volume
of the pipette.
Remember, only blowout a serological pipette
if it has a frosted band
or two thin rings.
HANDLING STERILE PIPETTES
HANDLING AND DISPOSING OF
PIPETTES
Chipped and cracked
pipettes should be
replaced as they are
unsafe and may affect
the accuracy of
measurements.
NEVER mouth pipette.
Hold the pipette by the
upper third of the tube
and keep the tip from
touching anything.
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OTHER PIPETTE TYPES
Transfer of uncalibrated volumes up to 2.5 ml can
be accomplished using glass “transfer” or “Pasteur”
pipettes shown below. These may be sterilized
before use.
Roughly calibrated volumes of 1 and 2 ml can be
transferred with the one piece plastic transfer
pipettes which may be purchased as sterile or nonsterile units.
TRANSFERRING A PRECISE
VOLUME OF LIQUID
To produce a vacuum
for aspiration,
squeeze valve “A”
with your thumb and
index finger of one
hand while using
your other hand to
squeeze the bulb. “A”
stands for “air” or
“aspirate.”
(Click on the picture to see
a movie on the SLC computer)
To “pull” the liquid up into
the pipette place the pipette
into the liquid and squeeze
the “S” or “suction” valve
until the liquid reaches the
desired level.
Touch the tip of the pipette
to the side of the vessel
containing the liquid to
remove any adhering drops.
To “expel” the
liquid, squeeze the “
E” valve. This will
allow the liquid to
flow out of the
pipette except for
the last drop.
If the last drop of liquid
that remains in the tip
must be expelled,
squeeze valve “E” with
your thumb and
forefinger, cover the
opening in the small
bulb with your middle
finger and squeeze the
small bulb.
Use and Maintenance of
Micro-pipettes
Parts of the Pipette
Pipette tips
Operating the Micropipette
Step 1: Set the Volume
Pipettors – 3 Volumes:
Digital Volume Indicator:
Volume Adjustment Knob:
Operating the Micropipette
Step 1: (Continued) Read the Volume
How to Read the Volume Indicator:
(a): P-20 Model
6.86 m l = 0.00686
or 6.86 x 10-3 ml
(b): P-200 Model
132.4 m l = 0.1324
or 1.324 x 10-1 ml
(c): P-1000 Model
262 m l= 0.262
or 2.62 x 10-1 ml
Operating the Micropipette
Step 2: Attach the Disposable Tip
Example of tip sizes:
Attaching the
disposable tip
Operating the Micropipette
Step 3: Depress the
Plunger to the First
Stop
Step 4: Immerse Tip in Sample
Step 5: Draw up the sample
To aspirate the sample into the
tip, allow the pushbutton to
return slowly and smoothly to
the fully extended UP
POSITION. NEVER LET THE
PLUNGER SNAP UP! This
draws the exact calibrated
volume into the tip if the tip
remains below the liquid
surface during withdrawal.
Step 6: Pause
Wait a few seconds to ensure that the full
volume of sample is drawn into the plastic tip.
WAIT LONGER FOR LARGER VOLUMES. WAIT
LONGER FOR MORE VISCOUS ("SYRUPLIKE") SUBSTANCES.
Operating the Micropipette
Step 7: Withdraw the Tip
Remove the tip from the sample liquid. No liquid should remain on the OUTSIDE of
the tip. Wipe away any droplets on the outside of the tip with a lint-free tissue,
such as KIMWIPES, but only wipe droplets from the side of the tip. NEVER TOUCH
THE TIP OPENING or you may absorb part of your sample.
Proper Droplet Removal
WRONG Droplet Removal
Operating the Micropipette
Step 8: Dispense the Sample
To dispense the sample from the pipette:
a) Touch the tip end to the side wall of the receiving vessel and
b) Depress the plunger to the FIRST STOP.
c) Pause for at least one second-- 1-2 seconds for P-1000, 2-3 seconds for P-5000,
or longer for viscous liquids.
d) Press the plunger to the SECOND STOP (the second point, of greater resistance,
at the bottom of the stroke) to expel any residual liquid in the tip (like "blowing
out" a glass pipette).
(a) Start
Dispensing
(b) 1st Stop = (c) 2nd Stop =
Dispense
Expel
Operating the Micropipette
Step 9: Withdraw the Pipette
With the plunger fully depressed, withdraw the pipet from the receiving vessel
carefully, sliding the tip along the wall of the vessel. Holding the tip against the
side of vessel is especially important when transferring small volumes of liquid.
Step 10: Release the Plunger
Gently allow the plunger to return to the UP position. DO
NOT allow it to SPRING BACK!
Step 11: Discard the Tip
Discard the tip by depressing the tip ejector button, as
shown below. A fresh tip should be used for each sample
to prevent sample carryover.
Press ejector button to discard tip.
Practice with Pipette
Review material •
and practice using
a pipette
Use proper •
pipetting
techniques
Equipment and Supplies
• 3 automatic pipettes
• Several capped sample vials
• Green sample solution to
practice volume transfer
Accuracy and Precision
Accuracy means the closeness with which the •
dispensed volume approximates the volume set
on the pipette
Accuracy is specified as mean error, the average •
deviation of replicate measurements from the
expected set volume
Precision is the "scatter" or reproducibility of •
individual measurements of the same volume
Precision can also be expressed as standard •
deviation
Accuracy and Precision
(Continued)
Relative accuracies are generally about •
1% or less
Precision is less than 0.5 % except when •
transferring the smallest recommended
volume for a given pipette model
Using the pipettes to transfer volumes •
which are below the recommended range
will introduce larger errors
Pippetting Guidelines and
Precautions
For optimal reproducibility,
use the following pipetting
procedures:
(1) Consistent SPEED and SMOOTHNESS when you
press and release the PLUNGER
(2) Consistent pressure on the PLUNGER at the FIRST
STOP
(3) Consistent and sufficient IMMERSION DEPTH
(4) Nearly VERTICAL POSITIONING of pipette
(5) AVOID ALL AIR BUBBLES: Since the plastic pipette
shaft can be damaged if liquids are drawn beyond the
tip into the shaft
(6) NEVER lay the pipette on its SIDE nor INVERT the
pipette if liquid is in the tip
Practice with Pipettes
Practice using the pipette •
Practice setting a few volumes •
Practice reading the digits of set •
volumes
Practice drawing up and dispensing •
samples
Get the "feel" of the 1st and 2nd •
stops
Practice "blowing out" the pipette •
Relative sensitivities of tests (approx)
Usual operating range
[Ab] or [Ag]
precipitation
immunoelectrophoresis
double/radial diffusion
immunofluorescence
10 mg/ml - 1 mg/ml
0.1 - 10 mg/ml
ELISA (colour)
(chemiluminescence)
0.1 - 10 ng/ml
0.01 - 10 ng/ml
radioimmunoassay
0.01 - 10 ng/ml
CALCULATING THE RATES
A test is used in 50 people with disease
and 50 people without. These are the
results:
Disease
Test
+
-
+
-
48
3
51
2
47
49
50
50
100
Disease
Test
+
-
+
48
3
51
-
2
47
49
50
50
100
Sensitivity = 48/50 = 96%
Specificity = 47/50 = 94%
Positive predictive value = 48/51 = 94%
Negative predictive value = 47/49 = 96%