Diagnostic Tests for Dengue at PUHCs

Dr Amita Raoot
DFW
Part I: Dengue Diagnostic Protocol

Part II: Making of Peripheral Blood Smear

Part III: Platelet Count Estimation from
Peripheral Blood Smear

Part IV: Dengue NS1 Antigen Test

Dengue Fever
Dengue Fever
Dengue fever also known as break-bone fever, is a
mosquito-borne tropical disease caused by Dengue Virus.
Symptoms include fever, headache, joint pains and rash.
The incubation period (time between exposure and onset
of symptoms) ranges from 314 days, but most often it is
47 days.
80% cases are asymptomatic or only have mild symptoms
such as an uncomplicated fever.
In a small proportion of cases (5%) the disease develops
into the life-threatening dengue hemorrhagic fever,
resulting in bleeding, low levels of platelets and blood
plasma leakage, or into dengue shock syndrome,
where dangerously low blood pressure occurs.
FEVER
RASH
MUSCLE &
JOINT PAINS
AEDES AEGYPTI MOSQUITO
Dengue fever is characterized by:
Importance of Platelet Count in
Dengue
On getting infected with Dengue, patients
PLATELET COUNT STARTS FALLING.
A platelet count BELOW 50,000 cumm is
ALARMING - immediate medical attention is
required.
A platelet count BELOW 5000 cumm is FATAL.
Importance of Platelet Count in Dengue

LAB
TESTS
Platelets
Dengue Diagnostic Protocol

Platelet count
& Hematocrit
NS1
Antigen
Detection
Peripheral Blood Smear
MAKING OF SMEAR
DRYING OF SMEAR
FIXATION
STAINING
EXAMINATION
1
2
3
4
5

Peripheral Blood Smear
A peripheral blood smear (peripheral blood film) is a glass
microscope slide coated on one side with a thin layer of
venous blood. The slide is stained with a dye and examined
under a microscope.
It is one of the SIMLPEST yet very informative investigation .
The steps involved are:
Specimen

1) EDTA-anticoagulated blood :
- Smears should be made within 1 hour of blood collection
from EDTA specimens stored at room temperature to
avoid distortion of cell morphology
2) Capillary blood (Finger prick) collected at the patient's
bedside (without Anti-coagulation)
During collecting the sample take following precautions:
-Don't squeeze the puncture area.
-The first drop formed should not be collected but wiped
away.
-The second drop used for platelet count and blood film.


Materials:
1. Glass microscope slides.
2. Bio-wipes
3. Markers/label
4. Gloves
5. Skin puncture devise (sterile blood lancet)
6. Microscope
7. Immersion oil
8. Manual cell counter designed for differential
counts

Steps in PBS PREPRARTION
Place a 1" x 3" glass microscope slide with a frosted
end on a flat surface (usually the counter top of a
laboratory bench).
Attach a label on the slide or write the patients name,
specimen identification number, and date of preparation
on the frosted surface.
Place a 2 - 3 mm drop of blood approximately 1cm
from the frosted end of the glass slide.
Hold the slide between the thumb and forefinger of one
hand at the end farthest from the frosted end.


Preparation of Peripheral Blood Smear
Step A. Placing
a small drop of
venous blood
on a glass
microscope
slide, using a
glass capillary
pipette. A
wooden
applicator stick
can also be
used for this
purpose.
Condt..
Grasp a second slide ("spreader slide") between the thumb
and forefinger of the other hand at the frosted end.
Place the edge of the spreader slide on the lower slide in
front of the drop of blood.
Step B.
A spreader
slide has been
positioned at
an angle and
slowly drawn
toward the
drop of blood.
Condt
Step C.
Pull the spreader
slide toward the
frosted end until it
touches the drop of
blood. Permit the
blood to spread by
capillary motion until
it almost reaches the
edges of the
spreader slide
Condt

Step D.
Now spread the
blood across the
slide in even,
single, clean
forward stroke
contd

End Result
A glass slide with
a well-formed
blood film. After
drying for about 10
minutes, the slide
can be stained
Steps in making a PBS

Drying of Blood Film

Air-drying without forced air
circulation is sufficient.

In humid conditions, forced air-
drying is recommended.

Slow drying causes cells to contract,
whereas water in excess can cause
gross morphological artefacts, such
as decreased crispness of cellular
appearance & the development of
artificial vacuoles.

Fixation

Fixation preserves the morphology of the cells.

Optimal results are obtained by fixing and
staining immediately after the blood film is
completely air-dried.

If slides cannot be stained immediately, fixation
in methanol is necessary within 4 hours, but
preferably 1 hour after air-drying.


Staining
Leishman Stain:
Preparation: 1.5 g Leishman powder (eosin-methylene blue
powder) dissolved in 1000 ml methanol. The mixture is warmed
to 50C for 10-15 minutes. It is then filtered and kept for 3
months to ripen in brown bottles.
Staining procedure:
The dried smear is placed horizontally on a staining rack.
Pour the stain drop by drop till it covers the smear.
Keep it for 1-2 minutes for fixing the smear.
Then dilute the stain using distilled water or Sorensens
phosphate buffer water (pH 6.8) double the amount of stain.
Mix gently by blowing air. A golden scum is seen to form over
the diluted stain.
Allow to stain for 1012 minutes (time may require adjusting).
Wash off the stain with clean (or filtered) tap water .
Wipe the back of the slide clean & place it in a draining rack to
dry.

Staining
Unstained
smear
Stained
smear
PERFECT PBS





The perfect blood smear has a feathered edge
that is nearly square, has a rainbow sheen when
reflecting the light and is exactly one cell thick in
the feathered edge when viewed microscopically

p
/
s

3
1
5

1
2
/
0
7
/
1
4

CHARATERSTICS OF WELL MADE SMEAR
Tongue shaped: smear is thick at the frosted end and
becomes progressively thinner toward the opposite end.
The "zone of morphology" (area of optimal thickness for
light microscopic examination) should be at least 2 cm in
length.
The smear should occupy the central area of the slide
and be margin-free at the edges .
Blood film should be smooth and free of serration.
Unstained smear should be transparent so that a
newsprint is visible through it.
Make at least two smears.
Unacceptable forms of PBS

Common causes of a poor blood smear

1. Dirty /greasy slide.
2. Drop of blood too large or too small.
3. Edge of the spreader was not smooth.
4. Spreader slide pushed across the slide in a jerky manner.
5. Failure to keep the entire edge of the spreader slide
against the slide while making the smear.
6. Failure to keep the spreader slide at a 30 angle with the
slide.
7. Improper drying.
8. Improper pH of the stain/ buffer.
9. Old stain.
10. Staining surface is not leveled.



Estimation of Platelet Count through PBS

Microscopic Examination of Stained Blood Film
1. Low power (10x) Examination: To
judge the quality of stain , cells
distribution & presence of platelet clump.
1. High power (40 x) examination:
Examine the cells for abnormalities.
3. Oil Immersion examination (100x):
For estimating Platelet Count
Scanning technique for
peripheral blood differential
count. (a) 10 microscopic fields
are examined in a vertical
direction from bottom to top (or
top to bottom). (b) The slide is
horizontally moved to the next
field (Apply thin layer of oil over
the smear & examine it under
oil.)

Parts of PBS

30 30
Thin part

Ideal part for examination
junction of body & tail
Thick part

Platelets
1-3m irregular outline .

Normal Count: 150- 400 X 10
9
/L
(150,000 to 400,000 per cubic millimeter )

Appear as dark purple spots, about 20% the
diameter of RBC.

Approximately 5-15/oil immersion or 1/10-
20 RBC


PBS with Normal Platelet Count

Evenly distributed platelets : Leishman stain (40X)
About 27 platelets are seen in the field
Smears with Normal Platelet Count

Platelet Clump
PBS with Low Platelet Count

1. Use the oil immersion field (OIF) to count the number
of platelets per field.
2. Count platelets in at least 10 FIELDS in OIF & take
the average number of platelets.
3.Estimated platelet count ( cu mm) is given by:
Average number of platelets per OIF multiplied
by 15,000
For e.g if average number of platelets per OIF is 8,
then Estimated Platelet Count is 120000/cu mm
Rough indication of Platelet count:
1) <8 platelets/OIF = decreased
2) 8 to 20 platelets/OIF = adequate
3) >20 platelets/OIF = increased

ESTIMATION OF PLATELET COUNT
NS1 Antigen Detection: Rapid
Kit Test

NS1 Antigen Test for Dengue
The Dengue NS1 Antigen Test is a rapid immuno-
chromatographic (strip/cassette) assay for the qualitative
presumptive detection of non-structural protein 1 (NS1) in
human serum.
This test should be done within 1-7 days of onset of
symptoms.


PRINCIPLE OF THE TEST
Drops of serum/ plasma are placed in the sample well. It then
migrates by capillarity in nitrocellulose membrane. The test is placed
into a well containing 3 drops of buffer. and utilizes a separate control
to assure assay flow & performance.
When the antigen is present, it binds the specific antibodies attached
to the membrane and a colored line appears (T).
Strip/Cassete test
Monoclonal anti-NS1
antibodies Deposit Test Sample
T C
migration
This is a membrane based immunoassay. The test membrane is pre-
coated with a NS1 specific antibody on the test line region.
The test sample is added directly to the sample well & the test is placed
into a well containing 3 drops of buffer.
The solution migrates upward on the membrane (via capillary action) to
react with the anti-NS1 antibody on the membrane.
If NS1 antigen is present, a red line will appear at the test line.
The red line at the control region should always appear if the assay is
performed correctly. If it is not present, the test is considered invalid &
must be repeated.

Interpretation of the Test
Invalid
T C
Positive Result
Negative Result
T
T C
C
serum or plasma
15 mn