Dengue Detection Tests at PUCHs

7/27/2019 Dengue Detection Tests at PUCHs

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Diagnostic Tests for Dengue at PUHCs

Dr Amita Raoot

DFW


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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


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Dengue Fever


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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 onsetof 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 bloodplasma leakage, or into dengue shock syndrome,where dangerously low blood pressure occurs.


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FEVER

RASH

MUSCLE &

JOINT PAINS

AEDES AEGYPTI MOSQUITO

Dengue fever is characterized by:


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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 isrequired.

A platelet count BELOW 5000 cumm is FATAL.


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Importance of Platelet Count in Dengue

LABTESTS

Platelets


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Dengue Diagnostic Protocol

Platelet count

& Hematocrit

NS1AntigenDetection


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MAKING OF SMEAR

DRYING OF SMEAR

FIXATION

STAINING

EXAMINATION

1

2

3

4

5


A peripheral blood smear (peripheral blood film) is a glass

microscope slide coated on one side with a thin layer ofvenous blood. The slide is stained with a dye and examinedunder a microscope.

It is one of the SIMLPESTyet very informative investigation .

The steps involved are:


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Specimen

1) EDTA-anticoagulated blood :

- Smears should be made within 1 hour of blood collectionfrom EDTA specimens stored at room temperature toavoid 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.


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Materials:

1. Glass microscope slides.2. Bio-wipes

3. Markers/label

4. Gloves5. Skin puncture devise (sterile blood lancet)

6. Microscope

7. Immersion oil8. Manual cell counter designed for differential

counts


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Steps in PBS PREPRARTION

Place a 1" x 3" glass microscope slide with a frostedend on a flat surface (usually the counter top of alaboratory bench).

Attach a label on the slide or write the patientsname,

specimen identification number, and date of preparationon the frosted surface.

Place a 2 - 3 mm drop of blood approximately 1cmfrom the frosted end of the glass slide.

Hold the slide between the thumb and forefinger of onehand at the end farthest from the frosted end.


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Preparation of Peripheral Blood Smear

Step A. Placing

a small drop of

venous blood

on a glass

microscope

slide, using aglass capillary

pipette. A

wooden

applicator stickcan also be

used for this

purpose.


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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 andslowly drawn

toward the

drop of blood.


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Condt

Step C.

Pull the spreader

slide toward the

frosted end until ittouches the drop of

blood. Permit the

blood to spread by

capillary motion untilit almost reaches the

edges of the

spreader slide


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Condt

Step D.

Now spread the

blood across the

slide in even,

single, clean

forward stroke


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contd

End Result

A glass slide with

a well-formed

blood film. After

drying for about 10

minutes, the slide

can be stained


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Steps in making a PBS


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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 causegross morphological artefacts, such

as decreased crispness of cellularappearance & the development ofartificial vacuoles.


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Fixation

Fixation preserves the morphology of the cells.

Optimal results are obtained by fixing andstaining immediatelyafter the blood film is

completely air-dried.

If slides cannot be stained immediately, fixation

in methanol is necessary within 4 hours, butpreferably 1 hour after air-drying.


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Staining

Leishman Stain:Preparation: 1.5 g Leishman powder (eosin-methylene bluepowder) dissolved in 1000 ml methanol. The mixture is warmedto 50C for 10-15 minutes. It is then filtered and kept for 3months 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 overthe 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.


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Staining

Unstainedsmear

Stainedsmear


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PERFECT PBS

The perfect blood smear has a feathered edge

that is nearly square, has a rainbow sheen whenreflecting the light and is exactly one cell thick inthe feathered edge when viewed microscopically

p/s

315

12/07/1

4


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CHARATERSTICS OF WELL MADE SMEAR

Tongue shaped: smear is thick at the frosted end andbecomes progressively thinner toward the opposite end.

The "zone of morphology" (area of optimal thickness forlight microscopic examination) should be at least 2 cm inlength.

The smear should occupy the central area of the slideand be margin-free at the edges .

Blood film should be smooth and free of serration.

Unstained smear should be transparent so that anewsprint is visible through it.

Make at least two smears.

bl f f S


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Unacceptable forms of PBS


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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.


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Estimation of Platelet Count through PBS


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Microscopic Examination of Stained Blood Film

1. Low power (10x) Examination:Tojudge 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 forperipheral blood differentialcount. (a) 10 microscopic fieldsare examined in a verticaldirection from bottom to top (ortop to bottom). (b) The slide ishorizontally moved to the nextfield (Apply thin layer of oil over

the smear & examine it underoil.)


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Parts of PBS

3030Thin part

Ideal part for examination

junction of body & tail

Thick part


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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 or1/10-20 RBC


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PBS with Normal Platelet Count

Evenly distributed platelets : Leishman stain (40X)About 27 platelets are seen in the field


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Smears with Normal Platelet Count

Platelet Clump


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PBS with Low Platelet Count


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1. Use the oil immersion field (OIF) to count the number

of platelets per field.2. Count platelets in at least 10 FIELDSin OIF & take

the average number of platelets.

3.Estimated platelet count ( cu mm) is given by:

Average number of platelets per OIF multipliedby 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) 20 platelets/OIF = increased

ESTIMATION OF PLATELET COUNT


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NS1 Antigen Detection: RapidKit Test


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NS1 Antigen Test for Dengue

The Dengue NS1 Antigen Test is a rapid immuno-

chromatographic (strip/cassette) assay for the qualitativepresumptive detection of non-structural protein 1 (NS1) inhuman serum.

This test should be done within 1-7 days of onset of

symptoms.


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PRINCIPLE OF THE TEST

Strip/Cassete test

Monoclonal anti-NS1antibodiesDeposit 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 placedinto 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.


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Interpretation of the Test

Invalid

T C

Positive Result

Negative Result

T

T C

C

serum or plasma

15 mn


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Dengue Detection Tests at PUCHs

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