DIRECT IMMUNOFLUORESCENT ANTIBODY (DFA) STAINING (Appendix V )

I. Introduction

The DFA staining technique is used to detect viruses either directly in patient specimens or which have been isolated in shell vial or tube cultures. The method consists of a single staining step using a virus-specific antibody which is conjugated with a fluorochrome. Viruses which we currently identify by DFA staining include HSV-1, HSV-2, VZV, CMV (late antigen) and respiratory viruses (SimulFluor stains for respiratory syncytial virus, parainfluenza, influenza, adenovirus).

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II. Reagents and Materials

FITC-conjugated virus-specific antibody

FITC/Rodamine-conjugated virus-specific antibody (SimulFluor)

Phosphate Buffered Saline (PBS)

dH₂O

cold acetone (4^oC)

mounting fluid

sterile pipettes

cytospin and accessories (for tube culture)

humidified chamber

glass slides

coverslips

paper towels for blotting

 

III. Procedure

 

1. Shell Vial

This procedure is for staining of cells directly in shell vial. If staining a cytospin slide or slide made directly from a patient specimen, follow the tube culture procedure below.

i. Discard cap. Remove maintenance medium from the shell vial using sterile pipette.

ii. Add 1 mL of cold acetone. Cover with tray lid and let sit for 10 minutes.

iii. Decant acetone and blot shell vial on paper towel.

iv. Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Decant PBS.

v. Add 75ml (2 drops from bottle) of appropriate FITC or Rodamine-conjugated virus-specific antibody. Cover with tray lid.

vi. Incubate at 36^oC for 30 minutes.

vii. Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Decant PBS. Repeat.

viii. Remove the coverslip from each shell vial and place cell side down onto a drop of mounting fluid on a glass slide.

ix. For HSV 1, HSV 2, VZ and CMV, read using fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.

x. For respiratory viruses, read using fluorescence microscope with the FITC/Evans Blue Rodamine bi-filter and the 40x objective.

 

2. Tube Culture

i. Prepare cytospin slide from cell culture tube as outlined in Appendix XX.

ii. Fix slide in cold acetone for 10 minutes in a coplin jar. Remove slide and air dry.

iii. Add 20ml of appropriate FITC or Rodamine -conjugated antibody onto the fixed cytospin slide.

iii. Incubate in a humidified chamber at 36^oC for 30 minutes.

iv. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.

v. Wash with distilled water for 1 minute in a coplin jar.

vi. Wipe excess water from the slide without touching the cytospin preparation.

vii. Mount using coverslip and mounting fluid.

xi. For HSV 1, HSV 2, VZ and CMV, read using fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.

viii. For respiratory viruses, read using fluorescence microscope with the FITC/Evans Blue Rodamine bi-filter.

 

Interpretation of Results

Positive: Bartel CMV monoclonal antibody: Bright apple green fluorescence of cytoplasmic inclusion (late antigen) and homogenous early nuclear antigen in CMV-CPE cells.

Chemicon SimulFluor Respiratory Screen:

All respiratory viruses except RSV show bright apple green fluorescence of the cytoplasm and/or nucleus of the infected cell.

RSV shows bright gold fluorescence of the cytoplasm and/or nucleus of the infected cell.

Chemicon SimulFluor Flu A/Flu B:

Influenzae A virus shows bright apple green fluorescence.

Influenzae B virus shows bright gold fluorescence.

Chemicon SimulFluor RSV/Para 3:

RSV virus shows bright apple green fluorescence.

Parainfluenzae 3 shows bright gold fluorescence.

Chemicon SimulFluor Para 123/Adeno:

Parainfluenza 1,2,3 viruses show bright apple green fluorescence.

Adenovirus shows bright gold fluorescence.

Chemicon individual monoclonal antibodies:

Parainfluenzae 1 and 2, and adenovirus show bright apple green fluorescence.

Negative: Red Cells with no apple-green fluorescence.

 

IV. Quality Control

Appropriate positive and negative control slides should be stained with each batch.

 

VI. Reference

Isenberg, H.D., 1992, ASM. Clinical Microbiology Procedures Handbook Vol. 2.

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DIRECT ANTIGEN DETECTION FROM SPECIMENS (Appendix VI)

I. Introduction

Immunofluorescent staining may be used to rapidly detect viral antigen directly in a clinical specimen. Smears prepared at the patient's bedside may be used, or a smear may be prepared in the laboratory from cellular material in a specimen. Specimens for which direct antigen detection may be requested include bronchoscopy or nasopharyngeal specimens for respiratory virus detection or vesicular lesion scraping for HSV/VZV antigen detection. Cells are stained using pooled or specific monoclonal antibody stains and examined under the fluorescence microscope looking for specific fluorescence of cell cytoplasm or nucleus.

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II. Reagents and Materials

Virus-specific or pooled FITC and Rodamine conjugated antibody stains (with Evans blue counter stain):

Respiratory Viral Screen/RSV panel

FluA/B panel

RSV/para3 panel

Para1,2,3/Adeno panel

Specific Parainfluenza 1

Specific Parainfluenza 2

Herpes simplex 1

Herpes simplex 2

Herpes simplex bivalent

Varicella zoster virus

Phosphate buffered saline (PBS)

Cold acetone (4^oC)

Distilled water

FA mounting fluid

Vortex

Sterile pipettes

Cytospin and accessories

Humidified chamber

Coplin jars

Fuorescence microscope with FITC/Rodamine/Evans blue filters

 

II. Procedure

 

A. Preparation of slide

a) For swabs:

i. Vortex patient sample in transport medium for 30 seconds.

Remove excess fluid from the swab and discard the swab.

ii. Transfer 0.5 - l.0 ml of this specimen to a microcentrifuge tube. Centrifuge 1 minute at 14,000 rpm.

iii. Remove supernatant (can be placed back with original specimen to be processed further) down to 400 ul (8 drops). Vortex 5-10 seconds.

iv. Pipette 200 ul (4 drops) of this sediment into funnel for each well. Prepare the appropriate number of cytospin wells according to table below.

v. Cytospin at 2000 rpm (700g) for 5 minutes.

vi. Remove slide and air dry.

vii. Fix in cold acetone for 10 minutes in a coplin jar.

viii. Remove slide and air dry.

ix. Proceed to staining. Refer to Appendix V (DFA) for staining procedures.

 

b) For Bronchoscopy Specimens (BAL,Washings):

i. Mix specimen gently and examine for cellular turbidity. Specimens which are more turbid than a 0.5 McFarland standard are diluted to that approximate turbidity using Hank's Balanced Salt Solution (Gibco BRL).

ii. Pipette 200 ul (4 drops) of specimen into funnel for each well. Prepare the appropriate number of cytospin wells according to table below.

iii. Cytospin 200 uL at 2000 rpm (700 x g) for 5 minutes. Prepare the appropriate number of cytospin preparations according to the table below.

iv. Remove slide and air dry.

v. Fix in cold acetone 10 minutes in coplin jar.

vi. Remove and air dry.

vii. Proceed to staining. Refer to Appendix V (DFA) for staining procedures.

 

c) For Smears Prepared Outside of the Laboratory

For smears which have been prepared outside of the laboratory, examine macroscopically for evidence of material on slide. Specimens for which only one slide is received may be stained with ONE monoclonal antibody only according to the physician's request. It is not possible to stain a negative control for such specimens. A swab for viral culture should be requested if not received along with the smear.

Draw a circle around material on slide with a diamond pencil prior to staining.

i. Fix in cold acetone 10 minutes in coplin jar.

ii. Remove and air dry

iii. Proceed to staining. Refer to Appendix V (DFA) for staining procedures.

 

1. Staining Protocol

Prepare appropriate number of cytospin wells and stain as per chart. Refer to Appendix V (DFA) for staining procedures.

Specimen	Monoclonal Antibody
Nasopharyngeal/ Bronchoscopy/ Throat	Respiratory screen a (November to April)	SimulFluor, Light Diagnostics
(prepare 2 cytospin wells)	Flu A/ Flu B	SimulFluor, Light Diagnostics
Vesicular Lesion Swab	HSV-bivalent	Bartels
(prepare 2 cytospin wells)
	VZV	Meridan Bioscience Inc.

^a If respiratory screen or Flu A/B is positive, prepare more cytospin preparations and stain according to interpretation chart that follows.

SimulFluor Respiratory Screen Staining Protocol and Interpretations – Scheme 1

Using filter for FITC/Evans Blue (Filter no. 3 in fluorescence microscope no.2, Leica I)

First double-well cytospin slide

SimulFluor Respiratory Screen Staining Protocol and Interpretations- Scheme 2

Using filter for FITC/Evans Blue (Filter no. 3 in fluorescence microscope no.2, Leica I)

First double cytospin slide

III. Interpretation of Results

Examine smear for adequate numbers and types of cells. Respiratory specimens should consist of columnar (ciliated or goblet) epithelial cells. Scrapings from lesions should contain basal epithelial cells. (See reference 2, page 69). Adequate interpretation of results requires a minimum of approximately 20-50 cells per smear. Samples with inadequate numbers of cells should be shown to a charge/senior technologist and reported as having insufficient cellular material.

Positive: Specific, apple-green fluorescence in the cytoplasm and/or nucleus of the exfoliated cells. This specific fluorescence must be absent in the negative control and/or smears stained with other antibodies.

Negative: Dull-red stained cells with no viral specific apple-green fluorescence.

OR

Dull-red stained cells with pinpoint non-specific nuclear staining.

Insufficient cells: Fewer than 20 epithelial cells per smear

 

IV. Quality Control

Reagent QC:

a. Check expiratory date.

b. Verify that Reagent QC is satisfactory for the reagent lot/kit being used (recorded in Reagent Log and/or on the kit).

c. If necessary, perform the Reagent QC procedure ( external QC slide with all components eg. Bion 14-well Respiratory Panel).

 

Failed Reagent QC:

Test is invalid without satisfactory Reagent QC results.

a. Do not release reagent lot for use pending resolution of QC error.

b. Inform charge/senior technologist.

c. Record in Reagent Log Chart, Instrument Maintenance Log if microscope/incubator is involved in the failure (and Incident Report if necessary).

d. Re-run failed control materials in parallel to fresh controls to evaluate the QC material itself.

a. If the re-run shows the old QC material still fails and fresh QC is satisfactory, the error may be attributed to the old QC material itself and the reagent is satisfactory.

b. If the re-run shows both the old and fresh QC material fail (or other QC not satisfactory), the error may be attributed to the reagent then the reagent cannot be released for use.

 

Daily QC:

a. Appropriate positive and negative control slides (eg. ATCC 4-well slide with RSV/Para3 for SimulF RS stain) should be stained with each batch.

b. Examine the negative control well first to establish the dull red colour (Evans blue counterstained) and to determine if there is any nonspecific staining.

The positive control must be clearly distinguishable from the negative control or the test is invalid.

 

Failed Daily QC:

a. Do not release patient results pending resolution of QC error.

b. Inform charge/senior technologist.

c. Record in Reagent Log Chart (and Instrument Maintenance Log if microscope/incubator is causing the failure).

d. Re-run failed controls in parallel to fresh controls (and/or external QC) to evaluate the QC material itself.

e. If the re-run shows the old QC material still fails and fresh QC passes and nothing else is wrong with the batch (only the old QC material failed, patient results valid) patient results may be released.

 

Marked decrease/absence in fluorescence can be due to:

a. Reagent deterioration/skipping (did not apply the correct stain)

b. Microscope (filter, bulb, alignment)

c. Other equipment, reagents or technique

V. Reporting

See individual specimen protocols.

VI. Reference

1. Wiedbrauk, D.L. 1993, Raven Press. Manual of Clinical Virology.

2. Rossier, E., Miller, H., Phipps, P. 1989, University of Ottawa Press. Rapid Viral

Diagnosis by Immunofluorescence: An Atlas & Practical Guide.

3. SimulFluor Product Insert for cat. no. 3296, June 2002, Revision C: 40729

Light Diagnostics Chemicon International Temecula, CA 92590

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