SHELL VIAL PROCEDURE (Appendix II)

I. Introduction

The shell vial method employs centrifugation of the patient specimen onto a cell monolayer contained in a vial. In general, the centrifugation step shortens the time to a positive culture result. Virus may be detected by direct fluorescent antibody (DFA) or indirect fluorescent antibody (IFA) staining within hours or days of inoculation. Currently, the MRC-5 (Human Fibroblast cells, Diagnostic Hybrid Inc. [DHI]) shell vial is used for the detection of CMV, HSV and VZ, the R-Mix (DHI) is used for the detection of respiratory viruses and the E-Mix (DHI) is use for the detection of enteroviruses.

 

II. Reagents and Materials

Fluorescence microscope with filter for FITC/Evans blue

Inverted microscope

FITC-conjugated virus-specific antibody stains (HSV1,2; VZV; CMV-IE; D3 enterovirus)

SimulFluor DFA Respiratory Viral Screen/RSV panel-(Chemicon)*

Phosphate buffered saline (PBS)

Distilled water

Cold acetone (4^oC)

Mounting fluid

Sterile pipettes

Cytospin and accessories

Humidified chamber

Sterile freezer vial

Sterile shell vials with round coverslips and caps

Needle with hooked end attached to syringe

Maintenance media

Glass slides

Coverslips

Paper towels for blotting

III. Procedure

1. Registration

i) Upon receipt of a shipment of cells, initial and date the record sheet accompanying the shipment. The record should contain vendor, lot number, passage number and QC data. File in the Cell Culture QC binder.

ii) Register the shell vial lots in the lab information system (LIS), this is necessary to generate the Shell Vial MRC-5, E-Mix or R-Mix QC procedures. Print labels. Refer to virology LIS manual for procedure.

 

2. Seeding (not in routine use) of MRC-5shell vials

i) Before seeding the shell vials, aspirate about 15 mL of MRC-5 cell suspension into a 125 cm² tissue culture flask, place label on the side of flask and/or write “MRC-5, date and ‘Pre’”.

ii) After seeding shell vials, aspirate about 15 mL of MRC-5 cell suspension into another 125 cm² tissue culture flask, label on the side of flask with “MRC-5, date and ‘Post’”.

iii) Aliquot the MRC-5 cells in 1 to 2 mL volumes into a sterile shell vials containing round cover-slips.

iv) Each shell vial is capped tightly (CO₂ produced by growing cells is needed to maintain proper pH for optimal cell growth) and incubated at 36ºC for 2-3 days to form a relatively confluent monolayer before use.

 

3. Incubation

i. For seeded (not in routine use) MRC-5 shell vials, incubate at 36ºC and use them on days 3-10.

ii. Store DHI (MRC-5, E-Mix and R-Mix) shell vials at room temperature in the dark. The DHI shell vials can be placed in service after pre-incubating for 2 to 4 hours.

Only sufficient shell vials for the day should be pre-incubated and the unused ones can be pre-incubated again the next day up to the allowed 24 hr limit..

 

4. Refeed

Shipping media in the shell vials must be replaced with the corresponding refeed media prior to inoculation. This is done by decanting or aspirating with a pipette and adding 1.5 mL of the appropriate aliquot maintenance media (each of MRC-5, E-Mix and R-Mix has its own refeed medium from DHI).

 

5. Inoculation of shell vials

i) Refer to specimen protocol or Appendix XXI Specimen Cell Line Stain Table for the number of shell vials to be inoculated.

ii) Prior to inoculation, check for confluent monolayer formation, sterility and for presence of a coverslip. Ensure that the shipping media have already been replaced with appropriate fresh maintenance media. Record results daily under Shell Vial MRC5, Emix or Rmix quality control procedures in the LIS.

iii) Apply a specimen label (LIS barcode) to the shell vial(s) and a corresponding plane glass slide. Label slides for HSV1; HSV2; VZ; CMV on MRC-5 shell vials and RS on R-Mix shell vials accordingly. E-Mix will be read for CPE as with tube culture and does not require a pre-labeled slide.

iv) Inoculate 0.2 mL for MRC-5, E-Mix and 0.3 mL for R-Mix of processed specimen into the shell vials. Inoculate one specimen at a time, recapping immediately afterward.

v) Centrifuge at room temperature for 15 minutes at 4300 rpm (3500 x g).

vi) Use a new, sterile pipette for each vial. Process one specimen at a time, recapping immediately afterwards. After set up is complete, discard any remaining aliquotted maintenance medium. For specimens that have excess blood or mucous, remove excessive sediment by refeeding shell vials after about 2 hours of incubation.

viii) Incubate the shell vials at 36ºC, lined up in rows of HSV1 and HSV2. CMV, R-Mix and E-Mix should be lined up in different cluster plates (CMV-IE requires an extra step in IFA staining and 2 days of incubation; R-Mix and VZ are DFA staining but require 2 and 4 days of incubation respectively; E-Mix is incubated and read for CPE):

Cell Line	Virus/Stain/Read for CPE*	# of Vials	Incubation Time
MRC-5 MRC-5 MRC-5 MRC-5 R-Mix E-Mix*	HSV 1, 2 HSV bivalent VZV CMV Resp virus Screen (RS) Enteroviruses* (read for CPE)	2 1 1 1 1 1	1 day 1 day 4 days 2 days 2 days 5 days*

 

4. Staining of shell vials

i) Prior to staining, examine the shell vial monolayer using the inverted microscope:

ii) If there is <75% CPE, perform IFA or DFA staining on the shell vial monolayer using the required antibody conjugate. For CMV, see shell vial staining under Appendix IV (IFA) and for HSV 1&2, VZV and RS, see shell vial staining under Appendix V (DFA).

iii) If >75% of the monoloayer has lifted from the coverslip, check the colour of the maintenance media and proceed as follows:

iv) If the maintenance media is bright pink (suggesting alkaline pH), yellow or cloudy, check with charge/senior technologist before proceeding further.

iii) If the maintenance media is appropriately coloured (salmon pink), perform IFA or DFA staining using cytospin preparations of scraped shell vial cells. Follow the staining procedure for prepared cytospin slides as outlined in the tube culture section in Appendix IV (IFA) and Appendix V (DFA).

iv) Discard cap. Remove maintenance medium from the shell vials, using a different sterile pipette for shell vials of the same specimen number.

v) Add 1 mL of cold acetone to each shell vial. Cover and fix for 10 minutes.

vi) Decant acetone and blot on paper towel.

vii) Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Make sure the stream is gentle enough not to flip the cover-slip. Decant PBS.

viii) Add 75 ml (2 drops from bottle) of HSV1, HSV2 and VZ to the appropriate row of shell vials in the DFA cluster plate (including QC shell vials, if done on that day). Cover.

ix) Add 75 ml (2 drops from bottle) of CMV-IE to the appropriate row of shell vials in the IFA (CMV, 2 day) cluster plate (including QC shell vials, if done on that day). Cover.

x) Incubate both DFA and IFA shell vials at 36^oC for 30 minutes.

xi) Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Make sure the stream is gentle enough not to flip the cover-slip. Decant PBS. Repeat.

xii) For the DFA shell vials (HSV1, HSV2, VZV) remove the coverslip and place cell side down onto a drop of mounting fluid on the pre-labelled glass slide.

xiii) For the IFA (CMV 2 day) shell vials, add 75ml (2 drops from bottle) of appropriate FITC-conjugated anti-mouse antibodies, cover and repeat the incubation and wash steps (i and j).

xiv) Remove the coverslip and place cell side down onto a drop of mounting fluid on a glass slide.

xv) Read using fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.

 

III. Reading of Stained Shell Vials

i) CMV – Immediate Early Antigen (CMV-IE)

Using the fluorescence microscope with the FITC/Evans Blue filter, scan the entire field using the 25x objective. Use the 40x objective to investigate any fluorescing cells.

POSITIVE: An even matte apple-green fluorescence covering the entire kidney bean shaped/oval nucleus. May include specks of brighter fluorescence.

NEGATIVE: No typical cells with apple-green fluorescence

INVALID: If no counterstain is visible or only the edge of cover slip is stained, inform senior/charge technologist. The cover-slip may have flipped before being stained

 

ii) HSV/VZV

Using the fluorescence microscope with the FITC/Evans Blue filter, scan the entire field using the 10x or 25x objective. Use the 40x objective to investigate any fluorescing cells.

POSITIVE: Distinct apple-green fluorescence of the cytoplasma and /or nucleus of the infected cells. Dull red Evans blue counter stain should be visible for stained nonfluorescent cells.

NEGATIVE: No typical cells with apple-green fluorescence.

Dull red Evans blue counter stain should be visible for negative cells.

INVALID: If no counterstain is visible or only the edge of cover slip is stained, inform senior/charge technologist. The cover-slip may have flipped before being stained.

 

iii) RS- (Respiratory virus Screen)

Using the fluorescence microscope with the FITC/Evans Blue and Rodamine bi-filter, scan the entire field using the 10x, and 25x objectives. Use the 40x objective to investigate any fluorescing cells.

POSITIVE for respiratory virus: i) green: Cells with apple-green fluorescence fluorescence.

POSITIVE for RSV: i) gold: Cells with gold fluorescence.

NEGATIVE: No typical cells with apple-green or gold fluorescence

INVALID: If no counterstain is visible or only the edge of cover slip is stained, inform senior/charge technologist. The cover-slip may have flipped before being stained.

 

IV. Shell Vial for CPE (E-Mix) can be referred to Appendix III (Tube Culture/Shell Vial for CPE)

IV. Quality Control

A. Shell Vial MRC-5 Quality Control: (unopened shell vial)

This is done weekly when cell shipments are received to monitor cell growth. Record daily in LIS.

Examine daily (for 7 days) for:	Expected results:	Shell Vial MRC5 QC- expected results (LIS entry):
Absence of contamination	Visual inspection: (1) medium colour not yellow (2) medium not cloudy	OK*
Healthy cell growth	Under inverted microscopy: (1) confluent monolayer (2) medium colour pink	OK*
Cover slip	Under inverted microscopy: cover slip present	OK*

At the end of 7 days, one unopened shell vial in good condition is used as “Previous lot MRC-5” for the following week.

 

B. Shell Vial Inoculation QC procedure (6 shell vials + 1 previous lot):

This QC procedure is performed once a week utilizing HSV-1 (ATCC 539) to:

1. Show that each MRC-5 lot supports the propagation of the intended viruses.

1. Monitor entire shell vial procedures from inoculation to reading including incubation, staining and reading (HSV1 & 2 are DFA, CMV-IE is IFA).
2. The inoculation part is done by Tube Culture bench, the Shell Vial bench completes the procedure including reporting in the LIS.

 

 

Gr* = stained positive for the intended virus

Pos*= stained positive with the specified stain

Neg*= stained negative with the specified stain

 

2. Daily Slide Shell Vial QC procedure:

Done and recorded each work day to monitor the staining of each batch (except the day when Inoculated Shell Vial QC procedure is done).

4-well HSV daily QC slide	2-well CMV daily QC slide	Well containing:	Stain with:	HSV1 daily SLIDE SV QC- expected results on Staining Reaction (LIS entry):
1		HSV-1 (ATCC 539)	HSV-1	Pos*
2		Uninoculated MRC-5 cells	HSV-1	Neg*
3		HSV-2	HSV-2	Pos*
4		Uninoculated MRC-5 cells	HSV-2	Neg*
	1	CMV (ATCC 807)	CMV-IE	Pos*
	2	Uninoculated MRC-5 cells	CMV-IE	Neg*

Gr* = stained positive for the intended virus

Pos*= stained positive with the specified stain

Neg*= stained negative with the specified stain

4-well R-Mix daily QC slide	Well containing:	Stain with:	R-Mix daily SLIDE SV QC- expected results on Staining Reaction
1	Flu A (ATCC)	RS	Pos*
2	Uninoculated cells		Neg*
3	RSV (ATCC)	HSV-2	Pos*
4	Uninoculated MRC-5 cells	HSV-2	Neg*
	UHN/MSH Microbiology Department Policy & Procedure Manual	Policy # MI/VIR/16/02/v03	Page 10 of 10
	Virology Manual

D. Reagent QC (HSV1, HSV2, HSV bivalent, CMV-IE and VZ stains):

a. Performed prior to patient testing and must pass before reagents are released for use.

a. Done on external QC slides.

b. Record QC results in Reagent Log and LIS.

Failed QCs:

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

b. Inform charge/senior technologist.

c. Record in Reagent Log Chart, Instrument Maintenance Log (if eg. microscope/incubator is involved in the failure) and file incident report if necessary.

d. Re-run failed controls in parallel to fresh controls (and/or external QC) to evaluate the QC material itself (already done routinely for MRC5 cells).

e. If the re-run shows the old QC material still fails, 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 primary/secondary stain)

a. Microscope (filter, bulb, alignment)

c. Other equipment, reagents or technique

V. Reference

1. Isenberg, H.D. 1992. Clinical Microbiology Procedure Handbook Vol. 2. ASM Press.

2. Gleaves, Curt A. et al, J Clin Micro., Feb. 1985. Comparison of Standard Tube and Shell Vial Cell Culture Techniques for the Detection of Cytomegalovirus in Clinical Specimens.

3. Engler, Howard D., Selepak, Sally T., J Clin Micro., June 1994. Effect of Centrifuging Shell Vials at 3,500 x g on Detection of Viruses in Clinical Specimens.

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TUBE CULTURE (not in routine use) or SHELL VIALS for CPE PROCEDURE (Appendix III)

I. Introduction

Tube culture is the conventional method used by diagnostic virology laboratories for virus isolation. Since there is no universal cell line for recovery of all clinically significant viruses, a combination of cell types is used routinely depending on the symptoms, clinical specimen type and specific viruses being sought. Shell Vials (E-Mix ) can also be adapted to extend their normal incubation times and continue as tube cultures.

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II. Reagents and Materials

Fluorescence microscope Leica DBRB with #2 filter for Rodamine/FITC Evans blue and #4 filter for FITC Evans blue or

Fluorescence microscope Leica DC300F with #3 filter for Rodamine/FITC Evans blue and #1 filter for FITC Evans blue

Inverted microscope

Control slides

Virus-specific antibody (eg. Enterovirus D3 stain, DHI)

FITC-conjugated antimouse antibody

Phosphate buffered saline (PBS)

Distilled water

Cold acetone (4^oC)

Mounting fluid

Sterile pipettes

Cytospin and accessories

Vortex

Sterile freezer vial

Glass slides

Coverslips

Paper towels for blotting

Humidified chamber

 

III Procedure

1. Registration

i) Upon receipt in the lab, register cell culture received from the supplier in the lab information system (LIS). Refer to virology LIS manual for procedure.

ii) File the vendor QC sheet (received with the shipment) in the QC binder.

iii) Randomly, select two tubes from each lot and check the monolayer microscopically for confluent growth and quality of cells. Use these two tubes as the “unopened controls” outlined under the Quality Control section below.

iv) MRC-5, R-Mix and E-MIx cell lines are stored for 18-24 hours at 36^o C in O₂ (or until cell lines reach >50% confluency) and then are kept at room temperature until expiry.

2. Inoculation of cell culture shell vials

i) Aliquot 50 mL maintenance medium and allow to come to room temperature before using.

ii) Refer to the protocol for each specimen type to determine the number of tubes and types of cell lines to be inoculated. Also refer to Appendix XV if needed.

iii) Prior to inoculation, check the cell culture tubes for acceptable confluent monolayer formation and sterility.

iv) Decant the medium from the tube.

v) Using a clean, sterile pipette for each tube, add 1.5 mL of the aliquotted maintenance medium to each tube and re-cap. After set up is complete, discard any remaining maintenance medium.

vi) Inoculate 0.2 mL (4 drops) of processed specimen into each tube, recapping immediately afterward.

vii) Incubate the tubes in the roller drum at 36^oC. Refer to the appropriate specimen protocol for the incubation time for each tube.

viii) Refeed MRC-5, R-Mix and E-Mix minimally once per 5 days. Shell vials showing signs of chemical toxicity (red media / sloughing cells), bacterial / fungal contamination (yellow / turbid media) or aging should be refed within the day.

 

III. Reading of Cultures (Shell Vials for CPE)

i) Cytopathic effect (CPE): E-Mix or other cell lines should be examined daily for CPE. Any culture demonstrating > 2+ CPE should be confirmed by staining. The cells should be scraped, a cytospin slide prepared and appropriate monoclonal antibody staining performed. If no CPE is present, refeed with corresponding maintenance medium and Reincubate.

 

ii) Enteroviruses (D3 enterovirus, DHI): Perform enterovirus stain when CPE is observed:

a. Prepare cytospin preparation from cell culture as outlined below:

b. Remove about 0.5 mL (leaving about 1 mL) maintenance media from the culture using a sterile pipette.

c. Scrape cells using a sterile pipette. Break up cell clumps by pipetting up and down several times.

d. Pipette 4 x 200 uL (4 x 4 drops) of scraped cells into 4 funnels on 2 double slides.

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

f. Remove slide and air dry.

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

h. An enterovirus QC control slide should be stained in parallel with the patient as follows.

i. Stain the 4 wells by adding 20 mL each of Enterovirus D3, ECHO, Coxsackie B and Polio stains onto the fixed cell spots.

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

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

l. Wipe excess PBS from the slide without touching the cell spot.

m. Add 20 mL of FITC-conjugated antibodies.

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

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

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

q. Wipe excess water from the slide without touching the cell spot.

r. Mount using coverslip and mounting fluid.

s. Read with fluorescence microscope Leica DC300F with #3 filter for Rodamine/FITC Evans blue and the 40x objective (warning:#1 filter is for FITC Evans blue only).

Interpretation of Results

Positive for enterovirus: Green fluorescence

Negative: Dull-red counterstained cells with no apple-green fluorescence.

Invalid: If no counterstain is visible, repeat staining

QC slide failed, report to senior/charge

If positive, record in freezer program and freeze cells and supernate. Refer to Appendix X and XII for procedure.

 

iii) Confirmation by PHL: Any culture demonstrating CPE for which a virus cannot be detected using monoclonal antibodies or other in-house methods and for which toxicity has been ruled out (see below) should be referred to the Public Health Laboratory (PHL) for further work-up. Pass cells to a new culture before sending. Scrape and add 0.2 ml (4 drops) of scraped cells to a fresh culture containing 2 mL of fresh maintenance media (1:10 dilution). Consult the charge/senior technologist or medical microbiologist before referring the specimen to PHL.

 

iv) Culture Toxicity: If chemical toxicity is suspected in a culture (rounding of cells, sloughing of cells, granular cytoplasm of cells or unusual CPE, consult senior/charge technologist if unsure), proceed as follows:

 

v) Pass cells by scraping and adding 0.2 ml (4 drops) of these scraped cells to a fresh culture containing 2 mL of fresh maintenance media (1:10 dilution). Proceed with tube culture method as outlined above.

 

vi) The effects of chemical toxicity would be reduced by dilution whereas the effects of CPE (caused by viral replication) would be the same, if not accelerated on passage. If CPE is suspected, identify virus by antibody stains. If chemical toxicity is suspected, continue to incubate (may need further refeeding to reduce toxicity). If unsure of cell toxicity or CPE , refer to the charge/senior technologist for review.

 

vii) Contaminated Culture: If the culture appears visibly contaminated (eg. cloudy and/or yellow medium) and thus uninterpretable, proceed as follows:

a. On 1^st or 2^nd reading - change the maintenance medium, and reincubate.

b. On 3^rd or later reading or recurrence - issue a final report stating:

“Virology culture: Specimen is heavily contaminated with bacteria and/or fungus. Unable to interpret virology culture.”

c. Replant if specimen is from a sterile site or contamination is attributed to the lab. If multiple specimens are contaminated, report to senior/charge.

 

IV. Quality Control (Tube Cultures are not in routine use)

Record all results of QC in LIS (or log). Refer to virology LIS manual for procedure. Report any abnormal results to charge/senior technologist.

Five tubes are reserved from each lot of cell cultures received, and used as controls as follows:

i) Negative controls: (tubes labelled N1, N2, N3)

On Wednesday, Friday and Monday, an uninoculated tube from each cell line used that day is placed in the roller drum with the inoculated specimens. These tubes are incubated, read and refed with the patient inoculated cultures to monitor the monolayer quality, medium toxicity/contamination. They can also be used

to provide a baseline for comparison for inoculated cultures when reading for CPE. HFF, CMK, HEp-2 and RD tubes are kept for 5, 2, 2 and 1 weeks respectively.

ii) Unopened Controls: (2 tubes labelled C and V respectively)

These tubes are not opened. One tube is kept at 36^o C in O₂ in the clean room (C)

and one is placed on the roller drum (V) at 36^o C in O₂. These tubes are observed

for 1 week to identify toxicity and contamination originating with the vendor.

iii) Positive Controls:

Each week HSV-1 ATCC strain # VR- 539 is scraped from the previous week’s positive control tube and used to inoculate a fresh HFF tube. If the control fails to propagate, a new vial can be retrieved from liquid N₂ tank MINS shelf 6.

Additional positive controls may be set up for the following reasons:

• · Low isolation rates
• · Comparison of cell lines
• · Vendor changes
• · Proficiency test failures
• · Training
• · Continuing problems with negative controls
• · Preparation of QC material (i.e. positive control slides)
• Consult a charge/senior technologist to determine the cell lines and viruses to be set up.

 

V. Reference

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

Press.

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