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.

http://bahankuliahkesehatan.blogspot.com/