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/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/shell-vial-procedure-appendix-ii.html

Read More

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.

http://bahankuliahkesehatan.blogspot.com/

 

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.

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/tube-culture-not-in-routine-use-or.html

Read More

INDIRECT IMMUNOFLUORESCENT ANTIBODY (IFA) STAINING (Appendix IV)

 

FOR VIRAL CULTURE CONFIRMATION

I. Introduction

The IFA technique is used to identify viral isolates in the cells obtained from shell vials and tube cultures. The indirect method consists of two steps. In the first step, primary antibodies are allowed to react with viral antigens in the cells. These specific complexes are detected in a second step using a species-specific antibody conjugated with a fluorochrome. Viruses which we currently identify by IFA staining include cytomegalovirus immediate early antigen (CMV-IE) and enteroviruses.

http://bahankuliahkesehatan.blogspot.com/

 

II. Reagents and Materials

Virus-specific antibody

FITC-conjugated antimouse antibody

Phosphate buffered saline (PBS)

Distilled water

Cold acetone (4^oC)

Mounting fluid

Sterile pipettes

Cytospin and accessories (for tube cultures)

Humidified chamber

Sterile freezer vial

Glass slides

Coverslips

Paper towels for blotting

Humidified chamber (for tube culture)

 

III. Procedure

1. Shell Vial

Follow outline in Appendix II to determine if staining should be done in the shell vial itself or if a cytospin needs to be prepared. If the staining is to be done in the shell vial itself, proceed to step i) below.

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

a) Add 1 mL of cold acetone. Cover and fix for 10 minutes.

b) Decant acetone and blot on paper towel.

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

d) Add 75 ml (2 drops from bottle) of appropriate antibody. Cover.

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. Add 75ml (2 drops from bottle) of appropriate FITC -conjugated antibodies, cover and repeat steps vi and vii.

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

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

 

2. Tube Culture (or Shell Vials for CPE)

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

ii. Add 20 ml of appropriate antibodies onto the fixed cytospin slide.

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

 

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

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

vi. Add 20 ml of appropriate FITC-conjugated antibodies.

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

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

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

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

xi. Mount using coverslip and mounting fluid.

xii. Read with fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.

 

Interpretation of Results

Positive: Enterovirus:

An 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.

 

V. Reporting

See individual specimen protocols.

 

VI. Reference

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

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/indirect-immunofluorescent-antibody-ifa.html

Read More

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).

http://bahankuliahkesehatan.blogspot.com/

 

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.

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/direct-immunofluorescent-antibody-dfa.html

Read More

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.

http://bahankuliahkesehatan.blogspot.com/

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

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/direct-antigen-detection-from-specimens.html

Read More

HEMADSORPTION OF TUBE CULTURE MONOLAYERS (NOT ROUTINELY DONE) Appendix VII

The hemadsorption (HAD) technique is used primarily to detect viruses that produce little or no cytopathic effect (CPE) in tube culture monolayers. Using guinea pig RBC, it is used to screen inoculated cell cultures for the presence of influenza, parainfluenza, mumps and Newcastle disease viruses.

http://bahankuliahkesehatan.blogspot.com/

 

I. Procedure

Reagents

guinea pig RBC in Alsever's solution

sterile phosphate buffered saline(PBS)

 

Materials

sterile pipettes

precision pipettes

inverted microscope

centrifuge

15 mL sterile centrifuge tube

 

Preparation of 10% stock guinea pig RBC suspension

The stock suspension should be prepared every Monday and stored at 4^oC and used within 7 days of preparation.

1. Transfer 5 mL of blood to 15 mL tube and add equal volume of PBS.

2. Centrifuge at 3000 rpm (700 x g) for 5 minutes at room temperature.

3. Discard the supernate and add 10 mL of PBS.

4. Centrifuge and wash the cells until the supernatant is clear. (approx. 2-3 washings).

5. Determine the packed-cell volume and add PBS equal to 9 times the packed-cell volume to yield a 10% suspension.

 

Preparation of 0.4% working guinea pig RBC suspension

The working suspension should be prepared from 10% suspension on the day of testing.

1. Add 0.4 mL of the 10% suspension to 9.6 mL of PBS.

 

HAD Test

The HAD test is performed on day 5 and day 10 for respiratory specimens with no evidence of CPE.

1. Select one RMK tube to be used for HAD. Transfer the medium in the tube to another sterile, labelled capped tube. Place medium at 4^oC pending HAD results.

2. Add 0.2 mL of the 0.4% RBC suspension to each culture tube, to be tested.

3. Incubate the tubes horizontally at 4^oC for 30 minutes. Make sure the RBC suspension is distributed over the monolayer.

4. Gently rotate or tap the tubes to resuspend nonadsorbed cells. Immediately examine the tubes with inverted microscope with the 40X objective. Do not handle the tubes in such a way that the monolayers will become warm.

 

Interpretation of Results

1. Positive HAD test should show RBCs firmly attached to the monolayer. Hemagglutinated cells (clumped RBCs) are also seen in the fluid overlaying the monolayer.

2. Negative HAD test should show no or minimal RBCs attached to the monolayers, with almost all cells floating above the monolayers.

 

II. Quality Control

Positive and negative controls should be set up for HAD test prior to the expected "flu" season.

 

III. Positive HAD Test

1. Place the tube in a 36^oC water bath for 15 minutes.

2. Wash the eluted monolayers twice with PBS.

3. Perform indirect immunofluorescence test for influenzae and parainfluenzae viruses. See Appendix III.

4. The culture medium harvested prior to the HAD test may be used for subpassage, storage or identification by hemagglutination inhibition.

 

IV. Negative HAD Test

1. Discard tube that is HAD negative at 10 days after inoculation.

2. Tube inoculated after 5 days should be washed 3 times with 5 mL Hank's Balanced Salt Solution to remove all RBCs.

3. Refeed tubes with 2 mL of maintenance medium and reincubate the cultures for another 5 days.

4. Discard the culture fluid harvested from negative cultures unless subpassage is to be performed.

 

V. Reference

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

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/hemadsorption-of-tube-culture.html

Read More

Appendix VIII MEDIA

GROWTH MEDIUM (Not in routine use)

 

500 mL Eagles' MEM with Hanks' salt/without glutamine

10 mL Vitamins

5 mL L-glutamine (200 mM)

50 mL Inactivated fetal calf serum

Store at 4^oC. Stable for 2 weeks.

http://bahankuliahkesehatan.blogspot.com/

 

MAINTENANCE MEDIUM (Made in-house, not in routine use)

500 mL Eagles' MEM with Hanks' salt/without glutamine (stored at 4^oC)

5 mL L-glutamine (200 mM, stored frozen at -20 ^oC in clean room freezer)

10 mL Inactivated fetal calf serum (stored frozen at -20 ^oC in clean room freezer)

5 mL Fungizone (250 μg/mL, stored frozen at -20 ^oC in clean room freezer)

*5 mL Gentamicin (1 mg/mL, stored frozen at -20 ^oC in clean room freezer)

**5 mL Vancomycin (10 mg/mL, stored frozen at -20 ^oC in clean room freezer)

Store at 4^oC. Stable for 2 weeks.

 

* Gentamicin Solution (1 mg/mL):

Dilute entire contents (10 mL) of 10 mg/mL Gentamicin Sulphate vial into 90 mL of d. H₂O to achieve 1 mg/mL. Filter sterilize, then dispense into 5 mL aliquots (stored frozen at -20 ^oC in clean room freezer).

 

** Vancomycin Solution (10 mg/mL):

Add contents of 1 gram vial of Vancomycin to 100 mL of distilled H₂O. Filter sterilize, then dispense into 5 mL aliquots (stored frozen at -20 ^oC in clean room freezer.

 

MAINTENANCE MEDIUM (Diagnostic Hybrids Inc., ready to use)

Media Quality Control:

a. A 1.5 mL aliquot each of the corresponding maintenance media is to be placed into each of MRC-5, R-Mix and E-Mix shell vials for a check on sterility/toxicity before the media are used on patient samples.

b. MRC-5, R-Mix and E-Mix Maintenance Media are to be registered into the LIS (micqc) as “on receipt” with lot numbers and expiry dates.

c. Maintenance Media QC results are all entered into the LIS daily (R-Mix for 2 days, MRC-5 for 4 days and E-Mix for 5 days).

d. Activate and inactivate the media lots in the LIS as appropriate.

Failed Media QC:

a. Any media/cell lines showing microbial contamination or toxicity is considered abnormal.

b. Do not release media lot for use pending resolution of the QC failure.

c. Inform charge/senior technologist to discuss further actions including the following.

d. Record in Incident Report (if necessary).

e. Repeat QC/re-make media to evaluate if it is the cell media, the supplement(s) or other materials causing the problem.

 

CELL WASHING MEDIUM (Purchased)

Hanks' Balanced Salt solution without CaCL₂, MgCL₂ and MgSO₄.7H₂O

 

CELL FREEZING MEDIUM (SIGMA C6164) (Purchased)

MEM supplemented with a mixture of fetal bovine serum and calf serum containing DMSO.

 

VIRAL FREEZING MEDIUM (SIGMA C6039) (Purchased)

MEM supplemented with a mixture of fetal bovine serum and calf serum containing 10% glycerol.

 

TRANSPORT MEDIA:

 

BARTEL'S VIRAL TRANSPORT MEDIUM (B1029-36A) (Purchased)

Eagles' MEM with nonessential amino-acid and L-glutamine in HBSS.

2% FBS

10 μg/mL Gentamicin

50 μg/mL Streptomycin

50 μg/mL Penicillin

4 μg/mL Amphotericin B

15 mM HEPES

9.5 mM Sodium bicarbonate

 

CHLAMYDIA TRANSPORT MEDIUM (Not in routine use)

500 mL Eagles' MEM with Hank's salt/without glutamine.

50 mL Fetal Calf Serum (normal)

5 mL 3M Glucose

5 mL Gentamicin (1000 μg/mL)

5 mL Vancomycin (1000 μg/mL)

5 mL Fungizone (250 μg/mL)

Combine all components aseptically. Dispense 1.5 mL transport medium into sterile screw-cap vials containing 3 glass beads. Store at -20^oC.

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/appendix-viii-media.html

Read More

TRYPSINIZATION AND MAINTENANCE OF MONOLAYER CELL CULTURES (Appendix IX)

Monolayer cell cultures may be kept active and available for seeding of tubes, vials, dishes and plates. A constant supply of cell cultures can be maintained by routine subpassage of cell lines to new flasks. (Generally research use only.)

http://bahankuliahkesehatan.blogspot.com/

1. Discard medium from the cell culture flask (125 cm²). Rinse monolayer with 15 mL of Hank's Balanced Salt Solution and discard.

2. Add 5 mL of trypsin EDTA mixture (pre-warmed to 36^oC) to flask.

3. Incubate the culture flask for 3 minutes (no more than 5 minutes) at 36^oC. Observe after 3 minutes to see whether the cell sheet is breaking loose from the flask surface. Tap flask sharply against palm of hand to aid in loosening tissue.

4. When tissue has loosened completely, add 15 mL of pre-warmed growth medium into the flask.

5. Mix cells by drawing cells and fluid up and down in a pipette.

6. Adjust the volume to 90 mL with growth medium.

7. Aliquot 30 mL of suspended cell mixture into at least one new 125 cm² culture flask. Aliquot the remaining to shell vials at 1.5 mL each or tube culture (16 x 125 mm) at 2 mL each.

8. Incubate the culture flasks, shell vials or tube cultures at 36^oC.

9. Observe daily for growth of cells (3 - 5 days) and for change in pH of medium. If the medium becomes acidic or basic, replace it with fresh growth medium.

10. Replace growth medium with maintenance medium when a confluent monolayer is obtained (usually after 2 - 3 days).

11. Replace with fresh maintenance medium once a week.

Note: Freeze first passage of cell culture whenever a new shipment is received. (See Appendix VIII).

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/trypsinization-and-maintenance-of.html

Read More

CRYOPRESERVATION OF CELL CULTURES (Appendix X)

Generally research use only:

1. When a new bottle of cells to be frozen arrives, transfer 30 mL aliquots of cells into 125 cm² flasks (about 3 flasks).

2. Incubate at 36^oC until a complete monolayer is obtained (approximately 4 - 5 days).

3. Follow steps 1 - 5 in Appendix IX (Trypsinization and maintenance of monolayer cell cultures).

4. Transfer the trypsinized monolayer to a sterile 15 mL centrifuge tube.

5. Centrifuge at 2000 rpm (700 x g) for 10 minutes.

6. Discard the supernate and resuspend the cells in 10 mL cell freezing medium.

7. Distribute 1.0 mL volume into freezing vials labelled with cell line information.

8. Place the vials into a "Mr. Frosty" freezing container and hold in -70^oC freezer for 2 - 3 hours.

9. Record in the computer Freezer Program details of the cell lines stored. Use study "CEL".

10. Transfer the vials to liquid nitrogen freezer.

http://bahankuliahkesehatan.blogspot.com/

 

Reference

Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infection. A.P.H.A. 1989. Sixth Edition. Pg. 72.

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/cryopreservation-of-cell-cultures.html

Read More

RECOVERY OF CRYOPRESERVED CELLS (Appendix XI)

Generally research use only:

1. Obtain the frozen cells from liquid nitrogen storage and immediately place the vial in a 37^oC water bath WITH A PROTECTIVE COVER. ** Allow to thaw for 2 minutes (no more than 3 minutes).

2. Wipe the outside of the vial with 95% alcohol.

3. Transfer the contents of the vial to a new 125 cm² flask using a sterile transfer pipette.

4. Gradually add 30 mL growth medium to the cells (slowly over 2 minutes) to dilute the cells.

5. Incubate at 37^oC and observe at 24 hours for cell adherence and growth.

6. Discard old medium and refeed cells with 30 mL of growth medium at 24 hours to remove all traces of DMSO and reincubate the cells.

7. Replace growth medium with maintenance medium when the monolayer is confluent (usually after 2-4 days).

8. Replace maintenance medium with fresh maintenance medium once a week.

Reference

Isenberg, HD: Clinical Microbiology Procedures Handbook. American Society of Microbiology, 1992. Pg. 8.20.7.

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/recovery-of-cryopreserved-cells.html

Read More

CRYOPRESERVATION OF VIRUS ISOLATES (Appendix XII)

1. Record in the computer Freezer Program details of the isolate stored.

a. Select "A. Clinical Records".

b. "Add new entries?": enter "Yes".

c. Enter lab number, ensure patient data is correct, enter date frozen, enter study (ie "VIR"), ensure specimen type is correct.

d. Enter name of isolate to be frozen once.

e. Note the freezer locations in which the frozen isolates are to be placed.

- Freezer "G" & "J" refer to Liquid Nitrogen freezers.

- Shelf no. refers to the canister number.

- Box no. refers to the cane number. Each cane holds 5 freezer vials.

 

2. Discard maintenance medium on monolayer to be frozen.

http://bahankuliahkesehatan.blogspot.com/

3. Add 1.0 mL viral freezing medium (see Appendix VII) to positive cells. Scrape monolayer and mix well with freezing medium.

 

4. Distribute into freezing vial labelled with LIS label.

 

5. Store the vial immediately in liquid nitrogen freezer in the location specified by the freezer program.

 

FREEZING CAP SPECIMENS AND ISOLATES:

1. After planting CAP specimens, add 1 mL of viral freezing medium to the sample and transfer to a freezing vial. Enter into the freezer program as described above using "VQC" for study. Enter "CAPSAM" as isolate #1.

 

2. Freeze one vial of CAP viral isolates after identification using steps 2 - 5 described above. The frozen isolate will be isolate #2 and is entered using the code for the name of the virus. (The original sample and frozen isolate may be on different canes, this is acceptable).

 

Reference

Laboratory Diagnosis of Infectious Diseases. 1988 Volume II. Pg. 268.

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/cryopreservation-of-virus-isolates.html

Read More

Appendix XIII PRESERVATION OF CELL CULTURE MONOLAYERS & Appendix XIV QUALITY CONTROL OF CELL CULTURES USED FOR ROUTINE VIRUS ISOLATES

1. Aspirate the medium from the cell culture tubes to be preserved.

2. Add 8 mL buffered formaldehyde preservative medium to each tube.

3. Record the following information on the tube:

- virus

- number of days incubation

- lab number

4. Store preserved culture at room temperature.

Formaldehyde Preservative

100 mL Formaldehyde Solution (37 - 40%)

900 mL Distilled Water

20 mL Phenol Red (0.5%)

4.0 g NaH₂PO₄ - H₂O

6.5 g Na₂HPO₄

Materials

Vortex

Sterile pipettes

10 - 100 uL Eppendorf pipette

Humidified chamber

Coplin jars

Fluorescent microscope

 

Appendix XIV

QUALITY CONTROL OF CELL CULTURES USED FOR ROUTINE VIRUS ISOLATES

Tube Culture (Not in routine use)

Upon receipt of cell culture tubes, the record of the date received, vendor, lot number, and passage number is kept in the QC binder for cell lines. The monolayer is checked microscopically for sterility and appearance of an acceptable confluent monolayer.

 

A. Uninoculated Negative Controls:

Reserve 4 tubes of each lot cells for use as controls and label as follows:

N1, date;

N2, date;

N3 date;

C, date;

V, date

 

a. Negative Controls with refeed: (3 tubes, N1; N2; N3)

Select one tube each on Wednesday Friday and Monday to set up along with inoculated specimens. Incubate, observe and refeed these tubes in parallel with patient inoculated cultures to monitor monolayer quality, toxicity and sterility. They can also be used to provide a baseline for comparison of inoculated cultures when reading for CPE and immunostaining. CMK and HFF tubes are kept for 3 weeks, HEp-2 and RD for 2 weeks

b. Unopened Negative Controls without refeed: (2 tubes, C; V)

These tubes are left unopened and observed to identify toxicity and contamination originating with the vendor. All tubes, CMK, HEp-2 , HFF and RD tubes are kept for only one week. One tube (C) is kept in the clean room and one (V) is placed on the Virology drum.

 

Record sterility and cell appearance for these tubes into the LIS Manual (REGISTRATION OF TUBE CULTURE MEDIA)

B. Positive Controls:

Each week scrape from an HFF tubes containing the following QC strains of HSV-1 HSV-2 and CMV to propagate the QC strains in the new lot of HFF tubes, Use CMK tubes for RSV and nfluenza A. Examine microscopically for CPE and record the results into the LIS as growth control. At the same time also inoculate MRC-5 and R-Mix shell vials to perform quality control for MRC-5 shell vials (see II. Shell Vial Cell Lines (MRC-5 cell suspension below)

HSV-1 (ATCC VR-5539)

HSV-2 (ATCC VR-540)

CMV (ATCC VR-807)

RSV (ATCC VR-284)

Influenza A (ATCC VR-544)

The MRC-5 shell vials are then stained with HSV-1, HSV-2 and CMV IEA monoclonal antibodies at 24 hours, R-Mix shell vials with RS. Record the results into the LIS as shell vial growth control as well as stain controls.

Each month (and whenever necessary), remove from the liquid nitrogen storage, cryovials of above strains to propagate the QC strains in HFF tubes.

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

Coxsackie B1 (ATCC VR-28)

Parainflunza 3 (ATCC )

Varicella-zoster (ATCC VR-1367)

i) Low isolation rates

ii) Comparison of cell lines

iii) Vendor changes

iv) Proficiency test failures

v) Training purposes

vi) Consistent problems with negative controls

vii) Preparation of QC material (i.e. Positive control slides)

Consult a senior technologist to determine the cell lines and viruses to be set up.

II. Shell Vial Cell Line (Suspension seeding is not in routine use)

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 QC binder for cell lines.

Before seeding the shell vials, aspirate about 30 mL of MRC-5 cell suspension into a 125 cm² tissue culture flask, label on the side of flask with “MRC-5, date and ‘Pre’”.

After seeding shell vials, aspirate about 30 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’”.

Reserve 6 shell vials for use as negative and positive controls as follows:

 

A. Negative Controls for this week, also become Positive Old Lot for following week (3 Vials)

These are incubated at 36^oC and observed daily for one week or more to identify toxicity and contamination originating with the vendor. Results are recorded on the QC chart.

Three shell vials are reserved to QC the next shipment in parallel after the cells are added.

 

B. Positive Controls (3 Vials)

Each week, usually 2-3 days after seeding the shell vials, HFF tubes containing HSV-1 (ATCC VR-5539), HSV-2 (ATCC VR-540) and CMV (ATCC VR-807) are scraped from and used to inoculate 6 MRC-5 shell vials, 3 from the current lot and 3 from the previous lot (the same 3 vials were used as Negative Controls for a week). The shell vials are then stained with HSV-1, HSV-2 and CMV IEA monoclonal antibodies after 1 day incubation. Record the results into the LIS daily QC under the following codes:

a. VHSV1D HSV1 daily SLIDE SV QC

b. VHSV1D HSV1 daily SLIDE SV QC

c. VCMV-D CMV-IE daily SLIDE SV QC

d. VQCSV Shell Vial MRC-5 Quality Control

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/appendix-xiii-preservation-of-cell.html

Read More

Appendix XVI WEEKLY WORK SCHEDULE

1. Specimen Management Bench - Monday to Friday :

a) Accessioning and processing all Virology specimens.

b) For PCR testing: mix well and transfer CSF/ EDTA blood / Amniotic fluid to Blue-topped vial (e.g. HSV, EBV, CMV, Parvovirus B19, Polyomavirus, Enterovirus.)

b) Preparing and staining of Direct Smears, PCP slides.

c) Send out stool or Urine for EM.http://bahankuliahkesehatan.blogspot.com/

 

2. Shell Vial Bench – Monday to Friday :

a) Staining and reading of all shell vials.

b) Reading of all Direct Smears and PCP slides.

c) Read Super E-mix Shell vials daily.

d) Enter all shell vial related (e.g. new reagent lots, daily staining, weekly viral propagation and MRC-5 growth) QC results either in LIS and/or Data Sheet.

e) Tabulate and fax weekly viral isolates data (on/after Friday).

f) Maintain stocks of viral isolates as well as preparation of QC slide.

 

3. CMV Antigenemia Bench :

a) Monday to Friday: Record temperatures and humidity on freezers, fridges and room every morning.

b) Monday to Friday: Process, stain and read all CMV antigenemia specimens.

c) Perform RPR on Wednesday to Friday.

d) Perform VZ Ab testing using VIDAS.

e) Perform Monospot daily.

 

4. Molecular Bench:

a) Perform Tagman HCV RNA PCR using Ampliprep when there

are enough of specimens for a full run.

b) Perform Tagman HBV DNA Assay using Ampliprep when there are enough specimens for a full run.

c) Assist PCR bench with Chlamydia/GC testing.

d) Perform molecular bench inventories.

 

5. AXSYM Bench:

a) Perform all stat Hepatitis markers, HIV1&2, CMV IgG and Rubella

IgG and HIV assays and QC’s using AxSYM.

b) Perform weekly maintenance on Thursday or Friday.

c) Perform monthly maintenance at beginning of each month.

d) Perform RPR (syphilis) on Monday and Tuesday.

e) Do inventory on Wednesday/Thursday.

 

6. Eye Bank bench: Using NEXGEN:

a) Perform Eye Bank -BioRad EIA HBsAg &HIV and Ortho EIA HBcAb & HCV Ab daily.

b) Perform C. difficile Toxin A/B testing daily.

c) Perform EBV VCA IgG once a week.

 

7. PCR Bench :

a) Perform Chlamydia/GC using ProbTec.

b) Perform WNV RT-PCR for TGLN and CSF specimens; freeze samples and enter into LIS store.

d) Perform RNA and DNA extractions from CSF/whole blood.

e) Perform HSV PCR Tuesday and Friday.

f) Perform EBV PCR,CMV PCR and Parvo B19 PCR once a week.

g) Perform Enterovirus PCR when received.

 

8. P-Lab Bench :

a) Perform Ortho HTLV 1&2 Ab

b) Perform Immucor CMV Ab

c) Perform WNV IgM Ab

d) Freeze all transplant patients sera in freezer program under ‘Donor’, Inception Biosciences sera under ‘CTSCP’ and Cord Blood of Canada sera under ‘CBB’.

 

9. Evening Technologist:

a) Perform stat TGLN / stat recipient assays if requested.

b) Perform WNV IgM assay

c) Perform AxSYM assays, QC’s and maintenance

d) Process time-sensitive specimens (eg.molecular tests).

 

10. Night Technologist:

a) Perform stat TGLN / stat recipient assays if requested.

b) Continue WNV IgM assay if set up late.

c) Perform AxSYM assays, QC’s and maintenance.

d) Process time-sensitive specimens (eg.molecular tests).

 

11. Technician 1- Monday to Friday :

a) Disinfect, check centrifuges and document on sheet.

b) Spin and accession all Serology specimens.

c) Send out all PHL specimens and printing out manifest.

d) Process Viral load specimens.

e) Document pressure reading in LIS for Serology Accession Hood.

 

12. Technician 2- Monday to Friday :

a) Disinfect, check centrifuges and document on sheet if not yet done by Technician 1.

b) Process Viral load specimens.

c) Enter patients’ demographic in Viral Load program.

d) Spin, accession and freeze sera for HBV DNA and HCV RNA.

Help technician 2 to spin and accession all serology specimens

http://bahankuliahkesehatan.blogspot.com/

BAHAN KULIAH DAN MAKALAH KESEHATAN : http://bahankuliahkesehatan.blogspot.com/2011/08/appendix-xvi-weekly-work-schedule.html

Read More