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.

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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.

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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

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