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SAM 801.03

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 801

Supplemental Assay Method for Examination of Porcine Cells for Porcine 

Parvovirus Contamination

Date: November 28, 2014

Number: SAM 801.03

Supersedes: SAM 801.02, February 11, 2011

Standard Requirement: 9 CFR 113.51

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals:

/s/Geetha B. Srinivas Date: 22Jan15

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 26Jan15

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 26Jan15

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for Examination of Porcine Cells for Porcine Parvovirus Contamination

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Personnel qualifications/training

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions
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Supplemental Assay Method for Examination of Porcine Cells for Porcine Parvovirus Contamination

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro test procedure for examining 

porcine origin vaccine production cells for the presence of porcine parvovirus. The procedure 

uses several methods, e.g., immunofluorescence, hemagglutination, and aniline dye staining.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Four or more sterile plastic or glass tissue culture containers having at 

least 75-cm

2

surface area

2.1.2 At least 1 rack of sterile Leighton tubes with coverslips or 2 flats of Tech 

slides

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 Enough production cells and media to seed 2 containers from each 

production lot of cells to be tested

2.2.2 A supply of pretested serum that is normally used by the manufacturer. In 

the case that porcine serum is used, it shall be negative by either serum 

neutralization (SN) or hemagglutination inhibition (HI) for porcine parvovirus 

antibodies.

2.2.3 A supply of medium that is normally used to propagate production of 

swine cells and will grow a monolayer in 6 to 7 days

2.2.4 An anti-porcine parvovirus conjugate. An initial supply will be furnished 

by the National Veterinary Services Laboratories (NVSL) for reference.

2.2.5 An aniline dye stain that will demonstrate intranuclear inclusion bodies 

(e.g., May Gruenwald-Giemsa or Shorr’s)

2.2.6 Guinea pig red blood cells
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Supplemental Assay Method for Examination of Porcine Cells for Porcine Parvovirus Contamination

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2.2.7 A supply of pretitered porcine parvovirus. The seed will be furnished by 

the NVSL.

3. Personnel qualifications/training

Personnel performing the test must have experience or training in this protocol. This includes 

knowledge of aseptic biological laboratory techniques and preparation, proper handling and 

disposal of biological agents, reagents, tissue culture samples, and chemicals. Personnel must 

also have knowledge of safe operating procedures and policies, as well as training in the 

operation of the necessary laboratory equipment listed in Section 2.1.

4. Performance of the Test

4.1 At least 2 cell culture containers are seeded with each lot of porcine cells to be 

tested. (A lot shall consist of equal aliquots of cells from pigs in 1 litter only.) The 

trypsin used to disperse these cells shall be from a pretested negative lot or the trypsin 

shall have been sterilized with Beta-propiolactone. The cells are incubated at 35°- 37°C 

until a complete monolayer has formed.

4.2 After the monolayer has formed, remove the cells either with BPL treated trypsin 

in ATV or by scraping. The cells from the 2 flasks are pooled and redispersed in fresh 

growth medium. The cells are reseeded in fresh sterile containers at a 1:1 ratio, unless the 

manufacturer has a ratio that grows better in their experience. There shall be at least 2 

containers to continue the test. Incubate as before in Section 4.1.

4.3 At the time that the cell spread is essentially complete, the cells are removed from 

the containers as described in Section 4.1 and redispersed (1 container is reseeded into a 

sterile fresh container). At this time, the equivalent of 1 container is seeded onto at least 

20 Leighton tubes with slides or 10 Tech slides. At this time, at least 10 of these Leighton 

tubes or 4 Tech slides are inoculated with 0.1 mL of swine parvovirus diluted to contain 

approximately 100 TCID50s per tube. All vessels are incubated as before.

4.4 On the 2nd day postseeding, at least 2 Leighton tubes or 1 Tech slide from the test 

cell tubes and 2 Leighton tubes or 1 Tech slide from the positive control tubes are 

removed and fixed for fluorescent antibody staining. This procedure is repeated on the 

3rd, 4th, and 5th day. The slides may be stained every day or may be held at 

-20°C until all have been fixed.

On the 7th day, the fluid from 1 container is removed and a hemagglutination test is 

conducted using the 1:2 through 1:32 dilutions. At least 2 Leighton tubes or 1 Tech slide 

of test cells and 2 positive control Leighton tubes or 1 Tech slide are removed on the 1st 

day the positive slides show fluorescence. The slides are fixed for analine dye staining 

and stained.
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5. Interpretation of the Test Results

The cells are satisfactory for freedom of porcine parvovirus if they do not fluoresce specifically 

after FA staining. The positive control cell shall stain positively.

The fluids taken for the HA test shall not show any hemagglutination. The aniline dye stained 

coverslips shall not exhibit any intranuclear inclusion bodies. The positive control cells may or 

may not show inclusions.

6. Report of Test Results

Report results of the test(s) as described by standard operating procedures.

7. References

Title 9, Code of Federal Regulations, section 113.51, U.S. Government Printing Office, 

Washington, DC.

8. Summary of Revisions

Version .03

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .02

 The Contact information has been updated.