Center for Veterinary Biologics SAM 313.05

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Supplemental Assay Method for the Detection of Extraneous Hemadsorbing Agents in Master Seed Virus

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 313

Supplemental Assay Method for the Detection of Extraneous Hemadsorbing 

Agents in Master Seed Virus

Date: August 23, 2023 

Number: SAM 313.05

Supersedes: SAM 313.04, November 13, 2014

Standard Requirement: 9 CFR, parts 113.46 and 113.55

Contact: Email: Methodsrequest.notification@usda.gov

Phone: Center for Veterinary Biologics, 515-337-6100

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.
Center for Veterinary Biologics SAM 313.04

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Supplemental Assay Method for the Detection of Extraneous Hemadsorbing Agents in Master Seed Virus 

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of RBC

3.3 Preparation of the sample

4. Performance of the Test

4.1 Vessels incubated at each temperature separately

4.2 Separate vessels incubated simultaneously at each temperature

5. Interpretation of the Test Results

6. Report of Test Results

7. References 

8. Summary of Revisions

Appendices

I. Macroscopic view of hemadsorption

II. Microscopic view of hemadsorption
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Supplemental Assay Method for the Detection of Extraneous Hemadsorbing Agents in Master Seed Virus 

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1. Introduction

This Supplemental Assay Method (SAM) describes a method for detection of extraneous 

hemadsorbing (HAd) agents in master seeds (MS) used in the production of veterinary vaccines. 

Not all viruses cause a noticeable change in the cellular appearance, but can be detected by HAd 

of red blood cells (RBC) to virus-coded glycoproteins incorporated in the cell membrane. To 

perform the test, cell monolayers are washed and covered with a suspension of guinea pig and 

chicken RBC. Following incubation, uninfected cells will appear normal, while RBCs will 

adhere to cell monolayers that contain HAd surface viral antigens. Detection is accomplished by 

both macroscopic and microscopic reading methods. 

2. Materials 

2.1 Equipment/instrumentation

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

below. Equipment is calibrated and certified according to current standard operating 

procedures.

2.1.1 Microscope, inverted bright light

2.1.2 Illumination box

2.1.3 Low speed, refrigerated centrifuge (Beckman J-6B centrifuge with JS 4.0

rotor or equivalent).

2.1.4 Refrigerator, 2°-7°C

2.1.5 Micropipettors and appropriate tips

2.1.6 Vacuum source for aspirating liquids (vacuum pump with side arm flask 

of Chapman-type filter pump attached to a water line)

2.1.7 Laminar Flow Biological Safety cabinet (NuAire, Inc. Labgard or 

equivalent )

2.2 Reagents/supplies

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

reagents and supplies must be sterile.
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Supplemental Assay Method for the Detection of Extraneous Hemadsorbing Agents in Master Seed Virus 

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2.2.1 Appropriate vessels at least 7 days from the last subculture containing:

• MS-inoculated 

• negative control cell monolayers. 

The preferred vessel is 25-cm2 flasks , but any suitable alternative may be used if

the surface area is ≥ 6 cm2 each.

2.2.2 A vessel containing a monolayer of Vero cells, ≥ 6 cm2 to be used in 

preparation of positive control (PC).

2.2.3 PC virus (current approved lot of Bovine Parainfluenza type 3)

2.2.4 Alsever’s solution

1. 8.0 g sodium citrate (C6H5Na3O7•2H2O)

2. 0.55 g citric acid (C6H8O7•H2O)

3. 4.2 g sodium chloride (NaCl)

4. 20.5 g glucose (C6H12O6)

5. Q.S. to 1000 mL with deionized water (DI).

6. Filter sterilize with a 0.22-μm filter.

7. Store at 2°- 7°C.

2.2.5 Guinea pig and chicken RBC in equal volumes of Alsever’s Solution. 

Store at 2°- 7°C.

2.2.6 0.01 M Phosphate buffered saline (PBS)

1. 1.33 g sodium phosphate, dibasic, anhydrous (Na2HPO4)

2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)

3. 8.5 g NaCl

4. Q.S. to 1000 mL with DI.
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5. Adjust pH to 7.2-7.6 with 0.1 N sodium hydroxide (NaOH) or 1.0 N 

hydrochloric acid (HCl).

6. Sterilize by autoclaving at 15 psi, 121°± 2°C for 35 ± 5 minutes.

7. Store at 2°- 7°C.

2.2.7 Pipettes: 1-mL, 5-mL, and 25-mL

2.2.8 Conical tubes: 50-mL

2.2.9 Cell culture flasks: 25-cm2 (or appropriate vessel)

2.2.10 Graduated cylinder

3. Preparation for the Test

3.1 Personnel qualifications/training

Laboratory personnel must be trained in the operation of instruments and equipment used 

prior to performing this test. Personnel must be familiar with proper use of test reagents 

and biological materials. Personnel must have knowledge of safe operating procedures 

and policies.

3.2 Preparation of RBC

3.2.1 Wash guinea pig and/or chicken RBC

1. Dispense 10-20 mL of guinea pig and chicken blood preserved in 

Alsever’s solution into separate 50-mL conical centrifuge tubes.

2. Fill the tube with Alsever’s/PBS and gently invert tube several times 

to wash RBC.

3. Centrifuge RBC for 15 ± 5 minutes at 400-800 x g (e.g. 1500-1800 

rpm, J6-B centrifuge with a JS-4.0 or JS-4.2 rotor) at 4°± 2°C.

4. Aspirate PBS and buffy coat (bone colored layer of cells on top of 

RBC) using a pipette or vacuum type aspirator.
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5. Repeat Steps 2 through 4 for a total of 3 or more washes (until 

supernatant is clear). Store the packed guinea pig and chicken RBC at 2°-

7°C for up to 1 week. RBC should be discarded if hemolysis is observed.

3.2.2 Preparation of 0.2% RBC on day of test.

1. Dispense 49.8 mL of PBS into a suitable container.

2. Using 1-mL pipettes, add 100 μL of washed guinea pig RBC and 

100 μL of chicken RBC to PBS. Rinse pipette(s) thoroughly to dispense

all RBC. Gently swirl to mix.

3. RBC suspension should be discarded following completion of test.

3.3 Preparation of the sample

For each cell line, seed at least one 25-cm2 flask with MS inoculated cells and at least one 

25-cm2 flask with un-inoculated cells to be tested sequentially at 4°C and 25°C. In 

addition, seed two 25-cm2 flasks of un-inoculated Vero cells to serve as positive controls, 

one flask for each temperature. The positive control flasks are seed-inoculated with 

bovine parainfluenza virus 3 at the time of the last subculture. 

4. Performance of the Test

The HAd test is conducted at 2 temperatures: 4°± 2°C and room temperature (23°± 2°C) for MS

and NC flasks of each cell type. Two PC flasks (Vero cell monolayers) are tested (one at 4° and 

one at room temperature). Alternatively, MS, NC and PC flasks of each cell type may be 

incubated simultaneously at each temperature (Section 4.2).

4.1 Vessels incubated at each temperature separately

4.1.1 Decant medium from MS, NC and PC flasks.

4.1.2 Add 5 mL of PBS to each of the MS, NC and PC flasks, swirl gently and 

decant. Repeat for a total of 3 washes. 

4.1.3 Gently swirl the 0.2% RBC mixture and add 5 mL into each MS, NC and 

PC flasks (Section 3.2.2).

4.1.4 Incubate all flasks except one PC flask at 4°± 2°C for 25 ± 5 minutes.

4.1.5 Decant the 0.2% RBC mixture. 
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4.1.6 Wash vessels 3 times as described in Section 4.1.2.

4.1.7 Using an illumination box, compare each MS flask to the corresponding 

NC and PC control flasks. Look for areas of HAd on the cell monolayer (see 

Appendix I).

4.1.8 Using an inverted microscope at 100X, compare each MS flask to the 

corresponding NC and PC flask(s). Look for HAd of individual RBC to cell 

membranes (see Appendix II).

4.1.9 If HAd is not detected, add 0.2% RBC mixture to MS, NC cell monolayers

and remaining PC flask as described in Section 4.1.3.

4.1.10 Incubate all vessels at room temperature for 25 ± 5 minutes.

4.1.11 Repeat Sections 4.1.6 through 4.1.9.

4.2 Separate vessels incubated simultaneously at each temperature

4.2.1 Repeat Sections 4.1.1 through 4.1.3 for duplicate sets of vessels.

4.2.2 Incubate MS and NC flasks for each cell line, as well as a PC flask (Vero)

at 4°± 2°C for 25 ± 5 minutes. 

4.2.3 Incubate separate MS and NC flasks for each cell line, as well as a PC 

flask (Vero) at room temperature for 25 ± 5 minutes. 

4.2.4 Repeat Sections 4.1.6 through 4.1.9 for each set of vessels.

5. Interpretation of the Test Results

For a valid test, the PC cell culture must demonstrate HAd while the NC cell culture must be free 

of HAd activity.

HAd activity in the MS inoculated cell monolayers other than the non-neutralized MS is 

indicative of infection by extraneous viruses, and the MS is determined unsatisfactory. The MS 

may be retested to confirm original results if warranted. Additional investigative work may be 

warranted to determine cause of HAd.

6. Report of Test Results 

All records are kept in accordance with current recordkeeping practices
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7. References

Title 9, Code of Federal Regulations, parts 113.46, 113.51, 113.52 and 113.55, U.S. Government 

Printing Office, Washington, DC. 

8. Summary of Revisions 

Version .05 

• The coversheet and contact information has been updated.

Version .04

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

to keep the same next review date for the document. 

Version .03

• The Contact information has been updated. 

Version .02 

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document: 

• 1: An overview of the test method has been added. 

• 2.1: More current and appropriate equipment has been added. 

• 2.2: Surface area criteria to test and a Positive Control have been added to the test.

• 3.1: Personnel qualifications and training have been clarified.

• 3.3: Additional detail to washing and preparation of 0.2% RBC has been added. 

• 3.4: A Positive Control has been added to the test system. 

• 4.2: The Positive Control flask has been added to the incubation step. 

• 5.1: This section has been condensed and the roles of the Positive and Negative 

Control vessels have been clarified. 
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Supplemental Assay Method for the Detection of Extraneous Hemadsorbing Agents in Master Seed Virus 

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• The refrigeration temperatures have been changed from 4° ± 2°C to 2°-7°C

throughout the document.

• Appendices containing macro- and microscopic images depicting hemadsorbtion have 

been added.
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Supplemental Assay Method for the Detection of Extraneous Hemadsorbing Agents in Master Seed Virus 

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Appendix I

Macroscopic view of hemadsorption
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Supplemental Assay Method for the Detection of Extraneous Hemadsorbing Agents in Master Seed Virus 

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Appendix II

Microscopic view of hemadsorption