Opti-Spot^® Collection Guidelines

Opti-Spot provides the following benefits:

• Small sample size – only one drop of blood is required; more feasible for ante-mortem sampling.

• Saves technician time – no clotting or centrifugation required; no transfer of serum.

• Reduced sample preparation time – only an hour or two of drying time required.

• Costs savings in supplies – no serum tubes or pipettes; requires only standard overnight envelope at ambient temperature, no ice or insulation necessary.

Opti-Spot Collection Protocol

1. Materials required:

a. Opti-Spot card (available from IDEXX BioAnalytics for no charge).

b. Lancet or needle.

2. Sample labeling:

a. Label one Opti-Spot card with a unique identification number for each sample strip.

3. Opti-Spot cards and strips

• The Opti-Spot cards are constructed with 5 individual strips separated by perforations. The strips are easily separated along the perforation lines for individual use.

• On each strip, a 1.0 cm² circle is printed on the face of the Opti-Spot membrane as a guide. It is not necessary that the blood spot be centered within the circle.

b. Opti-Spot sample collection:

• Ante-mortem blood samples may be obtained by lancing the lateral saphenous, facial or the temporal vein. Touch the Opti-Spot membrane to blood drop as it forms on the surface of the skin.

• If the blood sample is taken by cardiocentesis, quickly dispense one drop of whole blood onto the Opti-Spot strip.

• The blood spot should be of sufficient size (approximately 1 cm²) to nearly fill the printed circle and be of sufficient volume to saturate the membrane. The blood spot will appear similar on both sides of the membrane.

• Allow the blood spot to dry for a minimum of one hour.

• Once dried, fold the protective upper tab over the blood spot and tuck under the lower tab.

4. Sample Shipment:

a. It is important that the Opti-Spot strip is protected from moisture. Once dry, place Opti-Spot samples in a water-tight plastic bag containing the provided silica gel desiccant pack, and ship samples in a standard overnight envelope.

Blood Serum Preparation and Shipping SOP

Materials and Equipment

• Blood sample.
• Collection tubes (not containing an anticoagulant or clot activator).
• Benchtop centrifuge (NOT refrigerated).

 

Procedure

1. Allow whole blood without anti-coagulant to clot: overnight at 4^o C or for 2-4 hours at room temperature.

2. Centrifuge for 10 min at manufacturer’s recommended speed for separation of serum (usually 1000-2000 RCF). Do not use brake to stop centrifuge.

3. Carefully aspirate the serum and dispense into a fresh tube.

4. Add 4 volumes of normal saline diluent, achieving a final dilution of 1:5. For example, add 400 µl of diluent to 100 µl of serum. Note: Alternatively, you may achieve an approximate 1:5 dilution by adding 1 volume whole blood to 2 volumes ambient temperature saline (allow diluted blood to clot prior to centrifugation as described above).

5. Five microliters of diluted serum is required for primary evaluation by Multiplex Fluorescent Immunoassay (MFI), irrespective of the multiplicity of the serological assays; however, an additional 75 µl may be required for confirmatory immunofluorescence assay (IFA) or Western blot (WB) testing. Seventy five microliters (75 µl) of 1:5 diluted (or 20 µl of undiluted) serum is the ideal minimum volume needed for primary and confirmatory testing. 

Serum samples should be shipped frozen using an overnight service. One or two pounds of ice packs are generally adequate depending on the type of container, the number of samples, and the ambient temperature. Optimal shipping conditions are achieved when the entire package with its lid open is frozen overnight at or below -20° C. The use of dry ice is not required. 

 

Pooled Serological Samples SOP

 

Timely and accurate diagnosis of infectious disease in rodent colonies is critical to the success of biomedical research. To this end, vivarium managers closely monitor the health of research animals through periodic, systematic, examination of representative groups of research and sentinel animals against a predetermined list of infectious agents. As the number of agents on our biosecurity exclusion lists has grown over the past several decades, so has the cost of health monitoring programs. 

The use of pooled serological sample testing is one potential way to reduce the monetary cost of health monitoring programs. The rationale behind pooled testing is based upon disease prevalence. The majority of infectious diseases in our rodent colonies are of low prevalence and, thus, there is a high probability that all serological samples are negative for antibodies to these low prevalence pathogens. Accordingly, testing a single pooled serological sample provides the same data as testing multiple individual samples, resulting in a potentially significant cost savings. 

As with most cost-cutting measures, however, there is a potentially detrimental tradeoff when pooling serological samples. An adaptive immune response is triggered when an infectious agent overwhelms or eludes the innate immune system and T cells and B cells are activated by their specific antigen. T and B cells then differentiate into effector cells, proliferate, and migrate to their sites of action. The process of activation, proliferation, and initiation of antibody production by B cells occurs over a period of approximately one to six weeks depending upon the pathogen. During this time antibody levels are relatively low with a weaker affinity for their antigen. When pooling serological samples there is a serious concern that if the pool size is too large, especially during the early stage of seroconversion, an antibody positive serological sample will be sufficiently diluted beyond the level of detection, resulting in a false negative finding. Therefore, pooling serological samples from disparate populations of animals is not recommended because of the potential dilution effect. Pooling samples from related animal groups, such as sentinel cage-mates, is an acceptable practice as these animals are likely to have been exposed to the same pathogens over the same time-period, thus, effectively eliminating the dilution effect. 

Pooling blood samples from related animal groups for preparation of dried blood spot samples may be accomplished by premixing samples, superimposing samples on the Opti-Spot^® membrane, or pooling eluted samples by the testing laboratory: 

1. Blood samples collected from individual animals may be premixed in EDTA tubes. One large drop (>25μl) is then placed on the Opti-Spot membrane. 

2. Two medium sized blood spots may be superimposed on the Opti-Spot membrane. To allow proper mixing of the samples, it is important to overlay the second blood drop on the Opti-Spot membrane while the first blood spot is still wet. 

3. Eluted individual dried blood spot samples can be pooled by the testing laboratory per client request on the submission form. This approach has the added advantage of allowing retesting of individual samples should the pooled sample result in equivocal or positive findings. This can be done by sending in an Opti-Spot for each sample that is to be pooled or by putting two separate spots (1 from each animal) on one Opti-Spot membrane.