PROTOCOL FOR SEPARATION OF PLASMA AND PERIPHERAL
MONONUCLEAR CELL FRACTION FROM WHOLE BLOOD

BD Vacutainer CPT tubes are used for blood draws. Instructions on the package insert should be followed for tube storage and blood collection procedures. For example, CPT tubes should be stored at room temperature.

At Study Site:

1. Collect 8ml of blood via venipuncture directly into a BD Vacutainer CPT tube
2. Invert the tube 10 times immediately after the draw (no shaking)
3. Keep the tube upright and at room temperature
4. Send tube to Johns Hopkins within 2 hours of blood draw. Alternatively, the tube can be spun at the study site and delivered to Johns Hopkins within 24 hours. The centrifuge used must achieve the recommended 1500-1800xg spin. If the tubes are spun at the study site, the tube should be inverted 5-10 times before transport to Johns Hopkins.
5. See note below concerning the organization of case and control samples.

Hopkins Address:

Johns Hopkins University
Stanley Division
600 N. Wolfe Street, Blalock 1105
Baltimore, MD 21287
410-614-0004

SHIPMENT OF SAMPLES

We will accept serum or plasma samples for antibody testing.

We prefer to receive a minimum of 0.5ml of plasma or serum. However, we can work with smaller volumes on request.

We employ a number of robotic systems for the processing of samples. It thus is a great help if the samples are sent in a way that is compatible with the systems which we use.

There are several ways in which samples can be sent.

Microtiter plates: The most expeditious for us is to have the samples sent in deep well microtiter plates, since the samples can then be tested with a minimum of handling. The sending of the samples in microtiter plates generally results in the shortest turnaround times.

If you send the samples in this way, please use a plate similar to the 1.1ml capacity plate made by Axygen (P-DW-11-C-S).

The plate should be covered with a sealing mat or alumina foil sealer plus VWR* Hi-Tack Tape.

1. 1. The sealing mat should be similar to AxyMat AM-2ML-RD-S. These are available from suppliers such as VWR (catalogue numbers 47734-788 and 10011-946
   (http://vwrlabshop.com/product.asp_Q_pn_E_0063725).
2. The alumina foil sealer from suppliers from suppliers such as USA Scientific (http://usascientific.com/index.asp?PageAction=VIEWPROD&ProdID=526) and wrapped over with VWR* Hi-Tack tape (5 inch width) (https://www.vwrsp.com/catalog/product/index.cgi?partnumber=5WH-92B&sim_code=1.0&catalog_number=68400-660&inE=1&highlight=68400-660&reference_type=1)

THIS IS A PICTURE OF THE PLATE PREFERRED

Samples in plates:

When sending samples in plates, please make sure the number order of the samples are vertical. The first, last or any well can be empty (marked “blank” on the list), but the main order of the samples goes from Row A through H.

Sample Plate Type I:

Sample Plate Type II:

Tubes:

The tubes that work best for us are ones which are compatible with microtiter formats. The best ones are ones such as M52006 from Micronics, which are available from supplies such USA Scientific (catalog # 1765-2006) (http://www.usascientific.com/index.asp?PageAction=VIEWCATS&Category=709)

THIS IS A PICTURE OF THE TUBES PREFERRED

We will accept samples in other types of tubes as well, although it will take longer to process them.

1. The best tube is the one with external screw cap
2. For tubes with internal screw caps - please be sure that the volume of sample should not be more than it is designed.

In any case, please be sure that the tubes are clearly labeled with a code number which matches the number on the sample list. It is strongly recommended that pre-printed or typed labels be used since hand-written ones are often difficult to read and identify. Note that samples whose labels come off are or difficult to read are a common cause of unidentifiable samples. We will not run any samples from a set until all samples have been identified, so problems with sample labeling can subsequently delay the completion of a study.

Note that samples are run in groups of 88, so sample sets which are in multiples of 88 are the most efficient ones for us to run.

We generally run assays in panels. The composition of the panels may vary depending upon availability of the assays reagents and the interests of the investigators. All assays are for IgG class antibodies unless otherwise indicated.

Primary Panel: Herpes Simplex Virus Type 1 (HSV-1)

Herpes Simplex Virus Type 2 (HSV-2)
Cytomegalovirus (CMV)
Toxoplasma gondii IgG

Secondary Panel

Epstein Barr Virus (EBV-EBNA)
Varicella Zoster Virus (VZV)
Toxoplasma gondii IgM
Human Herpes Virus Type 6 (HHV-6)
Influenza A Virus (H3N2)
Influenza B Virus
Gliadin IgG

Additional assays may be performed based on the interest of the investigators.

Results are reported electronically on a sheet which contains our laboratory ID number as well as the ID number provided by the investigator. The results are generally presented as quantitative values which are derived from the level of reactivity and standards run with each assay, as well as qualitative results listed as “positive”, “negative”, or “equivocal”. We also generally provide an overall estimation of the prevalence of antibodies in your population and comparison with other populations which we have tested.

Shipment of Samples

Please contact us before shipping any samples to ensure than someone will be present to receive the samples and to track them down if there is a problem.

Please ship the samples to the following address:
Robert H. Yolken
Johns Hopkins University
600 N. Wolfe Street
Blalock 1105 (NOTE: THIS IS THE MOST IMPORTANT PART OF THE ADDRESS)
Baltimore, MD 21287
410-614-0004

Samples should be shipped on dry ice, with enough dry ice for at least 3 days in shipping and storage. Federal Express is the preferred shipper.

It is recommended that you ship samples on Monday or Tuesday to avoid problems associated with the location of samples over the weekend.

Before the samples are shipped, please send to us by email a list which contains a spreadsheet which contains the code numbers of the samples, sample volume, sample location and other relevant information. Please do not include names, social security numbers, birthdates, or other identifying information. Send the list to:

Ann Cusic (acusic@jhmi.edu)

Robert Yolken (yolken@jhmi.edu)

Please also include a copy of the list in the box with the samples. We will check the samples when they arrive against this list and notify you if there are samples which are missing, broken in transit, or unidentifiable.

FILTER PAPER SAMPLES

We can also measure antibodies in whole blood samples which have been put onto filter papers. These are commonly used for the measurement of metabolites in neonates. We will accept filter paper samples which are at least 5mm in diameter.

We have found that "deep well" plates work better for us since we can add more diluents. The best ones are:

1011-940 1.1ml Deep well plates from VWR (http://www.vwrsp.com/catalog/product/index.cgi?catalog_number=10011-940&inE=1&highlight=10011-940)

Also, the best cover for these plates is one which has a slit which allows us to add diluents without disturbing the filter paper. This is:

101-11-126 96 well sealing mat from VWR (http://www.vwrsp.com/catalog/product/index.cgi?catalog_number=10011-126&inE=1&highlight=10011-126)

Please leave the first column of the plate empty so that we can use this for running our standards. Also, if possible please include blank filter paper samples in the plate since this assists in orienting the plate and performing our quality control determinations.

Please make sure that the plates are clearly marked in terms of plate identification and orientation.

Please send an electronic version of a sample list which matches the plates in terms of plate number and orientation.

Organization of Samples

Samples are run on microplates in sets of up to 88 samples. We make every effort to standardize the conditions of the runs across sets. However, it is difficult, if not impossible, to totally eliminate such differences, which are similar to “platform” effects seen in most solid phase assays. It is particularly difficult to eliminate the platform effects in large-scale studies where large numbers of samples are run over an extended period of time. In such cases, it is crucial that there be no bias in terms of samples run early in the study versus those run late in the study. The best way to avoid the effects of such bias is to have the samples assigned to the plates in random order. An alternative in case-control studies is to assign the same number of cases and controls to each plate. This allows for the use of a number of normalization routines as well as statistical methods, such as conditional logistic regression, which allows for comparisons within each plate or run. What needs to be avoided are sample sets in which all of the cases are run on one set of plates and all of the controls on another, making meaningful standardization across plates difficult, if not impossible.