Koi Health - Information on Koi Herpesvirus

How to get a “worthless positive sample ” for a meaningless Virus Neutralization test:

Remember the test is only as good as the sample submitted.  Remember a positive test is one in which virus growth is inhibited by addition of the sample.  There are several ways in which a sample can be accidentally altered or contaminated to inhibit viral growth.  The Virus Neutralization assay is repeatable and measures inhibition of the viral growth.  If the sample is not properly prepared or contaminated, one can get inhibition of viral growth at low titers or dilutions. The result may be a positive test at low titers even though there is no antibody to virus present. Those in the medical field understand the need for perfect samples and take it for granted but perhaps it would be useful to review some ways in which blood specimens can be altered to provide worthless results.

Four ways to contaminate or alter your blood sample to inhibit viral growth.           

  1. Heat up your sterile sample.  The process may well cause enough changes to inhibit viral growth.  If your carefully prepared sample of serum or plasma sits over a weekend on a delivery truck in the heat, it may well be worthless.  From a practical viewpoint, one must pack samples with some form of ice packs in an insulated container and ship by a traceable overnight service such as FedEx.  For International samples requiring longer shipment times, more heavily insulated Styrofoam containers with more ice packs are usually used. A call to the receiving lab to let them know to expect your shipment or verify receipt may be in order.  We prefer our samples to arrive in the lab frozen. 

  2. Allow your specimen to hemolyze red cell rupture.  If the specimen is handled in such a way that a significant number of the red cells rupture, viral growth can be inhibited.  There are a number of powerful intracellular substances which can inhibit viral growth. If sufficient quantities of these are accidentally released into the serum or plasma, they can inhibit viral growth without the presence of antiviral antibodies.  This is usually pretty easy to detect.  When the red cells lyse, hemoglobin which is red is released into the serum or plasma specimen.  The specimen will be red stained after centrifugation.  A small amount of hemolysis is usually not critical but larger amounts can and do inhibit viral growth.  Four of the more common and preventable means of hemolyzing specimens include:

    1. Allowing the blood to rapidly transfer through a tiny needle into the vacutainer.  When the blood flows rapidly through the needle, some red cells rupture.  If the syringe plunger is forcibly pushed putting the cells under increased pressure, hemolyis occurs. 
    2. If the needle is inserted into the tube and blood is allowed to rapidly splash into the bottom of the tube, some hemolysis occurs. This can largely be prevented by holding the tip of the needle against the inside wall of the wall of tube when transferring blood.
    3. If the blood is not almost immediately transferred from the collecting syringe to the tube, blood may clot in the syringe.  Although this clotting may be prevented by using heparin in the syringe, use of heparin adds another potential contaminate and is not necessary when proficiency in veinipuncture is acquired.  The increased difficulty in transferring blood from a partially clotted syringe may introduce other unwanted components or produce hemolysis in the sample.
    4. After centrifugation, if the red cells and red cell fragments below the gel migrate above the gel, they can contaminate the sample.  Shipment could potentially jostle the sample such that the sample is contaminated.  Indeed laboratory couriers have repeatedly demonstrated the ability to “shake up”and contaminate  centrifuged samples in gel tubes  transporting samples across town from physician offices to the reference lab.  Imagine what cross country shipping can do to samples.  If the samples are frozen and arrive at the lab frozen, rough handling during shipment is usually not an issue.  An alternative is to transfer the plasma or serum to a second sterile transport tube (about $.30) prior to shipment.  The tops can be removed from the vacutainer and the sterile tube (top carefully placed with sterile side up to avoid contamination) and the sample is carefully dumped from one tube to the other and recapped with the sterile top for shipment.  The top is taped in place or wrapped in Parafilm.  Alternatively the sample can be withdrawn into a small syringe, the needle capped and the plunger taped to prevent movement.  The receiving lab technicians usually hate the latter.  If samples are being sent internationally, it is recommended the serum or plasma be separated into transport tubes.  If the sample is being transferred to transport tubes, it can be divided such that some sample remains in the freezer in the event of shipping mishap.
  3. Allow “tissue juice” or tissue to mix with your sample.  “Tissue juices” and ruptured tissue contains some very potent antiviral substances.  They cannot be mixed with your sample. Frequently, when an individual is learning to draw blood from a fish, there is a tendency to withdraw the plunger of the syringe while moving the needle. This may result in small amounts of tissue or various “tissue juices” being drawn into the hub of the syringe.  The correct technique involves placing the needle in the vein and then withdrawing blood by gentle backpressure on the plunger.  Blood should slowly flow into the syringe.   If the tip of the needle is not appropriately in the vein, pressure is released on the plunger, the needle is repositioned, and the plunger is again gently withdrawn.  Very little suction is required if the needle is properly positioned.   Novices will often continue to hold back pressure on the plunger when repositioning the needle. This frequently will result in tissue or tissue juice being withdrawn into the syringe.  If there is any doubt about the syringe being contaminated with tissue or “tissue juice”, it is tossed and a fresh needle and syringe is used.  A larger than needed blood sample will effectively dilute a small amount of “tissue juice”.  We prefer to withdraw at least one ml of blood in the syringe to minimize potential tissue contamination.

  4. Use inappropriate tubes for your sample.  There are many types and sizes of blood and serum collection tubes.  Most are a form of sterile plastic with additives in a vacumn .  Gold and red topped serum collection tubes frequently have a “clot activator”.  Plasma collection tubes usually have heparin.  Purple top tubes contain EDTA to prevent clots, etc.   For example, in a vaccine study, avian blood samples were collected by a professional in a 4 ml purple top tube ( which contains 7.2 mg of K2 EDTA to prevent blood clotting). A small amount of collected blood would be transferred to the tube, and the tube subsequently spun down in the centrifuge.  This resulted in the blood cells being spun to the bottom of the tube with the clear supernatant  on the surface.  The later was transferred to lab in transport tubes.  At this point the sample includes most of the 7.2 mg of K2 EDTA.  As you might imagine, the EDTA in the sample consistently inhibited viral growth.   You would not want to use a 10 ml serum collection tube containing a clot activator to hold 1 ml of blood before centrifugation.  The excess clot activator can be transferred to your sample and potentially inhibit cell growth.  A tube half that size is more appriate.  We do know that a light green capped 4.5 ml vacutainer containing gel and 76 units of lithium heparin works well with samples of 1 ml or greater.  The microtainers do well from a size stand point but they are not sterile and that may cause contamination problems.

Again, the virus neutralization test is worthless if the sample is not good.  As BODAD says “Garbage in, Garbage out."


Once the samples have been obtained and the plasma or serum separated, they are usually wrapped and shipped in a small insulated container to the labratory by means of a trackable overnight service. Shipping containers may include cardboard boxes holding small styrofoam containers, insulated freezer sacks, insulated childrens lunch sacks, etc. One or more ice packs are usually included. Care should be taken to avoid shipment arrival on weekends or holidays. If in doubt, it is recommended to contact the receiving labratory by phone before shipping. A completed sample submission form, a page indicating the numbering system utilized, and a check for the processing cost for each sample should be included with the samples.

Once your carefully prepared sample is received by the Infectious Disease Labratory, what happens?