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Issue: 66 - Jun 15, 2014
Ten tips to Ensure Accurate Blood Test Results
By: Marshfield Labs
Marshfield Labs

When dealing with laboratory species and research animals, limiting preanalytical variability in test samples (defined as variability occurring between when the test is ordered until the sample is ready for analysis) and ensuring accurate blood test results is of utmost importance because often the sample cannot be redrawn and/or is part of a study that cannot afford to have spurious results.  The majority of spurious results reported or inadequate samples received in our laboratory are related to unknowingly improper collection, processing, handling, and storing.  The following are tips that will help reduce preanalytical variability and ensure accurate blood test results when collecting, processing, handling and storing blood samples.

  1. Standardize the collection site, method, and type of restraint

Any and all sampling variables should be minimized and accounted for when collecting blood from any species.  Variables to consider include but are not limited to: the collection site, method of collection, equipment used to collect the sample, the type of restraint used, and the level of expertise of the person collecting the sample. 

  1. Ensure the correct blood draw order

If you will be collecting blood into more than one type of blood tube, be sure to collect samples in the appropriate order to prevent cross-contamination of additives between tubes.  The correct order of blood draw/tube filling is:  blue-topped citrate tubered-topped serum tubegreen-topped heparin tubelavender-topped EDTA tube.  If only a blue topped citrate tube is needed, draw a non-additive discard tube first to avoid contamination with tissue factor found in tissue fluids that can accelerate the clotting cascade and cause platelet clumping.  One example of cross-contamination artifact is that of serum contaminated with potassium-EDTA, which results in spuriously and markedly decreased calcium and increased potassium.

  1. Avoid hemolysis

The most common cause of hemolysis is traumatic venipuncture which is understandably difficult to avoid in fractious or excitable animals.  Other common causes of hemolysis include applying too much suction when collecting the blood, using too small of a needle, and filling tubes by squirting the blood through the needle.  Ideally, needles should not be smaller than 22g when collecting blood samples although some small rodents may require smaller gauge needles.  Needles should be removed from the syringe before filling the tube and the tube should be filled by removing the lid and allowing the sample to dribble down the side of a tilted tube.  

Using the instrumentation at Mashfield Labs, we have found that hemolysis can result in:  

  • Slight increase in: GLU, AST, CHOL, TP,CO2, AMY, and LIP
  • Moderate to dramatic increase in: TBILI, ALB, Ca, Na, K (especially equine and non-mammal), Cl, PHOS, CK, Mg, LDH, UA, SDH, and Fe
  • No change in: ALT and BUN
  • Moderate to dramatic decrease in: ALP, T4, Bile Acids,CREAT, and GGT
  1. Be sure to fill the tubes with the correct volume of blood

Underfilled tubes can result in dilutional effects when blood is added to liquid anticoagulant or preservative.  Dilutional effects are less of a concern when tubes contain lyophilized anticoagulant but both liquid and lyophilized anticoagulants will affect tonicity in underfilled tubes that can result in erythrocyte shrinkage.  Be sure to fill the tube to the indicated marker on the tube label and to invert the tube gently 3-5x to mix thoroughly. Underfilling blue-top sodium citrate tubes used for coagulation testing will lead to artifactual prolongation of clotting times.  

  1. If submitting blood for a CBC, always submit a fresh smear

Sometimes, despite our best efforts, there are delays in sample processing and shipping that can result in ageing artifacts within whole blood samples.  If a fresh smear is submitted along with the whole blood sample, the fresh smear can be used to evaluate cellular morphology and provide estimates of cell numbers.

  1. Process the samples in a timely manner

Delayed processing can alter analytes and blood cell morphology.  Ideally, samples should be processed within 1 hour.  Serum must sit for a minimum of 30 minutes to allow the sample to fully clot but then should promptly be spun to separate the serum and the serum should immediately be removed from the clot to prevent spurious results associated with metabolism of analytes, electrolyte leakage from the cellular clot into the serum, and deterioration of certain analytes. Delayed processing can lead to hypoglycemia, hyperkalemia, hyperphosphatemia, and decreased enzyme activities.

  1. Store and ship samples at the appropriate temperature

As a general rule, specimens should be stored and transported at refrigerated temperatures, avoiding excessive heat and cold, unless otherwise indicated.   Coagulation testing can be kept refrigerated if the specimen will arrive at the laboratory and analysis will be performed within 24 hours.  Otherwise, citrated plasma should be separated, transferred into a plastic tube, and frozen for submission to the laboratory. Be sure to verbalize temperature requirements to the courier.  Serum may be frozen for future evaluation but not all analytes are stable for the same length of time.  Stability of analytes is variable and depends to the freezing temperature and length of time frozen.  Be sure to also submit separate aliquots for each test requiring a frozen specimen. Blood smears and cytological specimens should be stored at room temperature in a slide holder. Do not refrigerate slides.

  1. Label all samples well

Tubes and slides should be labeled with the animal’s unique identifier, the date, and any other information pertinent to the study.   Be sure to label the sample type if submitting more than one; serum, plasma and urine all appear similar. Tubes may be labeled with indelible ink (e.g. a Sharpie pen) or labels. Slides should be labeled using a pencil on the frosted end of the slide. Do not use ballpoint ink or label/sticker to identify slides.

  1. Do not expose cytological samples (blood, fluid, or cytology smears) to formalin

Formalin cross-links proteins which interferes with stains used for evaluation of blood smears and cytological samples, oftentimes rendering the sample non-diagnostic.

10.Do not use serum separator gel-containing tubes for hormone or drug assays

Hormones such as TSH or progesterone and drugs such as phenobarbital bind to the gel resulting in spuriously low concentrations.