The effects of resting time, centrifugation time, and technician training on plasma sample quantity and quality: Implications for the Dog Aging Project

Author

Sydney Holland

Where was it published?

Veterinary Clinical Pathology
12 February 2025

 What is this paper about?

Members of our Precision Cohort participate in an annual collection of routine samples like bloodwork (similar to an annual check-up), urine, feces, and hair collected by their primary care veterinarian. In a previous paper where we investigated why different samples of our Precision Cohort sample kits might be missing, we found that samples like plasma and serum were missing more frequently than other sample types. Plasma and serum are two types of samples that can be isolated from blood through laboratory sample processing. Because these samples are collected and isolated by primary care veterinarians and not by DAP researchers, it is important for us to test our own sample collection instructions to ensure that any primary care veterinarians or technicians that follow them are able to yield the best possible results.

Sample “missingness” can happen in many different ways. When we refer to a sample as “missing”, this does not always mean that someone forgot to collect it or that the sample was misplaced from the sample kit. In this case, when a sample is considered “missing”, it means that we were unable to gather data from the test or analyses that we intended to perform with the sample. When it comes to our blood samples, different portions of blood samples allow us to perform different kinds of analyses, which is why we assess whole blood samples, plasma, and serum. When one tube of whole blood is collected, that one tube can be divided up to perform multiple different tests that tell us different things about the patient’s health. However, each of these different tests require different minimum quantities of each sample type. Frequently, when one or more of our plasma samples are considered “missing”, this means that the plasma sample collected from one dog was not enough to perform all of our different intended tests. That means that for that dog, any tests that could not be performed are missing data.

In this paper, we conducted an in-house study following our own blood collection and plasma isolation instructions on a small sample of volunteer dogs. We conducted three separate trials in which we compared the plasma collection procedure as listed in our sample kit instructions versus procedures with slight modifications. By making slight adjustments to different aspects of the collection procedure, we could assess whether any specific change caused us to collect more or less plasma than we expected. The variables we tested were the time that whole blood was allowed to rest before plasma processing, the amount of time that blood samples were spun in a centrifuge to isolate plasma, and whether the collection was performed by a trained laboratory technician versus a technician with no training.

Our study found that none of our experimental modifications to the plasma collection procedure allowed us to collect significantly more plasma than the current plasma collection instructions. However, we did see that not all dogs were able to produce as much plasma as our instructions were designed to isolate, even though the same amount of blood was drawn from each dog. These findings provided a few important takeaways to our researchers.

Our current plasma collection instructions allowed for the highest quality and quantity of plasma collection given the volume of whole blood that our plasma is isolated from. However, even under our highly-controlled plasma collection, we observed how differences from dog to dog can affect their individual plasma concentrations in their whole blood. This means that two different dogs can have the same amount of whole blood drawn, but the plasma isolated from one blood sample may be plenty to complete all of our intended analyses, while the plasma isolated from the other blood sample may be too low to complete all of the intended analyses.

What do these results mean for me and my dog?

While these results may not be the most exciting or groundbreaking experiment to come from our research, this experiment represents an important part of research, which is evaluating and validating your methods of research. As researchers, we value the time and effort that our Precision Cohort participants dedicate to participate in our sample kit collection. While missing data is an inevitable part of large-scale research projects, we want to ensure that the volunteer efforts of each of our citizen scientists in the Precision Cohort are able to be equally impactful towards our future scientific discoveries.

Where can I learn more?

Holland SN, Tinkle AK, Prescott JN, Blattman BL, Creevy KE, Fajt VR. 2025. The effects of resting time, centrifugation time, and technician training on plasma sample quantity and quality: Implications for the Dog Aging Project. Vet Clin Pathol. 2025; 00: 1-8. doi:10.1111/vcp.13413

Abstract

Background: The Dog Aging Project (DAP) is a large-scale longitudinal study of aging in dogs. For some dogs in the DAP, blood samples for plasma isolation are collected by non-DAP personnel. However, plasma samples are sometimes inadequate, which can lead to, for example, insufficient volume for assays.

Objectives: We aimed to examine three factors that may affect plasma yield: resting time after sample collection, centrifugation time, and level of operator or technician training.

Methods: We designed three experiments using a convenience sample of five dogs. Each experiment varied one of the three factors and held the other two constant. Experiment 1 examined five different resting times: 10 min and 1, 4, 24, and 72 h. Experiment 2 compared centrifugation times of 7 and 14 min. Experiment 3 compared trained and untrained personnel. The sample resting was always under refrigeration. Experimental outcomes were total plasma volume, number of successful aliquots, hemolysis, and lipemia.

Results: A resting time of 72 h yielded statistically significantly lower plasma volume than resting times ≤4 h. Resting times of 24 and 72 h also had statistically significantly higher hemolysis scores compared with other resting time points. In addition, trained operators or technicians yielded an average of 0.5 more aliquots. Outcomes were similar by centrifugation time in Experiment 2.

Conclusions: To mitigate sample loss, we recommend shorter post-collection resting times and ensuring technician proficiency. Additionally, increasing the requested whole blood volume may improve sample yield.