MSACL 2017 US Abstract

Do DBS and DPS Micro Sampling Techniques have a Place in the Clinical Laboratory?

Jack Henion (Presenter)
Q2 Solutions

Bio: Emeritus Professor of Toxicology, Cornell University CSO Advion, Inc. VP R&D, Q2 Solutions

Authorship: Jack Henion, Imelda Ryona
Q2 Solutions

Short Abstract

Dried blood spot (DBS) and dried plasma spot (DPS) micro sampling techniques offer several benefits for the clinical diagnostic laboratory. These include small sample volumes, minimally invasive sample collection, point-of-care/home patient sample collection and economical sample shipping/and storage. To-date DBS techniques for newborn screening are well accepted, but DBS techniques have not been generally accepted in the clinical laboratory. This presentation will show how DBS techniques and a novel book-type DBS/DPS card provides a dried spot of red blood cells and the corresponding dried spot of plasma from the same micro sample can be analyzed by LC/MS/MS bioanalysis.

Long Abstract

Introduction

Micro sampling technologies are of particular interest for neonatal patients, preemie babies or patients who are very sick and challenged to provide frequent venous blood samples for ongoing tests by their physicians. The most common approach for collecting a micro blood sample is a simple finger prick using a disposable lance or the virtually painless Genteel lancing device (https://www.mygenteel.com). This novel device is used for home sample collection by patients with diabetes or other diseases requiring frequent monitoring of blood samples where simple blood sugar tests, for example, can assist the patient with their optimal treatment. Alternatively, for those samples that require analysis by a central clinical laboratory the dried blood spot may be sent via the regular mail often without the need for extra cooling. Upon receipt at the clinical laboratory any of a variety of tests may be performed including LC/MS/MS bioanalysis.

Our laboratory has reported the development of a homemade dried plasma spot (DPS) card which has been used for collection, transport, storage and automated LC/MS/MS bioanalysis for a variety of drugs including opiates, stimulants, cannabinoids, beta blockers and steroids. In addition, for those instances where large numbers of DBS or DPS card samples require rapid analyses and short turn-around of results, these cards may be placed into a robot for on-line fully automated LC/MS/MS bioanalysis.

One of the challenges for DBS techniques has been for whole blood samples which exhibit a wide range of different hematocrit values. Our previously developed DPS card provided acceptable results for normal hematocrit blood, but did not provide acceptable precision and accuracy results for the extremes of hematocrit (30% and 60%). We have developed a new device which produces acceptable precision and accuracy bioanalytical results for whole blood samples which span this wide range of hematocrit. This presentation will describe how this may be done by focusing on several opiates and stimulant drugs determined together in the same micro blood sample.

These results are derived by employing a new ‘book-type’ DBS/DPS card. This card can accommodate the direct application of whole blood drops from, for example, a finger prick with a range of volume from 10-50 microliters. The device consists of two sequential membrane filters which filter out the red blood cells onto the upper membrane while allowing the plasma to flow through the second membrane to be collected onto a cellulose substrate. After drying for about three minutes to all completion of the plasma filtration, the ‘book-type’ DPS card is ‘opened’ and the respective collected biological samples allowed to air dry for 30 minutes. This sample set (usually 3-4 aliquots of the same patient) may be stored in a desiccated zip-lock bag at room temperature and/or shipped via the standard mail to a laboratory for later LC/MS/MS analysis.

Methods

Human control blood was collected from healthy volunteers. For studies on hematocrit effects human blood of known hematocrit (30, 45 and 60%) were purchased from Bioreclamation. In general, approximately 25 microliter samples of blood were applied to the filtration membrane/device. A series of opioid drugs was fortified into the control blood ranging from 1 ng/mL to 1000 ng/mL. Stable isotope-labelled internal standards for each of these opioids were spiked into the control blood at 200 ng/mL. The opioid drugs included morphine, hydrocodone, codeine, oxycodone and fentanyl. The blood samples were applied onto the upper filtration membrane surface, the red blood cells were then allowed to filter (3min), wherein the upper filtration membrane was separated from the lower cellulose layer of plasma by simply ‘opening the book’. This exposes each collected biological sample (e.g. the red blood cells and the plasma) to air dry for 30 min.

A Shimadzu binary Nexera UHPLC system coupled to a Spark-Holland DBSA automated DBS card robot equipped with an in-line SPE cartridge which, after interference elution, was switched to an analytical HPLC column (Raptor Biphenyl, 2.7 µm, 2.1 x 50 mm & a guard column (2.7 µm, 2.1 x 5 mm) for SRM LC/MS (Shimadzu 8050) bioanalysis using atmospheric pressure chemical ionization (APCI) in the positive ion mode. This ionization produced the optimal signal-to-noise at the LLOQ and demonstrated no significant matrix suppression of ionization in contrast to ESI. Quantitative determination of each opioid was determined using the Shimadzu software to produce linear calibration curves with a dynamic range of at least three orders of magnitude.

Results

There are significant potential benefits for a successful DPS device but there are lingering questions as to bioanalysis results compared to plasma obtained by traditional centrifugation. These include the potential for hemolysis during the filtration of the red blood cells, the hematocrit effects due to potential effects on the filtration process from the red blood cells, reduced interference in alternative assays such as ligand binding assays, and increased stability of drugs and their metabolites due to the dry plasma spot and the associated inactivated enzymes. Our results suggest that plasma obtained by our filtration process behaves the same as plasma produced by centrifugation for the drugs studied. The filtration of red blood cells must be ‘gentle’ or hemolysis may occur, but this is entirely possible to achieve via our described procedures.

Once the DBS sample is ready for analysis it is eluted directly from the resulting DPS substrate using an automated flow-through concept afforded by the Spark-Holland DBSA system. A clamp is placed firmly on or around the DPS such that as elution solvent passes through the spot such that no leakage occurs. This clamp may encircle a small portion (2 mm) within the plasma spot or a larger clamp (6 mm) may be employed which encircles the entire plasma spot. This latter approach precludes any issues associated with a partial spot elution. Both of these approaches were studied and provided satisfactory bioanalysis results. The larger clamp may be preferred if there are issues associated with adequately reaching the LLOQ; e.g. the larger clamp elutes more sample. This was not an issue in this work

Our studies resulted in satisfactory bioanalysis of micro samples of blood which produced dried plasma spots. Linear calibration curves were obtained with acceptable precision and accuracy with an LLOQ of 1 ng/mL with a dynamic range of three orders of magnitude. The method is fully automated once the DPS devices are placed into the rack of the DBSA system which accommodates 160 cards. It should be mentioned that we have not as yet performed any analyses of the collected dried red blood cells. However, this should be entirely possible and in fact initial promising results have been reported by two of our collaborators.

Conclusions

Clinical diagnostic laboratories are implementing modern LC/MS/MS bioanalytical methods into their workflow for the quantitative determination of drugs in biological samples. To-date most of these samples are serum, plasma, or whole blood, collected in vacutainer tubes or related containers. As such conventional sample preparation techniques such as SPE, liquid-liquid extraction, etc. are employed to produce and extract suitable for LC/MS/MS bioanalysis. This presentation will show that this process may be simplified by either off-line or on-line DBS or DPS automated sample preparation coupled with SRM LC/MS with equivalent precision and accuracy of bioanalytical results.

This capability is facilitated by a reliable and an easy means of collection and analysis of human blood samples.  It may be accomplished by sampling a finger stick of blood which is then placed onto the DPS card, shipped and stored until time of analysis. The described representative common opioid compounds were quantified using a novel DPS card coupled with a fully automated analysis based upon SRM LC/MS bioanalysis. The described methodology appears to offer a simple approach to facile collection of finger prick blood samples collected on a red blood cell filtration device which can produce replicate (3-4 samples) of dried plasma free of red blood cells.


References & Acknowledgements:

We acknowledge financial support for this research from the Partnership for Clean Competition and consignment of the instrumentation by Shimadzu and the Spark-Holland Company.


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