Fundamentals of Analytical Toxicology. Robin Whelpton

       2.2.3.4 Plasma

Separation of plasma from anticoagulated whole blood normally requires centrifugation, as do many of the phase separation procedures discussed in Chapter 4. The inter-relationship of centrifuge rotor diameter, speed of centrifugation, and relative centrifugal force (g-force) is set out below (Box 2.1). Swing-out rotors are preferred for separating liquid phases.

Box 2.1 Calculating relative centrifugal force

       The relative centrifugal force (RCF, g) depends upon of the speed of the centrifuge in revolutions per minute (RPM) and the effective radius of rotation, r

       The radius of rotation varies along the length of the centrifuge tube

       RCF may be quoted as maximum, minimum, or average

       Conversion tables and nomograms for each rotor are normally supplied by the manufacturer of the centrifuge

       Modern centrifuges have the facility to set the RCF directly

       The RCF will be maximal at the bottom of the tube

       RPM for a required RCF can be calculated from:where r is in mm

       RCF from RPM is given by:

      On centrifugation, anticoagulated whole blood (approximately 2000 g, 10 min; 2–8 °C if necessary) will separate into three layers: the bottom layer (normally 45 % or thereabouts by volume) consists of red cells. A thin intermediate layer of white cells and platelets called the ‘buffy coat’ is the next layer; and the upper, aqueous, straw-coloured layer is the plasma (about 50 % v/v). More plasma than serum can be separated from whole blood.

      Some commercial tubes contain agents such as plastic beads or a gel that sits at the interface between the cells and the plasma to aid plasma collection. Gel separators have caused problems with some drug analyses (Berk et al., 2006), and although reformulated gels have been shown to have little effect on clozapine and norclozapine measurement (Handley et al., 2018), tubes containing gel separators are best avoided (Schrapp et al., 2019). Modern blood-collection tubes may contain a range of additives including surfactants, which may interfere in immunoassays, for example (Bowen & Remaley, 2014). The use of such tubes will also invalidate many trace elements analyses (Chapter 21) and may impair analyses for solvents and other volatiles.

       2.2.3.5 Blood cells

      To collect erythrocytes, heparinized blood should be centrifuged (2000 g, 10 min), the plasma, buffy coat, and top 10 % of erythrocytes (mainly reticulocytes) removed, and the remaining erythrocytes carefully washed with isotonic phosphate-buffered saline (PBS), pH 7.4, to remove trapped plasma. The cells may be used directly or stored at –20 °C, either to cause haemolysis, or for storage. Platelets are usually isolated by the slow centrifugation (e.g. 300 g, 15 min) of anticoagulated whole blood to yield platelet-rich plasma, which is recentrifuged (2000 g, 10 min) to harvest the platelets. Other white blood cells are most commonly obtained either by centrifugation through media of appropriate density (according to manufacturers' instructions), or isolated by solid-phase antibody techniques.

      Erythrocyte:plasma distribution

It is easier to measure whole blood:plasma analyte distribution than to attempt to measure the erythrocyte:plasma distribution, a measurement that requires the preparation of washed erythrocytes. Analyte is added to a portion of analyte-free heparinized whole blood and, after allowing time for equilibration and controlling the pH (the pH of blood tends to fall in vitro), one portion of the blood is centrifuged to obtain plasma and a second portion is frozen and thawed to give haemolyzed whole blood. After measuring the analyte concentration in the plasma (C_P) and in the haemolyzed whole blood (C_B), the analyte erythrocyte concentration (C_E) can be calculated if the haematocrit, H, (the proportion of erythrocytes in the blood) is known:

      (2.1)

      

       2.2.3.6 Dried blood spots

      DBS are produced by the deposition of either haemolyzed whole blood, or capillary blood onto either filter paper, or dedicated paper cards. As compared with either whole blood or plasma, sample transport and storage are easier because refrigeration is not needed provided that the analyte is stable and can be recovered from the spot quantitatively (Wagner et al., 2016; Freeman et al., 2018). Problems are that (i) capillary blood is not venous blood, and (ii) volumetric blood collection directly from the patient onto the medium on which the blood is to be dried is extremely difficult (Sulochana et al., 2019).

      Thus, for qualitative work such as screening for inborn errors of metabolism, or for detection of substance misuse, DBS are acceptable provided that the analyte is stable in the spot, but reliable quantitative work means measuring a known volume of venous blood onto the storage medium before allowing it to dry (Section 20.3.1.1). This being said, in forensic work, measurement of the haemoglobin content of a blood spot found at a scene, for example, may serve to give a surrogate measure of the amount of blood deposited initially.

      

       2.2.3.7 Volumetric microsampling

Volumetric microsampling devices (Figure 2.1) aim to collect a fixed volume of capillary blood, which can then either be made available for analysis in its entirety or can be used to provide a fixed volume of plasma for processing. These devices promise advantages over DBS related to sampling volume accuracy, simplified sample pre-treatment, and ease of automation (John et al., 2016).

Figure 2.1 Volumetric blood microsampling devices. (a) Mitra (Neoteryx) uses an absorptive pad to collect a fixed volume of blood; (b) HemaXis DB uses a fixed volume capillary to transfer blood to a standard DBS card

      Volumetric absorptive microsampling (VAMS^®) aims to absorb a fixed volume of blood or other fluid (10 or 20 μL) using the Mitra^® (Neoteryx) device (Spooner et al., 2015; Protti et al., 2019). Although some aspects still need to be investigated in depth (Kip et al., 2017), VAMS^® may be a viable alternative to DBS. It simplifies sample collection and storage, and may allow sample collection in a patient's home (Kok & Fillet, 2018). The HemaXis^TM DB 10 (DBS System SA) uses microfluidics to provide 10 μL whole blood from a reservoir onto a DBS card (Leuthold et al., 2015; Verplaetse & Henion, 2016). The HemaXis^TM DX promises to produce plasma or serum without the use of a centrifuge, filtration membrane, or pump and with no haemolysis. The resulting sample can be stored in dried or liquid format. The Noviplex™ Plasma Prep Card gives a fixed volume of plasma from a reservoir of whole blood (Heussner et al., 2017).