O.I. Panasenko, T.O. Samura, O.O. Filatov, V.P. Buryak, S.M. Kulish. N.A. Postal, I.V. Melnik, O.A. Kremzer, T.V Panasenko

Zaporozhye State Medical University

SAMPLE COLLECTION, TRANSPORT, AND STORAGE

In analytical toxicology no matter how complex the equipment and careful the analysis, the results may be rendered worthless if sample collection, transport, and storage have not been performed with the analysis in mind.it is important to be familiar with the nature and stability of the analyses, the nature of the sample matrix, and the circumstances under which the analysis is to be performed. Proper documentation of the history of the sample origin made of collection, transport, storage, and the like is essential.

The analytic concentration in the specimen is generally assumed to be representative of the concentration in the particular fluid or tissue sampled. Whole blood, plasma the fluid obtained on centrifugation of anticoagulated whole blood , or serum the fluid remaining when blood has clotted are widely used in clinical work. This is because not only is blood relatively easy to collect, but also a quantitative analysis can often give useful information as to the magnitude f exposure and hence the severity of poisoning. Excretions (exhaled air, urine) or secretions (saliva bile) are often less useful as regards interpretation of quantitative data, but can be very useful in qualitative work;

Biological samples may contain infective agents and must be handled with care, especially if originating from drug abusers, and must always be treated as if they are infective. The major common risks are associated with tuberculosis, hepatitis B, and human immunodeficiency virus (HIV). Urine is least likely to be infective. It is thought very likely that following solvent extraction or other robust sample preparation procedures, infective agents will be inactivated, except for variant Creutzfeldt-Jakob Disease (CJD), but in homogeneous assays, such as immunoassays, samples may continue to be infectious after incubation, even though diluted. Indeed, incubation may increase the titer of the infective agent.

Table 1. Types of variables affecting clinical samples

Variable	Exple(s)
Physiological
Age	Markers of bone timber such as collagen cross-linkages are increased increases in childhood
Sex	Sex hormones
Body Weight	Urinary creatinine increases with muscle mass
Recent food intake	Plasma glucose, triglycerides, and so on, after a normal meal
Died	Malnutrition or fasting will reduce serum albumin, urea and phosphate
Menstrual Cycle	Plasma concentration of luteinizing hormone (LH)
Drugs	Drug treatment may alter concentrations of some plasma constituents even in apparently healthy subjects.
Sample Analyte variations
Incorrect specimen	Value differences between plasma and serum, venous and arterial blood, random and 24 urine samples
Incorrect collection	The absence of an appropriate enzyme inhibitor may allow continued enzyme action such as catabolism of glucose or neuropeptides
Hemolysis	Red cell lysis may lead to changes in plasma constituents, particularly potassium, phosphate, and same enzyme and may interfere with the analytical method
Collection during an infusion	Collection near to an infusion site will give misleading concentrations of the compound being infused or dilate other blood constituents
Drug treatment	Drug or metabolites may interfere in the assay

Staff in regular contact with potentially infectious materials must be properly trained in the safe handling and disposal of biological samples. Such staff should be vaccinated against hepatitis B, polio, tuberculosis, and tetanus and possibly other diseases in specific countries. Sample handling should be performed with due attention to preventing droplets splashing into the eyes and minimizing aerosol formation. Screw-capped sample tubes are preferable to use with push-in stoppers as there is less risk of aerosol formation when opening the tube [6].

Clinical samples can be divided into blood and related fluids, body fluids other than blood, excretory fluids/residues, and other clinical specimens (Table 2). A range of additional specimens may be collected for toxicological purposes. Special precautions will be needed with unstable analytes. Most compounds measured in urine can be considered stable for at least a few hours at room temperature as the urine may already have been held at body temperature for some time before it was voided [8].

Blood ("Whole blood") is the fluid that circulates through the arteries, capillaries and veins. The adult human body contains some 5-6 litres of blood. It is composed of plasma and blood cells. Normally venous blood is obtained. If whole blood is to be analyzed, then sample should be collected into an appropriate anticoagulant, mixed, and then frozen in order to lyse the cells before the analysis.

Blood cells include red cells and white cells, leukocytes. All may be harvested from freshly collected blood with appropriate procedures Body fluids other than blood.

Amniotic fluid is the fluid that surrounds the foetus in the amniotic sac.

Breast milk is the protein and fat-rich fluid produced by nursing mothers. The first expression of breast milk is especially rich in protein.

Lymph is a yellowish fluid derived from the lymph glands.

Peritoneal fluid is the fluid that accumulates in the peritoneum. Tears are the clear watery secretion of the tear ducts of the eye.

Even mild haemolysis will invalidate a serum iron or potassium assays, for other analytes concentrated in red cells such as chlortalidone. Leaving plasma or serum in contact with red cells can cause changes due to enzymatic activity or redistribution of an analyte between cells and plasma. In general, plasma or serum should be separated from the blood cells as soon as possible, If necessary, whole blood can be stored at- 20 C or below but freezing will lyse most cell typer [7].

It is important to use serum or the anticoagulant recommended for a particular measurement ( Table 3) and not to substitute an alternative without careful consideration. Sodium citrate tulles contain 0,5 or 1 ml of the anticoagulant in aqueous solution and so are unsuitable for quantitative work. Furthermore, dilution of the sample may reduce the degree of plasma protein binding and consequently the plasma: red cell distribution of the analyte. It should be ensured that lithium heparin anticoagulant is not used if plasma lithium is to be measured. Heparin too has been known to interfere in drug analysis.

Table 3. Anticoagulant for in vitro use

Anticoagulant	Concentration (ml^-1 blood)	Comment
Lithium heparin	10-20 units	General biochemistry
Sodium heparin	10-20 units	General biochemistry
Sodium fluoride which either	1-2 mg	Glucose (inhibits glycolysis)
EDTA or oxalate	6-10 mg	General anticoagulant
Sodium citrate	3 mg	Clotting studies not recommended for other purpose as the aqueous solution dilutes the specimen
EDTA	2mg	Hematology (stabilized readily oxidized compounds)

When whole blood is allowed to stand (15 min, room temperature ) in a plain tube ( no anticoagulant) a clot forms that will retract sufficiently to allow serum to be collected. For many analyses serum is preferred to plasma because it produces less precipitation (of fibrin )on freezing and thawing.

The inter-relationship of centrifuge rotor diameter, speed of centrifugation and relative centrifugal force (g-force) is set out in Box 1 . Swing-out rotors are preferred for separating liquid phases.

On centrifugation of anticoagulated whole blood (2000g, 10 min, 2-8 C if necessary), it will separate into three layers: the bottom layers ( normally 45% or there a bouth 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). Provided the analyte is stable, anticoagulated whole blood can be kept at room temperature or refrigerated (2-8C) for two days or so before harvesting plasma [3].

More plasma than serum can be separated from whole blood. Some commercial tulus contain agents such as plastic beads or a gel that sits at the interface between the cell and the plasma to aid plasma collection. Gel separators have caused problems with some drug analysis [1.5], although reformulated gels have been claimed to have little effect on therapeutic drug measurements [2]. However, this work has not been extended to other analytical toxicology tests and tulus containing gel separators are therefore best avoided.

Box 1. Calculating relative centrifugal force

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

2. The radius of rotation varies along th length of the centrifuge tube.

3 .RCF may be quoted as maximus,minimum or average.

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

5. Modern centrifuges have the facility to set the RCF directly.

6. The RCF-will be maximal at the bottom of the tube.

7. RPM for a required RCF can be calculated from:

RPM = where r is in mm.

8. RCF from RPM is given by: RCF = (1.118* )r (RPM)²?

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) removed, and the remaining erythrocytes carefully washed with isotonic, buffered saline to remove trapped plasma. The cells may be used directly or frozen, either to cause haemolysis, or for storage. Platelets are usually isolated by the slow centrifugation ( e.g. 300g 15 min )of anticoagulated whole blood to yield platelet-rich plasma, which is centrifuge ( 2000 g, 10 min) to harvest the platelets. Other white blood cells are most commonly obtained by centrifugation through media a appropriate density (according to be manufacturer's instructions) or isolated by solid-phase antibody techniques.

If measurement of red cell: plasma distribution is to be performed, it is easier to add the analyte to a portion of "blank" heparinized whole blood and after allowing time for equilibration and controlling the pH of blood tends to fall in vitro as oxygen is lost) to obtain plasma from one portion of the blood and to freeze and thaw a second portion(to give haemolyzed whole blood) and compare the results. Admittedly this gives the plasma : whole blood ratio, but it is technically for simple than preparing washed erythrocytes. The analyte erythrocyte concentration can be calculated if the haematocrit (the proportion of erythrocytes in blood ) is known:

C_E =

Where, C_E= erythrocyte concentration C_b= whole blood concentration, Cp= plasma concentration, and H=hematocrit[4].

Different urine specimens, for example random, early morning end-of-shift, 24 hour, may be collected in the course of metabolic or other studies, In metabolic studies, it is important to note time the time of the beginning and the end of the collection period so that the rate of urine production can be calculated. A random urine sample is a midstream specimen-any preservative, such 2 mol L^-1 hydrochloric acid is added after words. Fresh urine is yellow/yellow-green in colour, but on storage in acidic solutions the colour changes to yellow/brown and even to dark brown due to oxidation of urobilinogen to urobilin. Crystals, party cularly of uric acid and calcium oxalate may form causing turbidity.

When random, early morning , or end-of-shift specimens are collected, it is common practice to relate certain analytical results to a "fixed" urinary constituent such as creatinine, which is considered to be excreted at a relatively constant rate in normal subjects . However, as creatinine is derived from creatinine, there are situations, such as muscle wasting or in bodybuilders dosing with creatine, when this is not stricly true.

The concentrations of many drugs and metabolities, and of some endogenous constituents, will remain the same in acidified urine for over a week at room temperature, and for up to a month at 2-8 C. Unacidified urine undergoes microbiological attack and many changes accur, including the complete lass of amino aids. For long-term storage acidified urine can be frozen (-20 ºC) but it may be necessary to centrifuge the sample to remove any precipitate formed during storage prior to any analysis.

Stomach contents is specimen encompasses vomit , gastric aspirate and gastric lavage fluid as well as the contents of the stomach at postmortem. The nature of this sample can be very variable and additional procedures such as homogenization followed by filtration and/or centrifugation may be required to produce a liquid amenable to analysis.

The analysis of faeces is rarely performed in clinical chemistry, but sometimes drug and possibly metabolite analysis may be required in pharmacokinetic and metabolism studies. Analyses may also be requested if for example, the guestion of drug leakage from ingested packets of drug antemortem is raised. Unire plasma, unire, and other fluid samples, faeces are not homogenous, and thus it is often necessary to analyze the whole sample or homogenize the whole sample and prove. That the fraction taken for analysis is representative of the whole. It may take more than a day before an orally administered drug or a drug metabolite appears in faeces.

Histology specimens are usually collected into a preservative such as formalin (aqueous formaldehyde solution). such pretreatmeent must be borne if mind if toxicological analyses are requested sub-seqnently. Samples of tissue obtained postmortem are normally kept at 4 ºC prior to analysis.

S U M M A R Y

Variations in bioanalytical measurement may be subject-dependent and reflect normal physiological changes, whilst others may reflect sample collection and handling procedures. Postmortem specimens are a special problem because , generally, information on the analyte concentration in blood at the time of death is required. Postmortem blood concentrations may not accurately reflect perimortem blood concentrations for several reasons. Haemolysis is common, for example, whilst haemostasis may lead to changes in the cellular composition of the "blood" being sampled. There is also the possibility of contamination during collection , for example , with stomach contents, and of leakage of intracellular potassium into plasma, which begins soon after death, is such an example.

R E F E R E N C E S

1. Berk S.I., Litwin A.M., Du Y., Cruikshank G, Gourevitch M.N., Arensten I.M.. False reduction in serum methadone concentrations by BD Vacutainer serum separator tules (SST TM) Clin. Chem., 2006.-val 52.-pp 1972-1974

2. Bush V., Blennerhasset I, wells A, Dasgupto A. stability of therapeutic drugs in serum collected in vacutainer separator tulus containing a new gel (SST II), The Drug Monit., 2001.-val.23.-pp259-262

3. F Clarke's Analysis of Drugs and Poisons. fourth Edition/Editied by Anthony C Moffat, M. David Osselton, Brian widdop.-London: The pharmaceutical Press, 2011.-2609 p.

4. Fundamentals of analytical toxicology/ Edited by R.I. Flanagan.-New York: Yohn Wiley and sons 2007.-532 p.

5. Karppi I., Akerma K.K., Parviainen M. Suitability of collection tubes with separator gels fr collecting and storing blood samples for collecting and storing blood samples for therapeutic drug monitoring (TOM.) Clin. chem. Lab. Med., 2000.-val.28-p.p.313-320

6. Lam C.W., Yames Y.T. Mecluskey R., Hunter R.L. Pulmonary toxicity of single wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation. Tox. Sci., 200-val.77-pp126-134

7. Sporkert F., Brunel C., Angsburger M.P. Fatal talperisone poisoning: Autopsy and toxicology findings in three suicide casus.-2012-val. 215.-pp.101-104.

8. Warheit D.B., Laurence B.R. Reed K.L., Roach D.H., Reynolds G.a.m, Welb T.R. Comparative pulmatary toxicity assessment of single wall carbon nanotules in rats.