O. I. Panasenko, A. A. Safonov, T. O. Samura, I. V. Melnik, V. A. Salionov,
A. S. Gotsulya, V. P. Buryak, N. A. Postol, R. A. Sherbina, A. A. Kremzer, I. M.
Keitlyn, T.V. Panasenko
Zaporozhye State Medical University
COLLECTIONS
FOR ANALYTICAL TOXICOLOGY.
TRANSPORT
AND STORAGE
It is usually
advisable to contact the laboratory by telephone in advance to discuss urgent
or complicated cases. Most specimens, particularly blood and urine, may be sent
by post if securely packaged in compliance with current regulations. However, if
legal action is likely to be taken on the basic of the results, it is important
to be able guarantee the identity and integrity of the specimen from when it is
of the specimen from when it is collected through to the reporting of the result.
Thus, such samples should be protected during transport by the use of tamper-
evident seals and should, ideally, be submitted .In person to the laboratory by
the coroner’s officer or other investigating personnel. Chain of custody is a
term used to refer to the process used to maintain and document the history of
the specimen (see BOX 1).
BOX
1. Chain of
custody documents. Name of the individual collecting the specimen. Name of
each person or entity subsequently having custody of it, and details of how it
has been stored. Dates/times the specimen was collected or transferred. Specimen
or postmortem number. Name of the subject or deceased. Brief description of the
specimen. Record of the condition of tamper-evident seals.
Fully validated
assays must include data on the stability of the analyte under specified storage
conditions. In the absence of other information, biological specimens should be
stored at 2-8 °C prior to analysis, if possible, and ideally any specimen
remaining after the analysis should be kept at 2-8 °C for 3-4 weeks in case
further analyses are required. In view of the medico legal implications of some
poison cases (e.g. if it is not clear how the poison was administered or if the
patient dies) then any specimen remaining should be kept (preferably at -20 °C)
until investigation of the incident is concluded. With regard to drugs, some
compounds such as clonazepam, cocaine, nifedipine, nitrazepame, thiol drugs,
and many phenothiazines and their metabolites are unstable in biological samples
at room temperature. Exposure to sunlight can cause up to 99% lass of
clonazepam in serum after 1h at room temperature. Covering the outside of the sample tube in aluminum
foil is a simple precaution in such cases. N-Glucuronides such as nomifensine N-Glucuronide
are unstable and may be present in plasma at high concentration: on
decomposition the parent compound us reformed. Some compounds unstable in whole
blood or plasma are listed in (Table 1).
Solid sodium
fluoride (2%, w/v) may be added to inhibit microbial and some other derivative
enzymes. Esters (including carbonates and organophosphates) may be rapidly. Hydrolyzed
by plasma esterase’s, including cholinesterase. If physostigmine or cocaine is
to be measured accurately, the blood should be drawn into tubes containing an
excess of neostigmine. Storage at -20 °C or below is recommended if the
analysis cannot be performed immediately and if the stability of the analyte is
unknown. However, even this may not be ideal because N-and S-oxyde may be reduced
to the parent compounds.
Table
1
Some
drugs, metabolites and other poisons unstable in whole blood or plasma
|
Volatile
Compound(s) |
No-volatile
Compound(s) |
|
Aerosol
propellants, anesthetic gases, carbon monoxide, ethanol, chlorvynyl, hydrogen
cyanide, mercury, methanol, nicotine, OP nerve agents, organic solvents,
paraldehyde, volatile nitrites (amyl nitrite, etc.). |
Acyl
glucuronide metabolites, amiodarone, bupropion, carbonate esters, esters
(aspirin, benzocaine, cocaine), gluceryl trinitrate, N-glucuronide
metabolites, insulin, lysergic acid diethylamide, nitrobenzodiazepines, nitrophenyl-pyridines,
olanzapine, N-oxide metabolites, S-oxide metabolites, paracetamole,
peroxides, phenelzine, rifampicin, sirolimus, thalidomide, thiopental,
zopiclone. |
Quinols such as
4-hydroxypranolol and some sulfur-containing compounds such as olanzapine, on
the other hand, are readily oxidized and stabilization b addition of a reducing
agent such as ascorbate or sodium metabisulfite is necessary, but in the case
of olanzapine, for example, there is the possibility of reducing the N-oxide by
such an addition. Hydrolyzed by plasma esterase’s, including cholinesterase. If
physostigmine or cocaine is to be measured accurately, the blood should be
drawn into tubes containing an excess of neostigmine. Storage at -20 °C or
below is recommended if the analysis cannot be performed immediately and if the
stability of the analyte is unknown. However, even this may not be ideal
because N-and S-oxyde may be reduced to the parent compounds.
Quinols such as
4-hydroxypranolol and some sulfur-containing compounds such as olanzapine, on
the other hand, are readily oxidized and stabilization b addition of a reducing
agent such as ascorbate or sodium metabisulfite is necessary, but in the case
of olanzapine, for example, there is the possibility of reducing the N-oxide by
such an addition. Storage at -5 to -70 °C should b accompanied by basic
precautions to accompanied by basic precautions to preserve sample integrity
(see Box 2). The requirements of the local ethics committee and other guidance
on the retention and storage of clinical samples must be complied with.
Box
2. Guidance on
freezer storage of samples. Do not freeze whole blood if plasma or serum is
not be analyzed. Ensure that labelling is waterproof. Ensure tubes are tightly sealed
and well filled, but do not overfill tubes, especially glass tubes. Do not keep
too long to minimize freeze-drying effects. Keep a record of freezer contents. Keep
a continuous record of freezer temperature. Fit an alarm in case of freezer
failure.
Plasticizers,
particularly phthalates, may originate from plastic bags used to store
transfusion blood, infusion tubing, and
from soft plastic closures for blood tulles. Such compounds are often retained
on reversed phase HPLC systems and show good absorption at 254 nm and below.
Polyvinylchloride (PVC), for example, can contain up to (40%, w/w) di-2-ethylhexyl
phthalate and compound of up to about 0,5 gl-1 have been reported
after storage of plasma in PVC bags for 14 days [1]. A further consideration is
that postmortem specimens may contain putrefactive bases such as
phenylethylamines and indole, which may interfere in the analysis of
amfetamines and other stimulants. Hexenal may arise from breakdown of fatty acids.
Gas chromatography retention data and mass spectra of a number of plasticizers,
pollutants, and other substances that may be encountered in toxicological
analyses have been reported [2]. Modern blood-collection tubes may contain a
range of additives including surfactants, which may interfere in immunoassays,
for example [4].
Drug may arise from unexpected
sources including food and over-the-counted (OTC) medicines. Quinine may
originate from ingestion of domestic water, from example, caffeine from caffeinated
beverages (tea, coffee, cola) and some proprietary stimulants, chloraquine and
related compounds from malaria prophylaxis, and pholcodine and other opiate
analogues from cough and cold cures. Morphine is a constituent of some antidiarrheal
preparations. A caffeine metabolite, paraxanthine, may be a problem in the PLC
theophylline assays [3]. Lidocaine-contaiing gel is commonly is commonly used
as a lubricant during procedures such as bladder catheterization or
bronchoscopy, and measurable plasma concentrations of lidocaine and some metabolites
may be attained.
The alkaloids emetine
and cephalline, and their metabolites, have been detected in stomach contents,
plasma, and in urine other syrup of ipecacuanna (ipecac) was given to induce
vomiting, especially in children, although this form of treatment is no
longer recommended practice. Sedative (CT) seams, lumbar puncture, or other
investigations. Neuromuscular blocking agents such as atracurium, which is
metabolized to laudanosine, and vecuronium, may be encountered in samples from
patiens undergoing mechanical ventilation. 1.3-Propanediol (propylene glycol)
is used us a vehicle in i. v. infusions. Benzoic acid, which is metabolized to
hippuric acid, is used as a preservative in some drugs and foods. The
antibiotic metronidazole is often encountered in samples from hospitalized
patients. Iodinated hippuric acids are used as X-ray contrast media.
Alcohols may originate
from skin cleansing swabs. Such compounds and drugs given in emergencies,
anticonvulsants, for example, may not be recorded on record sheets. Some
compounds or their metabolites may have very long plasma half-lives. Chlorpromazine
metabolites, for example, have been reported in urine many months after
stopping therapy. Contamination with trace elements is a particularly difficult
area. Contamination with volatiles, such as solvents used in the laboratory,
must be guarded against if one of the solvents in question is to be tested for
in a biological or related sample. Glassware and other items must be kept clean
and tested regularly for contamination uva internal quality control.
SUMMARY. Although not in
the immediate control of the laboratory, every effort must be made to ensure
appropriate priority is given to sample collection and handling because if this
us not teepee properly all subsequent effort us
wasted. Care in sample collection is especially important in postmortem and
overt medico legal work, but even in clinical work effort in providing advance
information to clinicians and pathologists on sample requirements (side of
collection, volume, addition of sodium fluoride, etc.) and feedback on the
problems that will arise when mistakes are made can only prove beneficial.
REFERENCES
1. Dine T., Luyckx M., Cazin M., Brunet C., Cazin I. C, Goudaliez F. Rapid
determination by high performance liquid chromatography of di-2-ethylhexyl
phthalate in plasma stored in plastic bags // Biomed. Chromatogr., 1991. - Vol.
5. - P. 94 - 97.
2. Pfleger K., Maurer H. H, Weller A. Mass Spectral and GC Data of Drugs,
Pollutants. Pesticides and their Metabolites, 2007, 3rd edn,
Wiley-VCH, Weinheim.
3. Stankovic A. K., Parmar G. Assay interferences from blood collection
tulles: a Cautionary note. ClinChem., 2006. - Vol. 52. - P. 1627 - 1628.
4. Rowe D. I., Watson I. D., Williams I., Berry D. J. The clinical use and
measurement of theophylline Ann. Clin. Biochem., 1988. - Vol. 25. - P. 4 - 26.