Chapter three:

CLINICAL TOXICOLOGY
LABORATORY
Learning Objectives
At the end of this chapter the student will able to:
 Mention the basic necessary information for clinical toxicology
laboratory
 Explain the role of clinical toxicology laboratory
 Describe steps in undertaking analytic toxicological investigations
 Discuss about collection, transportation, storage, characteristics,
physical examination &analytical tests of laboratory specimens.
 Describe about apparatus, reference compounds & reagents used in
clinical toxicology laboratory
 Discuss the routine laboratory tests carried out in clinical laboratory
tests.
Outline

 Introduction
 The role of clinical toxicology laboratory
 Basic information necessary for toxicology Laboratory
 Steps in undertaking an analytical toxicological
investigation
 Laboratory specimens
 General laboratory tests in clinical toxicology
Introduction

Clinical toxicology
 The detection & Tx. of poisonings caused by,
 Household & industrial products,
 Animal poisons & venoms,
 Environmental agents,
 Pharmaceuticals, and
 Illegal drugs
Introduction…
Analytical toxicology
 The qualitative or quantitative determination of drugs &
other foreign compounds (xenobiotics) & their metabolites
in biological & related specimens

 Objective evidence of the nature & magnitude of

exposure to a particular compound or group of compounds
can be obtained
Introduction…

 Due to the complexity of the Dx. & Tx. of poisoning,

toxicology test results, if available at the appropriate time,
 Can influence the Dx. & subsequent Tx. of the
poisoned patient

 The goal of clinical toxicology is

 to provide clinically useful toxicology test results to
support the needs of the poisoned patients
Introduction…

 Toxicology laboratory must provide appropriate testing

in 3 general areas:
Þ Identification of agents responsible for acute or
chronic poisoning;
Þ Detection of drugs of abuse; &
Þ Therapeutic drug monitoring

 A good understanding of clinical chemistry,

pharmacology & pharmacokinetics is desirable.
Introduction…

It impossible to look for all potential drugs or toxins that

may be present in a sample.

 In most cases in clinical or hospital-based settings,

 tests are done for only a finite number of compounds;
generally the more common drugs of abuse.

 Ideally, a diagnosis of poisoning would be made clinically,

with the laboratory playing a confirmatory role.
The role of clinical toxicology
laboratory
 Most poisoned p/ts can be treated successfully without any
contribution from the laboratory other than routine clinical
chemistry & hematology tests

 Toxicological analyses can play a useful role, if

 The diagnosis of poisoning is in doubt,

 The administration of certain antidotes or protective agents

is contemplated,
 The use of active elimination therapy is being considered.
 Drug monitoring
Basic information necessary for
toxicology laboratory

 In order to maximize the value of toxicology lab. results

 Close communication b/n the clinicians & the
laboratory is essential
 At a minimum, the ordering requisition for a toxicology
test should contain:
A. Suspected agent(s)
B. Suspected dose
C. Time of ingestion and sampling
D. Clinical presentation
E. Location of the victim
Date/time of admission
Date/time of ingestion or exposure

Drugs prescribed or used in treatment

Drugs/poisons claimed or suspected

Clinical details/investigation required/priority

Steps in undertaking an analytical
toxicological investigation
 Analysis dealings with a case of poisoning are divided into:
Pre-analytical phase
 Obtain as much information about the victim as possible
 Medical, social & occupational history, Tx given, & the
results of lab. or other investigations.
 Procedures must be in place
 Sample collection & transport; Receipt & storage of
biological samples in the lab., &
 For arranging the priority for the analysis
Steps in undertaking an analytical
toxicological investigation….

Analytical phase
 Validated procedures must be used
 to perform the requested or appropriate analyses
 to the required degree of accuracy & reliability in an
appropriate, clinically relevant time-scale

Post-analytical phase
 Report & interpret the results, & discuss them with the clinician
 Perform additional analyses, if indicated, on the original
samples or on further samples from the victim.
 Store residues of samples appropriately
Laboratory specimens

 The most critical component for a successful

toxicological analysis is selection of appropriate
specimen

 No single specimen type is universally appropriate for

identification of toxic agents.

 The role of the clinical laboratory is to recommend

 the most appropriate samples to be collected & tested
for specific investigations
Laboratory specimens…

 The selection of specimen type is based on both the

 Toxicokinetics of the suspected agent &

 Laboratory methodology
 In general,
 Serum or whole blood – quantitative tests, &
 Urine & gastric contents – qualitative tests
 For the broadest possible screening (especially in
emergency toxicology),
 Minimally, blood & urine should be sent
Specimen collection
Blood
 Plasma/serum for quantitative assays, but for some poisons,
such as CO & cyanide whole blood has to be used for
qualitative tests.
 10 ml into heparinized tube; 2ml in a fluoride/oxalate tube;
& 10-ml in to plain tube
 Avoid the use of disinfectant swabs containing alcohols
(ethanol, propan-2-ol)
 In general, there are no significant d/c in the concentrations
of poisons b/n plasma & serum
Specimen collection…

Urine
 Is useful for screening tests as
 It is often available in large volumes &
 Contains higher conc. of drugs or other poisons than blood
& can be detectable for days
 50-100 ml (adult),
 In a sealed, sterile, plastic container
 No preservative should be added.

 In acid washed, metal free container for quantification of

heavy metals
Estimated Detection Periods for Selected Drugs in Blood and
Urine
Specimen collection…

Stomach contents
 May include vomit, gastric aspirate & stomach washings
 At least 20 ml is collected in plastic container;
 No preservative should be added.

 If obtained soon after ingestion,

 Large amounts of poison may be present
 Immediate clue to certain compounds may be obtained by
the Chx odor & smell;
 It may be possible to identify tablets or capsules simply by
inspection
Specimen collection…

Scene residues (non-biological)

 Suspected materials found with or near the victim (scene
residues, including foods, drinks & containers)
 are retained for analysis, if necessary; since they may be
related to the poisoning episode.

 A few mgs of scene residues are usually sufficient for the tests
 Dissolve solid material in water or other appropriate
solvent
Specimen transport and storage
 Specimens sent for analysis must be clearly labeled with the
 Victim's full name,
 Date & time of collection, &
 Nature of the specimen (if not self-evident)

 Record date & time of receipt of all specimens in the lab., &
assign a unique identifying No.
 Avoid the possibility of cross-contamination.
 All biological specimens should be
 stored at 4°C prior to analysis & post
 kept at -20°C in case for further analyses
Physical examination of the
specimen
Urine
 High concs. of some drugs or metabolites can impart Chx
colors to urine.
 Urine color may also provide a diagnostic clue

Color Possible cause

Orange to red-orange Phenazopyridine, rifampin, deferoxamine,
mercury, or chronic lead
Pink Ampicillin or cephalosporins
Brown Chloroquine or carbon tetrachloride
Greenish-blue Copper sulfate or methylene blue
Physical examination …

 Treatments given for poisoning may color urine

 Deferoxamine (in iron poisoning) – red
 Methylene blue (in nitrate poisoning) – blue

 Colorless crystals of Ca+2 oxalate may form at neutral pH

after ingestion of ethylene glycol

Fig: Calcium oxalate crystals, as found in urine by

microscopy in ethylene glycol poisoning
Physical examination …

Stomach contents and scene residues

 Some Chx. smells can be associated with particular poisons

Smell Possible cause

Bitter almonds Cyanide
Fruity Alcohols, esters
Garlic Organophosphate insecticides, arsenic
Gasoline Petroleum distillates
Phenolic Disinfectants, phenols
Rotten-egg Sulfur dioxide & hydrogen sulfide
Sweet Chloroform & other halogenated hydrocarbons
Physical examination …

 A green/blue color –
 suggests the presence of iron or copper salts

 Very low/very high pH –

 may indicate ingestion of acid/alkali

 Microscopic examination using a polarizing

microscope
 may reveal the presence of tablet or capsule debris
General laboratory tests in
clinical toxicology
 Since the immediate care of poisoned patients is largely
supportive:
 The routine use of drug/toxin screens in the acutely
poisoned patient is rarely beneficial

 Simple, readily available biochemical & hematological

tests are carried out initially & are usually of more valuable
 In the diagnosis of acute poisoning &
 To monitor the patient‘s clinical progress
Biochemical tests

Blood glucose (BG)

 Is essential to know those toxic substances that affect blood
glucose biotransformation
 Hypoglycemia –
 Insulin, Iron salts, acetyl salicylic acid etc

 Hyperglycemia –
 less common complication of poisoning than hypoglycemia
 has been reported after over dosage with acetylsalicylic
acid, salbutamol & theophylline.
 Biochemical tests…

Electrolytes, blood gases and pH

 Toxic substances or their metabolites, w/h inhibit key steps
in intermediary biotransformation,
 cause metabolic acidosis owing to the accumulation of
organic acids, notably lactate.
 Ms/t of serum or plasma Anion Gap (AG) can be
helpful.

 Direct ms/t of O2 saturation allows detection of

methemoglobin resulting from
 intoxication with various oxidizing drugs
Biochemical tests…

 In metabolic acidosis – resulting from severe poisoning

with CO, cyanide, ethylene glycol (glycolic acid),
methanol (formic acid), fluoroacetates, paraldehyde or
acetylsalicylic acid
 the AG typically exceeds 17 mmol/l

 Toxic metabolic acidosis

 also occur in severe poisoning with iron salts, ethanol
(acetic acid), paracetamol, isoniazid, phenformin &
theophylline
Biochemical tests…

Serum osmolality
 Normal plasma osmolality (280-295mOsm/L) is largely
accounted by Na+, urea & glucose
 Large es in plasma osmolality may follow the absorption
of osmotically active poisons in relatively large amounts
 Methanol, ethanol, or propan-2-ol

 Together with the standard chemistry panel, serum

osmolality allows identification of an osmolal gap,
 may indicate intoxication with ethanol or other alcohols.
Biochemical tests…

Table. Effect of some common poisons on plasma osmolality

Compound Analyte concentration (g/I)
corresponding to 1 mOsm/kg increase
in plasma osmolality
Acetone 0.055
Ethanol 0.046
Ethylene glycol 0.050
Methanol 0.029
Propan-2-ol 0.059
Biochemical tests…

Plasma enzymes
 Plasma activities of liver enzymes (AST, ALT) may
increase rapidly after absorption of toxic doses of
substances that can cause liver necrosis,
 Paracetamol, Carbon tetrachloride, & Copper salts.

 Increased plasma GGT activity –

 in Chronic ethanol abuse
Biochemical tests…
Cholinesterase activity
Acetylcholinesterase
Acetylcholine Choline + Acetic acid
 Plasma cholinesterase is a useful indicator of exposure to
organophosphorus & carbamate pesticides, & nerve agents
 A normal plasma activity, effectively excludes acute
poisoning by these compounds.
 Depression of AchE can persist for 2-6 wks post-exposure
 Plasma cholinesterase can also be decreased in other
circumstances (e.g. if liver function is impaired).
Hematological tests

Hematocrit (Erythrocyte volume fraction)

 Acute or acute-on-chronic over dosage with – iron salts,
acetylsalicylic acid, indomethacin, & other non-
steroidal antiinflammatory drugs
 may cause GI bleeding, leading to anemia

 Anaemia may also result from chronic exposure to toxins

that interfere with haem synthesis, such as lead
Hematological tests…

Blood clotting
 The prothrombin time & other measures of blood clotting
are likely to be abnormal in :
 Poisoning with metabolic toxins, such as paracetamol
 Acute poisoning with rodenticides, such as coumarin
anticoagulants, &
 After overdosage with heparin or other anticoagulants
Hematological tests…

Carboxyhaemoglobin & Methaemoglobin

 Ms/t of blood carboxy-Hgb can be used to assess the
severity of acute CO & chronic dichloromethane
poisoning.

 Met-Hgb may be formed after over-dosage with

 Dapsone & oxidizing agents (chlorates or nitrites), &
 Aromatic nitro compounds (nitrobenzene & some of its
derivatives).
 May be indicated by– dark chocolate-coloured blood
Exercise

1. What is the basic information necessary for clinical toxicology

laboratory?
2. What are the roles of clinical toxicology laboratory?
3. Mention the steps that are necessary to undertake analytic
toxicological investigations.
4. Describe specimen collection, transportation, storage, characteristics
& physical examination used in clinical toxicology laboratory.
5. Describe apparatus, reference compounds & reagents used in clinical
toxicology laboratory.
6. Describe the routine laboratory tests used in clinical toxicology
laboratory.
References

 Flanagan RJ et al. FUNDAMENTALS OF ANALYTICAL

TOXICOLOGY, John Wiley & Sons 2007

 Flanagan RJ. Developing Analytical Toxicology Services:

Principles and Guidance. WHO, 2005