PREANALYTICAL ERRORS

By Dr Sulakshmi B Kurlekar
INTRODUCTION

• Historical Perspective
• Laboratory and Diagnostic Errors
• Brain to Brain Loop– The Lundberg Concept
• The Total Testing Process
 • Introduction
 Diagnostics in the clinical chemistry laboratory is a pivotal part of clinical
decision-making but is not exempt from ‘human errors’.
 Scientific innovations such as automation and electronic order test requesting
have contributed to substantial improvements in the field of laboratory
science, but errors still occur.
 One major example of such failing is connected to the prevalence of errors
occurring in pre-analytical phase of the Total Testing Process(TTP).
 Pre- analytical errors can occur at the time of patient assessment, test order
entry, patient identification, sample collection, sample transport, or sample
receipt in the laboratory. Previous work and clinical insights suggest that
most errors in the TTP are extra-laboratory (i.e. they occur before the
samples reach the laboratory for analysis). Such errors are frequently the
results of human mistakes during phlebotomy practice. Therefore to reduce
these errors the pre-analytical phase of the TTP must be prioritized.
 Researchers observed that about two-thirds of errors in the
clinical chemistry laboratory occur in the pre-analytical phase,
when compared to the analytical or post- analytical phase. This is
plausible because the pre-analytical stage requires more human
intervention when compared to the analytical and post-analytical
phases of the TTP.
 Significant advances in automation; robotics; laboratory
information management systems (LIMS) and precision engineering
have remarkably simplified many laborious and cumbersome
procedures in the clinical chemistry laboratory. Therefore,
analytical errors are no longer the main factors influencing the
reliability of clinical application of laboratory diagnostics.
 Historical perspectives

In the 1970s a new term “pre-analytical phase” was introduced to the laboratory
medicine lexicon (Statland and Winkel, 1977). In the 1980s terminologies such as
interfering factors were introduced to professional training programmes,
culminating in the publishing of the first book on pre-analytical variables in 1987
(Einer and Zawta, 1987); and these have become part of the terminologies used in
clinical laboratory sciences vocabulary (Dybkaer, 1997) and international guidelines
(CLSI, 2004).
In 1981, the Clinical Science Laboratory Institute (CSLI) introduced pre- analytical
standards to examine pre-analytical procedures, and the European Committee on
Clinical Laboratory standards (ECCLS) followed this movement. The actions of
these bodies meant that the term ‘pre-analytical phase’ was included in training
and teaching programmes in clinical chemistry and laboratory medicine (Guder
and Wahlefeld, 1983; Hagemann, 2005; ISO, 2007).
 Historical examples of pre-analytical errors may help us understand
how ‘seemingly normal’ extra-laboratory routines may impact on
the analytical phase and lead to spurious results. For example in the
early 1960s, clinicians requested urinary amylase measurements in
order to exclude pancreatitis as the cause of acute abdominal pain.
Surprisingly most of the results showed increased amylase activity
even though some of the patients showed no signs of
‘pancreatitis’. It was soon discovered that because the urine
samples were collected in open vessels, they apparently became
contaminated by salivary amylase from drops of spittle from the
nursing staff as they held discussions over the urine samples when
transporting them to the laboratory for analysis (Guder, 2014). The
cause of the pre-analytical error was eventually eliminated by
collecting the samples in closed containers (Guder, 2014). Fluid or
blood amylase samples have now increasingly become the samples
of choice for pancreatic amylase activity investigation. 
 •Laboratory and diagnostic errors
 A laboratory error is defined as any defect, from test ordering to reporting test
results and appropriately interpreting and reacting on these results.

 Conventionally medical errors are grouped into 4 types namely: errors of diagnosis,
errors of treatment, errors of prevention and errors of miscellaneous origin. Errors
in the TTP are associated with all of the four error groupings mentioned, albeit most
medical errors are closely linked to ‘errors of diagnosis’.

 According to recent data, diagnostic errors rank as the most common source, most
costly and most precarious of medical mistakes for inpatients as well as patients for
attending outpatient clinics or departments. Despite the ubiquitous nature of
diagnostic errors that often lead to avoidable disability or death in some cases,
diagnostic errors still remain a relatively unmeasured subject area of patient safety.
Medical errors can result in physical and emotional suffering for the patient and
frequently lead to a number of mortalities annually, along with excess economic
burden and litigations.
 Medical errors that put patients at risk have generated a lot of
media attention in recent years, so much so that publications
such as that of the Institute of Medicine (IOM), estimate that
preventable errors leading to about 1.5 million adverse events
occur in the United States annually and that between 44,000 to
98,000 deaths occur annually as a result of medical errors,
excluding unreported events. About 55% of missed or delayed
diagnoses in the ambulatory setting and about 58% in accident and
emergency departments arise from failure to request appropriate
diagnostic and laboratory tests in the pre-analytical phase of the
TTP.
 • Brain-to-brain loop: The Lundberg concept

 It was Lundberg (1981, 1999), who first proposed the brain-to brain loop concept
(Figure 1.1) to describe the total testing cycle (Figure 1.2). According to
Lundberg, the activity loop begins outside the walls of the clinical chemistry
laboratory with a clinical question in the physician’s mind. This leads to test
requesting or ordering, collection of the patient’s sample and identification,
transportation of sample to the laboratory, sample separation (by centrifugation
to make it suitable for analysis), sample aliquoting and sorting into batches.
Next, the sample is delivered for analysis on automated platforms. In the final
step results are generated and reported, and action taken (interpretation and
decision making) by the physician. Each stage in this complex loop, when
performed correctly guarantees quality procedures in the clinical chemistry
laboratory, thus ensuring valuable medical decision making and effective
patient management.
It can be appreciated that several transitional steps are involved in
the loop, some of which are pre-analytic (before laboratory testing
of samples); some are analytic (relating to the actual laboratory
testing of samples) and a post-analytic step that involve the
transmission and interpretation of test results into the health
records. The introduction of the brain-to-brain model resulted in an
identification and classification of errors related to laboratory
testing. Previous studies (Plebani and Carraro, 1997; Carraro and
Plebani, 2007; Plebani, 2006, 2007, 2009, 2011) suggested that
most errors in the ‘Lundberg loop’ do not occur within the analytical
stage, nor do they frequently fall within the ‘pre-analytical’ or ‘post-
analytical’ stages under the control of biomedical scientists.
Figure 1.1: Brain-to-Brain Loop Concept for Laboratory Testing.
Adapted from Plebani et. al., (2011). Am J Clin Pathol 2011;
136:829-833.
 Sepulveda (2013) summarized the Lundberg loop in seven steps shown by the coloured
numbered boxes: 
1. The right question was asked from the patient by the clinician or physician 
2. The right test was ordered by the physician 
3. The right sample was collected on the right patient, at the correct time, with appropriate
patient preparation. 
4. The right technique was used collecting the sample to avoid contamination with
intravenous fluids, tissue damage, prolonged venous stasis, or hemolysis.
5. The sample was properly transported to the laboratory, stored at the right temperature,
processed for analysis, and analysed in a manner that avoids artifactual changes in the
measured analyte levels. 
6. The analytical assay measured the concentration of the analyte corresponding to its
“true” level (compared to a “gold standard” measurement) within a clinically acceptable
margin of error (the total acceptable analytical error (TAAE). 
7. The report reaching the clinician contained the right result, together with interpretative
information, such as a reference range and other comments, aiding clinicians in the
decision-making process.
 
 It has been established that most of the errors occur before
and after laboratory analysis (Plebani, 2010), and that the pre-
and the post analytical phase are responsible for up to 96% of
total Turn around Time (TAT) anomalies (Manor, 1999;
Rodriquez- Borja et al., 2014 Rodriquez-Borja, 2015).
Therefore incorrect interpretation of laboratory or diagnostic
tests in the final stages of the brain-to-brain loop (B-T-B) also
causes a large proportion of errors in the ambulatory and
emergency settings (Kachalia et al., 2007).
 
• The total testing process (TTP)

 The total testing process (TTP) is the sequence of events starting with the
ordering of a test by a clinician and ending with the interpretation of the test
result by the clinician (Figure 1.2). The TTP concept is similar to the previously
described Lundberg’s loop, the difference being that it occurs in phases. To fully
appreciate the processes involved in generating a result for a patient’s sample in
a clinical chemistry laboratory it is imperative to elucidate the three phases of
the TTP: Any analytical process in the clinical chemistry laboratory involves
these three major stages:
1. The pre-analytical stage, which involves patient specimen acquisition and
preparation.
2. The analytical stage, which is the measurement of the test analyte.
3. The post-analytical step, which consists of reporting results.
 Figure 1.2. Total Testing Process (TTP): Adapted from Boone (2007): Presentation at the Institute on
Critical Issues in Health Laboratory Practice: Managing For Better Health, September 23–26, 2007
Atlanta, GA, USA Centers for Disease Control and Prevention.
 Laboratory testing in clinical chemistry is a significant source of
medical errors affecting patient management and safety. Errors
generated in the pre-analytical phase decisively influence the total
error and consequently the diagnostic accuracy. The pre-analytical
phase is probably the most important step in the TTP in the clinical
chemistry laboratory and indeed in laboratory medicine, if total
laboratory quality is to be achieved. This assertion is true because
the pre-analytical phase is conceivably the most complex and highly
vulnerable to uncertainties (such as biological variations) and
untoward incidents. Recent studies carried out by a number of
investigators have shown that approximately 93% of errors
encountered within the TTP is due to the lack of standardised
operating protocols for sample collection, which includes patient
preparation, phlebotomy, sample handling/transportation and
storage. 
 Errors relating to pre-analytical stages of the TTP are arduous to
control and therefore require the adoption of drastic but suitable
approaches for the prevention (or reduction) of these errors. A
number of suitable strategies for error prevention have been
examined and streamlined into the TTP. The major challenge for
biomedical scientists and other laboratory staff has been the absence of
an effective communication system with which to engage with the end-
users of the clinical chemistry laboratory service (i.e. physicians, nurses,
consultants, phlebotomists and other healthcare professionals). A
good communication system is essential for prevention or reduction
of pre-analytical errors.
 Currently there has been increased focus on the reduction of pre-
analytical errors (by healthcare professionals working in the clinical
chemistry laboratory) and the possible impact on patient
management by development of quality improvement ‘tools’ such as
 1 .THE PRE-ANALYTICAL PHASE

This phase involves patient assessment, test order entry, request

completion, patient identification, specimen collection, specimen
transport, and sample receipt in the laboratory. Errors can occur at any
of these stages. Bonini et al., (2002) in a study in Italy, observed that
pre-analytical errors were widespread in the laboratory, ranging from
31.6% to 75%. In a separate study, Khoury et al., (1996) reported error
rates of 39% for ‘identification’ transcription in five clinical chemistry
laboratories in Australia. A transcription error rate of this magnitude
has the likelihood of seriously compromising patient identification data.
Several studies have recorded their findings under the following
categories of pre-analytical variables
 Incorrect patient identification 
 Improper sample labelling
 Improper test request
 Incorrect timing of sample
 Insufficient sample volume
 In-vitro haemolysis 
 Collecting sample into wrong tube or container
 Incorrect sample handling and transport
The most commonly reported types of errors from these previous studies, in the
pre-analytical phase are: a) missing sample and/or test request; b) wrong or
missing patient identification; c) contamination from intravenous infusion
route; d) hemolysed, clotted, and insufficient samples; e) inappropriate
containers; f) inappropriate ratio of blood to anticoagulant, and g)
inappropriate transport and storage conditions.
 Although the studies discussed in the preceding paragraph
describe alarming statistics relating to the category and
frequency of errors in the pre-analytical phase, the precise
magnitude of the error rate in the laboratory is still debatable and
difficult to estimate simply because ‘error’ has no ‘definite and
universally accepted definition’. A primary limitation identified in
some of these studies is the use of small sample size numbers .
Other issues relate to the acknowledgement by some authors
that some ‘errors’ may have gone unobserved. Reduction of
errors in the pre-analytical phase can be achieved by consciously
taking specific rigorous pre-emptive steps that are desirable for
good laboratory practice (GLP).
 2.THE ANALYTICAL PHASE

The analytical phase begins when the prepared patient sample is

delivered to the analytical platforms for testing, and it ends when the test
result is interpreted and verified by the biomedical scientist in the
laboratory. Validating assay methodology, performance specifications such
as sensitivity, specificity, accuracy, precision and linearity are crucial steps
along the continuum which (if ignored) can lead to irreversible errors in
the analytical phase of laboratory analysis in clinical chemistry. During the
analytical phase, it is particularly important to design the manual work
procedures such that the risk of placing patient samples in an incorrectly
labelled test receptacle is minimised. A valid result provided for the
wrong patient can have dire consequences including death. Many
resources have been harnessed to improve analytical quality in the
clinical chemistry laboratory by establishing and investing in internal
quality controls (IQC) and external quality assessment (EQA) schemes. 
The role of EQA and proficiency testing (PT) is to provide reliable information
allowing laboratories to assess and monitor the quality status of internal
procedures and processes, the suitability of the diagnostic systems, the
accountability and competence of the staff, along with the definition of
measurement of uncertainty (discussed in section in laboratory results.
Biomedical scientists working in the clinical chemistry laboratory are directly
responsible for appropriately analysing EQA materials. They report results,
identify trends or bias that may not be apparent in analytical runs, investigate
root causes producing unacceptable performances, apply and monitor
appropriate actions for eliminating the underlying causes, authenticate the
effectiveness, and, retrospectively determine whether the problem affected
the clinical decision. Potential sources of errors in the analytical phase may be
broken equipment, improper mixing of samples or reagents, interferences
(endogenous or exogenous), not detecting errors during equipment
calibration and quality control.
 3.The Post-analytical phase
 This phase involves validation of the patient test result and then transmission of the results to
the clinicians and other healthcare professionals, in order to arrive at a clinical decision to
manage their patients. Test turnaround time (TAT), an important aspect of the quality of the
testing process, relies heavily on both the pre-analytic and analytic phases, to the point that
any delays in these phases can create potential problems that adversely affect patient care. A
particularly important patient safety risk is the reporting of ‘critical limits’ defined as values
that represent situations that could be life threatening without treatment (Lundberg, 1981).
Result reporting is subject to specific communication and interpretation errors, which may
originate in the pre- analytic and analytic phases. Potential sources of errors in the post-
analytical phase are: incorrect validation of results, errors in reporting and delivering reports
and prolonged TAT. Incorrect validation and incorrect reporting may lead to incorrect
interpretation by the requesting physician. This may lead to wrong diagnosis or treatment and
may cause harm to the patient. Biomedical scientists must be aware of the importance of
relevant clinical information when validating and authorising results, especially when
cumulative records are available. An unexpected test result can highlight the possibility of an
incorrectly labelled sample or request form and should be investigated immediately.
 SOURCES OF UNCERTAINTY IN THE PRE-ANALYTICAL STAGE
 
 In clinical chemistry measurements, the uncertainty in patient results includes both pre-
analytical and analytical variation, as well as intra-individual biological variation.
 The assessment of uncertainty in the pre-analytical stages in sample preparation is often
essentially overlooked. Pre-analytical factors are diverse, ranging from biological and
physiological events to technical details of specimen collection and transport (ISO, 2012). When
standardized procedures are followed, pre-analytical variation can be minimized and the
number of errors decreased. Traditionally, laboratories have focused on the uncertainty in
the analytical phase, but characterization of uncertainty should include the whole process
from phlebotomy up to reporting of results. With all uncertainties quantified and presented
together in tabular form as an uncertainty budget, the laboratory will have a tool to identify
important uncertainty sources. The combined uncertainty is a function of the magnitude and
probability distribution of the different uncertainty sources and the number of such sources.
The uncertainty can be reduced, and laboratory quality improved, by focusing on the
sources that contribute most to the combined uncertainty.
COMMON ERRORS GENERATED IN PRE-ANALYTICAL PHASE OF TTP
 Inappropriate laboratory test requisition
The misappropriation of laboratory services through requesting inappropriate
laboratory tests requests impacts, on total costs, and the inherent increased risk of
diagnostic errors and injury. The estimations of inappropriate laboratory tests vary
from 11% to 70% for general biochemistry and 17.4% to 55% for cardiac enzymes and
thyroid tests.
 Incomplete laboratory test requisitions
One important source of pre-analytical error is incorrect or incomplete information
on the test request forms or labels which have been found in more than two thirds of
all rejected samples in the laboratory. Several other studies confirm that test
requests can be a clinically important source of errors. Paper - based test requests are
precarious as they can be incompletely filled, placed in the wrong collection box, or
simply be lost. Incomplete laboratory requests forms are rarely rejected at the service
point. In many instances, the phlebotomy section or reception staff in the laboratory
may not know the significance of the missing data. Specific missing information
included the physician’s name, misidentification of patient and requested tests.
 Wrong patient identification 

Patient identification is the cornerstone of patient safety. Correct patient

identification is the vital task in all laboratory medicine. Therefore, efforts to
ensure compliance with standardized identification routines should be prioritized.
Errors in patient identification before specimen collection is responsible for up to
25% of all pre-analytical errors. Critical patient identification errors occur in
approximately 1 out of 1200 tests requested. Mistakes in patient identification
often occur during manual tasks, which can be avoided, using electronic
technologies like barcodes, radiofrequency identification and wristbands.
Wristbands have patient’s name and identification number, and sometimes have
a barcode. A previous study by Howanitz et al.,(2002) has reported error rates of
3.05 – 7.40% while examining 1757,730 wristbands over 2 years, for identification
wristbands mostly comprising of missing or incomplete wristbands, and wrong
wristband on the patient.
  Wrong labelling of the sample containers

Labelling of specimen containers should always be done immediately

after sample collection while, labelling them before sample
collection increases the risk of the specimen collection from the
wrong patient. Mislabelling is responsible for 50% of all
identification errors. Information from SHOT (Serious Hazards Of
Transfusion) shows that mislabelled specimens are 40 times more
likely to contain the wrong patient’s sample (IBMS, 2014).
Ambiguous or erroneous identification of patients presents a risk
to patient's health and can result in misdiagnosis, mistreatment,
ill-health or mortality.
 Poor phlebotomy practice

Proper sample collection (phlebotomy) is an important part of good

laboratory practice and improper collection can lead to delays in
reporting, unnecessary re-draws and re- tests, decreased end-user
confidence, increased costs, incorrect diagnosis / treatment, injury and
occasionally loss of life. Studies have shown the importance of
checking for specimen adequacy as a critical factor in test result
accuracy and usefulness (Lippi et al., 2006). Samples that are
missing, coagulated, hemolysed, insufficient or inadequate volumes
due to inappropriate specimen collection and handling account for a
large percentage of pre-analytical mistakes.
Improper phlebotomy practices are the bane of the clinical chemistry
laboratory and are one of the main causes of pre-analytical errors,
which occur due to lack of knowledge or heavy workload/ tiredness.
This has put a strain on the ability of the clinical chemistry
laboratory to provide an excellent service to the users of its
services. The procedure should always be performed by trained
/qualified phlebotomists. In the clinical laboratory, venipuncture is
described as all of the steps involved in obtaining an appropriate and
identified blood sample from the vein. According to recommended
practice (CSLI, 2007, 2008), the phlebotomist should ensure that the
patient is comfortable and if appropriate should verify whether the
patient is fasting (for at least 12 hours or overnight as necessary for
some chemistry analytes such as glucose and lipids); what
medications are being taken or have been discontinued, as required.
 Inadequate/insufficient sample volume

 Insufficient sample volume is a major factor leading to rejection of

samples. The main reason for this anomaly is the lack of knowledge of the
phlebotomist, difficult sampling procedures relating to pediatric patients,
debilitated cases, those on chemotherapy, and those with difficult to
localize veins. Insufficient sample constituted the most frequent cause of
test rejection.
INFLUENCING AND INTERFERENCE
FACTORS
INFLUENCING FACTORS
 Influencing factors are the effects on laboratory results of biological origin that most
commonly occur in vivo but can also be derived from the sample in vitro during
transport and storage. Biological influence factors lead to changes in the quantity of
the analyte to be measured in a defined matrix. They modify the concentration of
the measured analyte in a method-independent way.
 In the healthy individual they refer to circadian rhythm; whereas in an unhealthy
individual they appear as side effect of the disease and its treatment.
 They can be modifiable, diet
time of the day
time of the year(season)
unmodifiable such as, gender
ethnicity
genetic background and so on.
 Their effects may be reduced by standardization of preanalytical condition.
DECISION ABOUT SAMPLE TYPE

The medical needs and clinical questions usually determine the source of sample
and time of sampling.
Besides medical knowledge about the physiology of analytes intended to be
measured, criteria such as practicability of sampling and risk of patient harm may
influence the decision
Most commonly used sample type used is Venous blood, followed by capillary
blood, which , because of the lower amount of sample needed, became the
preferred sample in newborns and young children. Arterial blood on the other
hand, is mainly used for analysing blood gases and acid base state. Other sample
types are urine, saliva and other body fluids.
 CONTROLLABLE VARIABLES

 Time of sampling
 Influence of Circadian rhythm
 Menstrual cycle
 Influence of Diagnostic and Therapeutic Procedures
 Diet
 Effects of Fluid Intake Before Sampling
 Smoking Tobacco
 Body position and Tourniquet
 Muscular activity
 Preparing for Blood sampling
INFLUENCE OF CIRCADIAN RHYTHM Several analytes tend to fluctuate in terms of their
plasma concentration over the course of a day
TIMING OF COLLECTION
 For cortisol, between 7 and 9 a.m.
 Before interfering diagnostic and therapeutic procedures
 In drug monitoring: consideration of the peak after administration and the steady state phase
before the next dose.
 Documentation of the exact time of sampling is very important.

DIURNAL VARIATION
OF SELECTED ANALYTES
MENSTRUAL CYCLE

Hormonal pattern contribute to the following changes.

For example , aldosterone concentration in plasma is twice as high
before ovulation than in the follicular phase
renin can show a preovulatory increase
cholesterol shows significant decrease during ovulation
phosphate and iron decreases during menstruation
INFLUENCE OF DIAGNOSTIC AND THERAPEUTIC
PROCEDURES:
They can show both in vivo and in vitro effect on the laboratory test
 Operation
 Infusion and transfusions
 Punctures, injections, biopsies, palpations and whole body massage
 Endoscopy
 Dialysis
 Physical stress ( eg- ergometry, stress and exercise)
 Function tests ( eg- glucose tolerance test)
 Immunoscintigraphy
 Contrast media drugs
 DIET : Long-term effects of diet

 Creatinine shows a increase of upto 20% , in its plasma concentration after

ingesting a cooked meat meal (kinetic jaffe method). Protien-rich food also
affects serum urea and urate levels.
 A diet rich in fat leads to increased serum triglyceride concentration
reduced serum urate
 A diet rich in carbohydrates decreases serum protein and lipid
concentrations
 Compared to omnivorous subjects, vegetarians tend to have lower
concentrations of plasma cholesterol, tryglycerides and creatinine, with
reduced urinary excretion of creatinine and a higher urinary pH as a result of
reduced intake of precursor of acid metabolites.
 Acute effects of Diet and other Influencing Factors

 Prior to blood sampling, the confounding influences of foods and fluid intake should be
excluded.
 The activity of some enzymes ( ALP, AST, ALT) increase upto 20% following a meal.
Triglyceride and glucose concentrations increase during the absorptive phase. It is the
turbidity of the plasma/serum samples, caused by chylomicrons following absorption of
lipids which interfere with various measurement procedures and causes apparent changes
in analyte concentrations.
 To avoid any misinterpretation , it is therefore recommended that the blood sampling be
done after 12 hours of fasting and reduced activity ( bed rest).
 In response to a meal, hydrochloric acid secretion in the parietal cells of the stomach is
coupled with extraction of chloride from and release of bicarbonate into plasma in order to
maintain electrical neutrality. Thus the venous blood leaving the stomach enriched with
bicarbonates, and this phenomenon is responsible for a mild postprandial metabolic alkalosis
( i.e the alkaline tide ) with concomitant increase of pCO2 and a subsequent reduction of
ionized calcium by 0.2 mg/dL.
 Effect of Fluid Intake Before Sampling

Caffeine containing drinks such as tea, coffee and cola drinks stimulate the
adrenal cortex and medulla, leading to the subsequent
increase of the concentration of catecholamines and their metabolites
free cortisol
11-hydroxycorticoids, and
5-HIAA in serum. They inturn increase the plasma glucose concentration
Plasma renin activity may also be increased following caffeine ingestion.
As caffeine is a diuretic inhibits the reabsorption of electrolytes, thus causing
transient increase in excretion ( calcium, magnesium, sodium, chloride,
potassium)
Ingestion of coffee increases the rate of lipid catabolism, thus leading to the
increase of plasma lipids, free fatty acids, glycerol, and lipoproteins.
ALCOHOL
Acute effects occur within 2-4 hours of ethanol consumption, causing
• decrease in plasma glucose and increase of lactate
• increases hepatic formation of uric acid, inhibits renal urea excretion, thus causing
an increase of uric acid in plasma.
• acetate decreases plasma bicarbonate, resulting in mild to severe metabolic
acidosis, depending on the amount of ingested alcohol.
• increases activity of serum GGT and some other enzymes ( eg, isocitrate
dehydrogenase, ornithine carbamoyltransferase)

Chronic effects include

• increase in serum triglyceride concentration due to decreased plasma triglyceride
breakdown and
• increase in the serum activity of many enzymes( GGT, AST,ALT)
• increased urine ethanol excretion leads to a decreased formation of vasopressin with
increasing diuresis
• increased secretion of renin and aldosterone.
SMOKING TOBACCO
Smoking increases the serum concentrations of fatty acids, epinephrine, free
glycerol, aldosterone and cortisol. These changes occur within 1 hour of smoking a
cigarette.(Through adrenal gland stimulation, nicotine causes the increase of
the concentration of epinephrine in the plasma and urinary excretion of
catecholamines and their metabolites.)
Smoking leads to acute increase in the serum triglyceride, LDL, and total
cholesterol concentrations
Glucose metabolism is also dramatically affected by nicotine. Within only 10
minutes of smoking a single cigarette, glucose concentration increases by up to 10
mg/dL. This increase may persist for 1 hour.
To avoid a risk of misinterpretation of laboratory results, smoking habits should
be documented in clinical records.
For CEA, different reference limits should be applied for smokers and non smokers
due to large differences between two groups. The higher concentration found in
smokers is caused by an increased synthesis and secretion of CEA in the colon.
INTERFERENCE FACTORS
 INTERFERENCE FACTORS

They have the ability to interfere with the analytical procedure and alter the test
results. Their effect depends on the method-that is, the same interferent may not
necessarily affect two different methods used to measure the same analyte.
The most common interference factors being
Hemolysis
Lipemia
Icterus
Drugs
Paraprotiens and
Various contaminants such as gels, tube additives and fibrin clots
Interfering factors are considered clinically relevant when the
bias caused by their interference is greater than the maximum
allowable deviation of a measurement procedure
They can be endogenous and exogenous. Endogenous
interferences originate from the substances present in the
patient sample, whereas exogenous interferences relate to the
effects of various substances added to the patient sample, such as
separator gels, anticoagulants, surfactants, and so on.
 HEMOLYSIS

Hemolysis is defined as a process of membrane disruption of erythrocytes and other

blood cells, accompanied by the subsequent release of cell components into the plasma
and red coloration of the serum ( or plasma) to various degrees after centrifugation.
Hemolysis is the most common preanalytical error and the most common cause of
sample rejection. It occurs with a frequency of up to 30% and accounts for almost 60% of
unsuitable specimens.
The highest frequency if hemolysis has been observed in samples from emergency
departments, pediatric departments and intensive care units.
The two major sources of hemolysis are in vivo hemolysis and in vitro hemolysis.
In vivo hemolysis is a result of a pathological condition and occurs within the body
before the blood has been drawn. It may occur as a result of numerous biochemical
[ enzyme deficiencies, erythrocyte membrane defects, hemoglobinopathies]
Physical [ prolonged marching, drumming, prosthetic heart valves]
Chemical [ethanol, drug overdose, toxins, snake venom]
Immunological [auto- antibodies] mechanisms
Infections [babesiosis, malaria ]
In vivo hemolysis can further be categoried as intravascular and extravascular,
depending on site of the destruction of red blood cells. Intravascular hemolysis
occurs as direct and immediate disruption of red blood cells due to cell injury
within the vasculature, whereas in extravascular hemolysis, red blood cell
membranes are damaged by the reticuloendothelial system, primarily in the
spleen. The most common causes of in vivo hemolysis are reaction to
incompatible transfusion and autoimmune hemolytic anemia.
In vivo hemolysis is not very common and accounts for only 3% of all hemolyzed
samples.
Decreased concentrations of haptoglobin in serum and free haemoglobin in urine
are the most pronounced and specific laboratory signs of in vivo hemolysis.
Haptoglobin is a protein that binds free haemoglobin in circulation to prevent
oxidative damage induced by haemoglobin. Once released from the erythrocyte
into the plasma, hemoglobin forms complexes with haptoglobin, and those
complexes are removed from the circulation by macrophages.
In more pronounced cases of in vivo hemolysis, haptoglobin in
serum can be undetectable, whereas its concentration in case of in
vitro hemolysis remains unchanged. When in vivo hemolysis is
confirmed, the laboratory should not reject hemolysed sample for
analysis, because parameters in hemolysed samples reflect the
actual patient condition and are extremely relevant for adequate
patient care [diagnosis, therapy management, monitoring]
In vitro hemolysis occurs outside the patient at many steps of the
pre-analytical phase : blood sampling,
sample handling
delivery to the laboratory
sample storage.
MECHANISMS OF HEMOLYSIS INTERFERENCES

Spectrophotometric Interferences: It occurs due to the ability of haemoglobin

to absorb light at 415-, 540-, and 570- nm wavelengths. This characteristic of
haemoglobin causes optical interferences that can lead to either falsely
increased or decreased concentrations of the measured parameters.

Release of the Cell Components Into the Sample: Some components are
present in blood cells in concentrations that are several times higher than
those in the extracellular space.
RATIO BETWEEN INTRACELLULAR AND EXTRACELLULAR CONCENTRATION OF
VARIOUS PARAMETERS IN RED CELLS
ANALYTE INTRACELLULAR CONCENTRATION
( Compared to extracellular )
Lactate dehydrogenase 160
Inorganic phosphate 100
Potassium 40
Aspartate aminotransferase 40
Folic acid 30
Alanine aminotransferase 7
Magnesium 3
 SAMPLE DILUTION

Some analytes are present in much higher in much higher concentrations

in plasma than in blood cells like albumin, bilirubin, glucose, sodium, and
a few others. For those parameters hemolysis can cause a dilution
effect, and their concentrations will be lower in hemolysed samples.
The effect of sample dilution causes clinically significant bias only at
higher degree of hemolysis.

For example, glucose is negatively affected by severe hemolysis (-8.3%)

only at the concentration of 3.34 g/L of free Hb if measured by the
Beckman Coulter chemistry analyser and reagents.
 CHEMICAL INTERFERENCE

Various blood cell components may affect the analyte measurement procedure by
directly or indirectly competing for molecules in the reagents, inhibiting indicator
reactions or modifying the analyte by complexation, proteolysis, or precipitation.
One such effect is caused by the adenylate kinase, which is present in both
erythrocyte and platelets. Adenylate kinase is an enzyme that catalyzes the
reversible conversion of ATP and AMP to two ADP molecules and maintains the
adenine nucleotide cell content. When released from the cells during hemolysis,
adenylate kinase may compete for ADP with creatine kinase in a creatine kinase
assay if inhibitors are not supplied in the reaction mixture.
Hemoglobin released from erythrocytes during hemolysis may interfere with
various assays through its pseudo-peroxidase activity. Pseudo-peroxidase activity
of free haemoglobin released from erythrocytes interferes in the assay for
measurement of bilirubin concentration through the inhibition of the formation of
diazonium salt.
Hemolysis may cause a clinically significant interferences on a wide
range of analytes in immunochemistry assays. This interference is
caused by modifying the reaction analytes ( antigens and
antibodies) by the proteolytic action of cathepsin E, the major
proteolytic enzyme in mature erythrocytes. Proteolytic enzymes
released from erythrocytes may mask or potentially enhance epitope
recognition in various immunoassays. Interference caused by
proteolytic activity may cause measurement bias of various
degrees and various directions, depending on the assays.

Hemolysis has been shown to cause negative interference with

concentrations of cTnT, insulin, cortisol, testosterone and vitamin
B12, false–positive increases for PSA and cTnI in a concentration-
dependent manner.
 LIPEMIA

Lipemia is defined as a turbidity of the sample visible to the naked

eye. Turbidity of the sample is caused by the light scattering due to
the presence of large lipoprotein particles. The increase in
concentration of lipoproteins in blood most commonly occurs due
to postprandial triglyceride increase, parenteral lipid infusions, or
some lipid disorders. Not all lipoproteins have equal contribution to
the sample turbidity. The effect of lipoprotein particles on the
sample turbidity depends on the size of the particles. Chylomicrons
and VLDL lipoproteins, the largest lipoprotein particles in the
circulation, have the greatest combination to the sample turbidity.
To avoid postprandial lipemia, patients are therefore requested to
fast for 12 hours before the blood sampling.
MECHANISMS OF INTERFERENCES CAUSED BY LIPEMIA

Spectrophotometric Interference: Lipemia causes interference by light absorbance

and light scattering. The lipemic sample absorbs light, causing a decrease in the
intensity of light passing through the sample. The ability of lipoprotein particles to
absorb light is manifested in the in the range of wavelengths (300-700 nm). Sample
absorbance rises with the decreasing wavelengths and is maximal in the ultraviolet
range. That is why many enzymatic methods in which the end product is measured at
340nm are strongly affected by lipemia.
Lipemia samples also cause light scattering. Light scattering occurs in all directions,
and its intensity depends on the number and the size of lipoprotein particles and the
wavelength of measurement. For this reason, light scattering of lipoprotein
particles causes significant interference with turbidimetry and nephelometry. In
methods where the transmittance of light is inversely proportional to the
concentration of the analyte, in the absence of the sample blank, sample turbidity
causes positive bias.
INTERFERENCE CAUSED BY THE VOLUME DEPLETION EFFECT:

Plasma in healthy individuals in the fasting state consists of only minor portion of
lipids (<10% of total plasma volume). The rest of the plasma is water. The increase
in the concentration of lipoprotein particles leads to an increase in the plasma
volume occupied by lipids. Particles that are not lipid soluble are displaced by the
lipids to the to the water part of the plasma. Therefore lipemia leads to a false
decrease in the concentration of the measured analyte in all methods in which
concentration of respective analyte is measured in the plasma volume.
One example of interference caused by the volume depletion effect is the bias in
electrolyte measurement, leading to so-called pseudo-hyponatremia. This type
of interference affects electrolytes only if measured by flame photometery and
by indirect measurement using ion-selective electrodes but not in direct
potentiometry. However , it must be noted that the volume displacement effect
of the lipemic sample will affect the electrolyte measurement only in grossly
lipemic samples with concentrations of triglycerides greater than 17 mmol/L
( 1504 mg/dL )
INTERFERENCE CAUSED BY PARTITIONING OF THE SAMPLE

Upon centrifugation of a lipemic sample, lipoproteins are not homogenously

distributed in the serum or plasma due to the lipid gradient. Water-soluble
analytes are more concentrated in the lower layer of the plasma or serum,
whereas lipids and lipid-soluble analytes, such as drugs and some lipid-soluble
hormones, are more concentrated in the lipid-rich layer. This is especially
important in automated chemistry analyzers with fixed path lengths of the
sample probe. The results may differ for those analytes that are not evenly
distributed between the lipid and water portion of the sample, depending on
the part of the sample from which the sample probe is taking the sample for
analysis.
INTERFERENCE CAUSED BY PHYSICOCHEMICAL MECHANISMS

An excess of lipoproteins in the blood may interfere in electrophoretic and

chromatographic methods by causing abnormal peaks. Elevated levels of
triglycerides and lipoprotein particles may disturb the electrophoretic
pattern and morphology, as well as falsely increase the relative percentage of
the prealbumin, albumin, and α1 and α2-globulin regions.
One additional complication of excessive lipemia is the increased sample
susceptibility to hemolysis leading to the specific turbid and reddish
appearance of the sample ( the so-called “strawberry milk” appearance).
This effect is most probably caused by the increased fragility of the
erythrocyte membranes due to alterations in the content of the
phospholipid membrane layer and is more pronounced with the increase in
lipid (particularly triglyceride) concentrations.
REMOVAL OF LIPIDS FROM THE SAMPLE
They most often originate from emergency departments, intensive care units,
and endocrinology and gastrointestinal clinics from patients suffering from
conditions that include acute pancreatitis, acute or chronic kidney failure,
thyroid disorders, and diabetes mellitus. Unlike hemolysis, the interference
caused by lipemia can be fully eliminated, or at least reduced, by removing
the excess of lipids from the sample. Still, even if lipids have been successfully
removed from the sample, any visible turbidity of a sample should be
documented and reported with the test results because it offers clinically
useful information about the patient. Moreover, lipid testing and testing for
lipid-soluble drugs (eg, benzodiazepines) and hormones (eg, thyroid
hormones) should always be done on the native sample, before delipidation.
Methods for lipid removal include ultracentrifugation, high-speed
centrifugation, and some lipid-clearing agents.
 LIPID REMOVAL BY ULTRACENTRIFUGATION AND HIGH-SPEED CENTRIFUGATION.
According to the CLSI C56-A standard for Hemolysis, Icterus, and Lipemia/Turbidity Indices as
Indicators of Interference in Clinical Laboratory Analysis, ultracentrifugation is the recommended
method for removal of the excess of lipids in the sample. Ultracentrifugation use the centrifugation
force of almost 200,000 g and are very effective in clearing lipemic sera by separating lipids,
especially chylomicrons (top layer) from aqueous part ( lower layer ) of the sample. After
centrifugation, the infranatant (lower part of the sample ) can be transferred into the clean tube
and analysed. It should be kept in mind that by removing the upper lipid layer, one also removes
the lipid-soluble analytes like drugs and hormones.
Though considered gold standard, ultracentrifugation is not widely available in many laboratories.
High- speed centrifugation using using the microcentrifuge with a maximum centrifugation speed
of up to 20,000 g may therefore serve as an acceptable alternative and is the method of choice for
most laboratories.

 LIPID REMOVAL BY LIPID-CLEARING AGENTS

Lipid –clearing agents are widely used in many laboratories due to their low cost, convenience, and
ease of use. Those agents (cyclo-dextrin, polyethylene glycol, dextran sulphate, hexane, and others)
may vary in their ability to extract lipids from a lipemic sample and may also lead to reduction of a
significant amount of protein from the sample.
 ICTERUS

The normal concentration of bilirubin in human plasma (or serum ) is up to 20µmol/L.

Change in the colour of the serum ( plasma ) becomes detectable when bilirubin
concentration exceeds 34µmol/L. Bilirubin concentrations above 100 µmol/L are
clinically defined as icterus. Icteric plasma is commonly seen in patients from
intensive care units, gastroenterology centers, and pediatric clinics.
Bilirubin interferes with numerous chemistry tests such as enzymes ( ALT , alkaline
phosphatase, creatine kinase, lipase), electrolytes, metabolites ( urea, creatinine,
glucose), lipids (cholesterol, triglycerides ), protiens (albumin, total protiens, IgG) ,
hormones (estradiol, beta-HCG, free T3), and even some drugs ( gentamicin,
phenobarbital, theophylline, tobramycin.)
MECHANISMS OF INTERFERENCE CAUSED BY ICTERUS
Icterus interferes through two mechanisms: spectrophotometric
interferences and by interfering with chemical reaction.

Spectrophotometric Interference of bilirubin : Bilirubin causes

spectrophotometric interference due to its ability to absorb light in
the wide range of wavelengths between 400 and 540 nm

Chemical Interference of Bilirubin : Bilirubin produces negative bias

on assays that involve H2O2 as an intermediate reaction ( eg,
cholesterol, glucose, uric acid, triglycerides )
Detection of Hemolytic, Icteric, and Lipemic Samples

Hemolysis becomes visible at the concentration of 0.3 to o.5 g/L of free haemoglobin, and the
intensity of the red colour of the serum or plasma further increases with the increase in
concentration of free serum haemoglobin.
Lipemia causes sample turbidity, which approximately corresponds to the concentration of
serum triglycerides.
Increased concentrations of serum bilirubin lead to yellow to orange colouration of the
serum, and the change of the colour correlates to the increasing concentration of bilirubin in
the serum.
Free hemoglobin, triglycerides, and bilirubin have characteristic absorption peaks in a wide
wavelength range of 300 to 600 nm. This is also the range where sample absorbance is
measured in spectrophotometric methods, and that is why hemolysis, icterus, an lipemia
cause spectral interferences
AUTOMATED SERUM INDICES

Most mainstream chemistry analyzers can detect serum indices by the use of
semiquantitative, spectrophotometric measurement and grading the
interfering substances into categories.
 MANAGEMENT OF HEMOLYTIC , ICTERIC, AND LIPEMIC
SAMPLES

There is large discrepancy among the ways results are reported from samples with
interferences.
When interferences from hemolysis, icterus, and lipemia are causing unacceptable
bias and results are clinically inaccurate, such results should not be reported and
sample redraw should be requested. Such a test report should always be
accompanied with comments informing the clinical staff about the reasons for not
reporting the originally requested test results. It is also important that a laboratory
notify the staff when sample appearance ( colour , turbidity) deviates from a
normal state by including a comment on a test report ( eg sample hemolysed,
icteric, lipemic or turbid. Comments should also indicate if the sample has been
treated in any way to minimize the effect of interfering substances ( eg
delipidation)
 DRUG INFLUENCES ( DRUG EFFECTS) AND INTERFERENCES
Mechanisms of Interferences
According to the mechanism of action, drug interferences on laboratory tests can be
categorized as either BIOLOGICAL or CHEMICAL
INDUCTION OF HEPATIC ENZYMES – Phenytoin raises levels of GGT through this mechanism
ENZYME INHIBITION – Finasteride and dutasteride cause a decrease in PSA concentration by
inhibition of 5α-reductase
All these drug actions occur in vivo, and thus the changes in parameters reflect a true state
in human body, thus ruling out analytical error.
Analytical ( chemical ) interference is caused when the parent drug or metabolite has
structural similarity to the analyte therefore interfering in the immunometric or photometric
methods.
Reaction interference can occur when the compound or its metabolite catalyse or inhibit the
steps of chemical reaction.In above cases Manufacturer Claims Regarding Drug Interferences,
can be of great use. And also the patients drug history.
 Creatinine is one of the most commonly measured parameters in a clinical chemistry
laboratory, the laboratories worldwide use either the Jaffe colorimetric assay with alkaline
picrate or an enzymatic assay for creatinine measurement.
Some commonly prescribed antibiotics and analgesic drugs can interfere with creatinine
measurement. Cephalosporin antibiotics cause falsely increased creatinine in sera shortly
drawn after intravenous administration of cefpirome.
Also subtherapeutic, therapeutic and toxic concentrations of acetaminophen, acetylsalicylic
acid and metamizole cause falsely raised creatinine when measured by jaffes method
High concentrations of catecholamines also show strong negative interferences with
creatinine enzymatic assay.
 Ethamsylate, a hemostatic drug causes a significant decrease in creatinine when
measured by enzymatic technique. Also evidences suggest ethamsylate causes
significant false decline of cholesterol, triglycerides and uric acid.
 The active metabolite of leflunomide, a immunosuppressant with antiviral
property shows interference with ionized calcium by falsely decreasing the
results. Commonly seen in kidney transplant patients. Similar interference is seen
with sodium perchlorate used as a oral drug in treatment of hyperthyroidism.
 Carbohydrate-deficient transferrin (CDT) is a biomarker for long term alcohol
consumption. The sensitivity of the test can be affected by number of drugs like
amlodipine, perindopril, atorvastatin, isosorbide mononitrate.
 Thiopental is a barbiturate used for treatment of elevated intracranial pressure,
falsely elevated sodium concentrations were seen when V-LYTE Integrated
multisensory technology system was used for electrolyte measurement
Supportive Medical Therapy
Iodine-based compounds ( iohexol, iodixanol, and ioversol) are mostly used in
x-ray methods can interfere with chemical reaction in several ways. Iodine
molecules have high density and can prevent proper formation of the barrier in
the serum gel separator tubes. Iopromide is used as contrast media agent in
coronary angiography. A false increase in troponin I concentration measured
by Opus Magnum reagent is detected if the sample is taken immediately after
the procedure. This interference is reagent specific.
Gadolinium contrast agents act as chelators, and that seems to be the main
interference mechanisms. Therefore calcium measurement is mostly affected
by the presence of these compounds. Other clinical chemistry assays are also
influenced by several different gadolinium contrast agents: angiotensin-
converting enzyme, total iron-binding capacity, zinc, magnesium and
creatinine.
 Natural Preparations
Chinese medicine like Chan Su, can contain some physiologically highly active
molecules like bufadienolides. The structural similarity of bufadienolides and
digoxin is responsible for both cardiotoxicity and interference in the
immunochemistry method. Ingestion of this medicine can cause false-positive
and false-negative results for the digoxin measurement depending on assay
format. Herbal supplements that are used widely worldwide, like ginseng, can
also interfere with digoxin measurement. Chinese herbal remedies may
contain steroids, with the potential to interfere with some assays and to
cause suppression of the hypothalamic-pituitary-adrenal axis.
Similarly, it is also well documented that some Ayurvedic herbal medicine
products may be contaminated with lead, with the potential to cause toxicity.
 Accidental Exposure and Poisoning
In case of accidental poisonings with herbs, household cleaning products, or any
other exogenous compounds, interferences on laboratory test results can
potentially prolong the diagnostic procedures in acute patient care and cause
harm to the patient.
Cardiotonic glycosides like Convallaria majalis and Nerium oleander, due to their
structural similarities to digoxin, can interfere with digoxin measurement.
Accidental intoxications often occur as a result of children ingesting cleaning
products. Miniature racing cars run on nitromethane, which has been shown to
interfere with creatinine measurement, producing falsely elevated
concentrations.
Extreme creatinine concentration without evident renal failure has been
observed in suicide attempt in which Blue Thunder fuel containing nitromethane
was ingested. In a jaffe method, nitromethane also reacts with alkaline picrate
and forms a red chromophore, thus causing falsely elevated creatinine.
 Sample Contaminant
Several components of the blood collection tubes can interfere with the measurement and
influence laboratory results.
Lubricants like silicone oils and glycerol facilitate insertion and removal of the stoppers. Glycerol
should not be used for the lubrication of stoppers in tubes that will be used for triglyceride
measurement because glycerol interferes with most triglyceride assay.
Silicone-based lubricants are less likely to interfere with assays, although silicone can falsely
elevate ionized magnesium and triiodothyronine levels.
Some clot activator based silica particles affect the measurement of some analytes like lithium
and testosterone.
Silicone surfactants used to decrease nonspecific adsorption of components on tube walls may
interfere with measurement of vitamin B12 and cancer antigen 15-3
Antibiotics are commonly added into reagents and buffer solutions to prevent microbial
growth.
Carryover from reagents containing gentamicin may cause spuriously high gentamicin results.
Gentamicin is present in some diagnostic reagents ( Glucose, urate, direct bilirubin, CK ALT, AST,
beta-hydroxybutyrate) as an antibacterial additive and is known to cause spuriously high
gentamicin results on the Beckman Coulter AU-480 analyser due to reagent carryover.
 INTERFERENCES IN IMMUNOASSAY
Potential exogenous sources of preanalytical interferences include
• Sample type (type of additive)
• Underfilling the tube
• Sample stability during transport and storage
• Hemolysis
• Lipemia or sample turbidity
• Icterus (hyperbilirubinemia)
• Fibrin clots
• Carryover
• Administration of radioactive or flourescent compounds
• Drugs
• Herbal medicine
• Nutritional supplements
 TUBE ADDITIVES AS POTENTIAL INTERFERING FACTORS IN
IMMUNOASSAY

HEPARIN - it interferes with numerous immunoassays by affecting the binding of

antibody and antigen and thus affecting the rate of reaction.
Heparin plasma has been documented to cause significant negative bias (up to 30%) in
cardiac troponin measurement. Heparin interference occurs due to its negative charge
and its binding to positively charged troponin. The binding of heparin and troponin
gives rise to conformational changes of troponin and affects the antigen-antibody
interaction. This interference was neutralized in the fourth-generation troponin T assay
by adding cationic heparin blocking agent.
EDTA ( ethylenediamine tetraacetic acid ) – is a commonly used additive, especially
in hematology and endocrinology because it offers increased stability of cells and
analytes. Most hormones ( except ACTH ) are stable up to 5 days in EDTA plasma if
they are kept refrigerated at 4°C. Its main action is chelation of cations. It shows
interferences in some chemiluminescence immunoassays that use conjugated
alkaline phosphatase as secondary enzyme in their reactions. For example , it has
been shown that underfilling the EDTA tube by half or more causes clinically
significant bias in the measurement of intact parathyroid hormone with the DPC
IMMULITE assay.
POTASSIUM OXALATE – Acts as calcium chelating anticoagulant and is often
combined with antiglycolytic agents ( sodium fluoride and sodium iodoacetate). As
with EDTA, oxalate can also inhibit some enzymes (eg. Amylase, lactate
dehydrogenase, alkaline phosphatase) by chelating bivalent cations that are
necessary for their activity.
 Interferences in immunoassays Caused by Hemolysis, Lipemia,
and Icterus

Hemolysis interfere with a wide range of immunochemistry assays

through the proteolytic action of cathepsin E, a major proteolytic enzyme
in mature erythrocytes.
Increased lipid concentration and consequent sample turbidity may affect
some immunochemistry assays by affecting the light scatter pattern or by
masking the binding sites on antigens and antibodies and thus physically
interfering with antigen-antibody binding
  Formation of fibrin clots

Under optimal clotting conditions, serum is considered to be free of fibrin, fibrinogen, and cells, and it is
a preferred matrix for most immunoassays.
To allow complete clot formation , serum tubes should be allowed to clot for a minimum of 30 minutes.
However, with new tube types, containing clot activators ( thrombin-based clotting agent), serum
clotting time is substantially reduced ( on average < 2.5 minutes) without compromising the sample
quality and stability for most chemistry analytes.
Blood from patients who are receiving heparin therapy may require a longer time to completely clot, and
there is a greater likelihood for latent postcentrifugation clot formation. Insoluble fibrin , fibrin strands,
and microclots may affect instrument performance and cause interferences. If fibrin is aspirated for
analysis and goes undetected, there is high likelihood of getting false-positive results for that sample.
This is manifested by duplicate measurement errors ( i.e unacceptable deviations in two measurements
on the same sample)
Such discrepancies has been repeatedly reported in cardiac troponin measurements, a possiblre
approach is to recentrifuge all positive troponin samples at high speed ( 6700 g for 5 min) and then
repeat the analysis
  Interference Caused by Separator Gels

Separator gels are used to ensure rapid and prolonged separation of serum/plasma
from clotted blood and cells, respectively. Separation of the sample is enabled due to
the specific gravity of the gel, its ability to undergo a temporary change in viscosity
during centrifugation, and its ability to lodge between the packed cells and the top
serum/plasma layer.
Hydrophobic compounds may bind to the gel, which is why tubes containing
separator gels are not appropriate for some hydrophobic drugs and hormones such as
–
DRUGS : phenytoin, phenobarbital, carbamazepine, tricyclic antidepressants,
quinidine, lidocaine
HORMONES : testosterone, estradiol, cortisol, free thyroxine, total triiodothyronine
At times when kept under improper storage conditions (time and temperature) the
gel may degrade and release small particles or globules into the supernatant.
  Interferences Caused by Sample Carryover

Sample carryover in automated analyzers occurs due to the inefficient probe and cuvette
washing and subsequent inability of an instrument to successfully remove any remnants of
sample or reagent. Due to improper washing, certain amount of reagent or analyte can be
transferred ( carried ) by the measuring system from one assay reaction to a subsequent
reaction thereby erroneously affecting test results. Additional probe and cuvette washing
may be performed after the extremely elevated results with an increased risk for carryover.
According to International Union of Pure and Applied Chemistry ( IUPAC) , sample carryover
testing is performed by running one sample with high concentration of an analyte at least
two times, followed by at least three runs of a sample with low concentration of the
analyte
According to CLSI, carryover testing for immunoassays is done by running four consecutive
analyses of two samples at different levels ( samples with extremely high analyte
concentration (A) followed by samples with very low (B) concentrations for the same
analyte)
  Interferences Caused by Paraprotiens

Paraprotien interferences are not uncommon. The frequency of paraprotien interferences has
been estimated to be as high as 3% to 4% in hospitals .
GROUP OF ANALYTES MOLECULE
Enzymes Alkaline phosphatase
GGT , LDH
Electrolytes, minerals, and Calcium
microelements Inorganic phosphorous , Iron
Metabolites Bilirubin , Cholesterol, Creatinine,
Glucose, Urea , Uric acid
Protiens C-reactive protiens , IgA , IgG
Hormones TSH , HCG
Drugs Gentamicin , Vancomycin , Valproic acid
Phenytoin
Cardiac markers Troponin I
Tumor markers Α-fetoprotein
CA-125
 Problems with sample preparation, storage and
transportation

 The length of time of sample preparation, including the speed and temperature of centrifugation, exposure
to light and preparing aliquots, are important factors that must be considered before analysis. Proper
sample storage condition is paramount (this relates to the length of storage, temperature, freezing and
thawing). However during transport, a sample may be exposed to shaking, changes in light conditions, and
changes in temperature. Transport delays to the laboratory can give rise to clinically important errors if
transport conditions are not optimized.
 The National Committee for Clinical Laboratory Standards (NCCLS) H5-A3 1994 recommended a maximum of
2 hours for transporting blood samples at a temperature of 10–22 °C. Delay in transporting the specimen to
the laboratory where processing begins, not only prolongs the time until the attending physician receives a
test result but also impacts specimen integrity that may lead to a false-negative or false-positive result of
analysis and a misleading answer. A delay in transporting a blood sample to the laboratory for measurement
of ‘blood sugar’ is likely to cause a ‘falsely reduced’ blood glucose result. Such errors that occur at this stage
cannot be corrected later.
These mistakes would require collection of fresh blood samples, which
could be quite distressing for the patient. The sample preparation steps
contribute to approximately 19% of the overall cost of analysing a single
sample and are time-consuming (37% of time spent in producing result).
 Clinical laboratory informatics

The Lord Carter (2008) Review of Pathology recommended that information

technology (IT) connectivity be put in place in all NHS pathology services as a
matter of urgency, to improve the way that pathology enabled decisions about
diagnosis and treatment are made by assemblage and analysis of data retrieved
from LIMS. Recently ‘Order Communications’ have been comprehensively
promoted as a way of improving efficiency and effectiveness of laboratory
testing services (Yorkshire Centre for Health Informatics, 2014) through a
number of perceived improvements in resource utilisation. Effective electronic
communication between the clinical chemistry laboratory on the one hand and
healthcare providers on the other hand is considered as an indispensable
element of any efficient and effective pathology service, as it would help
address gratuitous demands (such as unnecessary test requesting) and reduce
or eliminate the risk of errors (Health Commissions Report, 2007).
 Informatics relates to gathering, management, and processing of information
usually involving computing. Medical informatics involves the design,
management, and the study of systems that store and communicate medical
information. It follows therefore that Clinical Informatics is an offshoot of
medical informatics and is primarily concerned with the communication and
management of information associated with laboratory analysis, generation of
results and interpretation of test results . Efficient clinical laboratory operation
relies heavily on informatics, since the function of the clinical laboratory is the
creation and communication of information for patient diagnosis and
management. Clinical informatics relates to processes that extend beyond the
confines of clinical chemistry laboratory and may involve diverse ‘extra-
laboratory’ processes, which embraces the support of correct test ordering by
healthcare professionals, the accurate communication and storage of order
requests, correct test interpretation and finally the management of information
necessary for optimal performance by the clinical laboratory. 
 Laboratory information systems
Laboratory information systems (LIS) form a dynamic connection between the
biomedical scientist, the clinical chemistry laboratory analytical platforms and the
healthcare professionals. The clinical chemistry laboratory at uses microcomputers in
every phase of the TTP including pre-analytical workstations, analytical platforms,
reagent inventory control, quality control, data interpretation, and online monitoring of
analytical performance. All these aspects are integrated through a common database
and communications network and in this way it is able to support the extensive
laboratory information system. Presently the total management of the pre-analytical as
well as the post-analytical processes are entirely dependent on computer applications.
The use of barcode technology has facilitated the automation of all the processes from
patient and sample identification to sample testing on the analyser platforms.
Sophisticated and intelligent computer software programmes are now able to produce
further information that may be used for identifying pre-analytical errors in the TTP,
leading to better diagnoses, which maybe useful for managing patients and thus
improve their wellbeing and safety. A key role of the LIS is to help reduce chances for
errors, be it technical or human in origin.
The information generated by laboratory workflow is received, processed and
stored and archived by the LIS. The LIS supports a myriad of laboratory functions
by automating the flow of virtually all the extensive database applications that
are embedded in clinical laboratory operations. It is becoming very common in
most health institutions to interface LIS with Electronic Health Records (EHR) in
order to link patient registration information with electronic test requesting
(commonly referred to as ‘Order coms’). The amalgamation of the LIS and EHR is
commonly designated as Laboratory Information Management Systems (LIMS).
A well-designed health information management system, which incorporates
reliable, accurate and timely availability of data is widely acknowledged as a
foundation of a robust public health system. The development of LIMS as part of
the NHS has helped to support a variety of programmes and functions such as
internal and external quality assurance, research and information dissemination
and governance. Specifically the LIMS systems perform a myriad of functions
from pre-analytics (test ordering and specimen collection), analytics as well as
post analytics (reporting of test results) to healthcare professionals. 
Laboratory test ordering: Manual versus ICE Order entry

Conventionally healthcare providers have always ordered laboratory tests using manual requisitions
(paper-based orders). Healthcare providers would place a tick or write the name of the requested
test on a pre-printed hospital laboratory order form. The sample would then be collected, labeled by
a phlebotomist and placed in a specimen bag along with the requisition, and delivered to the clinical
chemistry laboratory for analysis. However due to increasing pre-analytical errors, complexities,
challenges, and resource constraints of modern healthcare systems, paper orders are becoming
increasingly unsuitable. Manual order requests are gradually being replaced by electronic order
entry systems, which allow direct healthcare provider input of diagnostic testing orders into
requests into a computer system are known as Computerized Provider Order Entry (CPOE) systems.
Implementation of laboratory CPOE systems may offer healthcare centres many benefits, including
reduced test turnaround time, improved test utilization. Electronic requesting should improve the
accuracy of patient demographic information (Yorkshire Centre for Health Informatics, 2014); allow
better tracking of results and allow laboratories to report the results of tests they send away for
analysis via their normal reporting systems.
 Approaches for reducing pre-analytical errors in clinical
chemistry

A comprehensive and methodical approach to reducing pre-analytical errors has

been previously described and consists of five interrelated stages listed below:
 Developing clear written standard operating procedures (SOPs) for routine and
urgent clinical work.
 Improving healthcare professionals’ training.
 Acquiring automated technologies / information systems, both for support
operations and for executive operations.
 Developing and monitoring quality indicators (QIs)
 Improving communication between laboratory staff and other healthcare
professionals.
  Developing clear written standard operating procedures
(SOPs)

Standard Operating Procedures (SOPs) should be controlled and must be unambiguous.

Trained laboratory staff charged with performing the pre-analytical procedures must
understand the procedures in place and follow them exactly. They need to be aware of
possible errors that may occur if the operating procedures are not followed, and what
consequences these errors can have on sample analysis and eventually on patient
management. The ISO 15189 Standard (2012) instructs laboratories to prepare a manual for
the pre-analytical procedure that will give clear instructions to the patient before the
collection of biological samples. Among other things, and depending on the type of
analysis, patients would be instructed to control their diet, physical activities, stress, use of
medications etc. This problem is most easily solved by the application of computerized
referrals, where all the necessary data are obtained from the patient; however, the
problem is much bigger when samples are collected at sites distant from the laboratory.
  Improving healthcare professionals training
Biomedical scientists and other laboratory staff have their training/competencies
reviewed annually as part of a professional development portfolio (PDP) and Knowledge
and skills framework (KSF). Biomedical scientists need to apply the knowledge and skills in
a number of dimensions to achieve the expectations of their post

Acquiring automated technologies and laboratory information

systems
The introduction of information technology (IT) and automated platforms has lead to
an appreciable reduction in pre-analytical errors in the clinical chemistry laboratory.
Computerized order entry (CoE) simplifies test ordering and eliminates a second person
from transcribing the orders. The introduction of sample barcoding (to simplify specimen
routing and tracking) and pre-analytical automated workstations abridge the process of
sample separation, aliquoting and sorting into batches and thus reduce the number of
manual steps and less staff and helps to reduce human induced errors.
 Developing and monitoring quality indicators, QIs

Performance and outcome measures can significantly improve the quality of patient
care (Plebani, 2011). Such measurements of improvement can be achieved through the
development and monitoring of specific quality indicators (QI) in the TTP. The pre-
analytical phases in clinical chemistry are more prone to errors than the analytical and
post-analytical phase. Evidence abounds that before the turn of the century, reliable
QIs and quality specifications have been developed and introduced for effective
management of analytical processes. The development of these specific QIs were an
extension to the Internal Quality Control (IQC) systems coupled with availability of
different External Quality Assessment (EQA)1 schemes already in place, which have
made it possible for the clinical chemistry laboratory to measure, monitor and improve
their analytical performance on a monthly basis, although retrospectively
 QIs have been defined as vital tools (IOM, 2000) for enabling clinical chemistry
laboratory staff to quantify the quality of a selected aspect of patient care by comparing
it against a defined criteria (ISO 15189: 2012). A QI objectively measures and evaluates all
of 6 critical domains as outlined by the IOM, namely:
a) Patient safety, b) Patient-centeredness, c) Effectiveness, d) Efficiency, e) Equity and f)
Timeliness. Assessment of QI is therefore based on the evidence associated with the
aforementioned domains and can be implemented in a constant and comparable
manner across settings and over a period (IOM, 2000); the identification of a reliable QI
represents an essential stage in the schemes targeting evaluation and improvement of
the quality of patient care. Although many authors are in agreement that QIs are
important in providing information on continued improvement it has two major
shortcomings: firstly, it is often difficult to compare data reported in the literature
because of differences in QIs us and the methods of collecting these data. Secondly,
while the most common errors in the pre- and post- analytical phases are considered,
available QIs do not include appropriate choice and selection of test as well as
appropriate interpretation and utilization of laboratory results at the right time.
 Improved communication between clinical chemistry
laboratory staff and other healthcare professionals.

Many mistakes generated in the TTP may be due to poor communication, action taken by others involved
in the testing process (e.g., physicians, nurses and phlebotomists), or poorly designed processes, all of
which are beyond the laboratory's control (Plebani, 2006). Inter-professional working to improve service
delivery involves several healthcare professionals with expertise; knowledge and skill bases and
experience being drawn together in a structure to provide services. These different professionals make
adjustments and allowances in their responsibilities to take account and relate with the roles of others.
Many workers value inter-professional team working as a source of mutual support in the face of internal
and external pressures (Payne, 2000) and particularly the demands of working in the NHS and spending
cuts. Failure to initiate and maintain communication between the laboratory and the users of its service
may be responsible for causing certain medical errors. Clinical communication is highly complex and
prone to errors especially during transitions of patient care and emergency situations. Standardised
approaches and tools may provide potential solutions to improve the quality of communication and
prevent subsequent patient harm.
 Quality indicators in the pre-analytic phase

IOM (2000) defined quality as: “The degree to which health services for individuals and
populations increase the likelihood of the desired health outcomes and are consistent
with current professional knowledge”
As stated above, the pre-analytical phase should be subdivided into an extra-
laboratory pre-analytical phase and a ‘true’ pre-analytical phase, which is undertaken
within the laboratory after sample reception. The former phase, which comprises
initial procedures usually performed neither in the clinical laboratory nor undertaken,
at least in part, under the control of laboratory personnel, includes test requesting,
patient and sample identification and sample collection. The latter phase involves the
steps required to prepare samples for analysis (order entry into computer interface,
centrifugation and aliquotting into secondary tubes etc.). In a patient-centred
scenario, QIs should be designed to cover all steps of the pre-analytical phase,
including the appropriateness of test selection, which is a key issue in projects aiming
to ensure clinical effectiveness. The IFCC Working Group in a recent study have
identified 16 Quality Indicators to monitor the pre-analytical phase of TTP
Q I-1 : A p p ro p ria te n e ss o f te st re q u e st N u m b e r o f re q u e sts w ith c lin ic a l q u e stio n (% )

Q I-2 : A p p ro p ria te n e ss o f te st re q u e st N u m b e r o f a p p ro p ria te te sts w ith re s p e c t to th e c lin ic a l q u e stio n (% )

Q I-3 : E x a m in a tio n re q u is itio n N u m b e r o f re q u e sts w ith o u t p h y s ic ia n ’s id e n tific a tio n (% )

Q I-4 : E x a m in a tio n re q u is itio n N u m b e r o f u n in te llig ib le re q u e s ts (% )

Q I-5 : Id e n tific a tio n N u m b e r o f re q u e sts w ith e r ro n e o u s p a tie n t id e n tific a tio n (% )

Q I-6 : Id e n tific a tio n N u m b e r o f re q u e sts w ith e r ro n e o u s id e n tific a tio n o f p h y s ic ia n (% )

Q I-7 : T e s t re q u e st N u m b e r o f re q u e sts w ith e r ro rs c o n c e rn in g te s t in p u t (% )

Q I-8 : S a m p le s N u m b e r o f sa m p le s lo s t/n o t re c e iv e d (% )

Q I-9 : S a m p le s N u m b e r o f sa m p le s c o lle c te d in in a p p ro p ria te c o n ta in e rs (% )

Q I-1 0 : S a m p le s N u m b e r o f sa m p le s h a e m o ly s e d (h a e m a to lo g y , c h e m istr y ) (% )

Q I-1 1 : S a m p le s N u m b e r o f sa m p le s c lo tte d (h a e m a to lo g y , c h e m is try ) (% )

Q I-1 2 : S a m p le s N u m b e r o f sa m p le s w ith in s u ffic ie n t v o lu m e s (% )

Q I-1 3 : S a m p le s N u m b e r o f s a m p le s w ith in a d e q u a te s a m p le -a n tic o a g u la n t ra tio (% )

Q I-1 4 : S a m p le s N u m b e r o f sa m p le s d a m a g e d in tra n s p o rt (% )

Q I-1 5 : S a m p le s N u m b e r o f im p ro p e rly la b e lle d s a m p le s (% )

Q I-1 6 : S a m p le s N u m b e r o f im p ro p e rly s to re d sa m p le s ( % )
The lack of patient identification or patient misidentification have serious
consequences for reaching the final conclusion and clinical decision, as well as for
patient safety, thus this is one of the key indicators in the process. Errors in
patient identification may also occur during the procedures of sample
preparation. Ordering inappropriate tests is another pre-analytical variable with a
negative impact on patient safety. It is the cause of unnecessary test repetitions
in up to 30% of cases (Plebani, 2006). Biomedical scientists in conjunction with
clinical scientists should acquaint the clinician with the importance of biological
variations, potentially significant in the pre- analytical process (test selection) but
also in the post-analytical process (interpretation of test results). It is necessary
for the clinician to understand the concept of biological variations that include
intra-individual biological variations (deviation of results in relation to the
person’s homeostasis) and inter-individual biological variations (variations
between different persons in relation to the established homeostatic value).
Based on such knowledge it is possible to select between two tests, the best one
with the highest diagnostic value.
 
In the pre-analytical phase, major sources of variability - which includes biological
variability, environmental conditions, postural changes, patient identification and
sample labelling tourniquet time, type of container (e.g., vacutainer tube),
phlebotomy procedure (order of sample draw, contamination, mixing); sample
transportation (time, pneumatic tube transport systems from wards and clinics);
sample preparation for analysis (length, temperature of centrifugation,
preparing aliquots, can occur during patient preparation for phlebotomy. Errors
that may occur in this process often become obvious in the analytical and post-
analytical phase as well. For instance, the effects of interferences may be
discovered during analysis or the clinical interpretation of results (for example a
falsely elevated potassium result is observed in ‘hemolysed’ samples; similarly a
sample with a high degree of lipemia or protein concentration will adversely
affect the ‘true’ sodium result). For these reasons identification of quality
indicators is necessary in order to avoid potential errors in the pre-analytical
phase.