INTRODUCTION TO

CHEMICAL PATHOLOGY
S. AMETEPE
 OUTLINE
 Introduction to chemical pathology

 Specimen collection & handling

 Side room testing/point of care testing/ near patient

testing
 OBJECTIVES

• Describe the biochemical and pathophysiological mechanisms of

diseases and the biochemical principles underlying their treatment.

• Select appropriate laboratory test and interpret the results to confirm or

refute a provisional clinical diagnosis and to monitor progress during
treatment.

• To understand the potentials and limitations of various laboratory tests.

• Collect the right type of specimens for laboratory investigations under

the right conditions.
 CHEMICAL PATHOLOGY
 Clinical Biochemistry

 Clinical Chemistry

 Medical Biochemistry

 Pure Blood Chemistry

 Physiological Chemistry
DEFINITION FOR CHEMICAL PATHOLOGY

The systematic study of

biochemical processes associated with
health & disease &
the measurement of constituents in body
fluids or tissues to
facilitate diagnosis of disease
 CHEMICAL PATHOLOGY

 Chemical Pathology is the study of the biochemical basis of disease,

and the application of biochemical and molecular techniques in
diagnosis

 Constant changes in the chemical constitution & biochemical

mechanisms of the body as a result of disease.

 Chemical pathology is a sub-specialty within pathology which

extends across most medical specialties and involves the chemical
analysis of bodily fluids
 CHEMICAL PATHOLOGY
Primarily involves the chemical analysis of the following bodily
fluids:
 whole blood
 serum or plasma
 urine
 cerebrospinal fluid
 Faecal material
 effusions
 seminal fluid
 sweat and amniotic fluid to assist in the diagnosis of various
disease
 ROLE OF CHEM. PATHOLOGY IN
HEALTHCARE

Diagnosis: used to help differentiate between

several possibilities based on the initial history
and examination

Monitoring: to check disease progression or

response to therapy e.g. monitoring DM
patients
 ROLE OF CHEM PATH IN HEALTHCARE

Screening: to screen for the presence of disease in an

apparently healthy population or detection of disease before
it is clinically evident

Prognosis: providing information on disease susceptibility

e.g. cholesterol can predict the risk of coronary artery
disease.

Serves as tools to assist clinicians in the diagnosis of various

disorders, as well as management and follow-up of patients
 WHY CHEM PATH?
 Providing a consultation service to clinicians to advise on the most
appropriate testing within specific clinical situations

 Interpretation of a wide variety of clinical laboratory tests

 Advice regarding the limitations of laboratory tests in specific

circumstances

 Advice on the influence of "pre-analytical" factors, medications and

other factors on laboratory tests which may influence clinical decision
making
 WHY CHEM PATH
 Ensuring the quality of laboratory testing through internal and external
quality assurance

 Introduction of new tests, since medicine and clinical testing is a rapidly

evolving science and new tests are developed continuously

 Training of medical students in the appropriate selection of clinical

laboratory testing and the most cost effective and appropriate use of the
clinical laboratory
 WHY CHEM PATH
 Selecting the right test, at the right time, for the right patient. After
making the decision that an investigation is necessary, and
selecting the most appropriate test,

 Consideration to factors present that may affect the interpretation

of results, or even the decision to proceed with the test at that time
 Analyses performed within the Chemical
Pathology laboratory include:
Whole blood, serum, or plasma tests
 albumin, to investigate disorders of water balance, liver diseases, protein energy
malnutrition.
 urea or preferably creatinine, to investigate disorders of renal function and monitor
uraemia.
 glucose, to diagnose and monitor diabetes.
 bilirubin, to diagnose and monitor jaundice, e.g. in newborn.
 amylase, to assist in the diagnosis of acute pancreatitis.
 alanine aminotransferase, to investigate liver disease.
 potassium and sodium, to investigate and monitor disorders causing electrolyte
disturbances.
 Analyses performed within the Chemical
Pathology laboratory include:
 Cardiac markers for detection of cardiac damage
 Iron and porphyrins
 Endocrinology, including:
 Pituitary function
 Sex hormones
 Thyroid function
 Adrenal hormones
 Analyses performed within the Chemical
Pathology laboratory include:
 Inherited metabolic diseases
 Therapeutic Drug Monitoring (TDM)
 Drugs of abuse testing
 Allergy testing
Specialised testing including:
 Occupational Health testing (monitoring of exposed workers)
 Insurance testing
 Environmental testing (water analysis, etc.
 Tumour markers such as Prostate specific antigen (PSA) and various other
markers used in the detection and management of cancer patients
 Analyses performed within the Chemical Pathology
laboratory include:
Urine clinical chemistry tests
 protein, to diagnose and monitor proteinuria, e.g. in pregnancy or nephrotic
syndrome.
 glucose, to screen for and monitor diabetes.
 bilirubin, to assist in the diagnosis of hepatocellular and obstructive jaundice.
 urobilinogen, to investigate haemolytic jaundice.
 ketones, to detect and monitor ketonuria, e.g. in diabetes.
 haemoglobin, to investigate intravascular haemolysis, microbial infection and
glomerulonephritis.
 nitrite and leukocyte esterase, to assist in the diagnosis of urinary tract infection.
 relative mass density (specific gravity), occasionally needed to investigate the
concentrating and diluting power of the kidneys.
 Analyses performed within the Chemical Pathology
laboratory include:
Faecal clinical chemistry tests
 Occult blood, to investigate bleeding lesions of the gastrointestinal
tract.

 Lactose, to investigate lactase deficiency.

 Examining faeces for excess fat to investigate

disorders of fat absorption.

 Analyses performed within the Chemical
Pathology laboratory include:
Cerebrospinal fluid (c.s.f.) clinical chemistry tests

 Protein to investigate meningitis and African trypanosomiasis.

 Glucose, occasionally needed to assist in diagnosing meningitis .

 QUALITY ASSURANCE (QA) IN CHEMICAL PATHOLOGY

QA has been defined by WHO as the total process whereby

the quality of laboratory reports can be guaranteed. It has been
summarized as this:
● right result, at the
● right time, on the
● right specimen, from the
● right patient, with result interpretation based on
● correct reference data, and at the
● right price.
QUALITY ASSURANCE (QA) IN CHEMICAL PATHOLOGY

 Quality assurance (QA) is the overall program that

ensures that the results reported by the testing
laboratory are correct.

 Quality Assurance Programmes ensure laboratory test

results are reliable, reproducible, and relevant.
QUALITY ASSURANCE (QA) IN CHEMICAL PATHOLOGY

 It is an ongoing, comprehensive program, which

analyzes every aspect of an entire operation; it involves
determining a quality goal, deciding whether the goal
has been achieved, and implementing corrective action
if the goal has not been reached.

 In the laboratory, quality assurance involves the entire

testing process: pre-analytical, analytical (testing), and
post-analytical processes.
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS
 The pre-analytical stage covers the reasons for performing a
particular test, preparation of the patient, collection and transport of
the specimen, the request form, and checks which need to be made
when the specimen reaches the laboratory.
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS
 Collection of blood specimens
 Factors regarding the collection of blood specimens that can affect the
correctness of clinical chemistry test results include:
 incorrect venipuncture technique,
 haemolysis of red cells,
 collection into the wrong container,
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS
Venipuncture technique
The following precautions need to be followed when collecting venous blood:
 Do not apply the tourniquet too tightly or for too long a period because this will
cause venous stasis leading to a concentration of substances in the blood such as
haemoglobin, plasma proteins, potassium, and calcium.
 Do not collect the blood from an arm into which an intravenous (IV) infusion is
being given.
 If an anticoagulated specimen is required, add the correct amount of blood to the
tube or bottle and mix the blood with the anticoagulant by gently inverting the
container several times.
 Follow a safe technique and wear protective gloves
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS
Avoiding haemolysis
 The haemolysis (rupture) of red cells can be a serious source of
unreliable test results. If red cells are haemolyzed, substances from the
cells are released into the serum or plasma leading to a false increase in
the concentration of analytes, e.g. potassium. Haemolysis also
interferes with many chemical reactions.
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS
Haemolysis can be avoided by:
 Checking that the syringe and needle are dry and that the barrel and
plunger of the syringe fit well.
 Not withdrawing the blood too rapidly or moving the needle once it
is in the vein.
 Remove the needle from the syringe before dispensing the blood
into the specimen container. Allow the blood to run gently down the
inside wall of the container.
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS

Haemolysis can be avoided by

 Adding the correct amount of blood to anticoagulant.
 Do not shake the blood vigorously but gently mix it with the anticoagulant.
 Using clean dry tubes or bottles for the collection of blood from which serum is
required and by allowing sufficient time for the blood to clot and clot retraction to take
place. Red cells are very easily haemolyzed by the rough use of an applicator stick to
dislodge a clot
 Centrifuging blood samples for a minimum period of time. Centrifuging for 5 minutes
at about 1000 g is adequate to obtain serum or plasma.
 Not storing whole blood samples in, or next to, the freezing compartment of a
refrigerator
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS

collection into the right container:

 Blood glucose (non-urgent or urgent): Dispense the correct amount of
blood into a tube containing fluoride-oxalate.* Gently mix the blood with
the anticoagulant.

 Centrifuge the specimen to obtain plasma.

 Fluoride is an enzyme inhibitor. It prevents the break down of glucose to

lactic acid by enzyme action (glycolysis).
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS

Stability of analytes in blood specimens:

Some of the chemical changes which may occur in blood specimens
within a few hours of being collected include:
 Diffusion of potassium and some enzymes through the red cell membrane
into the serum or plasma.
 Decrease in the concentration of glucose by glycolysis (when fluoride-
oxalate is not used).
 Reduction or loss in the activity of certain enzymes, for example acid
phosphatase.
 Decomposition of bilirubin in daylight or fluorescent light .
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS

Stability of analytes in urine

The chemical changes which may occur in urine specimens stored at
room temperature include:
 Breakdown of urea to ammonia by bacteria, leading to an increase in the
pH of the urine. This may cause the precipitation of calcium and
phosphates.
 Oxidation of urobilinogen to urobilin.
 Destruction of glucose by bacteria.
 Precipitation of urate crystals in acidic urine
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS

Collection of cerebrospinal fluid

 The biochemical analysis of cerebrospinal fluid (c.s.f.)
includes the measurement of total protein and occasionally,
glucose. c.s.f. for measuring glucose
 To prevent the breakdown of glucose in the c.s.f. (leading to a
falsely low value), collect 0.5–1.0 ml of the fluid into
fluoride-oxalate preservative
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS

A specimen should be rejected by the laboratory if :

 It is unlabelled or the identity on the form does not match that on the specimen.
 It is not correct for the test being requested or it has been collected in the wrong
container, e.g. containing the incorrect anticoagulant.
 The specimen container is leaking.
 There is evidence of contamination.
 A blood sample appears haemolyzed or an anticoagulated specimen contains clots.
 There is too long a delay in the specimen reaching the laboratory or it has not been
transported correctly.
 QUALITY ASSURANCE: PRE-ANALYTICAL STAGE OF
CHEMICAL PATHOLOGY TESTS
Abnormal appearances of specimens
 A dark coloured urine may be positive for bilirubin or haemoglobin.
 A urine that contains whole blood may contain S. haematobium eggs.
 A black faecal specimen may contain occult blood due to gastrointestinal bleeding.
 A dark brown serum may indicate intravascular haemolysis due to sickle cell disease,
severe malaria, or an incompatible blood transfusion.
 A lipaemic (fatty) serum is associated with raised triglycerides (above 3.4mmol/l).
 A deep yellow (icteric) serum indicates that a patient is jaundiced.
 A blood sample that contains a high concentration of red cells from which little serum or
plasma can be obtained indicates severe dehydration or a blood disorder .
 FACTORS TO CONSIDER AT THE TIME OF
COLLECTING THE LAB SPECIMEN
 Patient posture :proteins and protein-bound constituents
change with posture e.g. Albumin, calcium, cholesterol,
cortisol and protein bound iodine.
 Venostasis: raise the conc. of plasma proteins,
haemoglobin, hormones, calcium & lipids. Remove the
tourniquet soon after puncturing the vein
 Site of venipuncture: e.g.. site of infusion-fluid is likely not
have mixed with the entire blood volume.
 FACTORS TO CONSIDER PRIOR TO SPECIMEN
COLLECTION
 Patient’s Diet: Ca, FBS.

 Patient’s current medication:

Oral Contraceptives, Cough Mixtures

 Time of day:
Iron & Corticosteroids
 FACTORS TO CONSIDER AT THE TIME OF
COLLECTING THE LAB SPECIMEN
 Identification of specimen-
 Patient’s name
 Location/ward
 Identifying number
 Date
 Time of specimen collection
 Suspected pathology/Diagnosis
 PRESERVATION OF SPECIMEN IN TRANSIT
Specimen for hormonal assays e.g. gastrin, rennin and
parathyroid hormone must be separated from the cells
in a refrigerated centrifuge.

Specimen for bilirubin and carotene must be protected

from both sunlight and fluorescent light to avoid photo
degradation.
 VACUTAINER/EVACUATED TUBES
 These are tubes for blood collection which are color-
coded based on the anticoagulant present. They
come in various sizes; 2, 5, 7, and 10 ml.

 Blood is drawn in this order: Blood culture tubes, red

top, blue top, green top, lavender top, and gray top

 If multiple tubes are needed, there should be a proper order of

draw to avoid cross-contamination and erroneous results.
 Commonly used blood tubes and preservatives.
TUBE TYPE CAP COLOUR DESCRIPTION

Plain serum Red Serum obtained by centrifugation which prevents the exchange of analytes with
clot. Used for most routine chemistries

Serum separator Yellow Coated with a clot activator. The gel barrier allows for primary sampling and
storage. Used for most routine chemistries

Lithium heparin
(plasma)
Green Available with and without a gel barrier. The anticoagulant heparin allows the
sample to be centrifuged immediately, so whole blood or plasma can be tested.

Fluoride oxalate
(Plasma)
Gray The anticoagulant oxalate binds calcium and the fluoride inhibits glycolysis, thus
maintaining blood glucose for several days
Osmotic effects of oxalate may cause haemolysis.
Used for blood glucose measurements

Ethylenediamine
tetraacetic
Pink EDTA chelates calcium-preserving cell
morphology so that: blood films can be used for haematological investigations,
acid (EDTA) analytes requiring whole blood, for example, haemoglobin A1c and erythrocyte
Vacutainers used for blood collection and storage
 Sample collection

 Whatever, the nature of the sample, it must be accompanied by a request form, which
must be completed in full to allow the sample to be positively identified and linked to
any previous reports.
 Each form should state the patient’s name, address, hospital number, date of birth, time
and date of sampling, the requests, and any other information which might help
interpretation.
 This could include, for example, the patient’s sex, last menstrual period, ethnic origin,
drug monitoring information, such as the time of last treatment, and symptoms.
 When a sample reaches reception, the package must be opened and the samples checked
against the request forms.
 This ensures the correct patient is booked into the system and the sample is of the type
appropriate for the chosen analyses.
 Blood Collection

 Blood is a body fluid containing plasma, red blood cells (RBCs), white blood
cells, and platelets.
 Blood is specialized for performing various functions such as the transport of
nutrients and oxygen to various body organs, transportation of antibodies,
transport of waste products to kidneys, and regulation of body temperature.
 Normally, blood Ph is maintained in a narrow range of 7.35–7.45.
 Blood for biochemical investigations may be drawn from arteries, veins, or
capillaries.
 Venous blood is commonly used for the majority of biochemical investigations.
 It can be drawn from any prominent vein. Arterial blood is mostly used for blood
gas analysis.
 Blood Collection

 Radial, brachial, or femoral arteries are the most common site for
arterial blood.
 Capillary blood is collected by puncture in infants or when very
little blood is required.
 Blood is collected in various collection tubes called vacutainers
which are sterile glass tubes with a colored rubber stopper.
 Blood contains various chemical constituents such as glucose,
proteins, lipids, globulin, fibrinogen, urea, amino acids, uric acid,
creatinine, hormones, vitamins, electrolytes, etc.
 Collection of Specimen
 Many factors need to be considered when collecting lab
specimens.
 In some cases, preparation of the patient prior to the test
may be required.
 The sample volume to be collected depends upon the
number and type of tests being performed.
 Generally, 3–5 ml of blood is required for many
investigations.
 If the tests are run on automated instruments, then less
volume of blood may be sufficient.
 Procedure for Plasma Preparation

 Draw blood from the patient and pour it into a vacutainer with an
appropriate anticoagulant.
 Mix blood with anticoagulant properly and allow the tubes to stand for
10 min.
 Then, the sample is centrifuged for speed separation and packing of
cells.
 The supernatant is the plasma.
 Serum composition is the same as that of plasma except that serum lacks
fibrinogen.
 For many laboratory biochemical tests, plasma and serum both can be
used interchangeably.
 Procedure for Serum Preparation
 Draw blood from the patient.
 Select vacutainer without anticoagulant.
 Allow the vacutainer to stand for 20–30 min so that a clot is formed.
 Centrifuge the sample at 3000 rpm which affects a greater packing of
cells.
 Various cells along with clots will settle in the form of pellets at the
bottom of the tube.
 The supernatant is the serum.
 QUESTION

 A sample from a 65-year-old male arrived in the laboratory at 9 am from a well-

man clinic. The potassium is 8.5 mmol/L (range=3.5 – 5.0 mmol/l) which is
dangerously high. The sample is repeated and the same result is obtained. All other
laboratory checks have been carried out and the result is analytically valid. The
result is telephoned to the Medical Officer who reveals that the sample was taken at
4 pm the previous day by the nurse.

 (a) What is the most likely cause of the high result?

 (b) What would your recommended course of action be?
 QUALITY ASSURANCE: ANALYTICAL STAGE OF
CLINICAL CHEMISTRY TESTS

 The analytical stage covers the principle of the test method,

the reagents, standards, control materials and equipment
used, details of the test method, and quality control
procedures
Quality Assurance Programme

The QA program consists of the following components:

1. Internal Quality Control (IQC)
2. External Quality Assessment (EQA)
3. Quality Management
 Quality control
 Quality control is a set of procedures that maintain the validity of the clinical biochemical
results.
 The clinician needs to know that he or she can confidently rely on the results of any
diagnostic test leaving the laboratory.
 Thus, they can be used to support diagnoses and in the monitoring of patients in care.
INTERNAL QUALITY CONTROL
 Ideally, every test would be 100% accurate and have absolute precision.
 In practice, this ideal state can never be achieved because there are too many variables
associated with the process of analysis, including the workings of the laboratory staff, the
instrument used, the steps involved in the analytical method, the reagents used in the
method and in calibrating the instrument.
 However, it is necessary to know the accuracy and precision of any test so that its results
can be correctly interpreted when making a diagnosis or monitoring the treatment of a
patient.
Aims of Internal Quality Control (IQC)

 Ensure that test results are reliable

 Ensure that test results are reproducible
 Quality Control of daily routine work
 Selection of quality control materials
 Quality control materials are the substances used to assess the quality control of
clinical methods.
 They are usually serum containing a known quantity of the analyte in question.
 The main aim when choosing a material to use for internal quality assurance
procedures is to have one that reacts in the same way as the serum or urine of a
patient.
 Most major quality control manufacturers will provide, whenever possible, material
based on a human matrix.
 Whatever, the QC material, it should be treated in exactly the same way as would a
patient sample.
 For qualitative assays, positive and negative QCs should be used with the negative
control set just below the cut-off value for the assay.
 Levy-Jennings or Shewart chart

 An assay is in control when results are distributed either side of, but close to the
mean.
 Values within +/-2 SD are generally regarded as acceptable.
 The presentation of QC results in this way makes it possible to see developing
trends, such as sudden shifts in precision or accuracy, relatively easily.
 Any change from this pattern, such as a series of results that are evenly distributed
but extend to +/- 3 SDs would result from a decrease in precision, and therefore
indicate a deterioration in the performance of the test that requires investigation.
 When six or more results show a consecutive move in the same direction, whether
up or down from the mean, it is called a trend. Trends are suggestive of a
developing problem, such as a deterioration in the quality of the reagents used in
the test or in the QC material, or may indicate the analytical instrument requires
maintenance or has laundry problems.
 Levy-Jennings or Shewart chart
 If six or more results occur on one side of the mean, rather than scattered about it as
in a trend, then this is called a shift.
 Shifts can be positive or negative depending on whether they occur above or below
the mean respectively.
 In both cases, they indicate a change in accuracy of the assay.
 This may be caused by one of a number of factors, including changing a calibrator
or a reagent by reconstituting them incorrectly, using materials from a different
batch.
 Changes in the temperature at which the analysis is performed or simply due to a
fault in the analytical
 instrument, such as a wrongly calibrated pipettor or faulty lamp in the
spectrophotometer can also be causes.
 Multirules RULE!
 The “multirule” procedure was developed by Westgard and Groth to further judge
whether control results indicate out-of-control situations.
 These rules established a criterion for judging whether an analytic process is out of
control.
 To simplify the various control rules, Westgard and Groth used abbreviations for the
various control rules .
 Control rules indicate the number of control observations per analytic run, followed by
the control amount in subscript.
 For example, the 12s rule indicates that a data point cannot exceed 2s more than once. If
a method is in control, ideally none of the control rules should be violated and the
analytic run will not be rejected
 MULTI-RULE PROCEDURES
 12s One control observation exceeding the mean 2s. A warning rule that initiates
testing of control data by other rules. •
 13s One control observation exceeding the mean 3s. Allows high sensitivity to
random error.
 22s Two control observations consecutively exceeding the same 2s . Allows high
sensitivity to systemic error.
 R4s One control exceeding the 2s and another exceeding the 2s. Allows detection of
random error. •
 41s Four consecutive control observations exceeding 1s or 1s. This allows the
detection of systemic error. •
 10x Ten consecutive control observations falling on one side or the other of the mean
(no requirement for SD size). This allows the detection of systemic error.
WESTGARD RULES

 12s One control observation

exceeding the mean 2s. A
warning rule that initiates
testing of control data by other
rules.
WESTGARD RULES

 22s Two control observations

consecutively exceeding the
same 2s . Allows high
sensitivity to systemic error.
WESTGARD RULES

 13s One control observation

exceeding the mean 3s.
Allows high sensitivity to
random error.
WESTGARD RULES

 41s Four consecutive control

observations exceeding 1s or
1s. This allows the detection
of systemic error.
WESTGARD RULES

 R4s One control exceeding the

2s and another exceeding the
2s. Allows detection of random
error.
WESTGARD RULES

 10x Ten consecutive control

observations falling on one side
or the other of the mean (no
requirement for SD size). This
allows the detection of systemic
error.
 External quality control
 The purpose of an external quality control is to assess the performance of a
laboratory, compare it to that of other laboratories that use the same analyser
and method, and against other methods/analysers measuring the same
analyte.

 The information gained from participation in such a scheme gives assurance

that every laboratory achieves the same result as those using the same
methodology and also indicates whether the method applied in one laboratory
has a bias or reproducibility problem.
 Reproducibility of laboratory estimations
 Most laboratory estimations should give results that are reproducible to well within 5
per cent; some, such as those for sodium and calcium, should be even more precise, but
the variability of some hormone assays, for example, may be greater. Small changes in
results produced by relatively imprecise methods are not likely to be clinically
significant.
 Imprecision is the term used to describe the random changes that reduce the agreement
between replicate assay measurements.

 This can be considered in terms of the within-assay precision, which is the assay
variability when the same material is assayed repeatedly within the same assay batch, or
day-to-day precision, which is the variability when the same material is assayed on
different days.
 Reproducibility of laboratory estimations

 The assay coefficient of variation (CV) is used to express

imprecision and can be calculated by the following equation:

 CV% =

 Reproducibility is the degree of consensus between successive

measurements achieved on the same sample with different
conditions (e.g. different analyzer, different user, and different lot of
reagents) in a long time
 QUESTION

 Calculate the mean, SD, and CV for the data shown below, which
are a range of results obtained for the concentrations of oestradiol in
pmol/L obtained using a new analytical method.
240, 260, 230, 280, 240, 220, 240, 230, 260, 250.
 Plot the levy-jenning’s control chart and state whether any westgard
rule is violated.
 ACCURACY
 Accuracy is the Closeness of the agreement between the result of a
measurement and the true value of the measured. Or
 Describes how a given test result is close to the provided true value.
 Usually expressed in the same units as the result.
 The closeness of measurements to the true value is indicative of the
“accuracy "of the assay.
 Reference samples with known values are needed to check accuracy.
 Which procedure is the most ACCURATE,
given the data below ?
 A hemoglobin reference standard has a known value of 15.0 g/dl. When tested in
two procedures, the following values are obtained for the hemoglobin
concentration:

PROCEDURE A 14.2 g/dl

PROCEDURE B 15.5 g/dl

 PRECISION

 Precision is the Closeness of agreement between the measured values

obtained by replicate measurements of an analyte under specified
conditions. Or

 Describes how a test results are close to one another or to the

calculated mean (x) when repeated analysis of the same material is
performed.
 Repeated measurements for the Red Cell Count on a patient
sample were as follows for two automated methods

INSTRUMENT 1 INSTRUMENT 2
 4.1  6.2
 4.4

 4.2
4.3
 4.2  5.3
 6.2
 which machine appears
to be most precise?
Types of laboratory errors and mistakes

 Errors-Non-conforming results with “statistical

meaning”. This category includes all the “wrong”
laboratory measures due to non-human action.

 Mistakes -Non-conforming results with “no statistical

meaning”. This category contains all the human errors e.g.
mixing up samples
ERRORS AND MISTAKES
 Another classification of errors and mistakes is based on
the time and the stage they appeared in laboratory practice.

 The pre-analytical errors that involve all the errors happen

before the analysis of the patients’ samples.

 The analytical errors that include all the errors, takes

place during the analytical process of the patient’s
samples.
ERRORS AND MISTAKES

 The post-analytical errors refer to reading and transmission of the

patient’s results from analyzers to the results record, validation of
results that have been produced and posting of the results to
physicians or patients.

 The majority of pre-analytical and post-analytical outliers are

“mistakes” in contrary to analytical outliers that are considered as
“errors”.
 Types of Laboratory Errors

There are three major errors that may occur in a laboratory.

 They are: Random, systematic, and Gross errors or total analytical error
RANDOM ERRORS:
 Random errors are the errors that arise due to statistical fluctuations in the
observations and lead to inconsistent measurement values of a constant attribute.
 A random error is associated with the fact that when a measurement is repeated, it
will generally provide a measured value that is different from the previous value.
 Random errors are caused by uncontrollable variables, which cannot be defined or
eliminated. These are errors that may arise due to bubbles in reagents or reagent lines,
instrument instability, temperature variations, and operator variability, such as
variation in pipetting.
 SYSTEMATIC ERROR
 Systematic errors cause inaccurate results that are consistently low or
high.

 This error is reproducible and predictable and can be easily identified and
corrected.

 These errors are caused by insufficient control of analytical variables, e.g.,

impure calibration material, change in reagent lot, change in calibration,
assigning the wrong calibrator values, improperly prepared or
deteriorating reagents, etc.
 Systemic errors arise due to three factors:
Instrument errors:
 Instrument errors are errors associated with instrument functioning.
 These arise due to power fluctuations, defects in any parts of the instrument,
temperature variation, or when the instrument is not calibrated.
 The instrumental errors can be removed by proper calibration or maintenance of
the instrument.
Method errors:
 These are errors that arise due to the use of non-ideal physical or chemical
methods. For example, the speed of reaction, problems associated with sampling,
and interference from side reactions can lead to such errors.
 The development and use of the proper method can help to correct these errors.
Systemic errors arise due to three factors:

Personal errors:
 These are caused by an observer’s personal habits or
mental judgment, a wrong judgment of dimensional
values, color acuity problems, etc.
 It may be accidental or systematic. Proper training and
experience can help to eliminate personal errors
effectively.
GROSS ERRORS OR TOTAL ANALYTICAL ERROR

 Such errors arise due to equipment failure or observer’s

carelessness.
 LABORATORY ERRORS
 The laboratory errors may also be grouped into pre-analytical, analytical, or post-
analytical errors according to the time of occurrence.
 Pre-analytical errors arise before the analysis of the sample takes place.
 Common examples of pre-analytical errors are a mismatch of sample and
laboratory data.
 The analytical errors occur during analytical methods and include errors related to
expired or spoiled reagents, use of controls or calibrators that have expired,
sampling errors, and changes in the analyzer’s measuring unit.
 Post-analytical errors arise during the transmission of data from analyzers, result
validation, and dispatching/communicating results to physicians or patients.
 Common post-analytical errors are loss of the results, inappropriate specimen or
anticoagulant, error in the storage of the sample, or mistakes in patients’
identification.
 Common Pre-Analytical Errors

 Inappropriate specimen (e.g. wrong specimen-anticoagulant

ratio)

 Wrong anticoagulant (e.g. sodium citrate in place of EDTA)

 Improper conservation method

 Inappropriate patient’s preparation (e.g. wrong diet)

 Mistakes in patients’ identification

 Common Analytical Errors
 Expired reagents which may lead to erroneous results.
 Expired controls or calibrators.
 Incorrect pipetting of patient’s sample or reagents.
 Changes in analyzer’s photometric unit / flow cell /
measuring unit
 Analytical errors influence the repeatability,
reproducibility, precision and accuracy of the
analytical methods.
 Common Post-Analytical Errors
 Wrong matching between sample and laboratory’s
files.

 Wrong copy of results from the analyzer’s report to the

laboratory report (in cases of manual transfer),

 Delay in delivering the results to the physicians,

clinics or patients.

 Loss of the results, 5. Incorrect result reporting or

writing,
 QUALITY ASSURANCE: POST-ANALYTICAL STAGE OF
CLINICAL CHEMISTRY TESTS
 The post-analytical stage includes the reporting, checking (verifying),
timeliness, and interpretation of test results and procedure to follow should
a result be seriously abnormal or unexpected. Clinical chemistry test reports
must include the following:
 Type of specimen analyzed, e.g. whole blood, serum, plasma, urine, c.s.f.,
faeces.
 Analyte (substance) measured.
 Test results clearly and informatively presented using SI units (with
conversion factors to former units if needed).
 Reference range for quantitative tests.
 INTERPRETING RESULTS

 When interpreting laboratory results, the Lab Scientist should ask the following
questions:
 Is the result the correct one for the patient?
 Does the result fit with the clinical findings?
 If it is the first time the test has been performed on this patient, is the result normal when
the appropriate reference range is taken into account?
 If the result is abnormal, is the abnormality of diagnostic significance or is it a non-
specific finding?
 If it is one of a series of results, has there been a change and, if so, is this change clinically
significant?
 Abnormal results, particularly if unexpected and indicating the need for clinical
intervention, are best repeated.
 Test reference ranges
 By convention, a reference (‘normal’) range (or interval) usually includes 95 per cent of
the test results obtained
from a healthy and sometimes age- and sex-defined population.
 For the majority of tests, the individual’s results for any constituent are distributed around
this mean in a ‘normal’ (Gaussian) distribution, the 95 per cent limits being about two
standard deviations from the mean. For other tests, the reference distribution may be
skewed, either to the right or to the left, around the population median.
 All that can be said with certainty is that the probability that a result is abnormal increases
the further it is from the mean.
 There is a 5 per cent chance that one result will fall outside the reference range.
 No result of any investigation should be interpreted without consulting the reference range
issued by the laboratory carrying out the assay. Some analytes have risk limits for
treatment, such as plasma glucose or target or therapeutic limits, such aa plasma
cholesterol.
GAUSSIAN DISTRIBUTION CURVE
 Physiological factors such as the following affect the
interpretation of results.
Age-related differences
 These include, for example, bilirubin in the neonate and plasma alkaline phosphatase
activity, which is higher in children and the elderly.
Sex-related differences
 Examples of sex-related differences include plasma urate, which is higher in males,
and high-density lipoprotein cholesterol, which is higher in premenopausal women
than in men.
 Obviously, sex hormone concentrations also differ between the sexes
Ethnic differences
 These may occur because of either racial or environmental factors, for example
plasma creatine kinase may be higher in black than in white people.
 QUESTION
 A blood sample from a person with abdominal pain was sent to the laboratory
from an accident and emergency department. Some of the results were as
follows:
Plasma
 Bilirubin 14 μmol/L (< 20)
 Alanine transaminase 14 U/L (< 42)
 Alkaline phosphatase 326 U/L (< 250)
 Albumin 40 g/L (35–45)
 g-Glutamyl transferase 14 U/L (< 55)
 Albumin-adjusted calcium 2.34 mmol/L (2.15–2.55)
COMMENT ON THE RESULT
 Clinical sensitivity and specificity

 Diagnostic sensitivity is a measure of the frequency of a test being positive when a

particular disease is present, that is, the percentage of true-positive (TP) results.

 Diagnostic specificity is a measure of the frequency of a test being negative when a

certain disease is absent, that is, the percentage of true-negative (TN) results.

 Ideally, a test would have 100 per cent specificity and 100 per cent sensitivity.
 In numerical terms, clinical sensitivity is defined as the number of samples that give a
true positive (TP) result, divided by the number of expected positives, which is the
number of TP plus the values of false negative (FN) results, expressed as a percentage,
that is:
 Clinical sensitivity = TP × 100 / TP + FN
 Clinical sensitivity and specificity
 Clinical specificity can also be expressed as a percent value. It can be calculated by
dividing the number of measured true negative (TN) results by the number of expected
negatives, which is the number of TN, plus the number of false positives (FP), which is
again, multiplied by 100:
 Clinical specificity = TN × 100 / TN + FP

 An assay may be excellent at detecting all of those who have the disease under
investigation but if it also has a high rate of FPs, this will lead to many patients being
erroneously classified as having the disease.
 This, of course, results in anxiety to the patients and further investigations by the clinical
laboratory, both of which are unnecessary.
 In a similar manner, an assay which has a high rate of FNs will not detect all of those
who have the disease.
 Positive and negative predictive values

 The predictive value of a negative result is the percentage of all negative results
that are true negatives, that is, the frequency of subjects without the disorder in
all subjects with negative test results.

 A high negative predictive value is important in screening programmes, if

affected individuals are not to be missed.

 This can be expressed as

 The predictive value of a positive result is the percentage of all positive results
that are true positives: in other words, the proportion of screening tests that are
correct.
 Positive and negative predictive values
 A high positive predictive value is important to minimize the number of
false-positive individuals being treated unnecessarily.

 This can be expressed as:

 The overall efficiency of a test is the percentage of patients correctly

classified by the test.

 This should be as high as possible and can be expressed as:

 QUESTION

 One hundred patients with chest pain were screened with a new biochemical test that
showed 80 to be positive for chest pain. What is the test’s sensitivity?
Answer: 80/100 X 100% = 80%

 The same test was used on 100 patients without chest pain, and 95 had a negative
screening result. What is the test’s specificity?

 Answer: 95/100 X 100% = 95%

 QUESTION

 A new test is developed for the detection of coronavirus. When it

is tested in a group of 113 patients with coronavirus, 79 have a
positive test. In a group of 217 individuals without
coronavirus,10 have a positive test.
 What is the specificity and sensitivity of the test?
 QUESTION
 Ovarian cancer has an incidence of approximately 0.2% in the normal female population. A new tumour
marker assay, called Q, for use in detecting ovarian cancer has been developed. A controlled clinical trial
involving 100,000 females to evaluate its effectiveness is given in the table below.
 (a) What is the positive predictive value of assay Q?
 (b) What is the negative predictive value of assay Q?
 What is the overall efficiency of the test?
 (c) Comment on your findings.
Disease present

YES (DISEASE PRESENT) NO (NO DISEASE)

POSITIVE TEST 196 (TP) 6986 (FP)

NEGATIVE TEST 4 (FN) 92814 (TN)

 Point-of-care Testing (POCT)

 Also known as near-patient testing, alternate-site testing or

patient-focused testing

 Used in emergency dept., operating suites, clinics, health

maintenance organization (HMO), physicians, offices &
nursing homes

 Addresses acute patient needs

 Instrumentation includes portable chemistry analyzers,

glucometers, BG Analyzers, hemoglobin meters & coagulation
testing
 SIDE ROOM TESTING/POINT OF CARE TESTING/ NEAR PATIENT TESTING

 Refers tests performed outside the hospital

laboratory by untrained healthcare professionals
or non-laboratory personnel.

 Bedside
 Clinic
 By patients themselves at home
 SIDE ROOM TESTING/POINT OF CARE TESTING

There are four strands to POCT, which differ in who performs

the test and where the test is performed. Thus tests may be
performed:
 outside the laboratory but by laboratory staff
 outside the laboratory but by nursing and medical staff
 outside the laboratory by the patient
 in pharmacies, supermarkets, internet vendors
 CATEGORIZATION

Simple side-room tests: qualitative or semi-quantitative:

mostly performed on urine or. eg. urine dipsticks tests

Simple side-room test, semi-quantitative or quantitative:

mostly performed on blood specimens. e.g. Glucometers

Quantitative tests performed with equipment that have

microprocessor-controlled operations. can be quickly and
reliably operated after little instructions.
 ADVANTAGES OF SRT/NPT

 Turn Around Time- Relatively short analysis time.

 Early treatment and shorten the patient wait .

 Ease of use– can be perform by less trained personnel or
by the patients themselves

 Prompter stabilization of life-threatening crises (eg drug

overdose)
 ADVANTAGES OF SRT/NPT

 Closer therapeutic management (e.g. diabetes)

 Better patient compliance with therapy (diabetes, hyperlipidaemia)

 Reduce:
– Repeat clinic/patient visits
– Length of stay in hospital
– Use of blood products (implantable biosensors)
 DISADVANTAGES OF SRT/NPT

 Analytical performance can be inferior to lab (eg. some

glucometers)

 Risk of poor operator competence

 Risk of poor equipment maintenance

 Cost per test relatively more expensive.

 SETTINGS FOR NPT DEVICES

ACCIDENT & EMERGENCY

 Quick turnaround time for results

e.g. Diagnosis of acute MI- whole blood troponin NPT device.

Drug overdoses- plasma p’mol, cocaine
 SETTINGS FOR NPT DEVICES

 DRUG ADDICTION CLINICS

 measure misused drugs and alcohol( Alcohol breath test)
 screen workers for substance abuse.

Roche Diagnostics– for qualitative testing for ethanol in

either saliva or urine.
 SETTINGS FOR NPT DEVICES

 NEONATAL CARE AND ADULT INTENSIVE CARE

Neonatal units: determination of blood bilirubin using
NPT bilirubinometers.

 PATIENT SELF-TESTING
eg. Pregnancy self-testing using over-the-counter
pregnancy test kits
 NEAR PATIENT TESTING & DIABETES MELLITUS

NPT self monitoring is often used in the management of

diabetes mellitus.

 glucose determinations in urine

 ketones in urine or plasma
 blood glucose measurements
 urinary microalbumin tests