INTRODUCTION, ROLES OF BIOCHEMICAL

LABORATORY. MECHANIZATION AND

AUTOMATION IN CLINICAL

BIOCHEMISTRYCLINICAL

BIOCHEMICAL ANALYSES OF

PROTEINS, PLASMA PROTEIN SPECTRUM.

Introduction to Clinical biochemistry

• CLINICAL BIOCHEMISTRY (also known as clinical chemistry or chemical pathology) is the laboratory service absolutely essential for medical practice or branch of laboratory medicine in which chemical and biochemical methods are applied to the study of disease.• The results of the biochemical investigations carried out in a clinical

chemistry laboratory will help the clinicians to determine the diseases (diagnosis) and for follow-up of the treatment/recovery from the illness (prognosis).

Introduction to Clinical biochemistry

Introduction to Clinical biochemistry

• The use of biochemical tests:• Biochemical investigations are involved in every branch of clinical

medicine. • The results of biochemical tests may be of use in:• diagnosis and in the monitoring of treatment. • screening for disease or in assesing the prognosis. • reseach into the biochemical basis of disease • clinical trials of new drugs • Biochemical investigations hold the key for the diagnosis and prognosis of

diabetes mellitus, jaundice, myocardial infarction, gout, pancreatitis, rickets, cancers, acid-base imbalance etc. Successful medical practice is unimaginable without the service of clinical biochemistry laboratory.

Introduction to Clinical biochemistry

• In general, biochemical tests can be broadly divided into two groups: • In discretionary or selective requesting, the tests are carried out on the

basis of an individual patient's clinical situation. The case for discretionary requesting has been put admirably (Asher, 1954):• 1. Why do I request this test?• 2. What will I look for in the result?• 3. If I find what I am looking for, will it affect my diagnosis?• 4. How will this investigation affect my manage ment of the patient?• 5. Will this investigation ultimately benefit the patient?• In contrast, screening tests are used to search for disease without there

being any necessary clinical indication that disease is present.

Introduction to Clinical biochemistry

Test selection for the purposes of discretionary testingCategory Example

To confirm a diagnosis Plasma (free T4) and (thyroid-stimulating hormone, TSH) in suspected hyperthyroidism

To aid differential diagnosis To distinguish between different forms of jaundice

To refine a diagnosis Use of ACTH to localize Cushing's syndrome

To asses the severity of disease Plasma (creatinine) or (urea) in renal disease

To monitor progress Plasma (glucose) to follow of patients with diabetes mellitus

To detect complications or side effects ALT measurements in patients treated with hepatotoxic drug

To monitor therapy Plasma drug concentration in patients treated with antiepileptic drugs

Introduction to Clinical biochemistry

• Table 1.4 Examples of tests used in case-finding programmes.Programmes to detect diseases in Chemical investigations

Neonates: PKA (phenylketonuria) Serum [phenylalanine]

Hypothyroidism Serum [TSH] and/or [thyroxine]Adolescents and young adults: Substance abuse

Drug screen

Pregnancy: Diabetes mellitus in the mother

Plasma and urine [glucose]

Open neural tube defect (NTD) in the foetus

Maternal serum [a-fetoprotein]

Industry:

Industrial exposure to lead

Blood [lead]

Industrial exposure to pesticides Plasma cholinesterase activityMalnutrition Plasma [albumin] and/or [pre-albumin]

Thyroid dysfunction Plasma [TSH] and/or [thyroxine]

Introduction to Clinical biochemistry

• ADVANTAGES OF SCREENING• First, an uncommon or unexpected disease may be found and

created. Second, the early requesting of a battery of tests might be expected to expedite management of the patient. Most studies have not shown this to be so.

Disease Unexpected abnormal test results

Hyperparathyroidism Raised plasma calcium

Hypothyroidism Raised plasma TSH and/or a low T4

Diabetes mellitus High random plasma glucose

Renal tract disease Raised plasma creatinine or urea

Liver disease Increased plasma ALT, AST

Introduction to Clinical biochemistry

• DISADVANTAGES OF SCREENING• It is easy to miss significant abnormalities in the 'flood' of data coming from the

laboratory, even when the abnormalities are 'flagged' in some way. Most of the abnormalities detected will be of little or no significance, yet may need additional time-consuming and often expensive tests to clarify their importance (or lack of it).

• In other instances, to simplify requesting, a wide range of tests are routinely requested on all patients in a particular category, for example, admission screening on all those admitted through the Accident and Emergency (A&E) Department. Mention should also be made of batteries of tests which are generally requested on a discre tionary basis but where the test group collectively provides information about an organ system (e.g. tests for liver disease) or a physiological state (e.g. water and electrolyte status). Many laboratories analyse and report these functional or organ-related groups. For example, a 'liver function test' group might consist of plasma bilirubin, alanine aminotransferase (ALT), alkaline phosphatase (ALP), f-glutamyltransferase (GGT) and albumin measurements.

Introduction to Clinical biochemistry

Introduction to Clinical biochemistry

• Clinical biochemical tests comprise over ⅓ of all hospital laboratory investigations.• • Core biochemistry: Most biochemistry laboratories provide the "core analyses", commonly

requested tests which are of value in many patients, on a frequent basis.• Core biochemical tests:• Sodium, potassium, chloride and bicarbonate• Urea and creatinine• Calcium and phosphate• Total protein and albumin• Bilirubin and alkaline phosphatase• Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST)• Glucose• Amylase…….

Introduction to Clinical biochemistry• Specialized tests:• Not every laboratory is equiped to carry out all possible biochemistry requests.• Large departments may act as reference centres where less commonly asked for tests

are performed. • Specialized tests:• Hormones• Specific proteins• Trace elements• Vitamins• Drugs• Lipids and lipoproteins• DNA analyses

Introduction to Clinical biochemistry• The emergency lab:• All clinical biochemistry laboratories provide facilities for urgent tests.

An urgent test is designated as one on which the clinician is likely to take immediate action. The main reason for asking for an analysis to be performed on an urgent basis is that immediate treatment depends on the result.

Introduction to Clinical biochemistry• TYPES OF LABORATORY TESTS:• The biochemical investigations (on blood/ plasma/serum) carried out in the

clinical biochemistry laboratory may be grouped into different types.• 1. Discretionary or on-off tests : Most common clinical biochemistry tests that

are designed to answer specific questions, e.g., does the patient have increased blood urea/glucose concentration? Normally, these tests are useful to support the diagnosis.• 2. Biochemical profiles : These tests are based on the fact that more useful

information on the patients disease status can be obtained by analysing zor more constituents rather than one e.g., plasma electrolytes (Na+, K+, Cl-, bicarbonate, urea); liver function tests (serum bilirubin, ALT, AST).• 3. Dynamic function tests : These tests are designed to measure the body's

response to external stimulus e.g., oral glucose tolerance test (to assess glucose homeostasis) : bromosulphthfein test (to assess liver function).

Introduction to Clinical biochemistry• 4. Screening tests : These tests are commonly employed to identify

the inborn errors of metabolism, and to check the entery of toxic agents (pesticides, lead, mercury) into the body.• 5. Metabolic work-up tests : The programmed intensive

investigations carried out to identify the endocrinological disorders come under this category.• The term emergency tests is frequently used in the clinical

laboratory. It refers to the tests to be performed immediately to help the clinician for proper treatment of the patient e.g., blood glucose, urea, serum electrolytes.

Introduction to Clinical biochemistry• There are over 400 different tests which may be carried out in clinical

biochemistry laboratories. They vary from the very simple, such as the measurement of sodium, to the highly complex, such as DNA analysis, screening for drugs, or differentiation of lipoprotein variants. Many high volume tests are done on large automated machines. Less frequently performed tests may be conveniently carried out by using commercially prepared reagents packaged in "kit" form. Some analyses are carried out manually.

Introduction to Clinical biochemistry• Specimen collection:• The biological fluids employed in the clinical biochemistry laboratory

include blood, urine, saliva, sputum, faeces, tissue and cells, cerebrospinal fluid, peritoneal fluid, synovial fluid, pleural fluid, stones.• Among these, blood (directly or in the form of plasma or serum) is

frequently used for the investigations in the clinical biochemistry laboratory.

Introduction to Clinical biochemistry• Identification of patients and specimens• The correct patient must be appropriately iden tified on the specimen and

request form, as follows:• 1. Patient identification data (PID). This usually comprises name plus

unique number.• 2. Test request information. This includes relevant clinical details

(including any risk of infection hazard), the tests to be performed and where the report is to be sent.• 3. Collection of specimens. In the correct tube and the appropriate

preservative.• 4. Matching of specimens to requests. Each specimen must be easily and

unequivocally matched to the corresponding request for investigations.

Introduction to Clinical biochemistry• Some commoner causes of errors arising from use of the laboratory.

Error Consequence

Crossover of addressograph labels between patients

This can lead to two patients each with the other's set of results. Labels between patients Where the patient is assigned a completely wrong set of results, it is important to investigate the problem in case there is a second patient with a corresponding wrong set of results

Timing error There are many examples where timing is important but not considered. Sending in a blood sample too early after the administration of a drug can lead to misleadingly high values in therapeutic monitoring. Interpretation of some tests (e.g. cortisol) is critically dependent on the time of day when the blood was sampled

Sample collection tube error For some tests the nature of the collection tube is critical which is why the Biochemistry Laboratory specifies this detail. For example, using a plasma tube with lithium-heparin as the anticoagulant invalidates this sample tube for measurement of a therapeutic lithium level! Serum electrophoresis requires a serum sample; otherwise, the fibrinogen interferes with the detection of any monoclonal bands. Topping up a biochemistry tube with a haematology (potassium-ethylenediamine tetraacetic acid (EDTA) sample) will lead to high potassium and low calcium values in the biochemistry sample

Sample taken from close to the site of an intravenous infusion

The blood sample will be diluted so that all the tests will be correspondingly site of an intravenous (IV) infusion low with the exception of those tests which might be affected by the composition of the infusion fluid itself. For example, using normal saline as the infusing fluid would lead to a lowering of all test results but with sodium and chloride results which are likely to be raised

Analytical error Although comparatively rare, these do inevitably happen from time to time and any result which is unexpected should lead the requesting clinician to discuss the matter further with the Laboratory. Transcription errors within the Laboratory are increasingly less common because of the electronic download of results to the Laboratory computer as a source of the printout or results on the VDU. Most errors generated within the Laboratory occur at the Reception as a result of mislabelling of samples within the Laboratory

Introduction to Clinical biochemistry• COLLECTION OF BLOOD:• Venous blood is most commonly used for a majority of biochemical

investigations. It can be drawn from any prominent vein (usually from a vein on the front of the elbow). • Capillary blood (<0.2 ml) obtained from a finger or thumb, is less

frequently employed. • Arterial blood (usually drawn under local anesthesia) is used for blood gas

determinations.• • Precautions for blood collection : Use of sterile (preferably disposable)

needles and syringes, cleaning of patients skin, blood collection in clean and dry vials/tubes are some of the important precautions.

Introduction to Clinical biochemistry• Biochemical investigations can be performed on 4 types of blood

specimens – whole blood, plasma, serum and red blood cells. The selection of the specimen depends on the parameter to be estimated. • 1. Whole blood (usually mixed with an anticoagulant) is used for the

estimation of hemoglobin, carboxyhemoglobin, pH, glucose, urea, non-protein nitrogen, pyruvate, lactate, ammonia etc. (Note : for glucose determination, plasma is prefered in recent years).• 2. Plasma, obtained by centrifuging the whole blood collected with an

anticoagulant, is employed for the parameters—fibrinogen, glucose, bicarbonate, chloride, ascorbic acid etc.

Introduction to Clinical biochemistry• 3. Serum is the supernatant fluid that can be collected after

centrifuging the clotted blood. It is the most frequently used specimen in the clinical biochemistry laboratory. The parameters estimated in serum include proteins (albumin/globulins), creatinine, bilirubin, cholesterol, uric acid, electroylets (Na+, K+, Cl-), enzymes (ALT, AST, LDH, CK, ALP, ACP, amylase, lipase) and vitamins.• 4. Red blood cells are employed for the determination of abnormal

hemoglobins, glucose 6-phosphate dehydrogenase, pyruvate kinase etc.

Introduction to Clinical biochemistry

Introduction to Clinical biochemistry• Collection and preservation of blood specimens• Lack of thought before collecting specimens or carelessness in collection may adversely

affect the interpretation or impair the validity of the tests carried out on the specimens. Some factors to consider include the following:

• 1. Diet Dietary constituents may alter the concen trations of analytes in blood significantly (e.g. plasma [glucose] and [triglyceride] are affected by carbohydrate and fat-containing meals, respectively).

• 2. Drugs Many drugs influence the chemical compo sition of blood. Such effects of drug treatment, for example, antiepileptic drugs, have to be taken into account when interpreting test results. Details of rel evant drug treatment must be given when request ing chemical analyses, especially when toxicological investigations are to be performed.

• 3. Diurnal variation. The concentrations of many substances in blood vary considerably at different times of day (e.g. cortisol). Specimens for these analyses must be collected at the times specified by the laboratory, as there may be no reference ranges relating to their concentrations in blood at other times

Introduction to Clinical biochemistry• Care when collection blood specimens• The posture of the patient, the choice of skin-cleansing agent and the

selection of a suitable vien (or other source) are the principal factors to con sider before proceeding to collect each specimen:• 1. The skin must be clean over the site for collecti ng the blood specimen.

However, it must be remembered that alconol and methylated spir its can cause haemolysis, and that their use is clearly to be avoided if blood [ethanol] is to be determined.• 2. Limbs into which intravenous infusions are being given must not be

selected as the site of venepuncture unless particular care is taken. The needle or cannula must first be thoroughly flushed out with blood to avoid dilution of the specimen with infusion fluid.

Introduction to Clinical biochemistry• 3. Venepuncture technique should be standardised as far as possible to enable

closer comparison of successive results on patients.• 4. Venous blood specimens should be obtained with minimal stasis Prolonged

stasis can markedly raise the concentrations of plasma proteins and other non-diffusible substances (e.g. protein-bound substances). It is advisable to release the tourniquet before withdrawing the sample of blood.• 5. Posture should be standardised if possible When a patient's posture changes

from lying to standing, there may be an increase of as much as 13% in the concentration of plasma proteins or protein-bound constituents, due to redistribution of fluid in the extracellular space.• 6. Haemolysis should be avoided, since it renders specimens unsuitable for

plasma K+, magne sium and many protein and enzyme activity mea surements.• 7. Infection hazard High-risk specimens require special care in collection, and

this danger must be clearly indicated on the request form.

Introduction to Clinical biochemistry• Care of blood specimens after collection• Blood specimens should be transported to the lab oratory as soon as

possible after collection. Special arrangements are needed for some specimens (e.g. for acid-base measurements, or unstable hormones) because of their lack of stability. Most other analytes are stable for at least 3 h in whole blood, or longer if plasma or serum is first sepa rated from the cells. As a rule, whole blood specimens for chemical analysis must not be stored in a refrigera tor, since ionic pumps that maintain electrolyte gradients across the cell membrane are inactive at low temperatures. Conversely, separated serum or plasma is best refrigerated, to minimize chemical changes or bacterial growth.

Introduction to Clinical biochemistry• Several changes occur in whole blood specimens following collection. The

commoner and more important changes that occur prior to the separation of plasma or serum from the cells are:• 1. Glucose is converted to lactate: this process is inhibited by fluoride;• 2. Several substances pass through the erythrocyte membrane, or may be

added in significant amounts to plasma as a result of red cell destruction insufficient to cause detectable haemolysis. Examples include K+ and lactate dehydrogenase;• 3. Loss of CO2 occurs, since the Pco2, of blood is much higher than in air;• 4. Plasma [phosphate] increases due to hydrolysis of organic ester

phosphates in the red cells;• 5. Labile plasma enzymes lose their activity.