Medicine

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 dise

INTRODUCTION, ROLES OF BIOCHEMICAL LABORATORY. MECHANIZATION AND AUTOMATION IN CLINICAL BIOCHEMISTRY

CLINICAL BIOCHEMICAL ANALYSES OF PROTEINS, PLASMA PROTEIN SPECTRUM. ANALYTICAL METHODS.

ANALYSES USED IN DIAGNOSTICS OF DIABETES MELLITUS.

 

 

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).

 

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:

1.     diagnosis and in the monitoring of treatment.

2.     screening for disease or in assesing the prognosis.

3.     reseach into the biochemical basis of disease

4.     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.

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 manage­ment 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.

 

The situations in which discretionary test requests are undertaken are listed in Table 1.2

 

Table 1.2 Test selection for the purposes of discretionary testing

Category

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

 

Screening may take two forms: 1. Well-population screening in which typically a spectrum of tests is carried out on individuals from an apparently healthy population in an attempt to detect presymptomatic or early disease. The value of well-population screening has been called into question and certainly should only be initiated under certain specific circumstances which are listed in Table 1.3.

Table 1.3 Requirements for well-population screening

The disease is common or life-threatening

The tests are sensitive and specific

The tests are readily applied and acceptable to the population to be screened

Clinical, laboratory and other facilities are available for follow-up

Economics of screening have been clarified and the implications accepted

 

2. Case-finding screening programmes perform appropriate tests on a population sample known to be at high risk of a particular disease.

These are inherently more selective and yield a higher proportion of useful results (Table 1.4).

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 activity

Malnutrition

Plasma [albumin] and/or [pre-albumin]

 

Thyroid dysfunction 

Plasma [TSH] and/or [thyroxine]

 

ADVANTAGES OF SCREENING

First, an uncommon or unexpected disease may be found and created (Table 1.5). 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.

 

 

 

Table 1.5 Advantages of screening in identifying unexpected test results

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

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 discre­tionary 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.

 

 

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…….

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

 

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.

Emergency tests:

Urea and electrolytes

Blood gases

Amylase

Glucose

Salicylate

Paracetamol

Calcium

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).

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.

 

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.

 

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.

Identification of patients and specimens

The correct patient must be appropriately iden­tified 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.

 

 

Table 1.1 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

 

 

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.

 

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.

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.

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 concen­trations 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 compo­sition of blood. Such effects of drug treatment, for example, antiepileptic drugs, have to be taken into account when interpreting test results. Details of rel­evant drug treatment must be given when request­ing 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

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 con­sider before proceeding to collect each specimen:

1.  The skin must be clean over the site for collect­ing the blood specimen. However, it must be remembered that alconol and methylated spir­its 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.

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+, magne­sium and many protein and enzyme activity mea­surements.

7. Infection hazard  High-risk specimens require special care in collection, and this danger must be clearly indicated on the request form.

Care of blood specimens after collection

Blood specimens should be transported to the lab­oratory 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 sepa­rated from the cells. As a rule, whole blood specimens for chemical analysis must not be stored in a refrigera­tor, 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.

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.

 

ANTICOAGULANTS

Certain biochemical tests require unclotted blood. Serum from coagulated blood is the specimen of choice for many assay systems.

Heparin (inhibits the convension prothrmobin to thrombin) is the most widely used anticoagulant for clinical chemical analysis. Heparin  is an ideal anticoagulant, since it does not cause any change in blood composition. However, other anticoagulants are prefered to heparin, due to the cost factor.

Ethylene diamine tetra acetic acid (EDTA) is a chelating agent, and is  particularly useful for hematological examination because it preserves cellular components of the blood. It chelates with calcium and blocks coagulation. EDTA is employed to collect blood for hematological examinations It may affect some of the clinical chemistry tests.

Sodium fluoride is usually used as a preservative for blood glucose by inhibiting the enzyme systems involved in the glycolysis. Without an antiglycolytic agent, the blood glucose concentration decreases about 10 mg/dl per hour and false results may be obtained. Fluoride is also anticoagulant. It should not be used for enzyme assays, as well as when the test involves enzymatic analysis.

Citrate is widely used for coagulation studies.

Oxalate inhibits blood coagulation by forming insoluble complexes with calcium ions. Potassium oxalate may be used at a concentration of 1 -2 mg/ml blood. At concentration of > 3   mg/ml, oxalate may cause hemolysis.                

Potassium or sodium oxalate : These compounds precipitate calcium and inhibit blood coagulation. Being more soluble, potassium oxalate (5-10 mg per 5 ml blood) is prefered.

Potasium oxalate and sodium fluoride : These anticoagulants are employed for collecting blood to estimate glucose. Further sodium fluoride inhibits glycolysis and preserves bfood glucose concentration.

Ammonium oxalate and potassium oxalate : A  mixture of these two compounds in the ratio 3 : 2 is used for blood collection to carry out certain hematological tests.

HEMOLOYSIS

The rupture or lysis of RBC, releasing the cellular constituents interferes with the laboratory investigations. Therefore, utmost care should be taken to avoid hemolysis when plasma or serum are used for biochemical tests. Use of dry syringes, needles and containers, allowing slow flow of blood into syringe are among the important precautions to avoid hemolysis.

PRESERVATION OF BLOOD SPECIMENS

Plasma or serum should be separated within 2 hours after blood collection. It is ideal and advisable to analyse blood, plasma or serum, immediately after the specimen collection. This however, may not be always possible. In such a case, the samples (usually plasma/serum) can be stored at 4°C until analysed. For enzyme analysis, the sample are preserved at -20°C.

 

Sampling Errors:

1. Blood sampling technique. Difficulty in obtaining a blood specimen may lead to haemolysis with consequent release  of potassium and other red cell constituents. Results for these will be falsely elevated.

2. Prolonged stasis during venepuncture.  Plasma water diffuses into the interstitial space and the serum or plasma sample obtained will be concentrated. Proteins and protein-bound components of plasma such calcium or thyroxine will be falsely elevated.

3. Insufficient specimen. Each biochemical analysis requires a certain volume of specimen to enable  the test to be carried out.

4. Errors in timing. The biggest source of error in the measurement of any analyte in a 24-hour urine specimen is in the collection of an accurately timed volume of urine.

5. Incorrect specimen container. For many analyses the blood must be collected into a container with anticoagulant and preservative. For example, samples for glucose should be collected into a special container containing fluoride which inhibits glycolysis; otherwisethe time taken to deliver the sample to the laboratory can affect the result.

6. Inappropriate sampling site. Blood samples should not be taking downstream from an intravenous drip.It is not unheard of for the laboratory to receive a blood glucose request on a specimen taken from the same arm into which 5% glucose is being infused.

7. Incorect specimen storage. A blood sample stored overnight before being sent to the laboratory will show falsely high potassium, phosphate and red cells enzymes such as lactate dehydrogenase, because of leakage into the extracellular fluid from the cells.

 

          Many hormones show circardian rhythm. For example, ACTH has maximum peak at early morning, and minimum level at afternoon. Maximum level of growth hormone is during night and minimum is in the day time. Many  reference values are age related; e.g., levels of urea and cholesterol are more in geriatric patients. Exercise will increase the level of transaminases and creatinine. Triglyceride level is to be done in fasting condition. Caffeine (coffee and tea) will increase the levels of free fatty acid, glycerol, total lipids and glucose. Smoking will increase the levels of GH, cortisol and triglycerides.

 

 

Collection of urine:

An early morning fasting specimen is generally the most concentrated specimen. Therefore, this is preferred for microscopic examination and for the detection of proteins, beta chorionic gonadotropin and other metabolites.  

Urine, containing the metabolic waste products of the body in water is the most important excretory fluid. For biochemical investigations, urine can be collected as a single specimen or for 24 hours. Single specimens of urine, normally collected in the morning, are useful for qualitative tests e.g., sugar, proteins. Twenty four hour urine collections (done between 8 AM to 8 AM) are employed for quantitative estimation of certain urinary constituents e.g., proteins, hormones, metabolites.

Depending on the test, either a random or a com­plete timed collection of urine is needed. The timed collection is obtained as follows:

1. Just before the collection period is due to start, the patient empties his/her bladder. This urine must be discarded.

2. Thereafter, from the start (e.g. at 8 am) to the end of the collection period, all urine passed by the patient must be added to the container. If this con­tains preservative, the specimen must be mixed gently each time more urine is passed and added to the collection.

3. At the end of the period (e.g. 8 am the next day, in the case of a 24-h collection), the patient emp­ties his/her bladder. This urine must be included in the collection.

4. The period over which the collection was made must be recorded and written on the specimen container and the request form.

For large volumes, an aliquot (e.g. 25 mL) may be sent to the laboratory, but the complete speci­men must first be mixed and its volume recorded on the container and the request form.

Urine specimens tend to deteriorate unless the correct preservative is added from the start, or the specimen is refrigerated throughout the collection period. The changes include:

1. destruction of glucose by bacteria;

2. conversion of urea to ammonia, by bacte­ria, with fall in [H+] and precipitation of phosphates;

3. oxidation of urobilinogen to urobilin and porphobilinogen to porphyrins.

 

Preservatives for urine : The preservatives are used (1) to reduce bacterial action; (2) to minimise chemical decomposition, and (3) to decrease atmospheric oxidation of unstable compounds. The most satisfactory form of preservation of urine specimen is to refrigerate it during the collection. Formalin, thymol,  chloroform, toluene, concentrated HCI and glacial acetic acid are the commonly used urine preservatives.

For the collection of 24 hr urine samples, preservatives have to be used or else urine undergoes changes due to bacterial action. Hydrochloric acid, toluene, light petroleum, thymol, formalin etc., are among the common preservatives used.

 

Timed Urine Specimen

Usually, urine sample is collected for the 24 hour period. This will minimise the influence of short-term biological variations and diurnal rhythms. Generally, collection of urine samples are done from 6 AM to next 6 AM.  The bladder should be emptied when the collection is started (6 AM), and this urine is discarded. Thereafter all the urine  should be collected. The next day urine is voided at 6 AM and this sample is also collected.

 

 

Cerebrospinal Fluid:

CSF is a fluid of the nervous system. It is formed by a process of selective dialysis of plasma by the choroid plexuses of the ventricles of the brain. The total volume CSF is 100-200 ml.

Collection of CSF : CSF is collected by puncturing the interspace between the 3rd and the 5th number vertabrae, under asepetic conditions and local anesthesia.

Biochemical investigations on CSF : Protein, glucose   and   chloride   estimations   are usually performed in the clinical biochemistry laboratory.

 

The interpretation of results:

Most biochemical analyses are quantitative. Many tests measure the amount of the analyte in a small volume of the sample (blood, plasma, serum, urine or some other fluid or tissue). The tests results are commonly expressed in molar units. A mole of any compound always contains 6* 1023  molecules. Describing how much of an analyte is present in moles indicates how many molecules of the substance are present. Molar units can be converted to mass units: one mole is the molecular weight of the substance in grams. Results are reported as concentrations, usually in terms of the number of moles in one litre (mol/l).

 

Molar units:

Mole

Abbreviation

Definition

Milimole

mmol

*10 -3 of a mole

Micromole

µmol

*10 -6 of a mole

Nanomole

nmol

*10 -9 of a mole

Picomole

pmol

*10 -12 of a mole

Femtomole

fmol

*10 -15 of a mole

 

Enzymes are not usually expressed in moles but as enzyme activity in "units". Large molecules such as proteins are reported as grams or milligrams. Blood gas results (PCO2 or PO2) are expressed in kilopascals (kPa), the units in which partial pressures are measured.

 

Biological factors affecting the interpretation of results:

1.     Sex of the patient. Reference ranges for some analytes such as serum creatinine are different for men and women.

2.     Age of the patient. There may be different references ranges for neonates, children, adults and the eldery.

3.     Effect of diet.The sample may be inappropriate if taken when the patient is fasting or after a meal.

4.     Time when sample was taken. There may be variations during the day and night.

5.     Stress and anxiety.

6.     Posture of the patient. Redistribution of fluid may effect the result.

7.     Effects of exercise. Strenuous exercise can release enzymes from tissues.

8.     Medical history.Infection or tissue injury can affect biochemical values independently of the disease process being investigated.

9.     Pregnancy. This alters some references ranges.

10. Menstrual cycle. Hormone measurement will vary through the menstrual cycle.

11. Drug history.

 

The clinician may well ask the following questions on receiving a biochemistry report:

1. Does the result fit in with what I expected on the basis of the clinical examination and history of the patient?

2. If the result is not what I expected, can I explain the discrepancy?

3. How can the result change my diagnosis or the way I am managing the patient?

4. What should I do next?

 

Quality control:

Quality control in clinical biochemistry laboratory refers to the reliability of investigative service. Any error in the laboratory will jeopardize the lives of patients. It is therefore utmost important that the laboratory errors are identified and rectified.

Quality control comprises of four interrelated factors namely precision, accuracy, specificity and

sensitivity.

Precision refers to the reproducibility of the result when the same sample is analysed on different occasions (replicate measurements) by the same person. For instance, the precision is good, if the blood glucose level is 78, 80 and 82 mg/dl on replicates.

Accuracy means the closeness of the estimated result to the true value e.g., if true blood urea level is 50 mg/dl, the laboratory reporting 45 mg/dl is more accurate than the one reporting 35 mg/dl.

Specificity refers to the ability of the analytical method to specifically determine a particular parameter e.g., glucose can be specifically estimated by enzymatic glucose oxidase method.

Sensitivity deals with the ability of a particular method to detect small amounts of the measured constituent.

METHODS OF QUALITY CONTROL

1. Internal quality control refers to the analysis of the same stored sample on different days in a laboratory, the results should vary within a narrow range.

2. External quality control deals with the analysis of a sample received from outside, usually from a national or regional quality control centre. The results obained are then compared.

 

Laboratory analytical performance:

A number  of terms describe biochemical results. These include:

1. Precision and accuracy

Precision is the reproducibility of an analytical method. Accuracy defines how close the measured value is to the actual value.

2. Sensitivity and spesificity

Sensitivity of an assay is a measure of how little of the analyte the method can detect. Spesificity of an assay relates to how good the assay is at discriminating between the requested analyte and potentially interfering substances.

3. Quality assurance

Every laboratory takes great pains to ensure that the methods in use continue to produce reliable results. Laboratory staff monitor performance of assays using quality control samples to give reassurance that the method is performing satisfactolily with the patients' specimens.

These are internal quality controls which are analysed every day or every time an assay is run. The expected values are known and the actual results obtained are compared with previous values to monitor performance. In external quality assurance schemes, identical samples are distributed to laboratories; results are then compared.

 

Quality control should become an integral part of the operation of a clinical chemistry laboratory. The purpose of quality control is to ensure the reliability of each measurement performed on a sample.

A central reference laboratory sends a serum sample containing known quantity of a substance; this is analysed in a peripheral (small) laboratory. If the result obtained in the peripheral lab is the same as that of the reference laboratory, then the arrangements available in the peripheral lab is said to be reliable. This is called external quality control. This type of checking is usually done once or twice in a month. Moreover, the peripheral laboratory itself makes a reference standard serum sample, and checks the results on the daily basis this is called internal quality control.

Accuracy

It is the closeness of a result to the true value. For example, if one technician performs a test on a serum which is known to contain 5,0 mmol/L glucose and obtains a result of 4.9 mmol/L.

A second technician does the same test on the same sample, and gets the result of 4.5 mmol/L. Then the value of the first technician is considered as accurate. Values farther away from the true value are less accurate than those closer.

Precision

This refers to the reproducibility of the result. If one technician performs glucose analysis on the same sample on three different occasions and obtains 4 mmol/L, 3.9 mmol/L, and 4.1 mmol/L, then the results have been reproduced very well, and the precision is very good. Precision depends on the technique, the reagents, as well as on the technician.

Specificity

Specificity of a reaction denotes that only one substance will answer that particular test. For example, in the case of glucose oxidase method, only glucose molecules are assayed. So it is a very specific method. But if the reducing property of the glucose is utilised for the assay purpose (e.g., Nelson Somogyi method), then other reducing agents in the blood will interfere in the reaction, and hence specificity is lowered.  Specificity is determined by the method of the analysis.

Sensitivity                                                  |

It indicates that whether the method could be utilised to test a very dilute solution. For example, biuret method is used for solutions having a few g of protein/dl. Spectrophotometric method is useful to detect a few mg of protein/dl, while ELISA method is employed if the solution has only microgram of protein/dl. Thus ELISA method is most sensitive. Generally speaking, as the sensitivity is increased, specificity is decreased.

Limit of Errors Allowable in Laboratory:

In a laboratory, error may not be totally avoided; but should be kept at a minimum. The limits are denoted by the term, percentage error. The percentage of allowable error in an assay is given by the formula:                           

Difference between maximum and minimum of normal range                     

—————————————————                                       x100

Mean of the normal range x 4

The percentage error, therefore, will vary from test to test. To take an example, in the case of blood glucose analysis, the normal range is 70 to 110 mg/dl. If these values are substituted in the formula:

 

(110 minus 70)

———————— x 100 = 10%

90x4

Now, in the case of blood urea (normal range of 20-40 mg/dl), the percentage error will be

(40 minus 20)

———————— x 100 = 16%

30-x4

 

 

Biochemical testing outside the laboratory:

The methods for measuring some biological compounds in blood and urine have become so robust and simple to use tht measurements can be made away from the laboratory – by the patient's bedside, in the ward, in the home or even in the shoping centre.

Tests performed away from the laboratory:

The most common blood test  outside the laboratory is the determination of glucose concentration, in a finger stab sample, at home or in the clinic. Diabetic patients who need to monitor their blood glucose on a regular basis can do so at home or at work using one of many commercially available pocket-sized instruments.

A portable bench analyser. This instrument may be used to monitor patients' glucose and cholesterol, and its frequently used in many outpatient clinics and in screening centres.

Common tests on blood performed away from the laboratory

 

Analyte

Used when investigating

A

Blood gases

Acid-base status

Glucose

Diabetes mellitus

Urea

Renal disease

Creatinine

Renal disease

Bilirubin

Neonatal jaundise

Therapeutic drugs

Compliance of toxicity

Salicylate

Detection of poisoning

Paracetamol

Detection of poisoning

Glucose

Diabetic monitoring

B

Cholesterol

Coronary heart disease risk

Alcohol

Fitness to drive/confusion, coma

C

 

Common tests on urine performed away from the laboratory

 

Analyte

Used when investigating

A

Ketones

Diabetic ketoacidosis

Protein

Renal disease

Red cells/haemoglobin

Renal disease

Bilirubin

Liver disease and jaundise

Urobilinogen

Jaundise/haemolysis

pH

Renal tubular acidosis

Glucose

Diabetes mellitus

B

hCG

Pregnancy test

 

The tests commonly performed away from the laboratory can be categorized as follows:

A.   Tests performed in medical or nursing settings.

They clearly give valuable information and allow the practitioner to reassure the patient or family, or initiate futher investigations or treatment.

B.    Tests performed in the home, shopping centre or clinical setting.

They can give valuable information when properly and appropriately used.

C.    Alcohol tests.

 

Methodology of sideroom tests

It is a feature of many sideroom tests that their simplicity disguises the use of sophisticated methodology. A home pregnancy test method involves an elegant application of monoclonal antibody technology to detect the human chorionic gonadotropin (hCG) which is produced by the developing embryo. The test is simple to carry out: a few drops of urine are placed in the sample window, and the result is shown within 5 minutes. The addition of the urine solubilizes a mono-clonal antibody for hCG which is covalently bound to tiny blue beds. A second monoclonal, specific for another region of the hCG molecule, is firmly attached in a line at the result window. If hCG is present in the sample it is bound by the first antibody, forming a blue bead- antibody-hCg complex.  As the urine diffuses through the strip, any hCG present becomes bound at the second antibody site and this concentrates the blue bead complex in a line – a positive result. A third antibody recognizes the constant region of the first antibody and binds the excess, thus providing a control to show that sufficient urine had been added to the test strip, the most likely form of error.

 

General problems

The obvious advantages in terms of time saving and convenience to both patient and clinician must be balanced by a number of possible problems in the use of these tests. They include:

1.     Cost. Many of these tests are expensive alternatives to the traditional methods used in the laboratory.

2.     Responsibility. The person performing the assay outwith the laboratory (the operator) must assume a number of responsibilities which would normally be those of the laboratory staff. There is the responsibility to perform the assay appropriately and to provide an answer that is accurate, precise and meaningful.

 

Analytical problems

The most commonly encountered analytical errors arise because of failure to:

1.     Calibrate an instrument

2.     Clean the instrument

3.     Use quality control materials

4.     Store reagents or strips in appropriate conditions

All of these problems can be readily overcome by following instructions carefully.

Point of care testing (Near-Patient Testing)

In contrast to the analysis of biochemical tests in a large hospital clinical biochemistry laboratory, it is also possible to carry out an increasing range of biochemical tests on small, bench-top analysers or using dipsticks methodology close to the patient, either at the bedside, in the clinic or surgery or even in the home.

Table 1.6 lists some of the tests which can be analysed on this basis.

Table 1.6 Examples of POCT

POCT application                                        

Example

     Side-room tests on urine and faeces

Still widely used for screening purposes in primary and secondary care. This includes the 'multistix' range of tests which include glucose, protein, ketones, bilirubin and urobilinogen, etc. The technology of dipstick testing can now also be extended to Drugs of Abuse and other tests and may have a place in an appropriate clinic setting

 

Blood gas analysers                                              

These are increasingly widely used in acute settings in hospital such as A&E, Acute Admissions Unit and Intensive Care Unit. Using appropriately trained staff and paying attention to quality issues, results are generally excellent and allow multiple sampling on fresh blood samples in acutely unwell patients or where frequent monitoring is necessary for management purposes (e.g. cardiotho-racic operations)

 

Small chemistry analysers (often providing blood gas results and common analytes   such as electrolytes, glucose, lactate bilirubin or various combinations of tests results               

 Small instruments which provide a wider repertoire of tests are  increasingly available and most blood gas analysers do now offer this wider repertoire of tests. The additional tests provided can, to   some extent, be tailored to the location of the instrument. For of test results)                                 example, a Neonatal Unit would require an instrument which deals with small volumes of samples and preferably provides electrolyte results, including calcium, as well as lactate and bilirubin.

 

Blood glucose analysers                    

 

These are widely used both in primary and secondary care and by patients themselves. Again, with appropriate training, results are accurate and compare well with corresponding main Laboratory results. From the individual patient perspective, an extended profile of glucose results allows rational adjustment of treatment to optimise blood glucose control, particularly in the insulin-dependent diabetic

 

A particularly important benefit of point of care testing (POCT) relates to the rapid availability of results to optimally manage the acutely ill patient. Examples would include the measurement of blood gases in the Intensive Care Unit to assist ventilation status or the regular monitoring of diabetic patients using blood glucose testing on the ward. Tests performed in the Out-patient or GP surgery can provide an immediacy of response allowing for reassurance or the initiation of further confirma­tory tests. Used within the home, the monitoring of blood glucose can allow the diabetic patient to make lifestyle adjustments appropriate to optimal diabetic care, as well as providing a record for informed decisions on treatment.

Point of care testing is not without its disadvan­tages. Test costs are invariably considerably higher than those undertaken by a central laboratory. When undertaken outside of the laboratory con­text, there is a risk of operator error, especially if training is inadequate. Issues also surround the requirements to maintain and calibrate instru­ments to ensure meaningful results, as well as the need to address quality control and matters of health and safety. Where it is appropriate to intro­duce point of care testing, it is critical that matters of training, analytical performance, quality con­trol, and health and safety are properly addressed and the central clinical biochemistry laboratory often has an important part to play in this aspect of point of care testing.

 

The future There is no doubt that in future, biochemical testing of patients outside the laboratory will become practical for many of the analytes currently measured in the laboratory.

 

BIOCHEMICAL INVESTIGATION OF BLOOD PLASMA PROTEINS

 

Total blood volume is about 4.5 to 5 litres.

 

 If blood is mixed with an anticoagulant and centrifuged, the cell components (RBC and WBC) are precipitated. The supernatant is called plasma. About 55-60 % of blood is made up of plasma.

If blood is withdrawn without anticoagulant and allowed to clot, after about two hours liquid portion is separated from the clot. This defibrinated plasma is called serum, which lacks coagulation factors including prothrombin and fibrinogen.

Plasma contains  about hundred of different proteins.

Total protein content of normal plasma is 6 to 8 g/ 100 ml (65-85 g/l). As a first step of study, the plasma proteins may be separated into Albumin (35-50g/l), Globulins (25-35 g/l) and fibrinogen (2-4 g/l).

The albumin : globulin ratio is usually between 1.2:1 to 1.5:1.

Almost all plasma proteins, except immunoglobulins are synthesized in liver.

In clinical laboratory, total proteins in serum or plasma of patients are estimated by Biuret method.

In clinical laboratory, electrophoresis is employed regularly for separation of serum proteins.

 The term electrophoresis refers to the movement of charged particles through an electrolyte when subjected to an electric field.

The positively charged particles (cations) move to cathode and negatively charged ones (anions) to anode. Since proteins exist as charged particles, this method is widely used for the separation of proteins in biological fluids. The technique was first used by Tiselius in 1937; named it as moving boundary or frontal electrophoresis (Nobel Prize in 1948).

Factors Affecting Electrophoresis

The rate of migration (separation of particles) during electrophoresis will depend on the following factors:

1. Net charge on the particles

2. Mass and shape of the particles.

3. The pH of the medium.

4. Strength of electrical field.

5. Properties of the supporting medium.

6. Temperature.

Types of Electrophoresis

There are mainly two types of electrophoresis—horizontal and vertical.

Different types of support media are used in horizontal electrophoresis, e.g. filter paper, cellulose acetate, agar gel, agarose gel, starch gel, etc. The vertical electrophoresis mainly uses polyacrylamide gel. The nature of the supporting medium will also influence the mobility.

Electrophoresis Apparatus

The electrophoresis system basically consists of the electrophoresis tank to hold the buffer and fitted with the electrodes, as well as a power pack to supply electricity at constant current and voltage. When the electrophoresis is carried out, the buffer is  chosen in such a way so as to ensure effective separatum of the mixture of proteins. The pH and ionic strength and nature of the buffer may be varied according to the proteins to be separated, e.g. serum proteins are separated at a  pH of 8.6 using barbitone buffer. At this pH all serum proteins will have a net negative charge and will migrate towards the anode.                                

Support Medium for Electrophoresis  

Filter Paper                                    

Cellulose Acetate Membrane

Agar or Agarose

Starch Gel

Polyacrylamide gel electrophoresis (PAGE)

Visualisation of Protein Bands

After the electrophoretic run is completed, the proteins are fixed to the solid support using a fixative such as acetone or methanol. Then it is stained by using dyes

(Amido Schwartz, naphthalene black, Ponceau S or Coomassie Blue) and then destained by using dilute acetic acid. The electrophoretogram can be scanned using a densitometer and each band quantitated. In the densitometer, light is passed through the agar gel plate; the absorption of light will be proportional to the quantity of protein present on a band. Another method is that the stain may be eluted from the support and each fraction quantitated colourimetrically.

 

Normal Values and Interpretations

In agar gel electrophoresis, normal serum will be separated

into five bands. Their relative concentrations are given

below:

Albumin                                 :  55-65%

Alpha-1 -globulin                  :     2-4%

Alpha-2-globulin                   :    6-12%

Beta-globulin                         :    8-12%

Gamma-globulin                    :   12-22%

 

 

 

 

 

ALBUMIN

The name is derived from the white precipitate formed when egg is boiled (Latin, albus = white). Albumin constitutes the major part of plasma proteins. It has one polypeptide chain with 585 amino acids and 17 disulfide bonds. It has a molecular weight of 69,000. It is synthesised by hepatocytes; therefore, albumin level in blood is decreased in liver cirrhosis. Estimation of albumin is a liver function test. Half-life of albumin is about 20 days. Liver produces about 12 g of albumin per day, representing about 25% of total hepatic protein synthesis. Albumin can come out of vascular compartment. So albumin is present in CSF and interstitial fluid. Over 80 allelic forms of albumin, all with normal function, but with altered electrophoretic mobilities are described. Albumin is coded by a gene on the long arm of chromo­some number 4.

Functions of Albumin

1. It contributes to the colloid osmotic pressure of plasma.

The total osmolality of serum is 278-305 m osmol/ kg (about 5000 mm of Hg). But this is produced mainly by salts, which can pass easily from intravascular to extravascu lar space. Therefore, the osmotic pressure exerted by electrolytes inside and outside the vascular compartments will cancel each other. But proteins cannot easily escape out of blood vessels, and therefore, proteins exert the 'effective osmotic pressure'. It is about 25 mm Hg, and 80% of it is contributed by albumin. The maintenance of blood volume is dependent on this effective osmotic pressure. According to Starling's hypothesis, at the capillary end the blood pressure (BP) or hydrostatic pressure expels water out, and effective osmotic pressure EOP) takes water into the vascular compartment. At arterial end of the capillary, BP is 35 mm Hg and EOP is 25 mm; thus water is expelled by a pressure of 10 mm Hg. At the venous end of the capillary, EOP is 25 mm and BP is 15 mm, and therefore water is imbibed with a pressure of 10 mm. Thus, the number of water molecules escaping out at arterial side will be exactly equal to those returned at the venous side and therefore blood volume remains the same. If protein concentration in serum is reduced, the EOP is correspondingly decreased. Then return of water into blood vessels is diminished, leading to accumulation of water in tissues. This is called edema. Edema is seen in conditions where albumin level in blood is less than 2g / dl.

2.         Another major function of albumin is to transport various hydrophobic substances.

3.          Being a watery medium, blood cannot solubilise lipid components. Bilirubin and non-esterified fatty acids are specifically transported by albumin. Drugs (sulpha, aspirin, salicylates, dicoumarol, phenytoin), steroid hormones, thyroxine, calcium, copper
and heavy metals are non-specifically carried by albumin. Only the unbound fraction of drugs is biologically active. The histidine residue at position 3 of albumin binds copper.

4.       All proteins have buffering capacity. Because of its high concentration in blood, albumin has maximum buffering capacity. Albumin has a total of 16 histidine residues which contribute this buffering action.

5.           All tissue cells can take up albumin by pinocytosis. It is then broken down to amino acid level. So albumin may be considered as the transport form of essential amino acids from liver to extrahepatic cells.

Clinical Applications

        1. Albumin-fatty acid complex cannot cross blood-brain barrier and hence fatty acids cannot be taken up by brain. The bilirubin from albumin maybe competitively replaced by aspirin and such other drugs. In newborns, bilirubin is already high, and if such drugs are given, there is a probability that free bilirubin is deposited in brain leading to kernicterus and mental retardation.

2.          When two drugs having high affinity to albumin are administered together, there may be competition for the available sites, with consequent displacement of one drug. Such an effect may lead to clinically significant drug interactions, e.g. phenytoin -dicoumarol interaction.

3.          Protein-bound calcium is lowered in hypo­albuminemia. Thus, even though total calcium level in blood is lowered, ionised calcium level may be normal, and so tetany may not occur. Calcium is lowered by 0.8 mg/dl for a fall of 1 g/dl of Albumin.

4.          Human albumin is therapeutically useful to treat burns, hemorrhage and shock..

Normal Value and Interpretation: Normal level of Albumin is 3.5-5 g/dl. Lowered level of albumin (hypoalbuminemia) has important clinical significance.

Hypoalbuminemia

       1.   In cirrhosis of liver and in chronic liver failure, albumin

synthesis is decreased and so blood level is lowered.

2.         In malnutrition and in malabsorption syndrome the availability of amino acids is reduced and so albumin synthesis is affected.

3.         In nephrotic syndrome, kidney glomerular filtration is defective so that albumin is excreted in large quantities. Increased loss, to a certain extent, is compensated by increased synthesis; but blood level of albumin is decreased.

4.         Presence of albumin in urine (albuminuria) is always pathological. Large quantities (several grams per day) of albumin is lost in urine in nephrotic syndrome. Small quantities are lost in urine in acute nephritis, and other inflammatory conditions of urinary tract. Detection of albumin in urine is done by heat and acetic acid test. In microalbuminuria or minimal albuminuria or pauci-albuminuria, small quantity of albumin (50-300 mg/ d) is seen in urine (Paucity = small in quantity). However, microalbuminuria is also clinically important, as it is a predictor of future renal diseases.

5.         In burns, albumin is lost through the unprotected skin surface.

6.         In protein losing enteropathy large quantities of albumin is lost through intestines.

7.         Hypoalbuminemia will result in tissue edema. It may be seen in malnutrition, where albumin synthesis is depressed {generalized edema), in nephrotic syndrome, where albumin is lost through urine {facial edema) or in cirrhosis of liver (mainly ascites). In chronic congestive cardiac failure, venous congestion will cause increased hydrostatic pressure and decreased return of water into capillaries and so pitting edema of feet may result.

8.         Albumin is a negative acute phase protein; level of albumin falls mildly in presence of inflammatory cytokines such as interleukin-6.

9.         Analbuminemia {absence of albumin) is a very rare genetically determined condition.

Albumin-Globulin Ratio: In all the above mentioned conditions of hypoalbuminemia, there will be a compen­satory increase in globulins which are synthesised by the reticuloendothelial system. Albumin-globulin ratio (A/G ratio) is thus altered or even reversed. This again leads to edema.

Hypoproteinemia: Since albumin is the major protein present in the blood, any condition causing lowering of albumin will lead to reduced total proteins in blood (hypoproteinemia). So it is observed in cirrhosis, nephrotic syndrome, malnutrition and malabsorption syndromes.

Hyper-gamma -globulinemias

        1. When  albumin  level  is decreased, body tries to compensate it by increasing the production of globulins from reticuloendothelial system. Thus, all causes for

hypoalbuminemia will result in albumin : globulin ratio reversal, and corresponding increase in percentage in globulins.

2.          In chronic infections, the gamma globulins are increased, but the increase is smooth and widebased.

3.          Drastic increase in globulins are seen in para­proteinemias, when a sharp spike is noted in electro­phoresis. This is termed as M-band. This is due to monoclonal origin of immunoglobulins in multiple myeloma.

Hyper-beta-globulinemia: It is associated with hyper­lipoproteinemia, atherosclerosis and other hyperlipidemic conditions.

Hyper-alpha-globulinemia: In nephrotic syndrome, small molecular weight proteins (including albumin) leak out through urine. But proteins with larger molecular weight remains in blood; so there is an increase in alpha globulin fraction, which contains alpha-2-macroglobulin.

TRANSPORT PROTEINS

Blood is a watery medium; so lipids and lipid soluble substances will not easily mix in the blood. Hence such molecules are carried by specific carrier proteins. Albumin is an important transport protein, which carries bilirubin, free fatty acids, calcium and drugs.

1.    Pre-albumin is so named because of its faster mobility in electrophoresis than albumin. It is more appropriately named asTransthyretin or Thyroxin binding pre-albumin (TBPA), because it carries thyroid hormones, thyroxin (T4) and tri-iodothyronine T3). It can bind loosely with all substances which are carried by albumin. Its molecular weight is lesser than that of albumin. It is rich in tryptophan. Its half-life in plasma is only one day.

2.    Retinol binding protein (RBP) carries vitamin A. It is a low molecular weight protein, and so is liable to be lost in urine. To prevent this loss, RBP is attached with pre-albumin; the complex is big and will not pass through kidney glomeruli. It is a negative acute phase protein. Zinc is required for RBP synthesis, and so RBP level and vitamin A level may be lowered in zinc deficiency.

3.  Thyroxine binding globulin (TBG) is the specific carrier molecule for thyroxine and tri-iodo thyronine. TBG level is increased in pregnancy; but decreased in nephrotic syndrome.

4.  Transcortin, otherwise known as Cortisol binding globulin (CBG) is the transport protein for Cortisol and corticosterone..

5.  Haptoglobin (for haemoglobin), Hemopexin (for heme) and Transferrin (for iron) are importantto prevent loss of iron from body.

6.  Cholesterol in blood is carried by lipoproteins, HDL and LDL varieties.

 

POLYMORPHISM

The term polymorphism is applied when the protein exists in different phenotypes in the population; but only one form is seen in a particular person. Haptoglobin, transferrin, ceruloplasmin, alpha-1-antitrypsin and immunoglobulins exhibit polymorphism. For example, Haptoglobin (Hp) exists in three forms, Hp1-1, Hp2-1, and Hp2-2. Two genes, designated Hp1 and Hp2 are responsible for these polymorphic forms. Their functional capabilities are the same. These polymorphic forms are recognised by electrophoresis or by immunological analysis. Study of polymorphism is useful for genetic and anthropological studies.

ACUTE PHASE PROTEINS

The level of certain proteins in blood may increase 50 to 1000-folds in various inflammatory and neoplastic conditions. Such proteins are acute phase proteins. Interleukins (ID, especially IL-1 and IL-6, released by macrophages and lymphocytes, are the primary agents which cause induction and release of these acute phase proteins. Important acute phase proteins are C-reactive protein, ceruloplasmin, haptoglobin,a1 -acid glycoprotein, a-1-anti-trypsin and fibrinogen.

C-reactive Protein (CRP):

It is thus named because it reacts with C-polysaccharide of capsule of pneumococci. CRP consists of five polypeptide subunits to form a disc-shaped cyclic polymer. It has a molecular weight of 115-140 kD. It is synthesised in liver. It can stimulate complement activity and macrophage phagocytosis. When the inflammation has subsided, CRP quickly falls. CRP level has a positive correlation in predicting the risk of cardiovascular disease.

Ceruloplasmin

Ceruloplasmin is blue in colour (Latin, caeruleus=blue). It is an alpha-2 globulin with molecular weight of 160,000. It is synthesised in liver. It contains 6 to 8 copper atoms per molecule. Ceruloplasmin is also called Ferroxidase, an enzyme which helps in the incorporation of iron into transferrin. Ninety per cent of copper content of plasma is bound with ceruloplasmin, and 10% with albumin. Copper is bound with albumin loosely, and so easily exchanged with tissues. Hence transport protein for copper is Albumin. Ceruloplasmin is an enzyme. It is an important antioxidant in plasma.

Clinical Application:

 Normal blood level of ceruloplasmin is 25-50 mg/dl. It is estimated either by its oxidative property on phenylene diamine, or by radial immunodiffusion. This level is reduced in Wilson's hepato­lenticular degeneration. Ceruloplasmin level less than 20 mg/dl is pathognomonic of Wilson's disease. It is an inherited autosomal recessive condition. Incidence of the disease is one in 50,000. The defect is associated with chromosome No.13. The basic defect is a mutation in a gene encoding a copper binding ATPase in cells, which is required for excretion of copper from cells. So, copper is not excreted through bile, and hence copper toxicity is seen. Increased copper content in hepatocyte inhibits the incorporation of copper to apo-ceruloplasmin. So ceruloplasmin level in blood is decreased. Accumulation in liver leads to hepatocellular degeneration andcirrhosis. Deposits in brain basal ganglia leads to lenticular degeneration and neurological symptoms. Another common finding is copper deposits as green or golden pigmented ring around cornea; this is called Kayser-Fleischer ring. Copper deposits in kidney may cause renal failure, and in bone marrow leads to hemolytic anemia. Treatment consists of a diet containing low copper and injection of D-penicillamine which excretes copper through urine. Since zinc decreases copper absorption, zinc is useful in therapy.

Lowered levels of ceruloplasmin is also seen in malnutrition, nephrosis, and cirrhosis. Ceruloplasmin is an acute phase protein. So its level in blood may be increased in all inflammatory conditions, collagen disorders and in malignancies.

Alpha-1 Anti-trypsin (AAT)

AAT is otherwise called a-anti-proteinase or protease inhibitor (Pi). It inhibits all serine proteases (proteolytic enzymes having a serine in their active centre), such as plasmin, thrombin, trypsin, chymotrypsin, elastase, and cathepsin. Serine protease inhibitors are abbreviated as Serpins. Binding of this inhibitor to protease is very tight; once bound it is not released. Normally, about 95% of the anti-protease activity in plasma is due to AAT. It is synthesised in liver. It has a molecular weight of 50,000 and has 3 polypeptide chains. It forms the bulk of moleculesjn serum having a-1 mobility. It is estimated by radial immuno-diffusion method. Normal serum level is 75-200 mg/dl. Electrophoretically, multiple allelic forms can be separated, the most common variety is PiMM determined by the genotype MM. More than 75 variants are known, out of which about 30 genetic variants show decreased or very low serum concentrations. Gene is located on the small arm of chromosome number 14.

 

AAT deficiency causes the following conditions:

1. Emphysema: The deficiency is inherited as a co-dominant trait. The incidence of AAT deficiency is 1 in 1000, and is one of the most common inborn errors. About 5% of total population carry the abnormal gene in heterozygous state. The total activity of a1-AT is reduced in these individuals. Bacterial infections in lung attract macrophages which release elastase. In the a1-AT deficiency, unopposed action of elastase will cause damage to lung tissue, leading to emphysema. About 5% of emphysema cases are due to a1-AT deficiency. The alpha-1 band on electrophoresis is reduced or absent. The methionine residue at 358 position of a1-AT is important in the enzyme binding. This methionine may be oxidised to methionine sulfoxide by smoking. So emphysema is very common in smokers with normal a1-AT level and smoking will worsen the situation in a1-AT deficient persons.

2.     Cirrhosis: AAT deficiency is also seen in persons with PiZZ genes. This genetic make up is associated with cirrhosis of liver. The ZZ protein has a substitution of glutamic acid by lysine at position 342. The protein is unsialylated and is not released from hepatocytes, causing death of cells with consequent fibrosis and cirrhosis.

3.     In Nephrotic syndrome, AAT molecules are lost in urine, and so AAT deficiency is produced.

Alpha-2-Macroglobulin (AMG): AMG is a tetrameric protein with molecular weight of 725,000. It is the major component of a-2 proteins. Gene is located in the long arm of chromosome number 12. It is synthesised by hepa­tocytes and macrophages. AMG inactivates all proteases,  and thus it is an important in vivo anti-coagulant. Proteases cleave the "bait" region of AMG, releasing a small unit, to provoke conformational changes in AMG, which then "traps" the enzyme. So proteolytic enzymes cannot function. AMG-protease complexes are internalised by a receptor mediated endocytosis by macrophages, and then degraded. AMG is the carrier of many growth factors such as platelet derived growth factor (PDGF). Normal serum level is 130^300 mg/dl. AMG contributes about 1% of all total plasma proteins. Its concentration is markedly increased (up to 2-3 g/d I) in Nephrotic syndrome, because other proteins are lost through urine in this condition.

Alpha-1-Acid Glycoprotein: It is otherwise known as Orosomucoid. It has a molecular weight of 44,000 and has a high content of about 45% of carbohydrates. Its isoelectric pH is 0.7-3.5. It is synthesised by hepatocytes. It binds lipophilic substances and various drugs. It binds with progesterone tightly. Normal serum level is 55-140 mg/dl, and its half-life is five days. It is increased in pregnancy. It is also an acute phase protein. It is a reliable indicator of clinical activity of ulcerative colitis.

 

NEGATIVE  ACUTE  PHASE  PROTEINS

During an inflammatory response, some proteins are seen to be decreased in blood; these are called negative acute phase proteins. Examples are albumin, transthyretin (pre­albumin), retinol binding protein and transferrin.

Transferrin: It is a specific iron binding protein. It is a negative acute phase protein; so the blood level is decreased in acute diseases. It has a half-life of 7-10 days and is used as a better index of protein turnover than albumin.

IMMUNOGLOBULINS

The terms gamma globulin and immunoglobulin are not synonymous. Antibodies are chemically called immuno­globulins; Ninty percent of these molecules move in the gamma region on electrophoresis, but some molecules have beta and some even have alpha mobility. Immunoglo­bulin is a functional term, while gamma-globulin is a physical term. Immunoglobulins are generally abbreviated as Ig. They are classified into five major classes. Their characteristics are described in detail in.

Plasma contains many enzymes  and protein hormone. A comprehensive list of normal values for the substances present in blood is given in the Appendix II.

CLOTTING FACTORS

The word coagulation is derived from the Greek term, coagulare = to curdle. The biochemical mechanism of clotting is a typical example of cascade activation. The coagulation factors are present in circulation as inactive zymogen forms. They are converted to their active forms only when the clotting process is initiated. This would prevent unnecessary intravascular coagulation. Activation process leads to a cascade chemical amplification effect, in which one molecule of preceding factor activates 1000 molecules of the next factor, and so on. Thus within seconds, millions or trillions of molecules of final factors are activated.

Several of these factors require calcium for their activation. The calcium ions are chelated by the gamma carboxyl group of glutamic acid residues of the factors, prothrombin, VII, IX, X, XI and XII The gamma carboxylation of glutamic acid residues is dependent on vitamin K, and occurs after synthesis of the protein (post-translational modification).

Prothrombin

It is a single chain zymogen with a molecular weight of 69,000 and plasma concentration around 10-15 mg/dl. The prothrombin is converted to thrombin by Factor Xa, by the removal of N-terminal fragment. The thrombin is a serine protease with molecular weight of 34,000. The Ca++ binding of prothrombin is essential for anchoring the prothrombin on the anion phospholipid on the surface of platelets. When the terminal fragment is cleaved off, the calcium binding sites are removed and so, thrombin is released from the platelet surface

Fibrinogen

The conversion of fibrinogen to fibrin occurs by cleaving of Arg-Gly peptide bonds of fibrinogen. Fibrinogen has a molecular weight of 340,000 and is synthesised by the liver. Normal fibrinogen level in blood is 200-400 mg/dl. The fibrin monomers formed are insoluble. They align themselves lengthwise, aggregate and precipitate to form the soft clot. Fibrinogen is an acute phase protein. The final step in clotting is the conversion of soft clot to hard clot by factor XIII or Fibrin stabilising factor. This occurs by formation of cross links between Lysine and Glutamine residues. The amide group of glutamine is replaced by epsilon amino group of Lysine by factor XIII which acts as a transglutaminase.

Platelets

Platelets form a plug in injured vessels. Platelets also provide factor XIII and phospholipids and platelet factor IV. The serine proteases, and thereby the clotting mechanism is normally inhibited by heparin antithrombin complex. The platelet factor now released will remove this inhibition, and causes initiation of thrombosis. The platelets that adhere to the endothelial layer are activated and they secrete ADP and Thromboxane A2  which favour platelet aggregation and formation of the plug.

Anticoagulants

In vitro anticoagulants removes Ca++ which is essential for several steps on clotting, e.g. oxalates, citrate, EDTA. Heparin and antithrombin III are the major in vivo anticoagulants. They exert an inhibitory effect on the serine protease which activates the clotting factors by partial proteolysis. Heparin is also used in dialysis and in the treatment of intravascular thrombosis. Since vitamin K is essential for coagulation, antagonists to vitamin K are used as anticoagulants especially for therapeutic purposes, e.g. Dicoumarol, Warfarin sodium. Alpha-2-macroglobulin also has anticoagulant activity.

Fibrinolysis

Unwanted fibrin clots are continuously dissolved in vivo by Plasmin, a serine protease. Its inactive precursor is plasminogen (90 kD). It is cleaved into two parts to produce the active plasmin. Plasmin in turn, is inactivated by alpha-2-antiplasmin. Tissue plasminogen activator (TPA) is a serine protease present in vascular endothelium. TPA is released during injury and then binds to fibrin clots. Then TPA cleaves plasminogen to generate plasmin, which dissolves the clots. Urokinase is another activator of plasminogen. Urokinase is so named because it was first isolated from urine. Urokinase is produced by macrophages, monocytes and fibroblasts. Streptokinase, isolated from streptococci is another fibrinolytic agent.

Clinical significance: Thrombosis in coronary artery is the major cause of myocardial infarction (heart attack). If TPA, urokinase or streptokinase is injected intravenously in the early phase of thrombosis, the clot may be dissolved and recovery of patient is possible.

ABNORMALITIES IN COAGULATION

 Hemophilia A (Classical Hemophilia): This is an inherited X-linked recessive disease affecting males and transmitted by females. Male children of hemophilia patients are not affected; but female children will be carriers, who transmit the disease to their male off spring. This is due to the deficiency of factor VIM (AHG). It is the most common of the inherited coagulation defects. There will be prolongation of clotting time. Hence, even trivial wounds such as tooth extraction will cause excessive loss of blood. Patients are prone to internal bleeding into joints and gastrointestinal tract. Treatment consists of administration of AHG, prepared from pooled sera every three months. Since this is not generally available, the usual treatment is to transfuse blood periodically, which may lead to eventual iron overload, hemochromatosis. Several hemophilia patients, receiving repeated transfusions became innocent victims of AIDS. Pure AHG is now being produced by recombinant technology and is the treatment of choice.

Hochstetter (1635) and Banyer (1743) had described hemophilia-like diseases; but the first clear account was given by John Otto in 1803. The disease has gained importance because it was present in the royal families of Europe. The gene for AHG has been cloned; it is one of the largest genes with 186 kb in size. The gene locus is in the long arm of X-chromosome at Xq-24q site. Prenatal diagnosis by DNA analysis of cells from amniocentesis is now possible.

Hemophilia B or Christmas Disease: It is due to factor IX deficiency. The Christmas disease is named after the first patient reported with this disease. Similar deficiencies of factors X and XI are also reported. In these diseases, the manifestations will be variations of classical hemophilia.

Von Willebrandt's Disease: This is an autosomal dominant defect leading to failure of platelets to adhere. The defect is in the glycoprotein platelet adherence factor (PAF) present in the plasma and platelets. The bleeding time is prolonged.

Other disorders: Acquired hypofibrinogenemia or afibrinogenemia may occur as a complication of prolonged labour (abruptio placenta). Proteolytic thromboplastic substances may enter from placenta to maternal circulation which set off the clotting cascade (disseminated intra-vascular coagulation or DIC). But the clots are usually degraded immediately by plasminolysis. Continuation of this process leads to removal of all available prothrombin and fibrinogen molecules leading to profuse postpartum hemorrhage. In some cases of carcinoma of pancreas, trypsin is released into circulation leading to intravascular coagulation. This may be manifested as fleeting thrombo­phlebitis. Trousseau diagnosed his own fatal disease as cancer of pancreas when he developed thrombophlebitis.

 The combination of carcinoma of pancreas, migratory thrombophlebitis and consumption coagulopathy is termed as Trousseau's triad.

 

Proteinuria:

Richard Bright established proteinuria as an indicator of renal disease in 1827. Proteinuria is an important index of renal diseases. In normal urine, protein concentration is very low (less than 100 mg per day), which cannot be detected by the usual tests. These proteins are secreted by the tubular epithelial cells.

 Proteinuria may be classified as:

a) Glomerular proteinuria: The glomeruli of kidney are not permeable to substances with molecular weight more than 69,000 and so plasma proteins are absent in normal urine. When glomeruli are damaged or diseased, they become more permeable and plasma proteins may appear in urine. The smaller molecules of albumin pass through damaged glomeruli more readily than the heavier globulins. So, when proteins appear in the urine, the albumin fraction predominates. Albuminuria is always pathological. Large quantities ( a few grams per day) of albumin are lost in urine in nephrosis. Small quantities are seen in urine in acute nephritis, strenuous exercise and pregnancy.

Microalbuminuria or minimal albuminuria or pauci-albuminuria) is identified, when small quantities of albumin (50-300 mg/day) is seen in urine. It is seen as a complication of diabetes mellitus and hypertension. It is an indicator of future renal failure.

b) Overflow proteinuria: When small molecular weight proteins are increased in blood, they overflow into urine. For example, hemoglobin having a molecular weight of 67,000 can passthrough normal glomeruli, and therefore, it it exists in free form (as in haemolytic conditions ), hemoglobin can appear in urine (hemoglobinurial). Similarly, myoglobinuria is seen following muscle crush injury. Yet another example is the Bence-Jones proteinuria. In about 20% cases of multiple myeloma, the light chains  of immunoglobulins are produced abnormally. Being of smaller molecular weight, they are excreted in urine. These are called Bence-Jones Proteins (monoclonal light chains produced by plasmacytomas). When the urine is heated, at 45°C they start precipitating, at 60°C there is maximum precipitation, at 80°C these proteins start re-dissolving, and will form a clear solution at 100°C. The precipitate re-forms on cooling.

c) Nephron loss proteinuria: This occurs when functional nephrons are reduced, GFR is decreased and remaining nephrons are overworking.

d) Tubular proteinuria: It is seen when tubular reabsorption mechanism is impaired, and low molecular weight proteins are excreted.

     e) Urogenic proteinuria: This is due to inflammation of lower urinary tract, when proteins are secreted into the tract.

Measurement of urinary proteins may be carried out:

a) to establish the presence or absence of renal disease;

b) to define the nature of renal disease; c) to define the degree of renal dysfunction and d) to monitor the response to treatment.

The proteinuria is commonly assessed by the heat and acetic acid test (Frederick Dekkers, 1694). Fill two-thirds of a test tube with the urine sample and heat the top of the column to boiling. Add 1 % acetic acid (3 drops). Compare the top portion with the lower. A cloudiness on the top shows the presence of proteins. Albumin is coagulated when heated, which is precipitated at the iso-electric point, when acetic acid is added. Calcium and magnesium phosphates are also precipitated on boiling, but they are soluble in acidic medium.

Esbach's Albuminometer is used for rough quantitative estimation of proteins in the urine. Add urine upto the mark 'U' and the Esbach's reagent up to the mark 'R' of the apparatus. Mix well. Keep for 24 hours. Read the amount of protein precipitated on the tube in grams of protein per liter of urine. Esbach's reagent is picric acid and citric acid.

Microalbuminuria is detected by radial immunodiffusion or by enzyme linked immunosorbent methods.

Laboratory diagnostics of diabetes mellitus

 

Diabetes is the most common metabolic disorder, and its incidence is increasing. Biochemical measurements are particularly important in detecting it, monitoring its control and treating its metabolic complications. Hypoglycaemia occurs in insulin-treated diabetic patients, but is other­wise rare. However, it is an important diagnosis to make because of its possible consequences. Other disorders of carbohydrate metabolism are uncommon.

Dietary carbohydrate is digested in the gastrointestinal tract to simple monosaccharides which are then absorbed. Starch provides glucose directly,while fructose (from dietary sucrose) and galactose (from dietary lactose) are absorbed and also converted into glucose in the liver. Glucose is the common carbohydrate currency in the body.

 

Insulin is the principal hormone affecting blood glucose levels. It is a small protein synthesized in the beta cells of the islets of Langerhans of the pancreas. It acts through membrane receptors and its main target tissues are liver, muscle and adipose tissue. the overall effects of insulin is to promote cellular uptake and storage of metabolic fuels. Insulin is synthesised in the ß-cells of the islets of Langerhans in the pancreas. It is formed as prepro-insulin, which is rapidly cleaved to pro-insulin. The pro-insulin is packaged into secretory granules in the Golgi apparatus and cleaved to insulin and C peptide. Insulin and C peptide are later released into the circulation in equimolar amounts. A rise in blood [glucose] is the main stimulus for insulin secretion. Some amino acids (e.g. leucine), fatty acids and ketone bodies also promote insulin secretion. The release of insulin in response to hyperglycaemia is enhanced by the presence of GIP or glucagon. GIP is probably the most impor­tant factor in the larger release of insulin that occurs in response to an oral glucose load, com­pared with the same dose of glucose given intra­venously. Vagal stimulation also promotes insulin release.The insulin receptor is located on the cell surface and is internalised after insulin binding. Within dif­ferent organs, target enzymes have been identified that serve to explain the known effects of insulin on intermediary metabolism. For instance, activation of glucose transport, induction of hexokinase (or glucokinase) and activation of phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase in the liver are all consistent with the actions insulin in promoting increased glucose uptake and glycolytic breakdown. Stimulation of glycogen synthase accords with the effects of insulin on glycogen formation in the liver.

 

The effects of insulin are opposed by other hormones, glucagon, adrenaline, glucocorticoids and growth hormone. The blood glucose concentration is the result of a balance between these different endocrine forces.

 

Diabetes mellitus may be defined as a syndrome characterized by hyperglycemia due to an absolute orrelative lack of insulin and /or insulin resistance.

 

Primary diabetes mellitus is subclassified into insulin dependent diabetes mellitus (IDDM or type I) and non- insulin dependent diabetes mellitus (NIDDM or type II).

The contrasting features of IDDM and NIDDM  are shown in Table 1:

 

Secondary diabetes mellitus may result from pancreatic disease, endocrine disease such as Cushing's syndrome, drug therapy, and insulin receptor abnormalities.

 

Insulin dependent diabetes mellitus (IDDM or type I) accounts for approximately 15 % of all diabetics. It can occur at any age, but is most common in the young, with a peak incidence between 9 and 14 years of age. The absolute lack of insulin is a consequence of the autoimmune destruction of insulin-producing beta cells.

 

Non- insulin dependent diabetes mellitus (NIDDM or type II) accounts for approximately 85 % of all diabetics and can occur at any age.It is most common between 40 and 80 years. In this condition there is resistance of periferal tissues to the actions of insulin, so that the insulin level may be normal or even high. Obesity is the most commonly associated clinical feature.

 

Late complications of diabetes mellitus

Diabetes mellitus is not only characterized by the presence of hyperglycemia but also by the occurrence of late complications:

1.     Microangiopathy is defined as abnormalities in the walls of small blood vessels, the most prominent feature of which is thickening of the basement membrane.

2.     retinopathy may lead to blindness because of vitreous haemorrhage from proliferating retinal vessels, and, maculopathy as a result of exudates from vessels or oedema affecting the macula.

3.     Nephropathy leads ultimately to renal failure. In the early stage there is kidney hyperfunction, associated with an increased GFR, increased glomerular size and microalbuminuria. In the late stage, there is increasing proteinuria and a marked decline in renal function, resulting in uraemia.

4.     Neuropathy may become evident as diarrhoea, postural hypotension, impotence, neurogenic bladder and neuropathic foot ulcers due to microangiopathy of nerve blood vessels and abnormal glucose metabolism in nerve cells.

5.     Macroangiopathy leads to premature coronary heart disease.The exact mechanisms for increased susceptibility to atherosclerosis.

 

Clinical symptoms of hyperglycemia include:

·        polyuria

·        polydipsia

·        lassitude

·        weight loss

·        pruritus vulvae

·        balanitis

These symptoms are common to both NIDDM and IDDM but are more pronounced in IDDM. It is important to remember that patients with NIDDM may be completely asymptomatic.

 

Diagnosis and monitoring of diabetes mellitus

The diagnosis of diabetes mellitus has serious consequences. It confers a risk of long-term diabetic complications, including blindness, renal failure and amputations, as well as an increased risk of cardiovascular disease. It also means a lifetime of dietary restriction and medications and can seri­ously curtail lifestyle and employment prospects. The diagnosis may be suggested by the patient's history, or by the results of dipstick tests for glu­cose on urine specimens. However, urine glucose measurements by themselves are inadequate for diagnosing diabetes. They potentially yield false-positive results in subjects with a low renal thresh­old for glucose, and in a patient with diabetes, they may yield false-negative results if the patient is fasting. A provisional diagnosis of diabetes mellitus must always be confirmed by glucose measurements on blood specimens.

The most recent criteria for the diagnosis of dia­betes mellitus have been laid down by the World Health Organization (WHO) in 1998, and by the American Diabetes Association in 1997. These are broadly similar but differ in some details. It is likely that the precise requirements for the diagno­sis of diabetes and the states of impaired glucose regulation will continue to evolve as knowledge of the relation between glucose regulation and the future development of complications accumulates. The following descriptions adhere to the WHO recommendations. Separate criteria are described depending on whether venous or capillary whole blood, or venous or capillary plasma specimens are used. In practice, results for this important diagnosis will usually come from a clinical labora­tory and use venous plasma. According to the criteria, a random venous plasma [glucose] of 11.1 mmol/L or more, or a fasting plasma [glucose] of 7.0 mmol/L or more, establishes the diagnosis. A single result is sufficient in the presence of typical hyperglycaemic symptoms of thirst and polyuria. In their absence, a venous plasma [glucose] in the diabetic range should be detected on at least two separate occasions on different days. Where there is any doubt, an OGTT should be performed, and if the fasting or random values are not diagnostic, the 2-h value should be used. In prac­tice, the diagnosis is often obvious clinically, and [glucose] is only needed for confirmation and is unequivocally high. The diagnosis should never be made on the basis of a single test in a patient without symptoms.                             

At present a raised HbA1c should not be used to make a diagnosis of diabetes. Standardisation of HbA1c analysis has been a problem in the past although this is now less of an issue. This has meant that it has not been possible to reliably determine a single cut-off level that would diag­nose diabetes in all laboratories.

 

Glucosuria allows for a good first-line screening test for diabetes mellitus; normally glucose does not appear in the urine until the plasma glucose rises above 10 mmol/l (renal threshold for glucose – the maximum amount of glucose in blood, which is completely reabsorbed in kidney tubuls).

Ketone bodies may accumulate in the plasma of a diabetic patient. Ketones may be present in a normal subject as a result of simple prolonged fasting. Dry reagent strips which detect acetoacetate might therefore provide an understimate of ketonaemia/ketonuria.

Blood glucose

Glucose is routinely measured in the laboratory on blood specimen which have been collected into tubes containing fluoride, an inhibitor of glycolysis. Becouse of the need sometimes to obtain rapid blood glucose results and the wide-spread self-monitoring of diabetic patients, blood glucose is also assesed outside the laboratory using test strips.

 

The World Health Organization has published guidelines for the diagnosis of diabetes mellitus on the basis of blood glucose results and the response to an oral glucose load.

 

 

Random blood glucose (RBG) is the only test required in an emergency. An RBG of less than 8 mmol/l should be expected in non-diabetics.

RBG higher than 11 mmol/l usually indicates diabetes mellitus.

 

Fasting blood glucose (FBG) is measured after overnight fast (at least 10 hours). An FBG is better than RBG for diagnostic purposes. In non-diabetics it is usually lower than 6 mmol/l. Fasting values of 6-8 mmol/l should be interpreted as borderline.

 

Oral glucose tolerance test (OGTT)

A baseline blood sample is first taken after an overnight fast. The patient is then given 75 g of glucose orally, in about 300 ml of water, to be drunk within 5 minutes. Plasma glucose levels are measured every 30 min for 2 hours.Urine may also be tested for glucose at time 0 and after 2 hours. The patient should be sitting comfortably throughout the test, should not smoke or exercise and should have been on normal diet for at least 3 days prior to the test.

 

Indications:

·        borderline fasting or post-prandial blood glucose

·        persistent glycosuria

·        glycosuria in pregnant women

·        pregnant women with a family history of diabetes mellitus and those who previously had large babies or unexplained fetal loss

 

Interpretation of an OGTT

If the patient has a normal fasting plasma glucose and only the 2 h value in the diabetic range, the test should be repeated after approximately 6 weeks. Impaired glucose tolerance (IGT) should not be regarded as a disease. It signals that the patient is at an intermediate stage between normality and diabetes mellitus and is at an increased risk of developing diabetes. Such patients should be followed yearly, and dietary treatment may be used.

 

 

 

 

 

 

Long-term indices of diabetes control

A high concentration of glucose in the ECF leads to its non-enzymatic attachment to the lysine residues of a variety of proteins. This is called glycation. The extent of this process depends on the ambient glucose level . The concentration of glycated protein is therefore a reflection of a mean blood glucose level prevailing in the extracellular fluid during the life of that protein.

 

Haemoglobin A1c  or glycated haemoglobin

Glycated haemoglobin reflects the mean glycaemia over 2 month prior to its measurement, the half-life of haemoglobin. This test is accepted as a good index of diabetic control and is used routinely in most diabetics clinics to complement the information from single blood glucose levels, or indeed a patient's log of his or her own blood glucose measurements.

 

Fructosamine

Many other proteins in addition to haemoglobin are glycated when exposed to glucose in the blood. As indication of the extent of this glycation can be obtained by measuring fructosamine, the ketoamine product of non-enzymatic glycation. As albumin is the most abundant plasma protein, glycated albumin is the major contributor to serum fructosamine measurements. As this protein has a shorter half-life than haemoglobin, fructosamine measurements are complementary to HbA1c providing the index of glucose control over the 3 weeks prior to its measurement.

 

Microalbuminuria

It may be defined  as an albumin excretion rate intermediate between normality (2.5-25 mg/day) and

macroalbuminuria (>250 mg/day). The importance of microalbuminuria in the diabetic patients is that it is a signal of early, reversible renal damage.

 

 

Diabetic ketoacidosis

All metabolic disturbances seen in DKA are the indirect or direct consequences of the lack of insulin. Decreased glucose transport into tissues leads to hyperglycemia which gives rise to glycosuria. Increased lipolysis causes over-production of fatty acids, some of which are converted into ketones, giving ketonaemia, metabolic acidosis and ketonuria. Glucosuria causes an osmotic diuresis, which leads to the loss of water and electrolytes – sodium, potassium, calcium, magnesium, phosphate and chloride. Dehydration, if severe, produces pre-renal uraemia, and may lead to hypovolaemic shock. The severe metabolic acidosis is partially compensated by an increased ventilation rate (Kussmaul breathing). Frequent vomiting is also usually present and accentuates the loss of water and electrolytes.

 

Laboratory investigations

Urine (if available) should be tested for glucose and ketones, and blood checked for glucose using a test strip. Venous blood should be sent to the laboratory for plasma glucose and serum sodium, potassium, chloride, bicarbonate, urea and creatinin. An  arterial blood sample should also be  sent for measurement of blood gases. Blood glucose should be monitored hourly at the bedsite until less than 15 mmol/l. Thereafter checks may continue 2-hourly. The plasma glucose should be confirmed in the laboratory every 2-4 hours.

 

Treatment

The management of DKA requires the administration of 3 agents:

·        Fluids. Patients with DKA are usually severely fluid depleted and it is essential to expand their ECF with saline to restore their circulation.

·        Insulin. Intravenous insulin is most commonly used.

·        Potassium.

 

Hypoglycemia is a laboratory diagnosis which is usually taken to mean a blood glucose level below 2.5 mmol/l.

A low blood glucose level normally leads to the stimulation of catecholamine secretion and stimulation of glucagon, cortisol and grows hormone.

Symptoms mosr commonly seen in hypoglycaemia:

·        sweating

·        shaking

·        tachycardia

·        nausea

·        weakness

·       

 

Laboratory investigation

Blood glucose. The detection of hypoglycaemia is by blood glucose testing. Urine testing cannot detect hypoglycaemia.

Plasma insulin. Insulin measurements can lead to the diagnosis or exclusion of insulinoma.

Insulin/glucose ratio.

Plasma C-peptide. insulin secretion in insulin-treated diabetics cannot be assesed by the measurement of plasma insulin since the insulin given therapeutically will also be measured. However, insulin and its associated connecting-peptide (or C-peptide) are secreted by the islet cells in equimolar amounts and thus measurement of C-peptide levels together with insulin can differentiate between hypoglycaemia due to insuloma (high C-peptide ) and that due to exogenous insulin (low C-peptide ).

 

Specific causes of hypoglycaemia

Over 99% of all episodes of hypoglycaemia occur in insulin-dependent diabetic patients.

Reasons for hypoglycaemia in the diabetics include:

·        insufficient carbohydrate intake

·        excess of insulin

·        strenuous exercise

·        excessive alcohol intake

Other causes of hypoglycaemia may be divided in two groups: those which produce hypoglycaemia in the fasting patient, and those in which the low glucose concentration is a response to a stimulus (reactive hypoglycaemia).

 

Fasting hypoglycaemia

Causes of fasting hypoglycaemia include:

·        Insulinoma. ß-cell islet tumors of the pancreas may produce insulin both inappropriately and in excess.

·        Cancer. hypoglycaemia is associated with advanced malignancy.

·        Hepatic disease.

·        Addison's disease

·        Sepsis

 

Reactive hypoglycaemia

In reactive hypoglycaemia patients may become hypoglycemic in response to:

·        Drugs.

·        Food: post-prandial hypoglycaemia. Accelerated gastric emptying after gastric surgery (dumping syndrome) may give rise to this condition.

·        Alcohol.

 

Neonatal hypoglycaemia

The diagnosis and treatment of hypoglycaemia in the neonate is particularly important because of the high risk  of hypoglycaemic brain damage. There are a number of important causes:

Babies of diabetic mothers. A fetus that is exposed to maternal hyperglycaemia will have pancreatic islet cell hyperplasia and elevated insulin levels. After delivery the neonate is unable to suppress its inappropriately high insulin levels and will develop hypoglycaemia.

Intra-uterine retardation. Small-for-dates babies may have inadequate liver glycogen stores.

Inborn errors of metabolism. Galactosaemia and glycogen storage disease are examples.