Reference Laboratory




Clinical Biochemistry

Tumor Markers
The first tumor indicator to be used by clinical laboratories is the Bence Jones protein, used to diagnose Multiple Myeloma. First used in 1846 and although various other hormones, enzymes and proteins were used as tumor markers along with it until 1963, the use of these markersin monitoring cancer begins beyond this date and after the alpha phetoprotein (AFP) was found in 1963 and following that in 1965 when the carcinoembryonic antigen, or CEA, was found. The ideal tumor indicator must have a specificity for the tumor diagnosed as well as the sensitivity to provide a means of diagnosis in the earlier stages of cancer and before clinical symptoms are apparent. 

Tumor markers, although their use may differ for each indication, are generally as follows:

  • Screening of the general population
  • Clinical categorisation of the cancer
  • Prognostic monitoring of the advance of the condition
  • Evaluation of success in treatment
  • Determining a recurrence of the cancer 
Tumor markers may be found to be enzymes, hormones, oncofetal antigens and proteins.

Principal among these:

Prostate Specific Antigen, or PSA: a very beneficial indicator in diagnosing prostatecancer; it is used in its prostate cancer diagnosis, staging and treatment. The most beneficial clinical advantage is its use in the monitoring of prostate cancer treatment.
Calcitonin: used in the diagnosis of thyroid medullary cancer.
Chorionoc gonodotropin (hCG): Is a very useful indicator which is used in the diagnosis of trophoblastic tumors and non-seminomatose testicular carcinoma. Shows an increases in nearly all rophoblastic tumors and an increase of 70% in non-seminomatose testicular cancer. Is beneficial in the tracking of trophoblastic disease advance as well as tracking of treatment.
Alpha fetoprotein (AFP): used in the diagnosis, identification of prognosis and tracking of treatment in hepatocellular carcinoma, and along with hCG it is used in the staging of germ celled tumors. It has provided successful results in high risk haepatocellular carcinoma regions as well as in screenings for high risk groups.
Carcino embryonic antigen (CEA): is an indicator for colorectal and gastrointestinal system and pancreas cancers. It may show an increase with conditions such as cirrhosis, ulcerative colitis and benign breast diseases and is used in the staging of colon cancer.
CA 15-3: is used in the staging of patients with metastatic breast cancers.
CA 125: is an indicator used in patients with ovarian or endometrial cancers.
CA 19-9: is an indicator for pancreatic and colorectal cancers; at the same time it will exhibit an increase with acute and chronic pancreatitis and cholangitis.
CA 72-4: an indicator for gastrointestinal carcinomas.NMP 22 (Nuclear Matrix protein): an indicator in transitional celled urinary tract carcinomas.


Plasma cell diseases are a group of neoplastic diseases characterised by theproliferation of a single clone of plasma cells secreting immunoglobins which are rooted in B cell series of immunocytes.

Classification of plasma cell proliferativdiseases:
  1. Malignant monoclonal gammopathy’s Multiple Myeloma Waldentstöm macroglobulinemia
  2. MGUS (Monoclonal gammopathy of undetermined significance)
  3. Light chaine myeloma
  4. Heavy chain diseases
  5. Cryoglobulinemia
  6. Primary amyloidosis
Multiple Myeloma is a type of cancer characterised by the abnormality and uncontrollable multiplication of plasma cells which are part of the bone marrow and an important part of the immune system.
Criteria in diagnosis of symptomatic multiple myeloma (Dimopoulos M, Kyle R, Fermand PJ, et al. 2011):

1.Clonal bone marrow plasma cells 10%
2.Serum and/or urinary monoclonal proteins (excluding non-secreteing MM)
3.Evidence of organ damage especially in lymphoproliferative diseases:
                                          Hypercalcemia; serum calcium 11.5 mg / dl; 
                                          Kidney failure; serum creatinine 2 mg/dl 
                                          Anemia, normochromic; Hb value 10 g/dl
4.Bone lesions, lithic lesions, osteopenia, pathologic fractures

Nomination of M Proteins

Electrophoresis tests are the most significant in identification of M proteins in serum and / or urine. This first step is indicating an abnormal concentration and composition of immunglobuline in serum is the serum protein electrophoresis.Should a suspect condition be evident in the beta or gamma regions during serum protein electrophoresis, identifying an increasing immunoglobulin type is made by immunefixation electrophoresis.An agarous gel (Helena SAS-1 Immunofix)is used for immunelectrophoresis. Serum proteins are separated by electrophoresis in the IFE. Immunoprecipation follows using specific antiserums against IgA, IgM, IgG, kappa and lambda. In the presence of M proteins a precipitin band is formed; it is washed with saline gel and unbonded proteins are separated. It is dyed once again and then dried.M protein is predominantly scanned in IgG, IgA, IgM followed by two light chains of kappa and lambda. Kappa/lambda ratio is 2:1. As a discrepancy in this ratio would indicate M proteins the IFE pattern must be examined more closely.

Capillary Electrophoresis (Immundisplacement)

This is a new and modern procedure being used in the past few years. With benefits such as the analysis of smaller volumes, ease of use and a lower per test cost all offered in a rapid procedure it has a number of advantages which allow for its widepsread use. The electrophoresis appartus is made up of 8 capillary tubes with 50 µm diameter and 300 mm length; two electrolytic buffer cells, a high voltage power source and a detector connected to a data evaluation unit. Monospecific antserums and the patients’ serum are mixed so as to precipitate each of the light and heavy chains while electrophoresis is done in the capillary tubes in the upper portion. In the presence of increases monoclonal structures they are bonded by antisera and precipitated with a reduction of the peak in the final report.This is why it is known as immundisplacement. Our laboratory uses capillary electrophoresis (immundisplacement) to identify proteins. Validation is done by immunfixation electrophoresis.When SPE, or serum protein electrophoresis, serum free chain measurements and serum immunfixation electrophoresis are used in conjunction 99% of paraproteins can be identified.

Free Light Chain Diseases in Urine (Bence Jones Protein)

BJP’s are monoclonal kappa or lambda immunglobulin light chains. They are not attached to the heavier chain portion of the immunglobulin molecule. They are identified by means of antiserums used against free light chains in immunifixation electrophoresis tests in urine. Kappa and lambda light chains, free kappa and free lambda light chains in urine may quantitatively be identified by nephelometric and turbidimetric means allowing for kappa/lambda values to be evaluated.

Hormone Tests

Hormones have aregulating effect on the body and are required in human growth, development andreproduction.Hormone tests are used in the treatment of tumors of the glands whichsecrete hormones or the diseases which result in these glands producing eitherinsufficient or excessive hormones for secretion. The primary secreting glandsare the hypothalamus, pituitary gland, thyroid, parathyroid glands, adrenalglands, the pancreas, the ovaries and the testicular glands.Diagnosis andmonitoring of many illness or diseases such as diabetes, osteoporosis,virilism, hirsutism, sexual function disorders, infertility, growth anddevelopment disorders, hypertension is made through hormone tests. Hormonetests are carried out in our laboratories using electrochemiluminencetechniques and test analysers.

 Hormonetests of the Pancreas, Stomach, Intestines and Diabetes

Gastrin is ahormone which regulates acid levels of the stomach. It is produced during thewhole of the digestive process by G cells which are found in the stomach.Rareoccurences of G cell hyperplasia and Zollinger- Edison Syndrome (ZE) can resultin the excess production of gastrins and stomach acids. This may result in pepticulcers, which are difficult to treat. Gastrin values are evaluated in itsdiagnosis.Diabetes is a systemic illness which increases blood glucose levelsresulting from insulin insufficiency, insulin resistance or the occurrence ofboth. It is the result of a lack of hormones secreted by the pancreas.In theabsence of complaints a diabetes screen is generally requested of thosecarrying certain risk factors.  In pregnant women and in the presence ofrisk factors, a screening should be made during the first physician’scontrol.The insulin hormone starved hypoglycemia, Type 1 and Type 2, insulinoma(Langerhans islet cell tumor) and insulin resistance is used to evaluatediabetes.During starved insulin or OGTT, per minute insulin values may bemeasured. Additionally, C-peptide tests are used to monitor insulin productionand determine the cause of hypoglycemia.


 Recently wehave been hearing more about the HOMA IR (Insulin Resistance); a test whichallows us to understand whether the body has a resistance to insulin. It isused to determine the surrounding tissues response to insulin. Factors such asbeing overweight, polycystic ovary syndrome, treatment with medicationscontaining cortizone, an increased waist-line, age and a less active life-stylecan lead to insulin resistance.

ThyroidHormone Tests

The thryoid glandis a two-lobed gland located in front of the trachea; its’ purpose is toproduce and secrete T3 and T4 hormones, otherwise known as thyroid hormones.These hormones are important in growth and development and are needed by thebody’s metabolism. A reduction in hormone levels causes a clinical table knownas hypothyroidy while an increase presents itself as a hyperthyroid. Tests fortotal thyroxine (TT4), total triiodothyronine (TT3), free thyroxin (ST4), freetriiodothyronine (ST3) and the thyrotropin (TSH) hormone (which stimulates thepituitary thyroid) are used to diagnose for these clinical tables.

ParathyroidHormone Tests

Parathyroidglands are small endocrine glands located on the neck and mostly behind thethyroid gland. They assume a role in the development of the body’s calciummetabolism and thereby in bone development. An excess of the parathyroidhormone results in loss of bone density, kidney stones and hypertension. Treatmentis generally through the surgical removal of the enlarged gland orglands.  Some patients who are not suitable for surgery or whoseparathyroid levels are considered not too high may be monitored. Tests in thiscase are the parathormon intact PTH and parathormon related peptid test.

These tests arebeneficial in differentiating the diagnosis of hyperparathyroidism. It is alsoused to differentiate it from its’ non-parathyroid forms resulting inhypercalcemia. Another hormone used in monitoring calcium metabolism disordersis calcitonine. Calcitonine is also used to monitor thyroid medullarycarcinomas.


Hormone Testsof the Hypophysis


The hypophysis,or pituitary gland, is situated inside the skull and rests upon the fossa, orbones, which make up the base of the skull itself. It is comprised of twoparts, the anterior pituitary, or “adenohypophysis” and the posteriorpituitary, or “neurohypophysis”, and is anendocrine gland. The hormonessecretedby the anterior pituitary are ACTH, FSH, LH, TSH, the growth hormoneand prolactin. The posterior pituitary gland secretes an antidiuretic hormone(ADH) and oxytocin.Of these hormone, ACTH regulates production of the stresshormone cortizoas produced by the renal glands, the TSH thyroid hormone and FSHand LH hormones in relation to the reproductivity of females and males. Thegrowth hormone is required for growth and development, ADH regulates thebalance of body fluids, oxytocin the contraction of straight muscles duringbirth and of the process of giving birth itself. These hormones are allperiodically required for proper body balance.

An excess ofthe Growth Hormone resultsin gigantism during childhood and acromegaly in adults. A lack of the growthhormone is the cause of dwarfism. As growth in height will be minimal,shortness will be evident; IGF-1, IGF-BP3 as well as growth hormone stimulationand inhibition tests assist in identifying growth anomalies and the evaluationof hypophysis function.

An excess ofprolactin is the earliestclinical symptom of hypophysis disorder. In women, it is characterised by alack of menstruation and evidence of breast milk, whereas in men it ischaracterised by a loss of libido and infertility. It is generally accompaniedby headaches and the impairment of vision.

While a lackof prolactin has no realclinical significance on those who are not pregnant, its’ most importantsymptom, following pregnancy, is an inability to produce breast milk. Theprolactin test is used in these clinical disorders. It is tested on patientswith clinical incompatability in macroprolactine hyperprolactinemia.

An excess ofTSH (Thyroid StimulatingHormone) increases the intake of iodine by the thyroid tissue and T3, T4synthesis is increased; symptoms can be similar to those seen inthyrotoxicosis. Whereas in a lack of TSH thyroid tissue iodine intake and areduction of T3, T4 secretions; the symptoms seen are as in hypothroidism.

LH(Luteinizing Hormone) andFSH (Folicule Stimulating Hormone) regulates testosterone production, testiculargrowth and sperm development in men. In women, it regulates ovary growth andmenstrual flow. A lack of these hormones results in effects such as decreasedsexual desire and infertility in men.In ACTH, or adrenocorticotropic hormonedeficiency, renal gland secretion of cortisol is reduced, resulting inhypocortisolism. This is exhibitted by severe fatigue and paleness. CushingSyndrome is observed with an excess of cortisol. Patients with symptoms ofCushing Syndrome exhibit obesity, hypertension, menstrual irregularity, lack oflibido, round “moon” face, thinning skin, an ability to bruise easily, muscleweakness, darkening of the skin, fungal infections and slow wound healing.

ACTHStimulation Test: iswidely used to evaluate patients suspected of hypocortisolism, adrenaldeficiency and Addisons Disease.  A subnormal response to the ACTHstimulation test is sufficient proof of adrenal deficiency, whilenormalresponses rule out adrenal deficiency. Aside from cortisol evaluation theACTH stimulation test may also be used to check per minute values of 17 alphahydroxyprogesteron DHEA-S levels. 

DexamethasoneSuppression Test: This isa screening test used to diagnoseCushing Syndrome. Dexamethasone, anadrenocortical steroid, is orally given to the patient on the night beforecortisol levels are tested. Cortisol levels are measured the following day. Innormal individuals cortisol levels are suppressed whereas with Cushing syndromethis suppression does not occur.Symptoms such as an excessive loss of bodyfluids, excessive intake of water and the frequent need to urinate are signs ofADH, or vasopresine deficiency. With an exagerated feeling of thirst, thesepatients will drink between 5-20 liters of water per day. Should they not drinkthis amount, an excessive increase in blood sodium levels may result inconfusion and coma. In an excess of ADH or vasopresine, an inappropriate ADHSyndrome table may be exhibited. A reduction of blood sodium levels, loss ofappetite, nausea, vomitting, confusion and coma may develop. Treatment isprimarily a limitation of water intake and a gradula replacement of the sodiumlost. In disorders of the ADH hormone, a Water Limitation Test is performed.The patient is administered ADH followed by the measurement of changes in urinedensity, osmolarity and serum osmolarity levels which are then used in thediagnosis of central DiabetesInsipidus.

Adrenal GlandHormone Tests

Adrenal glandsare situated over both kidneys. These glands secrete adrenalin, noradrenalin,cortisol and aldosterone. When adrenalin and noradrenalin are secreted into theflow of blood they will increase heart beat rates and blood pressure as well ashave an effect on other body functions. An excess in the secretion of adrenalinand/or noradrenalin will result in a condition known as pheochromocytoma.Hypertension or high blood pressure is evident in most patients with thiscondition. The condition may be diagnosed by checking adrenalin andnoradrenalin levels in blood and urine.

Clinical signsare exhibited as blood pressure which is permanently high or recurring suddenpeaks. Physical or emotional stresses will generally begin the secretionprocess. Aldosterone also plays a role in the body’s salt and fluid metabolismand therefore assumes an important role in the diagnosis and monitoring ofhypertension.

It is thoughtthat mineralocorticoid hypertension constitutes 10% of essential hypertension.Evaluation of hypertension is made on the basis of plasma renin and aldosteronelevels and plasma renin activity, or PRA. An excess of cortisol secretion bythe adrenal glands results inthe Cushingh Syndrome whose diagnosiscan be madeby tests for saliva cortisol, free urine cortisol and dexamethasonesuppression.

AddisonsDisease is a conditionseen when insufficient steroid hormones are secreted by the adrenal gland. Anadrenal deficiency crisis is a life threatening situation which needs to beaddressed by urgent medical care to include saline solutions and the injectionof steroid hormones. The ACTH stimulation test is used in its’ diagnosis.

GenderHormones and the Evaluation of Hypothalamus, Hypophysis, Gonad Axis As with most of the other systems of thebody, hormones secreted by the hypothalamus, hypophysis and gonads control thecycle of reproduction.

The first ofthese is the GnRH a gonadotropin secreteing hormone also known as LHRHor luteinizing hormone. This hormone activates the secretion of two hormones bythe hypophysis gland; these are the LH luteinizing hormone and FSH, foliculestimulating hormone the GnRH test is used in the diagnosis of hypophysis andgonadal deficiency.

The lack of aresponse to the test indicates damage in gonadtropic cells or long term GnRHdeficiency. Patients with primary hypogonadism exhibit an excess in FSH, LHresponse and is used in diagnosis of pubertas praecox, or early sexualdevelopment. These hormones both play a role in sperm development in males andovulation in females.

Androstenedions(androstenedion, DHEA and its sulphuric aesther DHEA-S) are precursors of testosterone andestrogens produced by the gonads and the adrenals. ACTH stimulates the adrenalsecretion of these hormones. Tests for androstenedions are used to determineandrogen deficiency or its excessive secretion for hirsutism and /orvirilisation.

A deficiency inhormonal secretions by the ovaries and testes, reproductive organs in femalesand males, exhibits itself by a reduction of sexual desire and sexualdysfunction. Not only do the testosterone and estrogens secreted by theseglands have an effect on sexual development, they have an effect on thedevelopment of the primary muscle and skeletal mass. A deficiency of thesehormones may be attributed to damage of the glands as well as to a deficiencyof the hormones which are secreted by would stimulate the ovaries.

Diagnosis andtracing of any disorders in this cycle is made by testing of FSH, LH,Estradiol, Progesterone, Prolactin/ Macroprolactin, Testosterone, SHBG,Anti-Mullerian Hormone, Free Testosterone, Androstendion, DHEA-S and beta HCGin the determination of pregnancy.



Our laboratoryuses test analysers with the immunochemiluminescence technique and the PRISCAprogram. These tests allow us to determine Trisomy 21 (Down Syndrome) openneural tube defficiency (NTD) Trisomy 18 and Trisomy 13 risks during pregnancy;they do not however provide for a definite diagnosis. Or laboratory is able toundertake dual, triple, quadruple, combined and integrated tests as well as theAFP test. These are non-invasive tests relying on ultrasound readings andsamples of blood taken from the mother to determine the levels of certainhormones in the blood at specific periods of the mothers’ term of pregnancy;they pose no risk in child loss. During weeks 11-14 in the dual test PAPP-A andfree β-hCG; while in the combined test and in addition to the PAPP-A and free β-hCG some ultrasound readings of the unborn infant are taken. In thetriple test during weeks 14-22 AFP, total hCG and uE3 is tested for; andfor the quadruple test following that in addition to the triple test inhibine–A values are identified. The integrated test is made consecutively; the first,during weeks 11-14 a PAPP-A measurement of the mothers blood sample and a necksize measurement (NT) of the infant. The second of these is done during weeks16-20 and checks for AFP, total hCG and uE3 with the addition of inhibin Ameaurements to obtain a risk value.

The AFP test,determines the risk of NTD (neural tube defect) by means of a blood sample andis obtained by comparing the AFP value to pregnacy term in weeks. In additionto the hormone values obtained through a sample of the mothers blood, as above,a form is prepared which outlines information regarding the expecting motherrace and whether she smokes,has diabetes, her number of pregnancies, if the pregnancy is through IVFmethods, if previous pregnancies had a chromosome or NTD history. Theprobability of risk is then calculated by a statistical program which uses thisinformation and the results obtained.

Determination of Oligoclonal Banding
Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the Central Nervous System. In clinically suspect cases, laboratory analysis will support diagnosis. Previously, an increase in the gamma globulin area was seen in protein electrophoresis on patient CSF (Cerebro-Spinal Fluid).However, as this finding was seen in a number of other diseases it did not support diagnosis of MS. With the use of high resolution agarous gel electrophoresis, the identification of oligoclonal bands in CSF – seen to be a more specific indicator – was realised. Today, the isoelectricfocusing (IEF) and immunblotting method is of note as the specific method in identification of oligoclonal bands.

IEF on agarous gel is done between 3-10pH. Following electrophoresis, a transfer to a blotting membrane is realised and the oligoclonal bands are made visible by a chromogen. IEF should be simultaneously done on both the CSF and serum samples. Four or more bands have to visible on the CSF with none visible on the serum for the test to be deemed positive. 

 On the other hand, proof of an IgG index is significant in MS patients with reports that it increased in a majority of patients. Our laboratory conducts oligoclonal band testing in conjunction with CSF and includes oligoclonal band testing in serum, CSF and IgG in serum and albumen tests, ratios and IgG indexing. Additionally and in using the data obtained, blood-CSF barrier irregularities on the Reiber graph or wheter an intratecal IgG synthesis exists are also reported.

Carbohydrate Metabolism Tests
Blood glucose concentration is regulated by the complex interaction of numerous processes managed by hormones. Countering insulin, which reduces blood glucose, these hormones are glucagon, epinephrine, cortisol and the growth hormone.The body can encounter a number of diseases with a change in glucose balance. The most important of these being Diabetes Mellitus. Accompanied by hyperglycemia, dyslypidemia and glucosuria, diabetes mellitus is a systemic, chronic metabolic disorder displaying a number of clinical symptoms. Prevalence increases with age, the frequency of obesity is higher than in the normal population The interval between an increase in blood glucose levels and the appearance of clinical symptoms is quite long. Therefore, those who are over a certain age and fall within the risk-bearing group should undergo controls at frequent intervals.
Symptomatic criteria for Diabetes Mellitus: 

  • Starvation plasma glucose levels over 126 mg/dl
  • 2 hour glucose levels over 140 mg /dl
  • Random plasma glucose levels over 200 mg/dl
  • Levels over 200 mg / dl at 2 hours following the OGTT (Oral Glucose Tolerance Test) 
  • HbA1c levels over 6.5%

Mention can be made of an impaired glucose tolerance in cases where the starvation glucose levels are between100-125 and are between 140-199 mg/dl with the 2nd hour plasma glucose levels following OGTT. Individuals who would fall under this group are considered prediabetic and are frequently checked for probable diabetes and/or cardiovascular risks. Specialists also indicate the need for individuals falling within the HbA1c 6.0-6.4% levels to be considered diabetic risks and that these persons be taken into appropriate protective programs. In terms of diagnosis and apart from glucose and HbA1c tests, a series of tests can be done to determine insulin and C-peptides (including starving and stimulated C-peptide levels when the symptoms of type 2 diabetes are seen prior to insulin treatment). Anti-Glutamic Acid Decarboxylase (Anti-GAD), Islet Cell Antibody (ICA), Insuline Auto Antibody (IAA) tests can also be availed of.

Lipid Metabolic Tests
As structural and functional components of membranes and the storage of energy; lipids, hormones and hormone precursors have assumed important roles in all aspects of life. Public health programs were developed following the definitive cause-result relationship in between blood fats and lipoproteins and atherosclerosis; clinical laboratories further developed reference methods to test and determine total cholesterol levels including HDL cholesterol, LDL cholesterol, triglycerides and lipoprotein (a). The relationship between hypercholesterolemia and atherosclerosis was first discussed by Thanhauser and Muller in 1938. Later studies also showed a direct relationship between high levels of cholesterol and the incidence and prevalence of coronary heart disease.Healthy individuals’ total cholesterol levels should be: 200 mg / dl, while LDL Cholesterol levels should be: 130 mg/dl. An individual is considered at risk limits with a total cholesterol level at 200-240 mg/dl and LDL cholesterol at 130-160 mg/dl, while they would be considered at high risk with total cholesterol levels at 240 mg/dl, LDL cholesterol level of 160 mg/dl and an HDL cholesterol level of 35 mg/dl.

Healthy individuals’ should have laboratory tests performed once every 5 years, those at medium risk once every two years and those in the high risk category should have their tests repeated once a year. Hypertension, physical activity and eating habits should be regulated for those individuals especially falling within the risk-carrying groups.