Clinical Biochemistry Laboratory

Biochemistry is the study of the structure and function of biological molecules such as proteins, nucleic acids, carbohydrates and lipids.

Biochemistry is the study of the chemistry of living things. This includes organic molecules and their chemical reactions.

Biochemistry deals with body substance like enzymes, carbohydrates, amino acids, fats, proteins, hormones, DNA, RNA, pigments etc.



The major objective of biochemistry is the complete understanding of all chemical processes associated with living cells at the molecular level. Some of the objectives can be listed as follows:

1. Isolation, structural elucidation and the determination of mode of action of biomolecules.
2. Identification of disease mechanisms.
3. Study of in born errors of metabolism.
4. Study of oncogenes in cancer cells.
5. The relationship of biochemistry with the genetics, physiology, immunology, pharmacology, toxicology etc.

Biochemistry is related to almost all the life sciences and without biochemistry background and knowledge, a through understanding of health and well-being is not possible.

1. CLINICAL BIOCHEMISTRY LABORATORY PRESENTED By : 1.Reshma Machamasi 2.Sarmila Timilsina 3.Tisha Shakya 4.Jharana Thapa 5.Karjit Dhami PRESENTED TO: Tapeshwar Yadav (Lecturer) BMLT, DNHE, M.Sc. Medical Biochemistry Department of Laboratory Medicine, Nobel College of Medical Sciences, Kathmandu, Nepal

2. contest Contents: Introduction. Objectives. Scope. Basic Equipment Used In Biochemistry lab. Collection And Precaution of Blood. General Tests. Other Tests or Special Tests. Safety Rules In Laboratory.

3. Introduction Biochemistry is the study of the structure and function of biological molecules such as proteins, nucleic acids, carbohydrates and lipids . Biochemistry is the study of the chemistry of living things. This includes organic molecules and their chemical reactions. Biochemistry deals with body substance like enzymes, carbohydrates, amino acids, fats, proteins, hormones, DNA, RNA, pigments etc.

4. Objectives of biochemistry The major objective of biochemistry is the complete understanding of all chemical processes associated with living cells at the molecular level. Some of the objectives can be listed as follows: 1. Isolation, structural elucidation and the determination of mode of action of biomolecules . 2. Identification of disease mechanisms. 3. Study of in born errors of metabolism. 4. Study of oncogenes in cancer cells. 5. The relationship of biochemistry with the genetics, physiology, immunology, pharmacology, toxicology etc. Biochemistry is related to almost all the life sciences and without biochemistry background and knowledge, a through understanding of health and well-being is not possible.

5. Scope: Biochemists work along with chemists, physicists, healthcare professionals , engineers and so on. They can also work for various organisatons including hospitals , universities , education , drug discovery as well many more professionals. They usually involved in research related works. They can be Biotechnologist , Research Scientist , Pharmacologist laboratory technician , Lecturer in an educational institution etc.

6. Basic Equipments Used In Biochemistry Lab

7. 1.Centrifuge: It is a laboratory device that is used for separation of serum or plasma from blood. Separation of sediment in urine etc.

8. 2. Dry Bath/Water Bath A dry bath is laboratory equipment that is used to heat samples. Water bath is used to incubate sample in water at a constant temperature over a long period of time.

9. 3.Colorimeter The device that measure the intensity of concentration of the colour .

10. 4.Electrolyte Analyzer: It is used to measure sodium (Na+),potassium (K+),chloride ( Cl -),Lithium (Li +) and Calcium ion(Ca++).

11. 5.Semi-analyzer: It perform all routine tests and most of special type of tests. Test results are display or print out.

12. 6.Pipettes: It is used to transport a measured volumne of liquid (reagents or specimen).

13. Collection and Precaution of blood

14. Blood Collection: Mainly there are three types of blood collection Methods. 1.Capillary puncture method: 2.Arterial puncture method: 3. Vein puncture method: For futher testing we need sample mainly blood are use.For Biochemistry lab we may required large amount of blood,hence vein puncture should be perform while collecting blood.

15. Gel tube EDTA tube Plane tube

16. PRECAUTION : 1. Data collection of patient must be done carefully. 2.Container and syringe should be dry. Otherwise, it causes haemolysis . 3.The blood in the anticoagulant should mixed carefully. 4.Blood collection should not attempts more than three times. 5.The blood should not be force through needle. 6. Plastic tube must not used for calcium test as it adsorbs calcium. 7. Rapid shaking of tube with blood may cause haemolysis .

17. General Tests Performed In Biochemistry Lab

18. Diabetes Mellitus: Absence or insufficient production of insulin  causes diabetes . There are  two  main types of diabetes: Type 1 or Insulin dependent diabetes. Type 2 or Non- insulin dependent diabetes.

19. Type 2 or non-insulin dependent diabetes mellitus : Type 2 diabetes mellitus,which is much more common occurs when the body cannot produce enough insulin or the insulin is not working efficiently enough. I t was formerly known as non-insulin dependent or adult – onset diabetes due to its occurrence mainly in people over 40. # Gestational diabetes mellitus occurs when pregnant women have high blood sugar levels due to hormones produced in pregnancy. # A diagnosis of gestational diabetes means a higher risk of developing type 2 diabetes in the future. Type 1 or insulin dependent diabetes mellitus: Type 1 diabetes mellitus occurs when the body cannot produce insulin which is needed to control blood sugar level. These type of diabetes occurs in children hence also known as juvenile diabetes.

20. Significance: Hyperglycaemia (increased level of glucose ) are seen in hyperthyroidism , hyperpituitarism , surgical removal of pancrease , haemorrhage etc. Hypoglycaemia (decreased level of glucose) are seen in hypothroidism , hypopituitarism , severe exercise , starvation etc. Normal value : Fasting: 60 -110mg/dl. Post Prandial : 70- 140 mg/dl. Random: 70-140 mg/dl.

21. Renal Function Test: The Renal function test or kidney function test  refers to a few commonly used tests to screen for renal function. Analysis of blood and urine samples can be essential for the evaluation of kidney (renal) function. It includes : Urea Creatinine Uric acid Sodium Potassium

22. A. Urea: Urea is the end product of protein metabolism which is synthesized in liver. Increased level of urea are in renal disease, urinary obstruction,shock burn . Decrease level of urea are found in liver failure. Normal value : 15-40 mg/dl.

23. B.Creatinine Creatinine is a waste product fromed in muscle by creatine metabolism. It is synthesized in kidney,liver and pancrease . Increased condition are in renal disease,starvation,hyperthyroidism etc. Normal value : In male= 0.7 - 1-.4 mg/dl In female = 0.4 – 1.2 mg/dl

24. C.Uric Acid: Uric acid is formed in body from the end product of the metabolism of purines (Adenine and Guanine) present in RNA and DNA . It is a metabolic waste product excreted by kidney . Increased level of uric acid ( hyperuricaemia ) are in gout ,Renal failure,starvation and so on. Decreased level of uric acid ( hypouricaemia ) are in liver cirrhosis etc. Normal value : In male= 3.5 – 7.0 mg/dl In female= 3.5 – 6.0 mg/dl

25. D. Sodium: It is a extracellular fluid . It maintain heart beat,osmotic pressure and regulates body acid-base balance. Increased level of sodium ( hypernatremia ) are loss of water from body , dehydration , diabetes mellitus etc. Decreased level of sodium ( hyponatremia ) are occur due to diarrhoea , vomitting ,edema, chronic renal disease. Normal value : 135-145 mEq /l.

26. E.Potassium It is a intra cellular fluid . It plays important role in tranmission of nerve impulse and also in cardiac muscle activities. Increased level of potassium ( hyperkalemia ) in renal failure ,depression , Anuria etc. Decreased level of potassium ( hypokalemia ) seen in cardio megaly , musclar weakness etc. Normal valve : 3.5 - 5.0 mEq / litre .

27. Liver function tests: Liver function tests measure various chemicals in the blood made by the liver. An abnormal result may indicate a problem with our liver and may help to identify the cause. Further tests may be needed to clarify the cause of liver problem .

28. There are six tests perform in LFT: Bilirubin SGOT(Serum glutamic oxaloacetic transaminase ) SGPT(Serum glutamate pyruvic transaminase ) ALP (Alkaline phosphatase ) Protein Albumin GGT(Gamma- glutamyl transferase )

29. 1)Serum Bilirubin : It is an orange or yellow pigment formed in the liver by the break down of haemoglobin and excreted in bile. There are two types of bilirubin . Unconjugated or Indirect bilirubin Congugated or Direct bilirubin . Increased in jaundice and haemolysis . Normal value: Total bilirubin =Up to 1 mg% Direct bilirubin =Up to 0.3 mg% Indirect bilirubin =Up to 0.7 mg%

30. 2) Serum glutamic oxaloacetic transaminase (SGOT) It is an enzyme that is normally present in liver and heart. Increased level of SGOT seen in myocardial infection, liver disease etc. Decreased level of SGOT seen in renal dialysis, vitamin B6 deficiency. Normal value : Less than 40 IU/L.

31. 3 )Serum glutamate pyruvic transaminase (SGPT): It is an enzyme that is normally present in liver and heart cells. SGPT is released into blood. Increased level of SGPT seen in liver disease such as cirrosis ,carcinoma, obstructive jaundice. Decreased level of SGPT seen in renal dialysis. Normal value : Less than 40 IU/L.

32. 4)Alkaline Phosphatase (ALP): It is an enzyme present in all tissues of the body It is produced by osteoblast of bone . It is localized in cell membrane and with transport mechanism in liver, kidney and intestine. Increased level of ALP in bone disease and obstructive jaundice. Normal value : 108-306 IU/L.

33. 5)Serum Total Proteins: The amount of proteins found in the fluid portion of our blood. Increased level of total protein in dehydration , chronic infection etc. Decreased level of total protein in liver disease,kidney disease,malnutrition etc. Normal value : 5.5 - 7.8 gm/dl

34. 6)Albumin Albumin is synthesized in liver and is present in plasma. Albumin transports hormones, fatty acids, and other compounds, buffers pH and maintains osmotic pressure among other functions. Increase level of serum albumin seen in dehydration, multiple myeloma etc Decrease level of serum albumin seen in malnitrition , cirrhosis of liver, nephrptic syndrome etc Normal value – 3.5 to 5.0 gm %

35. 7.GGT(Gamma- glutaml transpeptidase ) It is a enzyme that transfers gamma- glutamyl functional groups. It is present in the cell membrane of many tissues,including kidneys,pancrease,liver,spleen,heart,brain etc. GGT is elevated by large quantities of alcohol ingestion. Increased level of GGT are in disese of liver , pancrease etc. Normal value : 10-30 IU/L.

36. Lipid Profile Tests: A pattern of lipids in the blood. Serum lipid profile is measured for cardiovascular risk prediction and has now become almost a routine test.   Fasting blood for 9-12 hours (water only) is required. A lipid profile usually includes the levels of : Total cholesterol. High-density lipoprotein (HDL). Triglycerides. Low-density lipoprotein (LDL). cholesterol .

37. A)Serum Triglycerides : It is a type of fat found in our blood. They are used to give energy to our body. If we have extras, they are stored in different places in case they are needed later. Increased level of triglycerides are in risk of heart disease . Normal value : 10-190mg/dl. Lipemic serum

38. B)Serum total cholesterol: It is a fat like substance that is found in all body cells. Increased level in obesity, hypothyroidism, diabetes mellitus,cardiovascular disease. Decreased in level Haemolytic jaundice, intestinal obstruction etc. Normal value :150-260mg/dl.

39. C)HDL It is good or healthy cholesterol because it transport cholestrol from tissue to liver. Lower levels of HDL cholesterol indicate increased risk of coronary heart disease. Higher levels indicates negative risk factor of coronary heart disease. Normal value : 30 – 60 mg/dl.

40. D) LDL: LDL is a lipoprotein (a combination of fat and protein) found in the blood. It is called "bad" cholesterol because it picks up cholesterol from the blood and takes it to the cells. A high LDL level is related to a higher risk of heart and blood vessel disease. Normal value : Upto 150 mg/dl.

41. Pancreatic function tests: It is a endocrine and exocrine functions. Pancreatic function tests are useful for diagonsis of pancreatic disorders. Glucagon is produced by α -cells of pancreas which increase glucose level. Insulin is produced by β - cell of pancreas which decreases glucose level. Pancreatic function tests includes: Amylase Lipase

42. A)Amylase: It is a hydrolytic enzyme which hydrolyses starch into maltose. It presents in saliva and pancreatic juice where it is secreated by parotid gland. Increased level in acute pancreatitis , renal failure, Appendicitis. Normal value :150-340 U/L.

43. B)Lipase: It is a enzyme that catalyses the break down of fats of fatty acids. Lipase  is produced in the pancreas, mouth, and stomach. High amounts of  lipase  may be found in the  blood  when the pancreas is damaged or when the tube leading from the pancreas (pancreatic duct) to the beginning of the small intestine is blocked. Normal value : 0 – 160 U/l

44. 4.Thyroid Function Test(TFT): It is a collective term for blood tests used to check the function of the thyroid. A TFT panel typically includes thyroid hormones such as  Stimulating hormone ( TSH,thyrotropin ) Thyroxine  (T4) Triiodothyronine (T3).

45. T3 ( Triiodothronine ): A free or total triiodothronine test is used to assess thyroid function. It affects almost every physiological process in the body including growth and development,body temperature,heart beat. Normal value : 1.42 – 4.2 pg/ml. II.T4 ( Thyroxine ): Free thyroxine (free T4) tests are used to help evaluate thyroid function and diagnose thyroid diseases, including hyperthyroidism and hypothyroidism , usually after discovering that the thyroid stimulating hormone (TSH)  level is abnormal. Normal value : 0.65 – 1.97 ng /dl.

46. III.TSH(Thyroid-stimulating hormone): A thyroid-stimulating hormone (TSH) blood test is used to check for thyroid gland problems.  The cause of an hypothyroidism, TSH levels can help determine whether hypothyroidism is due to a damaged thyroid gland or some other cause (such as a problem with the pituitary gland or the hypothalamus). Normal value : 0.4 – 5.5 µIU/ml.

47. Free T4 Free T3 Interpretation High Normal Normal Mild (subclinical) hypothyroidism. High Low Normal or low Hypothyroidism. Low Normal normal Mild (subclinical) hyperthyroidism. Low High or normal High or normal Hyperthyroidism. Low Low or normal Low or normal Non-thyroidal illness; pituitary (secondary) hypothyroidism. Normal High high Thyroid hormone resistance syndrome (a  mutation in the thyroid hormone receptor decreases thyroid hormone function) TSH

48. Cardiac Function Test: The  diagnostic tests in cardiology  are methods of identifying  heart  conditions associated with healthy vs. unhealthy, pathologic heart function. It includes the tests below: CPK. CPK MB. LDH. GOT/AST. Troponine . Myoglobin .

49. 1.CPK( Creatinine phosphokinase ): Creatine phosphokinase (CPK) is an enzyme in the body. It is found mainly in the heart, brain, and skeletal muscle. This article discusses the test to measure the amount of CPK in the blood. 2.CPK- MB( isoenzyme of CPK found in heart): T he   CPK - MB  test is a cardiac marker used to assist diagnoses of an acute myocardial infarction. It measures the blood level of  CK - MB , the bound combination of two variants ( isoenzymes CKM and CKB) of the enzyme phosphocreatine kinase . 

50. 3.LDH(Lactate Dehydrogenase ): Lactate dehydrogenase (LDH) is a protein that helps produce energy in the body. An LDH test measure the amount of LDH in the blood. It is most often measured to check for tissue damage. It is in many body tissues,especially the heart,liver,kidney,muscles,brain,blood cells and lungs. 4.Troponine: Troponin is a complex of three regulatory proteins( troponin C,  troponin I, and  troponin T ) that is integral to muscle contraction  in skeletal muscle and cardiac muscle, but not smooth muscle.

51. 5.Myoglobin Myoglobin  (symbol Mb or MB) is an iron- and oxygen-binding protein found in the muscle tissue of vertebrates in general and in almost all mammals. It is related to  hemoglobin , which is the iron- and oxygen-binding protein in blood, specifically in the red blood cells .   In humans, myoglobin is only found in the bloodstream after muscle injury.

52. Other tests or special tests done in Biochemistry Lab

53. Serum calcium It is a major mineral element of body. It is present in bones and teeth also present in body fluids in small concentrations. It also involved in blood coagulation. Important role in muscle contraction . Based source of calcium in milk and their product Increased condition is Hypercalcaemia . Decreased condition in serum hypocalcaemia. Normal value : 8.5-11.0 mg/dl .

54. Serum phosphorus: Phosphorus is an essential structural component of cell membranes and  nucleic acids . Phosphorus is found in most food sources and is a component of many commonly used food additives.  Phosphorus is an essential mineral that is required by every cell in the body for normal function. Hyperphosphatemia is observed in fasting then in post prandial,renal failure,hypervitamininosis,cardiovascular disease etc. Hypophosphatemia is observed in rickets,hypoparathyroidism etc. Normal value : In adult= 2.5 – 5.0 mg/dl. In children= 4.0 – 7.0 mg/dl.

55. HbA1c (Hemoglobin A1c)  HbA1c is a form of hemoglobin that is bound to glucose. The blood test for HbA1c level is routinely performed in people with type 1 and type 2 diabetes mellitus. Blood HbA1c levels are reflective of how well  diabetes is controlled. The normal range for level for HbA1c is less than 6% . HbA1c is also known as glycosylated , or glycated hemoglobin.

56. Microalbumin A urine microalbumin test is a test to detect very small levels of a blood protein (albumin) in urine. A microalbumin test is used to detect early signs of kidney damage in people who are at risk of developing kidney disease. Microalbumin tests are recommended for people with an increased risk of kidney disease, such as those with type 1 diabetes, type 2 diabetes or high blood pressure. Normal value : Below 20 mg/dl.

57. Spot PCR ratio: Urine protein test is used to detect protein in the urine ( proteinuria ). 24 hrs urine protein or a random protein to creatinine ratio may be used to monitor a person with known kidney disease or damage. A urine protein to creatinine ratio may be ordered on a random urine sample if there is evidence of significant and persistent protein in the urine. Normal value : In male= less than 0.11. In female= less than 0.16.

58. 24 Hours Urine Protein test: The 24-hour urine protein test checks the function of the kidneys and helps detect disease. Urine samples are collected in one or more containers over a period of 24 hours. When higher than normal amounts of protein are in the urine, it’s called proteinuria . This is often a sign of kidney damage and disease. Normal value : less than 150 mg/day.

59. Body Fluid Fluid is a liquid that is made in the cavity to lubricate the surface of the tissue that lines and covers most of the organs in the cavities. It included: Synovial fluid : present in joints like knee joint,hip joint,elbow joint,shoulder joint etc. Pleural fluid : it is a fluid of pleural surface(around the lung). Pericardial fluid : fluid present in pericardial sac(around the heart). Peritoneal fluid : it is a straw coloured clear liquid that is made in the abdominal cavity.

60. ADA (Adenosine Deaminase )Test: ADA is a protein that is produced by cells throughout the body and is associated with the activation of lymphocytes , a type of white blood cell that plays a role in the immune response to infections. An ADA test may be ordered when a person has an accumulation of fluid in the chest cavity(pleural fluid) and has signs or symptoms that suggest TB. ADA activity is increased in various conditions such as liver disease , tuberculosis , typhoid. The average of serum ADA in TB and non-TB patients are 20.88U/L and 10.69 U/L respectively.

61. CSF EXAMINATION Cerebrospinal fluid (CSF) analysis is a way of looking for conditions that affect the brain and spine. It is a series of laboratory tests performed on a  sample  of CSF.  CSF  usually contains a small amount of  protein  and glucose and may have a few white blood cells.

62. Tumor Markers: A tumor marker is a substance present in or produced by a tumor or by the tumor’s host in response to the tumor’s. Hormones as tumor markers: ACTH ADH Calcitonin HCG etc Carbohydrates as tumor marker: CA 15-3 CA 125 CA 19-9 etc.

63. Name of test Normal test Significance(high) 1.RFT # Urea 15-40 mg/dl Renal failure , Dehydration, shocks, severe diarrhea, severe burns, etc. #Creatinine 0.4-1.4 mg/dl Renal failure, congestive heart failure, shock, Obstruction in urinary tract, etc. #sodium 135 to 145 mEq/L Thirst, fatigue, coma, rapid heart beat, weakness, etc. #potassium 3.5 to 5.5 mEq/L Weakness, tiredness, palpitation, etc. 2 .LFT #Bilirubin T.bili= upto 1mg% D.bili= upto 0.4mg% Jaundice, haemolysis, etc. #Total protein 6.3-8.4 gm% Dehydration, multiple myeloma, etc. #Total albumin 3.5-5.0 gm% Dehydration, multiple myeloma, etc. #SGPT/ALT <40 IU/L Liver disease, obstructive jaundice, etc.

64. Name of test Normal value Significance #ALP 108-306IU/L Pregnancy, hepatic disease, osteoblast, etc. 3.LIPID PROFILE #Triglyceride Upto 150 mg/dl Diabetes mellitus, liver disease, nephrotic syndrome, etc. #Total Cholesterol 150-250 mg/dl Diabetes mellitus, obstructive jaundice, nephrosis, etc. #HDL/Good Cholesterol 30-60 mg/dl Diabetes mellitus, obstructive jaundice, nephrosis, etc. #LDL/Bad Cholesterol Upto 150 mg/dl Heart and blood vessel disease. 4.TFT # Free T3 1.42 – 4.2 pg/ml. #Free T4 0.65 – 1.97 ng /dl. #TSH 0.4 – 5.5 µIU/ml.

65. Rules for working in a biochemistry laboratory There are two major concerns to be considered when working in a biochemistry laboratory. First is safety and second is efficiency in the laboratory work. There are general rules that we students are advised to follow: Keep the benches and shelves clean and well-organized. 2. Avoid contaminating the chemicals, use only clean glassware, label glassware in use. 3. Plan your experiments before starting to carry them out. 4. Pay attention to others in the laboratory.

66. Safety rules in the laboratory Safety rules in general Do not work alone in the laboratory. 2. Unauthorized experiments are not allowed. 3. Eating, drinking and smoking in the laboratory are strictly prohibited. 4. Become familiar with the location and the use of standard safety features in the laboratory. The laboratory is equipped with fire extinguishers, eye washes, safety showers and first-aid kits. Any question regarding the use of these facilities should be addressed to your instructor. 5. Special care for eye protection is required. Safety glasses must be used when certain procedures are being carried out.

67. B. Special safety rules While heating a solution one should make sure not to overheat it.The mouth of the glassware containing the solution to be heated should never be pointed toward anyone. 2. Handling of strong acids and bases requires special attention. When diluting concentrated acids, the acid should be poured into the water and never the opposite. 3. The pipettes should never be filled with solutions of toxic substances, biological fluids, strong acids and bases by mouth suction. Use either automatic pipettes or pipette pumps . 4.Volatile liquids and solids that are toxic or irritating should be handled under fume hoods. 5. While handling flammable liquids such as ether, alcohols, benzene, naked flame (burners, matches) must not be in use. The above liquids must not be stored near radiating heat sources, such as the laboratory oven. 6. Before using electrical appliances, make sure they are grounded. 7. Before leaving the laboratory, electrical equipment should be turned off, and gas burners extinguished. No tap water should be left running.

68. Thank you !!!!!!