Clinical laboratory science ppt for year

Clinical laboratory presentation

1. Chapter Four Basic serological tests Learning objectives; At the end of the session you will be apple to; recognize the basic principle of serological tests explain serological tests of syphilis, pregnancy, HIV interpret their results 5/29/2024 1

2. Syphilis Syphilis is a chronic systemic disease, which leads to lesions on the body. It is derived from a Greek word " syphilos " meaning crippled, maimed Is a systematic infection caused by the spirochate Treponema pallidium Transmitted by: Mainly Sexual contact (Venereal syphilis) Less commonly via the placenta (congenital syphilis) or By accidental inoculation from infectious material e.g. fresh blood transfusion 5/29/2024 2

3. Laboratory diagnosis of syphilis Serologic detection of syphilis Treponomal antibody detection Non treponomal antibody detection 5/29/2024 3

4. Antibodies in syphilis Two types 1. Non treponemal antibody ( reagin antibody ) are auto antibodies against tissue antigens. Are non specific are produced in patients with other infectious disease as measles , hepatitis, leprosy , malaria & tuberculosis . Are IgG & IgA class is identified by VDRL & RPR. 5/29/2024 4

5. 2. Treponomal antibody ( specific antibody ) antibodies directed against T. pallidum antigens (either to the spirochete it self or its components) Are IgM type Are screened by FTA –Abs Specimen for syphilis diagnosis Serous fluid from chancres & secondary skin lesion - to detect motile T. pallidum. Blood/Serum/ plasma - for antibody testing. 5/29/2024 5

6. Non- treponemal (Non-specific ) Tests 1. Venereal Disease Research Laboratory ( VDRL ) Both qualitative and quantitative tube tests Principle heat inactivated serum is added to a measured drop of buffered saline suspension of cardiolipin lecithin cholesterol antigen on slide & after rotating the slide , the mixture is examined using 10X objective.  Aggregation Interpretation…. No clumping/ very slight roughness = non reactive Medium/ large clump = reactive. Small clumping = weakly reactive 5/29/2024 6

7. For the test to be valid the negative control must be non-reactive and the positive control must be reactive 5/29/2024 7

8. 2. Rapid plasma reagin( RPR ) card test Principle A modified cardiolipin antigen containing cholesterol and lecithin in which micro particulate carbon particles are suspended. In the presence of reagin , clumps or flocculation appear which can be visualized as black clumps against white back ground . The result is read macroscopically . Specimen:- plasma or serum Reporting results -Small to large clumps - reactive -No clumping or slight roughness – non reactive 5/29/2024 8

9. Pregnancy test Detect the presence of Human chorionic gonadothropin (HCG) hormone which is produced by the trophoblastic tissue in the placenta to confirm pregnancy to diagnose ectopic pregnancy and trophoblastic tumors . HCG is a glycoprotein hormone two glycoprotein sub units  &  . Serum HCG first reaches detectable level within 24 hour after implantation. 5/29/2024 9

10. Methods of evaluation A. Direct agglutination slide test( direct latex slide test ) Principle : the latex reagent consists of particle coated with anti-HCG antibodies This reagent is mixed directly with the urine Presence of visible agglutination by naked eye is reported as positive for HCG If there is no visible agglutination report as negative 5/29/2024 10

11. If hCG is present in the urine, it will combine with the antibodies and causes agglutination of the latex particles. Urine latex reagent Result ( hCG antibody a gglutination Coated particles) Positive result 5/29/2024 11 hCG Antigen is present hCG antigen combines with hCG antibody on latex particles

12. If no hCG is present in the urine, there will be no agglutination of the latex particles. Urine latex reagent Result (HCG antibody No agglutination Coated particles) Positive result 5/29/2024 12 No hCG Antigen No hCG antigen combines with hCG antibody on latex particles

13. B. Strip test Principle :- the test strip is based on the reaction between the HCG in the urine and gold coated antibody on the strip . A positive control is also impregnated on the strip so that interpretation will be very easy.   5/29/2024 13

14. HIV TESTS HIV is a human immunodeficiency virus which causes chronic diseases with other infectious agent. Several laboratory methods are available to screen blood, diagnose infection, and monitor disease progression in individuals infected by HIV. HIV tests can be classified into: detect antibody identify antigen detect or monitor viral nucleic acids 5/29/2024 14

15. Common HIV test Enzyme Linked Immunosorbent Assays (ELISA) Rapid Tests HIV serology

16. Enzyme Linked Immunosorbent Assays (ELISA) ELISA rely on a primary antigen-antibody interaction Characteristics of ELISA Are best performed at a regional or national laboratory Why? Because They require well-trained and skilled laboratory technologist Technologically advanced equipment (incubators, Washers and spectrophotometers) that requires maintenance and a constant source of electricity. ELISA can be performed with serum, plasma, urine, oral fluids, or dried blood spots HIV serology

17. General steps for performing an ELISA Dilute the specimen in the specimen buffer and put it in a micro well plate containing HIV antigen already bound to the plate. Incubate the plate as per protocol and then wash as indicated. Add antihuman immunoglobulin-enzyme conjugate, which will react with the HIV specific antibody if present HIV serology

18. Incubate Wash the plate, add the substrate and incubate as prescribed Add a stopping solution to terminate the enzyme reaction and read the absorbance of the solution using spectrophotometer. HIV serology

19. A positive reaction has occurred if the specimen in the specimen well changes color or becomes colored Positive reaction indicates the presence of HIV-specific antibody in the specimen. The reaction is best read quantitatively with an ELISA plate using spectrophotometer (ELISA reader). HIV serology

20. Double antibody ELISA (antigen test) 5/29/2024 20 Add Specimen Containing Ag

21. Double antibody ELISA (antigen test)…. cont ELISA reaction 5/29/2024 21 EL-antibody-A Antigen –A Antibody-A Substrate Color  change +

22. Characteristics of Rapid Tests Rapid tests are useful for small laboratories that routinely perform fewer than 100 HIV tests per day Many rapid tests do not require electricity, special equipment, refrigeration, or highly skilled staff although a few require refrigeration for heat- sensitive reagents. Sensitivity approaches 100%; specificity is >99% B. HIV Rapid Tests

23. Rapid procedure An antigen is coated on the strip with positive control up on addition of serum /plasma or whole blood depending on the test procedure , there will be reaction between antigen and antibody if present in the sample. Reactive results : two colored bars (one for the control & the other for the patient ) Non- reactive : single colored bar ( positive control only ) Invalid result : without having any line. 5/29/2024 23

24. Test algorithm for HIV It is a series of HIV tests that are useful to avoid false positive & false negative results one can establish tests algorithms by combining the different tests. The limitation of screening test /Rapid test Are their inability to detect the virus in early infection or during window period. 5/29/2024 24

25. Test Procedure… 5/29/2024 26

26. Test interpretation (within 30 minutes) 5/29/2024 27 Reactive for HIV1/2 Non Reactive for HIV 1/2 Invalid Results

27. Chapter Five Immuno-hematology Immuno-hematology is a specialized branch of biomedical science & it study the immunologic reactions involving blood & blood components. Main application includes Safe blood transfusion and blood banking Prevent of Rh immunization associated with pregnancy Detecting the infection and disease 5/29/2024 28

28. Blood grouping The ABO –system and the Rh system remain the most important in transfusion therapy. Blood typing is done Before a person receives or donates blood Before a person donates an organ for transplantation When a woman is planning to become pregnant To help determine whether two people could be blood relatives 5/29/2024 29

29. The ABO blood group system Major blood groups are A, B, AB , and O O type RBCs lack A or B antigens These antigens are carbohydrates attached to a precursor backbone, may be found on the cellular membrane as glycoproteins, and are secreted into plasma and body fluids 3 allelic genes A, B & O control the synthesis of specific enzymes ( transferases ) 5/29/2024 30

30. Antibodies of ABO system They are naturally occurring, produced from fetal life They are present in reciprocal to the missing ABO antigen Persons with type AB are "universal recipients" because they do not have antibodies against any ABO phenotype while persons with type O blood can donate to essentially all recipients because their cells are not recognized by any ABO isoagglutinins (universal donor) They are IgM in nature, react at room temperature 5/29/2024 31

31. ABO blood grouping The main function to prevent transfusion reaction. There are two methods Forward ( cell typing ) Reverse (serum typing ) 5/29/2024 32

32. 1. Forward or cell typing Based on the presence or absence of the corresponding antigen on red cell surface. RBCs + Antiserum  Agglutination (Ag in Sample ) (rgt) ( Ag-Ab rxn ) Interpretation : Positive agglutination with specific antiserum indicates the presence of antigen and Negative agglutination indicates the corresponding antigen is not present . 5/29/2024 33

33. 2. Reverse ( serum) typing based on the presence or absence of antibodies in the serum. Serum + RBCs  + ve agglutination ( Ab )A/ B Ag ( Rgt ) (antigen -antibody reaction) Interpretation positive agglutination antibodies specific for A/B antigen is present . negative agglutination no antibodies are present for antigens 5/29/2024 34

34. Rh- blood group system The Rh system is the second most important blood group system in pretransfusion testing Some individuals posses a weak form of the D- antigen known as weak D or Du variants . The antigen is well developed before birth . 5/29/2024 35

35. Rh- antibodies Are usually made by exposure to Rh antigens through transfusion or through pregnancy and show similar serologic Rh- antibodies characteristics. IgG in type, can pass through placenta. They are clinically significant because the Rh (D) antigen is potent immunogen Anti -D antibody is common cause of sever hemolytic disease of the new born (HDN) 5/29/2024 36

36. Rh- typing The importance of Rh typing Administration of Rh + ve blood to Rh – ve may sensitize the person to form anti-D antibody Donation of Rh + ve blood to recipient having anti D could be fatal . RBCs + Anti –D  + agglutination D-Ag (antisera) ( ag- ab rxn) -This can be performed on slide test, saline tube test or modified tube test. 5/29/2024 37

37. Interpretation If there is agglutination of RBCs --------positive for D- antigen ( Rh positive ) If there is no agglutination of RBCs --------the D- antigen is not expressed .This can be negative. 5/29/2024 38

38. Illustration Of The Forward And Reverse Grouping Reaction Patterns Of the ABO groups 5/29/2024 39

39. Cross matching It is the last phase of pre transfusion testing. It will detect ABO or Rh incompatibility There are two kinds of cross match. 1. Major cross match 2. Minor cross match 5/29/2024 40

40. Major cross match Detects antibodies in the recipients serum that may damage the cells of proposed donor. Recipient serum + Donor RBC  agglutination or hemolysis Result : Agglutination / hemolysis ----- incompatible No agglutination/ hemolysis ------compatible 5/29/2024 41

41. Minor cross match Detect antibodies in donor’s serum capable of affecting the RBC of the recipient. It has minor importance. Donor serum + recipient RBC  agglutination/ hemolysis. Result can be interpreted like major cross match . 5/29/2024 42

42. Chapter six Routine urinalysis test Learning objectives: At the end of the session, you will be able to; Know how to collect, preserve and transport urine Describe the principle of Routine urinalysis test Interpret values of these tests Perform some of these tests 5/29/2024 43

43. Collection, handling and preservation of urine Container should be clean, dry, wide mouthed, sterile for culture. Depending on the type of investigation, a single specimen may be adequate or it may be necessary to collect urine over a 24 hour period. 5/29/2024 44

44. Types of urine sample First morning urine specimen- is a specimen obtained during the first urination of the day Generally contain the highest concentrations of substances to be tested. Preferable for routine analysis like nitrite, protein and microscopic examination of sediments, especially formed elements (RBC,WBC) B. Random specimen - a specimen collected at any time of the day and most common and convenient type of specimen. -It is preferable for chemical screening and microscopic examination C. Clean – catch- specimens –used for culturing of bacteria -Mid stream - a specimen obtained from middle part of the first urine. - catheterization and suprapubic aspiration 5/29/2024 45

45. D. 24 hrs urine – A specimen obtained within 24hrs and necessary for quantitative determination of the anaylates , especial protein determination . E. Post prandial specimen- a specimen obtained after 2hrs of meal and good for glucose determination . The collected urine must be processed within 30 minute 5/29/2024 46

46. Potential changes in unpreserved /delayed urine sample 1.Physical changes Colo r will be changed due to oxidation and reduction of substances Ph becomes alkaline due to decomposition of urea to ammonia by bacterial action Odor and clarity change may occur 5/29/2024 47

47. 2. Chemical changes Glucose- decrease due to bacterial & cellular glycolysis Nitrate- either increase if bacteria reduces nitrite or decrease if it is converted to ammonia 3. Microscopic changes RBC, WBC and casts decrease due to disintegration, bacteria increase. 5/29/2024 48

48. Methods for urine preservation There are two method for urine preservation A. Physical method Refrigeration :- The most common method -The sample can be kept usually from 6-8hrs without gross change at 2-8 c. -Although this method decrease bacterial growth and metabolism, the best way to preserve urine for bacterial examination ( urine culture) Freezing :- keep urine sample below -20 c. -This method is good for preserving bilirubin and urobilinogen - Disadv . destroy formed elements. 5/29/2024 49

49. B. Chemical method of urine preservation When a specimen must be transported over long distance & refrigeration is not possible , chemical preservatives must be added. The ideal preservative should be; - Bacteriostatic -Inhibit urease and -Preserve formed elements -Should not interfere with chemical test. E.g. Toluene, formalin, boric acid 5/29/2024 50

50. Routine urinalysis test Def - it is physical, chemical and microscopic examination of urine or analysis of urine by manual or automated method. Performed in order : To diagnose disturbance of kidney function & urinary systems. To diagnose the diseases that do not originate in the urinary system, like Diabetic mellitus To monitor different causes of diseases and evaluating efficiency of treatments . 5/29/2024 51

51. Urinary System The urinary system is made up of Two kidneys Two ureters One bladder and One urethra The two kidneys form and excrete urine . 5/29/2024 52

52. Examination Of Urine - There are three types of urine examinations 1. Physical examination 2. Chemical examination 3. Microscopic examination 5/29/2024 53

53. 1. Physical examination of urine It provides preliminary information concerning disorders such as glomerular bleeding, liver disease, urinary tract infection The first, simple and oldest part of urinalysis involving assessment of physical properties of urine such as; Volume - the normal value 600ml -1200ml/24hrs Odor - the normal value aromatic odor normally Color - the normal color pale or dark yellow . Specific gravity - 1.024 Transparency - Clear 5/29/2024 54

54. 2.Chemical Examination Of Urine It is procedure or examinations which are accomplished by chemical reaction and used to detect abnormal or pathological dissolved constituents of the urine Chemical constituents of the urine Slightly acidic (PH 5-6)and contains 95% water and remaining 5% are dissolved substances such as: (Fresh voided urine from healthy individuals) 5/29/2024 55

55. Abnormal or pathological constitutes of urine Ph , Hgb , glucose, proteins, ketones, bilirubin, urobilinogen , cellular elements, nitrite, parasites, bacteria, fungus, different types of casts Rgt strip test common for chemical examination of urine 5/29/2024 56

56. 3. Microscopic Examination Used to observe formed cellular elements, casts, bacteria, parasite, yeast, crystals in centrifuged urine sediment. Reporting Casts /LPF WBC, RBC, epithelial cell, yeast, bacteria, sperm cell, crystals reported per HPF 5/29/2024 57

57. Microscopic Exam Red blood cells Presence of a few is normal??? Higher numbers are indicator of renal disease Result of bleeding at any point in urinary system 58 5/29/2024 40x objective

58. Cont’d… Large number of WBC indicate bacterial infection of UT If the infection is in kidney WBCs may be associated with cellular and granular cast, bacteria, epithelial cell 5/29/2024 59

59. Unit Seven BASIC CLINICAL CHEMISTRY TESTS Liver Function Test Renal function test Carbohydrate metabolism disorders Plasma protein 5/29/2024 60

60. Liver Function Test Liver is the largest organ in humans covered by Glisson’s capsule Liver contains multiple parenchymal cells Hepatocytes (liver cells) have a unique regenerative capability and marked capacity to respond to increased metabolic demands of the organism. 5/29/2024 61

61. Function Of liver Liver directly receives, processes and stores materials absorbed from the digestive tract Many plasma proteins, clotting factors and transport proteins are formed in the liver. Liver is main site of detoxification. It synthesize bile acid from cholesterol It responds to multiple hormonal and neural stimuli to regulate blood glucose concentration. It regulates the plasma hormone level. 5/29/2024 62

62. Clinical manifestation of liver diseases Jaundice Portal hypertension Hepatic failure and encephalopathy Nutritional and metabolic abnormalities Immunoglobulin abnormalities Disordered hemostasis 5/29/2024 63

63. Laboratory assessment of liver function Liver function tests are used for recognition of hepatic dysfunction . Tests of hepatic function can be classified in to : 1.Tests based on substances produced by the liver. Albumin, coagulation factor, cholesterol esterase 2.Tests based on substances metabolized by the liver. Drug, antibiotic, bilirubin, cholesterol. 5/29/2024 64

64. 3.Tests based on substances released from the damaged tissue. Endogenous substances: released by damaged hepatocytes (ALT, AST) 4. Tests based upon substances cleared from plasma by the liver. Endogenous substances (bile acid, bilirubin, ammonia) 5/29/2024 65

65. Disturbance of bilirubin metabolism Classification of Jaundice Based on presumed physiological and anatomical abnormality, it is classified as three I. Prehepatic jaundice resulted when excessive amount of bilirubin is presented to the liver for metabolism E.g1 hemolytic anemia E.g.2 physiological jaundice Unconjugated bilirubin can reach to 40-50mg/l by 8 hours and decrease to normal value by 7-10 days. Characterized by Unconjugated hyperbilirubinemeia - The bilirubin will not appear in the urine 5/29/2024 66

66. II Hepatic jaundice resulted from impaired cellular uptake, defect in conjugation and abnormal secretion of bilirubin by liver cells III. post hepatic jaundice resulted from impaired secretion/excretion of bilirubin by obstruction of flow of bile to GIT conjugated bilirubin raise in serum & appear in urine urine urobilinogen decreases stool becomes clay-colored E.g. When bilary tree becomes blocked or abnormally permeable. Serum concentration of conjugated bilirubin cholesterol, bile acid will increase and also increase synthesis of GGT&ALP. 5/29/2024 67

67. Methods for determination of serum bilirubin 1. Jendrassic and Grof method Serum or plasma is added to a reagent and absorbance is measured at 520 nm Normal value for infants > 1 month & adults Conjugated bilirubin = 0-0.2 mg/dl Unconjugated = 0.2-0.8 mg/dl Total = 0.2-1mg/dl 5/29/2024 68

68. 2. The test based on substances released from damaged liver tissue Alanine transaminase (ALT), it is so-called serum glutamic pyruvate transaminase(SGPT). Involved in the transfer of amino group from alanine to 2-ketoglutarate . it is specific to liver. When hepatic cells damaged, they leak this enzyme to blood where it is measured. ALT dramatically increase in -acute liver disease & -viral hepatitis Normal value 5-60Iu/l 5/29/2024 69

69. Significance of the test Increasingly ALT is being measured to monitor patients receiving antiretroviral drugs associated with hepatotoxicity such as nevirapine (NVP) and stavudine (d47). Lower level may be caused by malnutrition. 5/29/2024 70

70. II. Aspartate amino transferase (AST ). It is so-called serum glutamic oxalo acetic transaminase (SGOT)& found in Hepatocytes, RBCs, cardiac cells, bone, skeletal muscle. Involved in the transfer of an amino group between aspartate as 2-keto acids(2-oxoglutarat) Normal value 6 – 25 Iu /l Significance of the test -help to diagnose liver disease ( it is increased in intracellular liver disease) -monitor recovery from or treatment for liver disease. An important cause of elevated AST activity is myocardial infarction 5/29/2024 71

71. Disorder of carbohydrate (CHO) metabolism Blood glucose main end product of carbohydrate digestion Insulin is secreted by  -cell of islets of langrehuns of the pancreas regulates the amount of glucose in the blood a hormone that helps move glucose from your blood into your cells, which use it for energy. Glucagon secreted from  -cell of pancreas Tend to elevate glucose level 5/29/2024 72

72. Blood glucose tests may be done at different times fasting blood glucose 2- hour post prandial blood sugar random blood sugar Significance of the test to diagnose diabetes. to monitor treatment of diabetes . 5/29/2024 73

73. Blood glucose meter Measuring glucose in capillary blood using a reagent test strip and glucose meter provides a rapid result Inexpensive, simple to use 5/29/2024 74

74. Normal value of glucose: FBS: < 110mg/dl 2-hour post prandial: < 140mg/dl Random: < 126mg/dl 5/29/2024 75

75. Diabetes mellitus Characterized by chronic hyperglycemia with disturbance of CHO, fat, and protein metabolism cause: -defects in insulin secretion, action or both. symptoms thirst, polyuria, polyphagia & polydypsia or excessive thirst, blurring of vision & weight loss. In sever case ketoacidosis or ketomia  coma & death. 5/29/2024 76

76. Classification of diabetes mellitus Type I (IDDM) is also called  insulin-dependent diabetes mellitus It used to be called juvenile-onset diabetes, because it often begins in childhood Type 1 diabetes  is an autoimmune condition It happens when your body attacks your  pancreas  with antibodies The organ is damaged and doesn't make insulin Your genes might cause this type of diabetes It could also happen because of problems with cells in your pancreas that make insulin Treatment for type 1 diabetes  involves injecting insulin into the fatty tissue just under your skin. 5/29/2024 77

77. Type II (NIDDM) Type 2 diabetes  used to be called non-insulin-dependent or adult-onset diabetes About 90% of people with diabetes have type 2 When you have type 2 diabetes, your pancreas usually creates some insulin. But either it’s not enough or your body doesn’t use it like it should. Insulin resistance, when your cells don’t respond to insulin, usually happens in fat, liver, and muscle cells. 3. Gestational diabetes mellitus (GDM) Normal pregnancy is associated with increased insulin resistance GDM develops due to pregnancy-related elevation of hormones antagonistic to insulin, leading to insulin resistance, increased insulin requirements, and increased glucose availability for the developing fetus. These mechanisms result in an elevated risk of abnormal glucose metabolism, especially in the presence of obesity. Risk factor for GDM obesity, advanced maternal age & familial history of DM

78. RENAL FUNCTION TEST Function of the kidney Removal of excess body water Elimination of waste products of metabolism Elimination / excretion of foreign substances like drugs Retention of substances necessary for normal body function Regulation of electrolyte balance and osmotic pressure of the body fluids 5/29/2024 79

79. Physiology of the kidney Glomerulus receive incoming blood filter the blood & most proteins After the blood have been filtered in the glomerulus, it will go to the next part , the proximal convoluted tube The filtrate contains both the waste products and substances that are valuable to the body. Proximal covulated tube will conserve most ions ( Na, K, Cl, HCO 3 ) , glucose ,amino acid & eliminate urea & creatinine 5/29/2024 80

80. Analytical procedure of renal function test Non protein nitrogenous substances waste products formed in the body as a result of metabolism of nucleic acid , amino acid & protein The three non protein nitrogenous substances are urea creatinine uric acid 5/29/2024 81

81. A. Urea synthesized in the liver from ammonia & CO 2 transported by plasma to the kidney to be excreted in urine Azothemia is increase level of urea Uremia is increase blood level of urea cause renal failure Analytical method Urea concentration of blood is often expressed in terms of blood urea nitrogen(BUN) 5/29/2024 82

82. B. Creatinine derived from creatine & creatine phosphate and excreted in the urine product of protein metabolism Plasma level of creatine are related to relative muscle mass & renal function sample serum, plasma, urine Laboratory methods Jaffe reaction Serum is mixed with alkaline picric acid and the rate of red color formation is read at 520  m . Normal value Men =0.7 - 1.2 mg/dl Women =0.6 - 1.1 mg/dl 5/29/2024 83

83. C. Uric acid Final break down of purine metabolism Are converted to uric acid mainly by the liver and excreted by the kidney . Disease correlation 1.Gout increased levels of uric acid in the blood painful swelling Patient with gout are risk of developing renal calculi. 2.Chronic renal disease elevated level of uric acid. 5/29/2024 84

84. Uricase - peroxidase method Absorbance read at 520. Normal value Men = 3.5 -7.5 mg/dl Women = 2.5 – 6.5 mg/dl  Clearance tests (urea, creatinine, uric acid) are performed for determination of early renal failure . 5/29/2024 85

85. Thank you End of session 5/29/2024 86