Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Laboratory diagnosis of Diabetes mellitus

15,201 views

Published on

Laboratory diagnosis of Diabetes mellitus

Published in: Education
  • How to start a wildly profitable 7 figure marketing business and get your first commission check tonight, click here ★★★ https://tinyurl.com/y3ylrovq
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • How long does it take for VigRX Plus to start working? ♥♥♥ http://t.cn/Ai88iYkP
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • DOWNLOAD FULL BOOKS, INTO AVAILABLE FORMAT ......................................................................................................................... ......................................................................................................................... 1.DOWNLOAD FULL. PDF EBOOK here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... 1.DOWNLOAD FULL. EPUB Ebook here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... 1.DOWNLOAD FULL. doc Ebook here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... 1.DOWNLOAD FULL. PDF EBOOK here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... 1.DOWNLOAD FULL. EPUB Ebook here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... 1.DOWNLOAD FULL. doc Ebook here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... ......................................................................................................................... ......................................................................................................................... .............. Browse by Genre Available eBooks ......................................................................................................................... Art, Biography, Business, Chick Lit, Children's, Christian, Classics, Comics, Contemporary, Cookbooks, Crime, Ebooks, Fantasy, Fiction, Graphic Novels, Historical Fiction, History, Horror, Humor And Comedy, Manga, Memoir, Music, Mystery, Non Fiction, Paranormal, Philosophy, Poetry, Psychology, Religion, Romance, Science, Science Fiction, Self Help, Suspense, Spirituality, Sports, Thriller, Travel, Young Adult,
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • Intimacy has never been so much fun! Buy the clinically proven men's natural supplement that helped guys increase satisfaction by 71.43%! ■■■ http://t.cn/Ai88iYkP
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • DOWNLOAD FULL MOVIE, INTO AVAILABLE FORMAT ......................................................................................................................... ......................................................................................................................... ,DOWNLOAD FULL. MOVIE 4K,FHD,HD,480P here { https://tinyurl.com/yybdfxwh }
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here

Laboratory diagnosis of Diabetes mellitus

  1. 1. Presented by Dr. Monika Nema Dr. Monika Nema
  2. 2. Introduction Etiology Impaired insulin secretion Impaired insulin function Diabetes is a group of metabolic disorder sharing the common features of hyperglycemia. Dr. Monika Nema
  3. 3. Dr. Monika Nema
  4. 4. Laboratory diagnosis Urine analysis Blood chemistry Immunological Assays • Glucose • Ketone • Microalbuminuria • Blood glucose estimation • Glucose tolerance test • Glycated hemoglobin measurement • Lipid profile • Serum insulin or C- peptide level Dr. Monika Nema
  5. 5. Laboratory diagnosis Diagnosis Screening Assessment of glycemic control Assessment of association of long term risk Dr. Monika Nema
  6. 6. Current criteria Dr. Monika Nema
  7. 7. Laboratory test for diagnosis Dr. Monika Nema
  8. 8. Laboratory test for diagnosis  1. Estimation of blood glucose.  2. Oral glucose tolerance test. Dr. Monika Nema
  9. 9. Estimation of blood glucose  Measurement of blood glucose is indicative of current state of carbohydrate metabolism.  Depending on time of collection: Fasting blood glucose- after an overnight fast. Post meal or postprandial blood glucose-2 hrs after the subject has taken a normal meal. Random blood glucose – Any time of the day. Dr. Monika Nema
  10. 10. Total glucose in 100 ml of plasma is about 15% greater than in 100 ml of whole blood. Plasma is prefered as whole blood is affected by concentration of proteins (especially haemoglobin). In capillary blood the value of blood glucose at rest is about 5 % higher than venous blood. Dr. Monika Nema
  11. 11. Whole blood left at room temperature Glycolysis @ 7mg/dl/hr Sodium fluoride Leucocytosis and bacterial contamination (-) (+) Dr. Monika Nema
  12. 12. Blood glucose estimation Enzymatic method Chemical method Glucose oxidase/Peroxidase. Hexokinase. Glucose dehydrogenase Folin wu Somogyi – nelson method Orthotoluidine method Dr. Monika Nema
  13. 13.  Based on the same principles.  Principle- Cupric ions ( alkaline cupric sulphate )  Intensity of colour is proportional to glucose present in the blood Blood glucose cuprous ions phosphomolybdic acid Blue coloured compound. Dr. Monika Nema
  14. 14. Principle  Glucose + orthotoluidine green coloured complex  The intensity of the final colour is measured at 620 – 660 nm.  The measured colour intensity is directly proportional to the concentration of glucose . hot acidic medium Dr. Monika Nema
  15. 15. Glucose Oxidase/Peroxidase method  Glucose + O2 gluconic acid + H2O2 2 H2O2 2H2O +2 O2  Intensity measured at 530 nm. peroxidase pink coloured compoundPhenol 4 aminophenazone GOD Dr. Monika Nema
  16. 16.  Principle – Glucose + ATP G6P + ADP G6P + NADP 6 PG + NADPH  Glucose concentration proportional to rate of production of NADPH. HexoKinase G6PD Hexokinase method Dr. Monika Nema
  17. 17.  Accurate, sensitive, specific and precise.  Reagents are safe to handle.  Very small serum & reagents are required.  Mono step method, carried out at room temperature.  Linear upto 700 mg/dl.  Sodium fluoride do not interfere in the assay. Dr. Monika Nema
  18. 18. Glucose tolerance test  Glucose tolerance means the ability of the body to utilize glucose in blood circulation.  American Diabetes Association -------- For routine diagnosis  WHO ------------For those with impaired fasting glucose.  American Diabetes Association and WHO Gestational Diabetes Dr. Monika Nema
  19. 19. Indication of Glucose tolerance test  In asymptomatic persons with sustained or transient glycosuria.  In persons with symptoms of diabetes but no glycosuria or hyperglycemia.  Persons with family history but no symptoms or positive blood findings.  In persons with or without symptoms of diabetes mellitus showing one abnormal blood findings.  In patients with neuropathies or retinopathies of unknown origin. Dr. Monika Nema
  20. 20. Contraindications of glucose tolerance test  There is no indication for doing GTT in a person with confirmed diabetics mellitus.  GTT has no role in follow-up of diabetics.  The test should not be done in ill patients. Dr. Monika Nema
  21. 21. Types of glucose tolerance test  Standard Oral glucose tolerance test  I/V Glucose tolerance test  Mini Glucose tolerance test Dr. Monika Nema
  22. 22.  Patient should on carbohydrate rich unrestricted diet for 3 days.  Patient should be ambulatory with normal physical activity.  Medications should be discontinued on the day of testing.  Exercise, smoking and tea or coffee are not allowed during test period.  OGTT carried out in the morning after patient has fasted overnight for 8-14 hours. Preparation of patient Dr. Monika Nema
  23. 23. Test  A fasting venous blood sample is collected in the morning.  Patients ingest 75 g of anhydrous glucose in 250-300 ml of water over 5 minutes. ( for children, the dose is 1.75 g of glucose per kg). Dr. Monika Nema
  24. 24. Test  In the classical procedures, the blood and urine samples are collected at half hourly interval of the next three hours.  A curve is plotted with the blood glucose levels on the vertical axis against the time of collection on the horizontal axis.  The curve so obtained is called glucose tolerance curve. Dr. Monika Nema
  25. 25. Normal Glucose tolerance curve Dr. Monika Nema
  26. 26. Diabetic curve Dr. Monika Nema
  27. 27. Intravenous Glucose tolerance test •This test is undertaken for patients with malabsorption (Celiac disease or enteropathies). •Under these conditions oral glucose load is not well absorbed and the results of oral glucose tolerance test become inconclusive. Dr. Monika Nema
  28. 28. I/V Glucose tolerance test- Procedure • I/V glucose tolerance test is carried out by giving 25 g of glucose dissolved in 100 ml distilled water as intravenous injection within 5 minutes. • Completion of infusion is taken as time zero. • Blood samples are taken at 10 minutes interval for the next hour. • The peak value is reached within a few minutes. Dr. Monika Nema
  29. 29. I/V Glucose tolerance test Interpretation • Normally, blood glucose level returns to normal range within 60 minutes. • In diabetes mellitus, this decline is slow. Dr. Monika Nema
  30. 30. Mini or Modern GTT  As per current WHO recommendations, in the mini or modern glucose tolerance test, only two samples are collected.  Fasting (zero hour) and 2 hour post glucose load.  Urine samples are also collected during the same time.  The diagnosis is made from the variations observed in these results. Zero Hour After 2 Hours Normal Person < 110 mg/dL < 140 mg/dL Increase Glucose Tolerance 110 – 126 mg/dL 140 – 199 mg/dL Dr. Monika Nema
  31. 31. GTT Under special conditions  Cortisone stress test- used for detecting pre diabetes or Latent diabetes  Extended GTT- To diagnose the cause of hypoglycemia especially 2-3 hours after meals. Dr. Monika Nema
  32. 32. Factors affecting GTT a) Acute infections- Cortisol is secreted, the curve is elevated and prolonged. b) Hypothyroidism-A flat curve is obtained in hypothyroidism. Thyroid hormone increases the absorption of glucose from the gut. c) Starvation- There is rise of counter regulatory hormones, which show increased glucose tolerance. Dr. Monika Nema
  33. 33. Gestational diabetes  Gestational diabetes is high blood sugar that develops at any time during pregnancy in a woman who does not have diabetes. Dr. Monika Nema
  34. 34. OGTT in gestational Diabetes  Impairment of glucose tolerance develops normally during pregnancy, particularly in 2nd and 3rd trimester. • <25 yrs age, Normal body weight before pregnancy, absence of DM in first degree relative, no h/0 poor obstetric outcome, no h/o abnormal glucose tolerance Low risk • Tested at 24-28 weeks of gestation. Average risk • Marked obesity, strong family history of DM, glycosuria, personal history of GDM.High risk Dr. Monika Nema
  35. 35. Fasting plasma glucose or random plasma glucose Deranged Normal Repeat testing on subsequent day OGTT indicated for average risk and high risk pregnant female Dr. Monika Nema
  36. 36. OGTT for GDM One step approach Two step approach 100 gm glucose is administered 3- hours OGTT is performed 50 gm glucose is administered irrespective of time of last meal After one hour, venous blood sample collected If glucose level exceeds 140 mg/dl Otherwise GDM is excludedDr. Monika Nema
  37. 37.  Gestational diabetes is diagnosed if the woman is at or exceeds any two of the following four plasma glucose levels during 100 gm test Fasting – 95 mg/dl 1 hr – 180 mg/dl 2 hr – 155 mg/dl 3 hr – 140 mg/dl Dr. Monika Nema
  38. 38. Laboratory test for screening Dr. Monika Nema
  39. 39. Laboratory test for screening  Recommended screening test is fasting plasma glucose.  American Diabetes Association recommends screening for Type 2 DM in all asymptomatic individuals >= 45 yrs of age using fasting plasma glucose.  If fasting test is normal, screening test should be repeated every three years.  If fasting blood glucose level is normal but there is strong clinical suspicion then OGTT. Dr. Monika Nema
  40. 40. Selective screening  High risk individuals ---Obese Family h/o DM Hypertension Dyslipidemia Impaired glucose tolerance Screening test is performed at earlier age ( 30 yrs ) and repeated more frequently Dr. Monika Nema
  41. 41. Laboratory test to assess glycemic control Dr. Monika Nema
  42. 42. Laboratory test to assess glycemic control  Periodic measurement of glycated haemoglobin.  Daily self assessment of blood glucose.  Others. Dr. Monika Nema
  43. 43. Glycated hemoglobin  Glycated haemoglobin covers a number of chemically different modification resulting from the non-enzymatic and irrevesibly binding of different sugars to different amino groups in the haemoglobin molecule. ( Maillard Reaction ) Hemoglobin + glucose Aldimine Glycated hemoglobin Dr. Monika Nema
  44. 44. TYPE COMPONENTS Glycated haemoglobin (Ghb) Haemoglobin in which glucose &/ any other carbohydrates are bound to free amino groups. HbA1( fast haemoglobin) Carbohydrate bound to N- terminal of the β- chain. HbA₁а₁ Fructose-1, 6-biphosphate bound to the N- terminal valine of the β-chain HbA₁а₂ Glucose-6-phosphate bound to the N- terminal valine of the β-chain HbA₁ь Unknown carbohydrate residue bound to the N-terminal valine of the β-chain HbA₁с Glucose bound to the N-terminal valine of the β-chain Dr. Monika Nema
  45. 45. Glycated hemoglobin  HbA₁с gives information about the average blood glucose concentration over a retrospective period of time.  Reflects the mean glucose concentration.  Normally, less than 5% of hemoglobin is glycated. Dr. Monika Nema
  46. 46. Glycated hemoglobin  About 50% HbA₁с values results from the blood glucose of the preceding 30 days , 40% from the preceding 31 -90 days and only 10% from the period between the 91 – 120 days.  No effect of diet, exercise & insulin on test results.  More informative.  Blood sample can be drawn at any time of day.  HbA1c of 6 % corresponds to mean serum glucose level of 135 mg/dl.  With every rise of 1.0%, serum glucose increases by 35 mg/dl. Dr. Monika Nema
  47. 47. INDICATIONS  In all diabetics to monitor long term blood glucose level control, index of diabetic control:- 7% HbA₁с – good 10% HbA₁с- fair 13-20% HbA₁с- poor.  To monitor patient compliance.  To predict development & progression of microvascular complication.  For determining the therapeutic option whether to use oral agents, insulin ,or β cell transplantation.  Also increasingly used for primary diagnosis of DM. Dr. Monika Nema
  48. 48. Methods used for determination of HbA₁с  HbA1c electrophoresis.  Cation-exchange chromatography,  Boronate affinity Chromatography  Immunoassays.  Colorimetric method Dr. Monika Nema
  49. 49. At what interval should HbA₁с be determined? Treatment by time of diabetes Recommended frequency Type-1 DM( minimal /conventional therapy) 4 times a year Type – 1 DM (intensified therapy) Every (1) -2 months. Type-2 DM Twice a year in stable patients. Dr. Monika Nema
  50. 50. Glycated hemoglobin High values Low values Diabetes Mellitus Haemolysed specimen Polycystic Ovarian Disease Hereditary HbF Hyperglycemia Neonate&Pregnancy Glycosuria Fetal maternal transfusion Dr. Monika Nema
  51. 51. Glycated hemoglobin Falsely high values Falsely low values Iron deficiency anemia Hemolytic anemia Post spleenectomy Chronic blood loss Alcohol poisoning Chronic Renal Failure Lead toxicity Pregnancy Dr. Monika Nema
  52. 52.  The goal of therapy should be to achieve HbA₁с values as close as possible to the refrence range but without losing sight of the increased risk of hypoglycemia.  Guideline by ADA:-  HbA₁с values <7% indicate good glycemic control.(normal range: 4.5% - 6.3%).  If HbA₁с values > 8% the treatment should be reconsidered. Dr. Monika Nema
  53. 53. Self monitoring of blood glucose  Regular use of SMBG devices by diabetic patients has improved the management of DM.  SMBG devices measure capillary whole blood glucose obtained by finger prick and use test strips that incorporate glucose oxidase or hexokinase.  SMBG devices yield unreliable results at very high and very low glucose levels.  It is necessary to periodically check the performance of glucometer by measuring parallel venous plasma glucose in the laboratory. Dr. Monika Nema
  54. 54. Fructosamine assay  Generic term for measurement of all serum glycated protein though the bulk being albumin.  Does not appear to be influenced by transient (stress) hyperglycaemia.  Unable to detect short term or transient abnormalities in the blood glucose concentration. Ex: hypoglycemia.  Reference range – in non diabetic- 2.4-3.4 mmol/l.  Fructosamine / albumin ratio:- 54- 86 µmol/gm. Fructosamine test HbA1c Measures average blood glucose level over the past two or three weeks Measures average blood glucose level over the past two to three months. Dr. Monika Nema
  55. 55. Glycosylated albumin  Half -life of albumin is approximately 15 days.  Glycated albumin level is believed to reflect the glycemic change over a 2-week period.  GA can be useful in evaluating the therapeutic effect of recently substituted hypoglycemic agents at an early stage.  GA can also act as a valuable glycemic control marker in diabetic patients with various comorbidities since it is unrelated to the metabolism of hemoglobin. Dr. Monika Nema
  56. 56. Insulin assay  Measurement of insulin level by radioimmunoassay & ELISA.  Crucial for type I DM. Dr. Monika Nema
  57. 57. Proinsulin Assay  It is precursor molecule for insulin.  Most proinsulin is converted to insulin and C-Peptide, which are secreted in equimolar amounts into the blood.  The biological activity of proinsulin is only about 10% of insulin, but the half life of proinsulin is three times as long as insulin. Dr. Monika Nema
  58. 58. Proinsulin Assay  Elevated in:- At onset of IDDM and in healthy sliblings of IDDM patients. With established NIDDM. Older patients. Pregnant . Obese diabetics. Insulinomas. Functional hypoglycemia. Hyperinsulinemia. Dr. Monika Nema
  59. 59.  Released in circulation during conversion of proinsulin to insulin in equimolar quantities to insulin.  Its level correlate with insulin level in blood.  Low C – peptide levels are characteristic of type I DM.  C-peptide levels are measured instead of insulin levels because C- peptide can assess a person’s own insulin secretion even if they receive insulin injections.  The test may be used to help determine the cause of hypoglycaemia, values will be low if a person has taken an overdose of insulin but not suppressed if hypoglycaemia is due to an insulinomas..  Factitious hypoglycemia may occur secondary to the surreptious use of insulin. Measuring C-peptide levels will help differentiate a healthy patients from a diabetic one. Dr. Monika Nema
  60. 60. Urine glucose estimation  Presence of chemically detectable amount of glucose in urine is called glycosuria.  Urine glucose test results correlate well with plasma or serum glucose values.  Presence of glucose in urine indicates that blood glucose level of the patient could have elevated > 180 mg/dl.  Normally less than 500mg/24 hrs or less than 15 mg/dl of glucose is present in urine. Dr. Monika Nema
  61. 61. Renal glycosuria  Blood Glucose level is normal but there is defect in the reabsorptive ability of renal tubule.  Non pathological causes Pregnancy Stress Anxiety  Pathological causes Cystinosis Heavy metal poisoning Fanconi’s syndrome Galactosemia Dr. Monika Nema
  62. 62. Alimentary glycosuria  Lag storage disorder.  Occur in gastrectomy, gastrojejunostomy ,hyperthyroidism.  Glucose tolerance test reveals a peak at 1 hour above renal threshold.  Fasting and 2 hours glucose value are normal. Dr. Monika Nema
  63. 63.  Qualitative test. Benedicts. Clintest tablet test. Reagent strip test  Quantitative test.  Benedicts. Dr. Monika Nema
  64. 64. Benedict’s test  Based on copper reduction method  Detect any reducing sugar in urine  Principle Cu 2+ Cu + Cu + + OH - CuOH 2CuOH Cu2O + H2O Hot alkaline solution Heat Dr. Monika Nema
  65. 65. Procedure Add 8 drops of urine Boil for 2 to 3 min CoolTake 5.0ml of Benedict’s reagent Observe Benedict reagent : sodium citrate 173 gm, sodium carbonate 100 gm, cupric sulphate 17.3 gm and distill water 900 ml. Dr. Monika Nema
  66. 66. Observations Color Sugar Blue Absent Green without precipitate Present, trace Green with precipitate 1+ (0.5 g/dl) Brown precipitate 2+ (1.0 g/dl) Yellow - Orange precipitate 3+ (1.5 g/dl) Brick red precipitate 4+ (≥ 2.0 g/dl) Dr. Monika Nema
  67. 67. False positive test  Ascorbic acid  Creatine  Uric acid  Homogentisic acid  Cephalosporins  Salicylates  Radiographic media Dr. Monika Nema
  68. 68. Clinitest tablet method  Modified form of Benedicts test in which reagents are present in tablet form.  Contains copper sulfate, citric acid, sodium carbonate and anhydrous sodium hydroxide. Dr. Monika Nema
  69. 69. Reagent strip method  Based on specific glucose oxidase and peroxidase method.  Specific for glucose.  Principle - Glucose + O2 Gluconic acid + H2O2 H2O2 + Chromogen oxidized chromogen + H2O Dr. Monika Nema
  70. 70. False positive : - Oxidizing cleaning agent in urine container. False negative : - Ascorbic acid Dr. Monika Nema
  71. 71.  Contents – Potassiun Thiocyanate , Potassium Ferrocyanide , Sodium Citrate , Sodium Carbonate , Copper Sulfate.  Principle : Cu 2+ Cu + Cu + +potassium thiocyanate Cu thiocynate Hot alkaline solution White precipitate Dr. Monika Nema
  72. 72. Procedure 2-3g of NaCO3 keep Boiling Add Urine drop by drop using 5 ml ppt Till blue colour disappear Take 5.0ml of Benedict’s Qt reagent Chalky white  Method – titration  Calculation Glucose in urine = 5 (g/100ml) urine used 10 g glucose reduces 5 ml of reagent Dr. Monika Nema
  73. 73. Benedicts qualitative tests Positive Glucose oxidase strip method Positive Negative Glucose Lactose Fructose GalactoseBenedicts quantitative test Dr. Monika Nema
  74. 74.  Semiquantitative urine glucose testing for monitoring of diabetes mellitus in home setting is not recommended.  This is because (1) Even if glucose is absent in urine, no information about blood glucose concentration below the renal threshold is obtained. (2) Urinary glucose testing cannot detect hypoglycemia (3) Concentration of glucose in urine is affected by urinary concentration. Dr. Monika Nema
  75. 75. Laboratory tests to assess long term risks Dr. Monika Nema
  76. 76. Laboratory tests to assess long term risks  1. Urinary albumin excretion.  2. Lipid profile. Dr. Monika Nema
  77. 77. Screening for proteinuria should be performed yearly in the following patients:  Type 1 DM : 5 yrs after diagnosis of DM, or earlier in the presence of other cardiovascular risk factors.  Type 2 DM : at the time of diagnosis of diabetes. Dr. Monika Nema
  78. 78.  Urine should be screened for proteinuria with conventional dipstick on an early morning urine specimen.  If urine dipstick for proteinuria is negative, screening for microalbuminuria should be performed.  If microalbuminuria is detected, confirmation should be made with two further tests within 3 to 6 month period. Dr. Monika Nema
  79. 79. Frank proteinuria  Precipitation test.  Heat test- precipitation of protein by heat. Not affected by radiographic contrast media.  Sulfosalicylic acid method- precipitation of protein by acid. False positive results are obtained in presence of radiographic contrast media.  Reagent strip test. Dr. Monika Nema
  80. 80. Fill the supernatant urine upto 2/3 clean test tube Boil the upper portion PROCEDURE OF HEAT TEST If turbidity develops add 1 to 2 drops of glacial acidic acid Phosphates will clear No turbidity – Proteins absent Presence of turbidity – Proteins presentDr. Monika Nema
  81. 81. Transfer about 5ml urine to a centrifuge tube Centrifuge Transfer 3.0 ml of supernatant urine in a clean test tube Add 2-3 drops of 30% sulfosalicylic acid or equal amount of 3% Mix well and Wait for 10 minutes Observe the degree of turbidity and flocculation PROCEDURE OF SULFOSALICYLIC ACID METHOD Dr. Monika Nema
  82. 82. 1. Negative – No turbidity (~5mg/dl or less) 2. Trace – Perceptible turbidity (~20 mg/dl) 3. 1+ - Distinct turbidity but no discrete granulation(~50mg/dl) 4. 2+ - Turbidity with granulation but no flocculation(~200mg/dl) 5. 3+ - Turbidity with granulation and flocculation(~500mg/dl) 6. 4+ - Clumps of precipitated protein, or solid precipitate (~1.0g/dl or more) Dr. Monika Nema
  83. 83. Reagent strip method Principle : Impregnated with bromphenol blue buffered to pH 3 with citrate 30 to 60 second urine application Variable sheds of green color formed Dr. Monika Nema
  84. 84. Microalbuminuria  Normally, only a small amount of albumin is fiiltered at the glomerulus, and most of that albumin is degraded and reabsorbed by the proximal tubule.  Defined as persistent proteinuria that cannot be detected by routine reagent strips but greater than normal.  Present in the very early stage of diabetes, at a time when GFR may be normal and when there is no evidence of glomerular lesion. Dr. Monika Nema
  85. 85. Microalbuminuria  Normalbuminuria- <20 microgram/minute or <30 mg/24 hrs.  Microalbuminuria- the range in between: Urinary excretion of albumin of 20-200 microgram/minute or 30-300 mg/24 hrs. • Macroalbuminuria- More than 200 microgram/minute or more than 300 mg/24 hrs. Dr. Monika Nema
  86. 86. Microalbuminuria Lower limit Upper limit unit 24 hour urine collection 30 300 mg/24 hr Short time urine collection 20 200 ug/min Spot urine albumin sample 30 300 mg/l Urine albumin creatinin ratio Women 3.5 30 35 300 mg/mmol mg/gm Men 2.5 30 35 300 mg/mmol mg/gm Dr. Monika Nema
  87. 87. Microalbuminuria  It indicates increase in capillary permeability to albumin.  Albumin is the first protein to enter the urine after the kidney is damaged.  Appearance of microalbuminuria is predictor of progression to overt proteinuria.(incipient nephropathy)  It is an independent risk factor for cardiovascular disease in diabetes mellitus. Dr. Monika Nema
  88. 88. Methods of detection include 1. Measurement of albumin creatinine ratio in a random urine sample 2. Measurement of albumin in an early morning and random 3. Measurement of albumin in 24 hr sample. Test strips that screen for microalbuminuria are available commercially. Detection of microalbuminuria Dr. Monika Nema
  89. 89. Albumin to Creatinine ratio  Reagent Strips are firm plastic strips that contain two reagent areas that test for albumin and creatinine in urine.  An albumin-to-creatinine ratio is also determined, which allows for the use of single-void specimens in testing. The ratio is given in milligrams albumin per gram or millimole creatinine (mg/g or mg/mmol).  This product provides semi-quantitative results and can be used for screening samples for microalbuminuria; positive results should be confirmed with quantitative methods for albumin. Dr. Monika Nema
  90. 90. Microalbuminuria strips  The strip is an immunochemical strip specific for albumin.  Albumin in the sample get bound to soluble conjugate of antibodies and marker enzyme b-galactosidase.  Conjugate-albumin complexes are separated and enzyme b-galactosidase reacts with a substrate to produce a red dye.   The reagent part of the test strip should be dipped into the urine for 5 seconds and then laid down horizontally and read after 5 minutes.  The intensity of the colour produced is proportional to the albumin concentration in the urine.  The colour formed is compared with the reference chart on the vial. Dr. Monika Nema
  91. 91. Quantitative test for microalbuminuria  Colorimetric test  ELISA.  Radioimmune assay.  Immunoturbidiometric assay.  Nephelometry.  Chemiluminescence. Dr. Monika Nema
  92. 92. DYE BINDING COLORIMETRIC METHOD  Pyrogallol red molybdate reagent complex react with protein to form a blue purple colour.  Optical density of the coloured complex can be measured at 600nm.  The measured O.D. is propotional to the protein concentration in the specimen. Dr. Monika Nema
  93. 93. ELISA  Uses antibodies and colour change to identify the substance.  The intensity of the color measured with microwell reader at 450 nm. Dr. Monika Nema
  94. 94. RADIOIMMUNOASSAY  Technique used for the detection of antibody or antigen.  Uses radioactive label or tracer .  Tritium, I-131, I-125 are commonly used tracers.  PRINCIPLE – competitive binding between radiolabelled & unlabelled molecules of antigen to bind with a high affinity , specific antibody.  The amount of unlabelled antigen is measured by its competitive effect on the labelled antigen for limited antibody sites. Dr. Monika Nema
  95. 95. TURBIDIMETRY  Measurement of reduction in light transmission caused by particle formation.  Light transmitted in forward direction is detected.  Amount of light absorbed by a suspension of particles depends on the specimen concentration & on particle size.  Not specific to protein . Nucleic acid can also precipitate. Dr. Monika Nema
  96. 96. NEPHELOMETRY  Measurement of light scattered by the particulate solution.  Nephelometer measure scattered light at 90 to the incident light. Dr. Monika Nema
  97. 97. CHEMILUMINESENCE  Chemiluminescence is the emission of energy with limited emission of heat (luminescence), as the result of a chemical reaction.  In immunoassay technology , the light produced by the reaction indicates the amount of analyate in the sample. Dr. Monika Nema
  98. 98. Triglycerides (mg/dl) Category <150 Low risk 150-199 Intermediate risk ≥ 200 High risk LDL cholesterol <100 Low risk 100-129 Intermediate risk ≥130 High risk HDL cholesterol <35 High risk 35-45 Intermediate risk >45 Low risk Dr. Monika Nema
  99. 99. Laboratory test in acute complication of Diabetes Mellitus Dr. Monika Nema
  100. 100. Acute complication of Diabetes Mellitus  Diabetic ketoacidosis.  Hyperglycemic hyperosmolar state.  Hypoglycemia. Dr. Monika Nema
  101. 101. Diabetic ketoacidosis  State of absolute or relative insulin deficiency aggravated by ensuing hyperglycemia, dehydration, and acidosis- producing derangements in intermediary metabolism.  Normally the blood level of ketone bodies is <1 mg/dl & only traces are excreted in urine.  Increased synthesis causes the accumulation of ketone bodies in blood.  More common in case of Type 1 DM. Dr. Monika Nema
  102. 102. Hyperglycemia Ketosis Acidosis * Definition of Diabetic Ketoacidosis  The normal gap is <12 mEq/L.  In ketoacidosis, the “delta” of the anion gap above 12 mEq/L is composed of anions derived from keto-acids Dr. Monika Nema
  103. 103. Symptoms of Diabetic Ketoacidosis Dr. Monika Nema
  104. 104. Lab Findings in DKA  Severe hyperglycemia  Increased blood and urine ketones (Acetone,. Acetoacetic acid , 3-hydroxybutyrate ).  Low bicarbonate  High anion gap  Low arterial pH  Low PCO2 (respiratory compensation) Dr. Monika Nema
  105. 105. Methods to detect ketone bodies 1. Rothera’s test 2. Reagent strip 3. Gerhardt ferric chloride test Dr. Monika Nema
  106. 106. Rothera test  Based on nitroprusside reaction  Procedure Take 5.00 ml urine and saturate it with ammonium sulphate. Add a crystal of sodium nitroprusside. Slowly pour concentrated ammonium hydroxide(1-2ml) by the side of test tube. Pink-purple ring Dr. Monika Nema
  107. 107. Reagent strip for ketonuria  Based on nitroprusside reaction  Principle: Sodium nitroprusside + Glycine acetoacetic acid and acetone in alkaline medium Violet color  Sensitivity: 25-50mg/dl Dr. Monika Nema
  108. 108.  Addition of 10% ferric chloride solution to urine causes solution to become reddish or purplish if acetoacetic acid is present. Dr. Monika Nema
  109. 109. Hyperglycemic Hyperosmolar State  Compared to DKA, in HHS there is greater severity of:  Dehydration  Hyperglycemia  Hypernatremia  Hyperosmolality  Because some insulin typically persists in HHS, ketogenesis is absent to minimal and is insufficient to produce significant acidosis.  More commonly present in Type 2 DM. Dr. Monika Nema
  110. 110. Hypoglycemia  Results from an imbalance between glucose utilization and production in such a manner that the rate of glucose utilization exceeds the rate at which glucose being produced.  Whipple’s triad:- Symptoms consistent with hypoglycemia. Plasma glucose level < 55 mg/dl. Relief of symptoms with correction of hypoglycemia. Dr. Monika Nema
  111. 111. Conclusion  Diabetes is a very complicated disease.  Anyone at any age can have diabetes despite of negative family history  Laboratory plays an important part in the diagnosis and care of diabetic patient. Dr. Monika Nema
  112. 112. Thank youDr. Monika Nema

×