Diabetic Profile - Lab Diagnosis

Is group of tests that are used to diagnose diabetes or its complications , it includes:

Blood glucose
4 types: FBS, PPBS, RBS, OGGT
Microalbuminurea
Ketones
HbA1C
Insulin
ICA (islent cell antibody) for type I
C-peptide

1. Sri Lanka Society for Medical Laboratory Science slsmls.org / [email protected] Diabetic Profile Ravi Kumudesh MSc / BSc /Dip(MLT)

2. Diabetic profile: Is group of tests that are used to diagnose diabetes or its complications , it includes: Blood glucose 4 types: FBS, PPBS, RBS, OGGT Microalbuminurea Ketones HbA1C Insulin ICA ( islent cell antibody) for type I C-peptide 2 slsmls.org / [email protected]

3. Diabetes Mellitus It is a chronic disease due to disorder of carbohydrate metabolism, due to insulin deficiency results in hyperglycemia (increased blood glucose level) & glucourea (presence of glucose in urine). Associated with several changes in metabolism; such as metabolism of proteins & fats. 3 slsmls.org / [email protected]

4. Clinical Biochemical Findings in Diabetes Glucosuria. Large volume of urine increase urination frequency ( Polyuria ) Polyphagia (eats more frequently) Several metabolic changes 4 slsmls.org / [email protected]

5. Metabolic changes in diabetes Include increase in: Fat catabolisim leads to increase in FFAs in blood & liver. Acetyl.coA leads to increase formation of cholesterol & risk of atherosclerosis. ketone bodies generation in blood and urine leads to acidosis. catabolism of tissue protein due to energy requirement (because glucose can't uptake by cells) lead to weight loss and increase in level of amino acids in blood & more formation of urea by deamination of amino acid. 5 slsmls.org / [email protected]

6. Types of diabetes Type I diabetes mellitus (TIDM) Type 2 diabetes mellitus (TIIDM) Gestational diabetes mellitus (GDM ) Other "due to drugs or chemicals" 6 slsmls.org / [email protected]

7. Blood Glucose Levels 7 slsmls.org / [email protected]

8. 1. Fasting blood sugar (FBS) Measures blood glucose after fasting for at least 8-12 hrs It often is the first test done to check for diabetes . patient with mild or borderline diabetes may present with normal FBG values. If diabetes is suspected, GTT can confirm the diagnosis . Normal levels: 70-110mg/dl 8 slsmls.org / [email protected]

9. 2. Post- Prandial Blood Sugar (2-hour PPBS) After the patient fasts for 12 hours, a meal is given which contains starch and sugar (approx. 100 gm). Then after 2 hours blood is collected to measure glucose level. home blood sugar test is the most common way to check 2-hour postprandial blood sugar levels. 9 slsmls.org / [email protected]

10. 3. Random blood sugar (RBS) measures blood glucose randomly at any time throughout the day without patient fasting . it is useful because glucose levels in healthy people don’t vary widely throughout the day. blood glucose levels that vary widely may indicate a problem. 10 slsmls.org / [email protected]

11. 4. Oral glucose tolerance test (OGTT) Glucose Tolerance is defined as the capacity of the body to tolerate an extra load of glucose or it measures the body's ability to use glucose. It is series of blood glucose measurements taken after drink glucose liquid It is considered as definitive diagnostic test for DM. It is ordered to: Confirm the diagnosis, in pre-diabetic Diagnose gestational diabetes (most commonly) Recommended if 100-126 mg/ dL ( 5.5 mmol/L-7.0 mmol /L) 11 slsmls.org / [email protected]

12. Arrive FBS: After an overnight fasting (10-12 hrs) Drink: 75-100g dissolved in 250-300ml of water and given orally. After drink: blood samples and urine are collected every 30min for 3hrs (1 hr, 1.5 hr , 2hr, 2.5hr, 3hr ) A curve between time and blood glucose concentration, is plotted. Procedure 12 slsmls.org / [email protected]

13. Extended GTT : Glucose measured for 4-5 hrs after giving glucose to see how the curve behaves below the normal fasting glucose limits. Done in some conditions causing hypoglycaemia . Cortisone Stressed GTT : Can be used for detecting latent DM. Intravenous GTT : Is done if oral glucose is not tolerated or oral GTT curve is flat. In these cases 20% glucose as 0.5g glucose/Kg body weight. Usually peak occurs within 30 min after infusion and returns to normal after 90 min. Other types of OGTT 13 slsmls.org / [email protected]

14. Interpretation Normal Response : FBS is normal. After 1 hr it will rise, returns to normal fasting level within 2 hours . Diabetic curve : FBS: 140mg/dl or 7.8 mmol/L. After 2 hr: 200mg/dl (11 mmol/L ) or more. Glucosuria is usually seen Impaired GTT: with 2hrs glucose level between 140mg/dl - 200mg/dl It is not abnormal but must be followed up for DM .   14 slsmls.org / [email protected]

15. Renal Glycosuria : Curve is normal due to lowered renal threshold one or more samples of urine contain glucose. Lag storage/Alimentary Type : FBS is normal. Due to rapid absorption, maximum level is found at 30 min (180mg/dl). glucosuria is seen hypoglycaemic levels may be reached at end of 2 hours.  Flat curve of enhanced glucose tolerance: FBS is normal. Throughout the test the level does not vary + 20mg%. Interpretation 15 slsmls.org / [email protected]

16. Hypoglycemia : When blood glucose falls below 60 mg/dl. Causes Most commonly seen in overdose of insulin in treatment of DM. Hypothroidism . Insulin secreting tumours of pancrease – rare. Hypoadrenahsm (Addison's disease) Hypopitruitism . Severe exercise. Starvation. 16 slsmls.org / [email protected]

17. Measuring glucose level Principle: Glucose + H 2 O + O 2 Gluconic acid + H 2 O 2 2H 2 O 2 + 4 aminoantipyrine + PHBS Quinoneimine dye + H 2 O Red color POD GOD 17 slsmls.org / [email protected]

18. Kit components Glucose Oxidase Reagent : mixture of: glucose oxidase + peroxidase + aminoantipyrine + buffer Glucose standard Reagent : conc. 100mg/dl or 5.55 mmol/L 18 slsmls.org / [email protected]

19. Procedure Sample Standard Reagent blank 1ml 1 ml 1 ml Glucose oxidase reagent 10 µl - Sample (serum) - 10 µl - Glucose standard Prepare the reaction as the following: Mix, incubates at 37 o C for 10min Read abs at 510nm 19 slsmls.org / [email protected]

20. Calculation Glucose conc. = Abs. Sample X Conc. Standard Abs. Standard .. 20 slsmls.org / [email protected]

21. Bedside Method 21 slsmls.org / [email protected]

22. Microalbumin 22 slsmls.org / [email protected]

23. Qualitative Dipstick method Quantitative Assays for Microalbuminurea Enzyme linked Immunosorbant assay Radioimmuno assay Immunoturbidometric assay 23 slsmls.org / [email protected]

24. Specimen Collection for Microalbimin Collect freshly voided urine in a clean, dry container Preservatives should be avoided Samples which cannot be tested within 3 days of collection should be refrigerated Samples should not be frozen The test should be free from significant interference from glucosuria , pH, ketonuria or bacterial contamination 24 slsmls.org / [email protected]

25. MICRAL Strips Micral strip screening tests offer a cost-effective method of screening Dip sticks show acceptable sensitivity (95%) and specificity (93%) All positive tests should be confirmed by more specific methods 25 slsmls.org / [email protected]

26. False Positives Hyper filtration (Newly diagnosed diabetes) Exercise Marked hypertension Congestive Heart Failure Urinary Tract Infection Acute febrile illness 26 slsmls.org / [email protected]

27. Ketone Bodies 27 slsmls.org / [email protected]

28. What Are Ketones ? Acids that result when the body does not have enough insulin and uses fats for energy May occur when insulin is not given, during illness or extreme bodily stress, or with dehydration Can cause abdominal pain, nausea, and vomiting Without sufficient insulin ketones continue to build up in the blood and result in diabetic ketoacidosis (DKA) 28 slsmls.org / [email protected]

29. Why Test for Ketones ? DKA is a critical emergency state Early detection and treatment of ketones prevents diabetic ketoacidosis (DKA) and hospitalizations due to DKA Untreated, progression to DKA may lead to severe dehydration, coma, permanent brain damage, or death DKA is the number one reason for hospitalizing children with diabetes 29 slsmls.org / [email protected]

30. When Should Ketones Be Checked? The DMMP should specify, generally: When blood glucose remains elevated During acute illness, infection or fever Whenever symptoms of DKA are present Nausea Vomiting or diarrhea Abdominal Pain Fruity breath odor Rapid breathing Thirst and frequent urination Fatigue or lethargy Common symptoms including fruity odor to breath, nausea, vomiting, drowsiness, abdominal pain 30 slsmls.org / [email protected]

31. How Quickly Does DKA Progress? An isolated high blood glucose reading, in the absence of other symptoms is not cause for alarm DKA usually develops over hours, or even days DKA can progress much more quickly for students who use insulin pumps, or those who have an illness or infection Most at risk when symptoms of DKA are mistaken for flu and high blood glucose is unchecked and untreated 31 slsmls.org / [email protected]

32. Checking for Ketones Urine testing Most widely used method Blood testing Requires a special meter and strip Procedure similar to blood glucose checks 32 slsmls.org / [email protected]

33. 1. Gather supplies 2. Student urinates in clean cup 3. Put on gloves, if performed by someone other than student 4. Dip the ketone test strip in the cup containing urine. Shake off excess urine 5. Wait 15 - 60 seconds 6. Read results at designated time 7. Record results, take action per DMMP How to Test Urine Ketones 33 slsmls.org / [email protected]

34. Test Results: Color Code no ketones trace small moderate large ketones present 34 slsmls.org / [email protected]

35. Considerations Colors on strips and timing vary according to brand If using a scale with “urine glucose” and “urine ketones ,” be sure to read the correct scale when testing for ketones Follow package instructions regarding expiration dates, time since opening, correct handling, etc., as incorrect results may occur 35 slsmls.org / [email protected]

36. How To Test for Blood Ketones Prepare lancing device Wash hands using warm soapy water and dry them completely Remove the test strip from its foil packet Insert the three black lines at the end of the test strip into the strip port Push the test strip in until it stops 36 slsmls.org / [email protected]

37. How To Test for Blood Ketones Touch the blood drop to the purple area on the top of the test strip. The blood is drawn into the test strip Continue to touch the blood drop to the purple area on the top of the test strip until the monitor begins the test The blood ß- Ketone result shows on the display window with the word KETONE 37 slsmls.org / [email protected]

38. Glycohaemoglobin Hb A1C 38 slsmls.org / [email protected]

39. Hb A1C HbA1C: is glucose bound to hemoglobin Measures blood glucose conc. over a longer period of time It indicates how well diabetes has been controlled in the 2-3 months before the test. The A1C level is directly related to complications from diabetes Type of sample: whole blood in EDTA tube Normal Values Glycohemoglobin A1c:4.5%-5.7% Total glycohemoglobin:5.3%-7.5 % 39 slsmls.org / [email protected]

40. Key Messages Glycated hemoglobin ( A1C )  measure every 3 months (6 months if stable at target) Self monitoring Blood Glucose ( SMBG ) is an aid to assess interventions and hypoglycemia Individualize the frequency of SMBG SMBG and continuous glucose monitoring ( CGM ) needs to be linked with structured educational program to facilitate behavior change 40 slsmls.org / [email protected]

41. Glycated Hemoglobin: A1C Reliable measure of mean plasma glucose over 3-4 months Valuable indicator of treatment effectiveness Measure every 3 months when glycemic targets are not being met or treatments adjusted Measure every 6 months if stable at glycemic targets 41 slsmls.org / [email protected]

42. Conditions that can Affect Value Factors affecting A1C Increased A1C Decreased A1C Variable Change in A1C Erythropoiesis B12/Fe deficiency Decreased erythropoiesis Use of EPO, Fe, or B12 Reticulocytosis Chronic liver Dx Altered hemoglobin Fetal hemoglobin Hemoglobinopathies Methemoglobin Altered glycation Chronic renal failure ↓↓erythrocyte pH ASA, vitamin C/E Hemoglobinopathies ↑ erythrocyte pH Erythrocyte destruction Splenectomy Hemoglobinopathies Chronic renal failure Splenomegaly Rheumatoid arthritis HAART meds, Assays Hyperbilirubinemia Carbamylated Hb ETOH Chronic opiates Hypertriglyceridemia 42 slsmls.org / [email protected]

43. Insulin Levels 43 slsmls.org / [email protected]

44. Insulin is the primary hormone responsible for controlling glucose metabolism, and its secretion is governed by plasma glucose concentration. The insulin molecule is synthesized in the pancreas The principal function of insulin is to control the uptake and utilization of glucose in the peripheral tissues. Insulin concentrations are severely reduced in insulindependent diabetes mellitus (IDDM) Other conditions, non-insulin-dependent diabetes mellitus (NIDDM), obesity, and some endocrine dysfunctions. Insulin Test - Clinical Relevance 44 slsmls.org / [email protected]

45. The Insulin ELISA is a two-site enzyme immunoassay utilizing the direct sandwich technique with two monoclonal antibodies directed against separate antigenic determinants of the insulin molecule. Specimen, control, or standard is pipetted into the sample well, then followed by the addition of peroxidase -conjugated anti-insulin antibodies. Insulin present in the sample will bind to anti-insulin antibodies bound to the sample well, while the peroxidase -conjugated anti-insulin antibodies will also bind to the insulin at the same time. After washing to remove unbound enzyme- labelled antibodies, TMB- labelled substrate is added and binds to the conjugated antibodies. Acid is added to the sample well to stop the reaction, and the colorimetric endpoint is read on a microplate spectrophotometer set to the appropriate light wavelength. Insulin - Test Principle 45 slsmls.org / [email protected]

46. Islet Cell Antibody (ICA) 46 slsmls.org / [email protected]

47. Using the indirect fluorescent antibody method enables serologic assessment or possible detection of pancreatic disease. The presence of a ( histologically defined) circulating antibody to one or more of the islet cell antigens can aid in patient diagnosis and prognosis. The substrate utilized in this kit is sections of monkey pancreas. Islet Cell antibodies have been associated with a group of "autoimmune" endocrine disorders, more specifically with insulin dependent diabetes. Organ-specific autoimmunity is characterized by the presence of antibodies in patients that can be detected years before the onset of the clinical symptoms. Patients with autoimmune thyroiditis , adrenalitis or gastritis have an increased risk of developing insulin dependent diabetes at any age. Demonstration of Islet Cell Antibody (ICA) 47 slsmls.org / [email protected]

48. The indirect fluorescent antibody test is used for the detection of human IgG antibody to the antigens of monkey pancreas islet cells. Tissue is placed in the wells of specially prepared microscope slides. Dilutions of patient sera are placed on the wells where antibody, if present, binds to the antigen. The reaction is visualized through the use of a conjugate. The conjugate is fluorescein isothiocyanate (FITC) labeled, anti-human IgG Excitation of the FITC by ultraviolet (UV) light causes this dye to emit longer, visible, wavelengths of light in the yellow-green portion of the color spectrum. The conjugate will bind with human IgG antibodies attached to the antigens causing fluorescence when viewed through a microscope equipped with a UV light source Test Principle and Procedure 48 slsmls.org / [email protected]

49. C-peptide Test 49 slsmls.org / [email protected]

50. C-peptide testing can be used for a few different purposes C-peptide is a substance produced by the beta cells in the pancreas when pro insulin splits apart and forms one molecule of C-peptide and one molecule of insulin Insulin is the hormone that is vital for the body to use its main energy source, glucose Since C-peptide and insulin are produced at the same rate, C-peptide is a useful marker of insulin production C-peptide Test 50 slsmls.org / [email protected]

51. Sweating Palpitations Hunger Confusion Blurred vision Fainting In severe cases, seizures loss of consciousness When is it ordered? 51 slsmls.org / [email protected]

52. What does the test result mean? A high level of C-peptide generally indicates a high level of endogenous insulin production. This may be in response to a high blood glucose caused by glucose intake and/or insulin resistance. A high level of C-peptide is also seen with  insulinomas  and may be seen with low blood potassium, Cushing syndrome, and renal failure When used for monitoring, decreasing levels of C-peptide in someone with an insulinoma indicate a response to treatment; levels that are increasing may indicate a tumor recurrence A low level of C-peptide is associated with a low level of insulin production. This can occur when insufficient insulin is being produced by the beta cells, with diabetes for example, or when production is suppressed by treatment with  exogenousinsulin 52 slsmls.org / [email protected]

53. Not Only Test, But ….? 53 slsmls.org / [email protected]