Corso di Medicina di Laboratorio Prof. Giuseppe Castaldo, a.a Valutazione di laboratorio del diabete

> 135 millions of patients in the world Variable prevalence in different ethnic groups Type 1: about 10% Type 2: about 90% Prevalence of diabetes: 5% of the general population

Type 1 diabetes (juvenile onset) Immune-mediated diabetes Idiopathic (no immunological involvement, rare) Type 2 diabetes Gestational diabetes Impaired glucose tolerance (IGT) Primary diabetes: classification

Molecular basis of type 1 diabetes (T1DM) Deficiency of insulin secretion due to autoimmune destruction of beta cells Molecular basis of type 2 diabetes (T2DM) Impaired activity of insulin due to peripheral resistance and/or beta cells dysfunction

Biochemical effects of insulin

Secondary diabetes Diseases of the exocrine pancreas: Pancreatitis, trauma, infections, neoplasia, surgical resection, cystic fibrosis … Endocrinopathies: Cushing, glucagonoma, pheochromocitoma … Drugs: Steroids, diuretics …

Diabetes: diagnostic criteria (ADA, OMS) Symptoms (polyuria, polydipsia and weight loss) associated to plasma glucose levels > 200 mg/dL Fasting plasma glucose > 126 mg/dL (7.0 mmol/L) Plasma glucose at 2h from charge > 200 mg/dL (11.1 mmol/L) Glycated (glycosylated) haemoglobin > 6.5% (48 mmol/mol)

Plasma glucose – daily changes M. Luzzana, 1999

Plasma glucose (EK, GO) 8 hours of fasting … 1)Glucose must be measured on plasma using a tube containing an anticoagulant that inhibits glycolysis 2)The sample can be stored in the tube at RT for <72h

Fluoride inhibits the glycolysis

Glucometers …

Thus, glucometers can be used for therapy monitoring -By trained patients -Using glucometers connected to the central Lab

Glycosuria … Glycosuria must not be performed for diagnosis of diabetes

-Low diagnostic sensitivity (because the renal threshold of glucose is 160 – 180 mg/dL) -Low analytical specificity (various substances may interfere with glucose determination)

However … the incidental finding of glycosuria can start the diagnostic process

Protein adducts

Glycated albumin (Fructosamine) Albumin half life: about 20 days

Non enzymatic, irreversible process The synthesis of HbA1c depends mainly on serum glucose levels and its degradation occurs with the degradation of erythrocytes Mean life of erythrocytes: about 120 days The amount of HbA 1c is proportional to the mean serum glucose levels in the last 6-12 weeks Production of glycated hemoglobin HbA 1c

HbA 1c labile reversibile veloce HbA 1c stabile irreversibile lenta HbA 1c : glycation reaction Non enzymatic condensation reaction between the aldehydic group of glucose and the N-terminal aminic group of beta chains of Hb

Elective biochemical parameter for the retrospective evaluation of the degree of glucose control in diabetic subjects Relevant in the monitoring of both type 1 and type 2 diabetic patients Useful to assess the efficacy of the therapy and to predict the development of complications HbA 1c : clinical significance

Differential diagnosis between type 1 and type 2 diabetes -Therapy -Serum insulin levels -Autoantibodies

Autoantibodies 1974, Islet cell autoantibodies (ICA) 1983, Autoantibodies to insulin (IAA) 1990, Autoantibodies to glutamic acid decarboxilase (GADA) 1994, Autoantibodies to insular tyrosine phosphatase (IA-2) 2007, Autoantibodies to Zinc transporter (ZnT8)

Diagnostic sensitivity of autoantibodies for T1DM - GADA, IA-2, IAA85% - GADA, IA-2, IAA, ICA o ZnT896-98% - All negatives (type 1B)*2-4% -

I)there is no evidence that immune system is attacking beta cells II)alternating cycles where the patient needs and then does’t need insulin replacement. Type 1B diabetes is unusual and most often diagnosed in subjects of African or Asian heritage. You have type 1B diabetes if:

It permits to measure the pancreatic functional reserve in patients during insulin therapy C-peptide

Impaired glucose tolerance Fasting glucose: mg/dL Glucose after 2h from OGTT: mg/dL

Poor reproducibility Bad tolerated Preparation Clinical interferences Physical exercise OGTT (Oral glucose tolerance test)

No OGTT -Acute diseases or infections -Diabetes -Relapse after surgery -Assumption of drugs that may interfere with glucose metabolism

3 DAYS BEFORE THE OGTT: Plan on eating three healthy meals and snacks for 3 days before the test. You do not need to buy special food, but you do need to make sure you have healthy foods to eat. Your meals should be balanced with plenty of carbohydrates. Foods containing carbohydrates include: Fruits, Breads, Cereal, Pasta, Rice, Crackers, Starchy vegetables (corn/peas/carrots) Make normal exercise 8-14 HOURS BEFORE THE OGTT: DO NOT eat, smoke, or do heavy exercise 12 hours before the test. Preparing for OGTT

A good … curve 75 g of glucose2 sample : T0 e 120’

OGTT -Basal glucose -75 g of glucose (oral) -2 samples: T0 and 120’

NormalDMIGT Fasting glucose h after charge

Acute complications Hypoglicemia, frequent, potentiallty lethal Diabetic ketoacydosis Non ketotic hyperosmolarity

Chronic complications Nephropaty (renal insufficiency) Cardiovascular diseases

Risk for diabetic complications DiseaseRisk Cecity20 times Renal insufficiency25 times Amputation40 times Myocardial infarction2 – 5 times Ictus2 – 3 times Nathan, N Engl J Med 1993

Survey of complications Serum Glucose Ketones Microalbuminuria Creatinine, Cystatin C HDL-LDL Clolesterol

Microalbuminuria risk for diabetic nephropaty In patients with type 1 diabetes the screening of microalbuminuria should start within 5 years from diagnosis 80 % of patients with type 1 diabetes (20 – 40 % type 2) with microalbuminuria will develop proteinuria within 10 – 15 years

Urine or blood ketones should be measured at home (or in the laboratory) in the suspect of ketoacidosis or in case of: Fever Stress Persistent hyperglicaemia Pregnancy

Gestational diabetes Ormonal changes during pregnancy cause the enhanced peripheral resistance to insulin; pancreas may be unable to increase the production of insulin …

At the first visit all pregnant women that do not have a recent blood glucose result should perform the analysis to exclude diabetes

The screening for gestational diabetes must not be performed before the 16th week of pregnancy, because ormonal changes do not start before such period

At the week of pregnancy, the screening must be offered in all the following conditions: 1)Gestational diabetes in a previous pregnancy 2)Body mass index before pregnancy >30 Kg/m2 3)Blood glucose between 100 and 125 mg/dL

The screening is based on the charge of 75 g of glucose (OGTT 75 g) If the screening is negative, it must be repeated at the 28 th week

At weeks of gestation the screening must be offered in all the following conditions: 1)Age > 35 years 2)Body mass index before pregnancy >25 Kg/m2 3)Fetal macrosomy in a previous pregnancy 4)Gestational diabetes in a previous pregnancy 5)Familial anamnesis of diabetes 6)Ethnic origin in a area with a high prevalence of diabetes

Geographic area with a high prevalence of diabetes Asia: India, Pakistan, Bangladesh Caraibi: for the black population Middle Orient: Arabia, Iraq, Giordania, Siria, Oman, Qatar, Kuwait, Egypr * * * * ** *

Decisional levels for gestational diabetes mg/dLmmol/L -Fasting glucose> 92> hour> 180> hours> 153> 8.5

To woman diagnosed for gestational diabetes, an OGTT 75 g must be performed 6 weeks after delivery to confirm that gestational diabetes is resolved and to exclude the development of diabetes

For the screening of gestational diabetes, avoid: - Fasting plasma glucose - “random” glucose - Glucose challange - Urine glucose - OGTT 100 g