Pathology of Genetic Disorders Objectives Know defect, inheritance, pathology, diagnostic tests as given in lecture for: PKU Galactosemia

mia Sickle Cell Anemia Congenital Adrenal Hyperplasia MCAD A-1-AT Deficiency Hemachromatosis Cystic Fibrosis Lysosomal Storage Diseases Tay-Sachs and Gaucher Diseases Glycogen Storage Diseases Von Gierkes, McArdles and Pompes Diseases Classification of Genetic Disorders Mendelian Single Gene Disorders Autosomal Dominant Autosomal Recessive X-linked Non-Mendelian Single Gene Disorders Multi factorial Disorders Chromosomal Disorders Genetic Disorders Autosomal Dominant: Neurologic: Huntingtons Disease Renal: Polycystic Kidney Disease GI: Familial Polyposis Coli Heme: Hereditary Spherocytosis Skeletal: Marfan Syndrome Metabolic: Familial Hypercholesterolemia Autosomal Recessive: Lecture Topics X-linked: Musculoskeletal: Muscular Dystrophy Heme: Hemophilia Metabolic: Lesch-Nyhan Syndrome Neurologic: Fragile X Syndrome Genetic Disorders: Mechanisms Enzymes - Amino Acid Metabolism Phenylketonuria, Maple Syrup Urine Disease, Homocystinuria Enzymes - Other Galactosemia Congenital Adrenal Hyperplasia MCAD (Fatty Acid Oxidation) Lysosomal Storage Diseases Glycogen Storage Diseases Enzyme Inhibitors Alpha-1-Antitrypsin Disorder Transport Sickle Cell Anemia Cystic Fibrosis Hemachromatosis

Mon. 08/30/10 Genetic Disorders: Detection/Diagnosis Objective/Rationales: Population Screening Newborns Family or At-Risk Testing Prenatal amniotic fluid cells Carrier Detection Clinical Diagnosis Methods: Clinical Findings Measure Abnormal Accumulations: Serum, Urine, RBC/WBC Measure Enzyme Activity or Phenotype: serum, RBCs/WBCs, fibrocytes, or amniocytes Morphologic Changes in Tissues Gene Defect or Mutation Karyotype (to look for chromosomal abnormalities) Newborn Screening Pathology - Newborn Screening Laboratory: (population based testing -all newborns) Phenylketonuria Homocystinuria Maple Syrup Urine Disease Galactosemia Hypothyroidism: measure Thyroid Stimulating Hormone (TSH) or Thyroxine (T4) Sickle Cell Anemia Congenital Adrenal Hyperplasia Biotinidase Deficiency Other Disorders (detectable by MS/MS techniques) Fatty Acid Metabolism defects Organic Acid Metabolism defects Other Amino Acid Metabolism defects Pediatrics Programs/Services: (selected testing) Others: Genetics, Pathology, Neurology, etc: (selected testing)

Babys heel is pricked fill up filter paper card w/5 drops of blood.
Test q: Newborn screening in IN tests for at least 40 diseases. The test is performed on: blood. Test q: In the state of IN, newborns are screened for inborn errors of metabolism such as PKU. In almost all cases, the specimen collected for analysis is: blood.

PHENYLKETONURIA Incidence: 1:12,000 Classical - 1:20,000 Hyper-phenylalaninemia - 1:20,000 Transmission: Autosomal Recessive Defect: Phenlalanine Hydroxylase (decrease activity) Accumulations of Phenylalanine and metabolites Pathology: Mental Retardation by 6 months Motor Dysfunction: failure to walk Seizures Hypopigmentation of skin/hair (melanin a derivative of tyrosine) Diagnosis: Phenylalanine (blood) Tyrosine (blood) Treatment: Restriction of phenylalanine in the diet PKU: Guthrie Testing Bacterial Inhibition Assay

Phenylalanine Metabolic Pathway:

Test q: A person with phenylalanine hydroxylase deficiency typically: has mental retardation if not treated.

Below: Newborn Screening Laboratory Automater puncher punches 1/8 blood dots.

^ Agar plates. Maple Syrup Urine Disease (MSUD) Homocystinuria (HCU) MSUD (Maple Syrup Urine Disease): Incidence: 1:250,000 Inheritance: Autosomal Recessive Defect: Branch Chain keto-acid decarboxylase Pathology: Acidosis, retardation, respiratory failure Diagnosis: Leucine/Isoleucine (screen), Valine levels HCU (Homocystinuria): Incidence: 1:60-150,000 Inheritance: Autosomal Recessive Defect: Cystathionine synthase Pathology: Cataracts, skeletal and vascular defects Diagnosis: Methonine (screen), Homocysteine levels GALACTOSEMIA Incidence: 1:35,000 Transmission: Autosomal Recessive Defect: Galactose-1-Phosphate Uridyltransferase (Gal-1PUT) Phenotypes: N (normal), G (Galactosemia), D (Duarte = 50% enzyme activity) GG (0 % activity); DG (25%); GN (50%); DN (75%) Pathology: Accumulation of Galactose/Gal-1-PO4 in liver, spleen, lens, kidneys, heart, brain, and RBCs Jaundice with hepatomegaly and later cirrhosis Sepsis (E. coli), acidosis Corneal opacity Diagnosis: Galactose, Galactose-1-Phosphate levels (RBC/serum) Gal-1-PUT Activity and Phenotype (RBCs)

Test q: A 2wk old breast-fed infant exhibits jaundice, acidosis, and E coli sepsis. You suspect: galactosemia. Test q: A newborn infant has been feeding poorly and presents to the ER with jaundice and E. coli sepsis. Serum galactose is elevated. Treatment of this baby will include: elimination of both cows and breast milk from the diet.

Galactose Metabolic Pathway

Galactosemia - Liver

Gal-1-PUT is the main enzyme deficiency. The other two enzyme deficiencies are not as severe.

Clear spaces = Accumulation of galactose material in the hepatocytes.

CONGENITAL ADRENAL HYPERPLASIA (CAH) Incidence: 1:12,000 Transmission: Autosomal Recessive Defect: 21-Hydrolase - 95% (also 17 and 3-Hydroxylases) Pathology: Adrenal Hyperplasia with Virilization (androgen excess), ambiguous genitalia Salt-wasting >> hypotension, death (salt wasting is main problem not able to absorb Na) Diagnosis: 17-OH Progesterone Level Other hormones, gene typing Treatment: Glucocorticoids Adrenal Steroid Synthetic/Metabolic Pathways:

21OH-lase 95% of defects. Stops the flowchart in two spots. Increases testosterone (androgen excess).

Above: Hyperplastic adrenal glands. Lack of cortisol, so no negative feedback mechanism excess of ACTH produced. Instead of normal adrenal glands, have markedly hyperplastic glands that are trying to generate cortisol but are unable to do so.

FATTY ACID OXIDATION DISORDERS MCAD Incidence: 1:15,000 Defect: Deficiency of Medium Chain Acyl-CoA Dehydrogenase Clinical Findings: Acidosis, liver dysfunction, coma, death Sequelae: developmental delay, retardation Findings: Abnormal increase in C8 and C10 fatty acids detected by MS/MS Other chain length Acyl-CoA DH deficiencies

LYSOSOMAL STORAGE DISEASES: Sphingolipidoses: GM2 Gangliosidosis: Tay-Sachs (Hexosaminidase A) Sulfatidoses Gaucher Disease (Glucocerebrosidase) Nieman-Pick Disease (Sphingomyelinase) Metachromatic leukodystrophy (Arylsulfatase A) Krabbe Disease (Galactosylceramidase) Fabry Disease (alpha-Galactosidase A) Mucopolysaccharidoses Hurlers Disease (alpha-L-Iduronidase) Mucolipidoses + Others

Lysosomes:
Enzymes created in ER, transported to Golgi, tagged w/M6P. M6P receptor fuse w/the lysosomes. They are responsible for breakdown of organelles w/in the cell, breakdown products from other cells, etc.

Test q: A 1y/o infant girl is brought to the physician because of failure to thrive, poor neuro development, and poor motor function. Phys exam shows a cherry red spot on the macular of the retina. The infants muscle tone is poor. Both parents and a brother and sister are healthy, with no apparent abnormalities. However, one brother w/a similar condition died at the age of 18mo. This genetic disorder most likely resulted from which of the following underlying abnormalities? Mutation in a gene encoding a lysosomal enzyme (CHERRY RED SPOT = TAY SACHS) REPEATED x3!!

Lysosome Enzyme Defect: Lysosomal Storage Diseases Lysosomal Storage Diseases: Neurologic Systems: Gangliosides (Tay-Sachs) Hexosaminodase A enzyme defect, brain rich in gangliosides High incidence in Ashkenazic Jewish Population: 1:30 carrier rate Deposition of GM2 gangliosides in heart, liver, spleen, brain Progressive neurologic damage with death at 2-3 yrs of age Measure enzyme activity in serum, white cells Sphingomyelin (Niemann-Pick) Galactosylceramides (Krabbes) Glucocerebrosides (Gaucher juvenile, Type II) Not related to Jewish population Hepatosplenomegaly, significant neural deposition Early death Hepatic/Splenic: Glucocerebrosides (Gaucher- adult, Type I) Glucocerebrosidase enzyme defect WBCs and RBCs with glucocerebrosides; accumulate in macrophages High incidence in Eastern European Jewish population Massive hepatosplenomegaly, adult presentation, normal life span Tay-Sachs Disease: Sphingosine makes up ganglioside

Tay-Sachs Disease:

Test q: The adult form of Gauchers disease: has marked splenomegaly due to glucocerebroside accumulation. REPEATED x3! Test q: A 22y/o woman delivers an apparently healthy female infant following an uncomplicated pregnancy. At 4yr of age, the girl is brought to the physician bc her parents have observed progressive, severe neurologic deterioration. Physical exam now shows marked hepatosplenomegaly. A bone marrow biopsy specimen shows numerous foamy vacuolated macrophages. Analysis of which of the following factors is most likely to aid in the diagnosis of this condition? Level of sphingomyelinase in splenic macrophages. (Other choices: number of LDL receptors on hepatocytes, rate of synthesis of collagen, level of g-6-phosphatase in liver cells, level of -1-antitrypsin in the liver.) Test q: A 25y/o AIDS patient presents w/massive spenomegaly requiring splenectomy. Histologic sections show sheets of plump macrophages filled w/pink, fibrillar material. Granulomas are not identified. These findings are consistent with: Gauchers disease. Test q: A 30y/o male presents w/hepatosplenomegaly. A splenectomy reveals macrophages filled w/pink, amorphous material that resembles the appearance of tissue paper. The prognosis for this patient is: an almost normal lifespan. Test q: Accumulations of gangliosides in lysosomes is seen with: Tay Sachs disease.

Gauchers Disease

Test q: A female infant shows failure to thrive and failure to achieve developmental milestones. A pedigree reveals only this child is affected out of 4 generations on both sides of the family. Tissue fibroblasts obtained from this child shows a 46,XX karyotype. Cultured fibroblasts have increased amounts of an intermediate product in a multiple enzymic metabolic pathway from substrate to end product. What is the most likely recurrence risk for this condition in siblings of this infant? 25%.

GLYCOGEN STORAGE DISEASES Hepatic: von Gierke Disease (Glucose-6-phosphatase) Hepatomegaly, hypoglycemia, hyperlipidemia Failure to thrive, stunted growth, most survive Feed them cornstarch bc they can break down glucose from that normal growth and survival Myopathic: McArdle Syndrome (muscle phosphorylase) milder Muscular weakness, cramps, normal life span Generalized: Pompe Disease (alpha-1,4-Glucosidase) Diffuse organ involvement, early death. Most severe.

Test q: The inheritance pattern of diseases which result from mutations affecting enzymes is likely to be autosomal recessive. Test q: A 2y/o child is brought to the physician after having convulsions. The child has a history of failure to thrive. Phys exam shows hepatomegaly and ecchymoses of the skin. Lab studies show a blood glucose levels of 31 mg/dl. A liver biopsy specimen shows cells filled w/clear vacuoles that stain positive for glycogen. Which of the following conditions is most likely to produce these findings? Von Gierke disease.

Glycogen Storage Diseases

Below: See accumulation of glycogen microscopically. Can see renal tubules here that have expanded due to glycogen deposition.

Diagnosis of Lysosomal/Glycogen Storage Diseases Enzyme Activity: (synthetic substrates) Serum, urine Leukocytes Cultured fibroblasts or amniotic fluid cells Gene probes: gene typing Tissue Biopsies: liver, bone marrow, muscle ALPHA-1-ANTITRYPSIN DEFICIENCY Incidence: 1% population with abnormal phenotype (1:1,800 with ZZ) Transmission: Autosomal recessive Defect: Deficiency or defective enzyme Anti-Protease: chymotrypsin, trypsin, plasmin, WBC elastase and collagenase, etc Pathology: Hepatic cirrhosis, pulmonary emphysema; variable in severity and time of presentation Diagnosis: A-1-AT Levels and Phenotype (Immunoassay and IEF) Pi: MM (100%); MZ (58%); ZZ (15 % - most severe.); SS (60%); MS (80%) Gene Defect

Test q: A 40y/o male presents w/emphysema and cirrhosis. A liver biopsy shows pinkish red globules in the hepatocytes that are PAS+. This patient suffers from: an a-1 antitrypsin deficiency. Test q: In -1 antitrypsin deficiency, the component of the ECM most susceptible to damage is: Elastin. Test q: If acute inflammatory responses were to proceed w/o inhibition, they would cause considerable tissue destruction and permanent loss of function of inflamed organs. Once important regulator of acute inflammation is the substance: alpha-1-antitrypsin.

Can see nodules of residual hepatocytes.

SICKLE CELL ANEMIA Incidence: 1:4,000 (Indiana), 8% carrier rate in black population. Transmission: Autosomal recessive Defect: Amino acid substitution Abnormal hemoglobin structure Pathology: Anemia, SS crisis, thrombosis, organ infarcts, infections Diagnosis: Hgb Phenotyping (electrophoresis) SS, SC, S/beta-thalassemia are sickling disorders Gene Defect SS = homozygous sickle cell disease SC = hemoglobin C trait sickling process disorder Treatment: Supportive, hydroxyurea to stimulate production of Hgb F; bone marrow transplant CYSTIC FIBROSIS Incidence: 1:2,000 Transmission: Autosomal recessive Defect: Sodium/Chloride Ion Transport System Multiple allele defects - delta F508 (95% in Indiana) Chloride transport across membranes Pathology: Pulmonary Brochiectasis, Pneumonias, Failure Hepatic Cirrhosis Malabsorption Infertility Diagnosis: Trypsinogen level (screen) - increased Sweat Chloride Test (increased) Gene Defects - 1000+ allele variants

Hemoglobin Electrophoresis IEF (pKa based)

Below: Sickle Cell Anemia/Hemoglobinopathies:

Test q: Choose the genetic disorder w/the highest incidence in the newborn population: Cystic fibrosis (other choices: PKU, MSUD, Niemann-Pick, or Wilsons)

Cystic Fibrosis:

CF Chronic Pulmonary Infections (bronchiectasis)

CF: Pancreatic fibrosis (below)

CF: meconium ileus (newborn)

HEMOCHROMATOSIS Incidence: 1:400 (whites, western europe) Transmission: Autosomal Recessive Etiology: Primary - hereditary disorder, excessive iron absorption Secondary - excessive iron overload states, example: beta-thalassemia with multiple transfusions Anatomical Findings: Micronodular cirrhosis - iron deposits Diabetes - iron deposits in Islets, fibrosis Skin pigmentation - increased melanin bronze Diagnosis: Iron levels, blood and liver Gene defect (s)

Below: Iron pigment in fibrous scarring. (iron in macrophages)

Test q: A 50y/o male presents w/a serum glucose of 400mg/dL. Physical exam shows a bronze color to the skin and a small, firm liver. He develops sepsis and dies. You would expect to see __ at autopsy. Iron in hepatocytes and islet cells. Test q: A 52y/o man present to his physician w/a peculiar bronze pigmentation of the skin and symptoms referable to what was eventually proven to be diabetes mellitus. He also was noted to have an enlarged liver with abnormal liver function tests. This clinical presentation is most suggestive of which of the following disorders? Hemochromatosis.

Diabetes Mellitus Objectives: Classification of Diabetes Mellitus Diagnostic criteria for Diabetes Monitoring Methods: Glucose, Glycosylated Hgbs Pathology of Diabetes Mellitus Pancreas - Islets Renal pathology Vascular pathology: Large and small vessels DM - Classification/Incidence Systemic Disease: Multiple Organ Involvement Primary (Idiopathic) Type 1 (IDDM) aka juvenile onset Type 2 (NIDDM) Genetic defects of beta-cell function Genetic defects in insulin action Gestational Diabetes: woman is typically predisposed to develop type II later in life Impaired Glucose Tolerance/Insulin Resistance: Metabolic Syndrome (5-10%/year progress to Type 2 DM) 25% of population Secondary Hemochromatosis, Pancreatitis, etc Incidence: 2-3 % of population (7th in cause of deaths) Type 1 5 - 10 % Type 2 90 - 95 % (half of cases undiagnosed)

Tues. 08/31/10

Test q: A 25y/o female is pregnant w/her 1 child. Her previous med history is unremarkable. At 20wk, she develops gestational diabetes. If a renal biopsy were performed, you would expect to see: normal kidney.

st

Diabetes - Etiology Type 1 (IDDM):Juvenile considered autoimmune disorder Destruction of beta-cells and absence of insulin Genetics (HLA linked), Multi-factors, Autoimmunity T-cell mediated, Environmental Insult (? Viral) Auto-antibodies: Islet Cell, GAD65 (glutamic acid decarboxylase), IA-2, IA-2 beta (tyrosine phosphatases), or Insulin; One or more present in 85-90% of patients Prone to ketoacidosis (presentation can involve hypovolemia, shock) Typically in children, youth, but can occur at any age Type 2 (NIDDM): Adult Onset Peripheral resistance to Insulin and decreased secretion by beta-cells, may not be insulin dependent Genetics (complex - multiple genes), Obesity, Gestational Diabetes, Metabolic Syndrome Typically in adults but is being seen with increase frequency in children

Test q: A 12y/o male presents to the ER w/disorientation, fruity breath, and a serum glucose of 500mg/dl. This patient is most likely suffering from: Ketoacidosis.

 Metabolic Syndrome ~ 25% of population, increases with age Criteria: Abdominal Obesity (waist > 40 in males; >35 in females) Triglycerides: > 150 mg/dL HDL-Cholesterol: < 40 mg/dL males; <50 mg/dL females Blood Pressure: >130/>85 mmHg Fasting Glucose: > 100 mg/dL Increased risk of cardiovascular disease Complications of DM Renal (Nephropathy) Polyuria/Polydipsia Hypertension Renal Failure Cardiovascular System Infarcts-heart, kidney, etc Hypertension Eye (Retinopathy) Blindness Cataracts Skin Ulcers Vascular Insufficiency/Gangrene Neurologic System (Neuropathy) Polyneuropathy-sensory, motor Strokes GI System Impaired motility Bacterial Overgrowth Urogenital System Impotence Bladder dysfunction Immune System Infections-kidney, skin, etc

DM - Biochemical Findings/Diagnosis: Hyperglycemia Abnormal Fasting Glucose or Oral GTT Increased Non-enzymatic glycosylation (Hgb A1c) Increased fatty acid metabolism -ketoacidosis Hyperlipidemia Increased LDL cholesterol Decreased HDL cholesterol Increased triglycerides Renal Dysfunction Glucosuria Proteinuria (albumin = micro-albuminuria) Azotemia ( creatinine/Urea) = renal insufficiency Diagnostic Criteria - Diabetes Mellitus 1998 CRITERIA FOR DIABETES CLASSIFICATION Symptoms of Diabetes + glucose > 200 mg/dL or Fasting glucose > 126 mg/dL or 75 gm OGTT, 2 hr glucose > 200 mg/dL Increased Hgb A1c (GLYCOSYLATED HEMOGLOBIN) > 6.5 % Note: replication of the above is required Gestational Diabetes uses slightly different criteria 1 Hour Glucose post 50 gm OGTT > 140 (screen) 3 Hour 100 gm OGTT: abnormal response Impaired Glucose Tolerance: those cases which are not normal but do not meet the above criteria Fasting glucose between 100 - 126 now being used as guide for future testing (part of metabolic syndrome criteria also) Increased Hgb A1c: > 6.0 but < 6.5 %

Test q: A 35y/o male presents for a medical exam after weight loss and fatigue. His Hgb A1c is measured to be 7.5%. You would also expect to find: proteinuria. Test q: A 72y/o woman is admitted to the hospital in an obtunded condition. Her temp is 37*C, pulse is 95/min, respirations are 22/min, and BP is 90/60mmHg. She seems to be dehydrated and has poor skin turgor. Her serum glucose level is 872 mg/dl. Urinalysis shows 4+ glucosuria, but no ketones, protein, or blood. Which of the following factors is most important in the pathogenesis of this patients condition? Insulin resistance (Other choices were: HLADR3/HLA-DR4 genotype, Autoimmune insulitis, Severe depletion of cells in islets, Virus-induced injury to cells in islets.)

Glucose Tolerance Testing:

Retinopathy % based on test criteria: Puma Native Americans

Diabetic Control Monitoring: Lab tests Measure Non-DM DM

Pre-meal glucose Bedtime glucose Hgb A1c (%) < 110 < 120 <6 80-120 100-140 <7

Actions
<80, >140 <100, >160 >8

Glucose meter - home use Hgb A1c - laboratory tests/Point of Care Test Urine protein/albumin Renal function tests Lipid status Blood Pressure Retinal Exam

Arrow = inflection point. Point at which there is increase in retinopathy. Blood Glucose Monitoring - DM Glucometers - Point of Care Testing on Whole Blood In home use (provides serial data which can be down-loaded to computer systems) In Hospital/Clinic use: provides rapid test results Limitations/Notes: Can be set to give whole blood values (lower) or Corrected to give serum/plasma values (higher) Linearity = 40 - 500 mg/dL glucose Less reliable than lab test for low glucose levels Accuracy: correlates within +/- 15% of lab values May not be accurate if Hct < 20 or > 60% Precision = +/- 5% CV Must follow good practices: reagent storage, QC, calibration, instrument maintenance, etc When in doubt? Re-check glucose on plasma at Lab Pathogenesis of DM Complications Advanced Glycosylated Endproducts (AGE): Glycosylation of compounds Activation of Protein Kinase C Hyperglycemia and Polyol Pathways Advanced Glycosylated End-products (AGE): Non-enzymatic Glycosylation: Glucose + Protein Schiff Base Amadori Product Glucose Meter: Monitoring

Glycosylated Hemoglobin Methods

HPLC gold standard, high performance liquid chromatography

Permanent linkage of glucose molecule to protein.

Test q: A study of patients recently diagnosed with type 2 diabetes mellitus follows them for 20 yr to determine the prevalence and severity of complications of the disease. The records of these patients are analyzed to identify the laboratory methods used to monitor patients ability to maintain disease control and reduce the potential for complications. Which of the following lab studies is most likely to afford the best method of monitoring disease control in these patients? Glycosylated hemoglobin. (Other choices: random plasma glucose, fasting plasma glucose, glycosylated serum albumin, serum fructosamine, microalbuminuria) REPEATED x4!!

AGE Effects Glycosylated Hemoglobins HPLC Gold Standard CHEMICAL Cross-link polypeptides (e.g. collagen) Traps non-glycosylated proteins (LDL, Igs, Complement) (CV risk) Confer resistance to proteolytic digestion Induce lipid oxidation (increases atherosclerosis process) Inactivate nitric oxide (risk for infections, risk for thrombosis) NO is key component in bacterial control, so risk for infections. Bind nucleic acids BIOLOGIC Bind to AGE receptors on endothelial, monocytes and mesenchymal cells Induces: Monocyte emigration (inflammation, atherosclerosis) Cytokines and growth factor secretion (IGF-1, TGF-b, PDGF, VEGF) Increased vascular permeability Pro-coagulant activity (thrombosis) Enhanced cellular proliferation Enhanced Extra-cellular matrix (ECM) production Pro-inflammatory factors Protein Kinase C Activation: Hyperglycemia stimulates production of diacyl glycerol (DAG) which activates Protein Kinase C: Production of pro-angiogenic factors (VEGF, vascular endothelial growth factor) neo-vascularization of retinal vessels Increased vasoconstrictor endothelin-1 and decreased activity of vasodilator endothelial nitric oxide synthase Profibrogenic factors (TGF-beta, transforming growth factor) increased ECM and BM material Pro-coagulant activity Increased Plasminogen Activator Inhibitor (PAI-1) = reduced fibrinolysis Pro-inflammatory factors cytokines in endothelial cells Diabetic Nephropathy Mechanisms Sorbitol Pathway in Hyperglycemia

DM - Anatomic Findings: Pancreas Pancreas: Islets of Langerhans (beta cells) No Change (in some) Beta cell de-granulation Inflammation (Insulitis) - lymphocytes (DM-1) Beta cell loss (DM-1, DM-2) Islet/Beta cell hyalinization/fibrosis (DM-1) Islet/Beta cell amyloid (amylin) deposition (DM-2) Beta cell hyperplasia - infants of diabetic moms
Test q: A 15y/o boy collapses while playing football. On phys exam, he is hypotensive and tachycardic with deep, rapid, labored respirations. Lab studies show serum Na+ of 151mmol/L, K+ 4.6mmol/L, Cl- 98mmol/L, CO2 7mmol/L, and glucose 521 mg/dl. Urinalysis shows 4+ glucosuria and 4+ ketonuria levels but no protein, blood, or nitrite. Which of the following is the most probable pathologic finding in the pancreas at the time of his collapse? Lymphocytic infiltration of islets. Test q: The type of amyloid found in the islets of Type II diabetes is: amylin, Test q: A 75y/o female is examined after developing polyuria, polydipsia, and a vaginal yeast infection. Her fasting plasma glucose is >300mg/dl on two occasions. If this patient underwent pancreatic biopsy, you would expect to see: Amylin. Test q: Deposition of pink material in pancreatic islet cells in Type II Diabetes: Is amyloid made up of amylin protien. REPEATED x3!

Schematic DM Complications

Below: Pancreas Islets. Normal.

Islets in Type 1 and 2 DM: Atrophy +

DM - Anatomic Findings: Vessels Diabetic Micro-angiopathy - diffuse small vessel Basement Membrane Thickening Plasma proteins, basement membrane material (glycosylated) Skin, muscles, retina, glomeruli, etc Hyalinization of capillary/arteriolar walls = glomerulosclerosis and arteriolosclerosis Kidney, skin, etc Aneurysm formation - structural cell loss Retinal aneurysms, hemorrhages, exudates, thrombosis Atherosclerosis - diffuse large vessel Atheromatous plagues, accelerated atherosclerosis Ischemia, infarcts - heart, brain, kidney, extremities Aneurysm/rupture - aorta Hypertension
Test q: The most common cause of death in diabetics relates most closely to the development of atherosclerosis.

Type 2 DM: Amyloid Deposition:

Microangiopathy Basement Membranes: DM

Proteinuria in Diabetes: Micro-albuminuria = albumin measured at low levels in urine Category 24 hr Timed Random
Units mg/24h mcg/min mcg/mg creat

Normal Micro Macro

< 30 30 - 300 > 300

< 20 20 - 200 > 200

< 30 30 - 300 > 300

Renal Micro-angiopathy: Renal - Glomerulosclerosis Diffuse glomerulosclerosis Nodular glomerulosclerois Renal - Nephrosclerosis (arteriolosclerosis) Afferent and efferent arteriolar hyalinization Increase in BM production. Markedly thickened. Makes glomeruli leakier will spill protein into the urine.
Test q: Increase in advanced glycosylated end products results in: diffuse basement membrane thickening of skin vessels.

Kidney: Nodular glomerulosclerosis. Kimmelstiel- Wilson lesion

Nodular/Diffuse Glomerulosclerosis

Kidney - Arteriolosclerosis/Nephrosclerosis (afferent and efferent arterioles = DM)

DM: Renal Injury Fate Glomerulosclerois/Nephrosclerosis (diffuse) Fibrosis: glomerular hyalinization/fibrosis Inflammatory infiltrate (lymphocytes) Atrophy of nephron unit Cortical Thinning (granular) End-Stage: Renal failure

Kidney - Nephrosclerosis/Glomerulosclerosis

Sclerotic Nephron Unit

Kidney - Cortical Atrophy 2 to Glomerulosclerosis/Nephrosclerosis

Test q: A 45y/o man is evaluation for hypertension. Arteriography shows marked stenosis of the left renal artery. Unfortunately, he has an acute MI and dies shortly after the procedure. At autopsy, the left kidney weighs only 30g and has a smooth capsule. The right kidney weighs 120g (normal is ~150) with a pitted surface. Microscopically the right kidney shows: Glomerulosclerosis.

DM-Anatomic Findings: Small Vessels Eyes - Retinopathy Basement membrane thickening Micro-aneurysm formation Dilated, tortuous vessels w/aneurysms. Hemorrhage, exudates, thrombosis Neovascularization proliferation of very small blood vessels has large effect on vision Cataracts Treated w/laser therapy
Test q: Biopsy of the retina of a 65y/o insulin dependent diabetic w/decreased visual acuity will show: neovascularization. Test q: A 66y/o woman has had diabetes mellitus for more than 30yr. She now has decreasing visual acuity. There is no eye pain. On phys exam, her intraoculat pressure is normal. She is 160 cm (52) and weighs 79kg (BMI 31). Which of the following lesions is most likely to account for her visual problems? Proliferative retinopathy. REPEATED x3.

Retinopathy:

DM: Papillary Tip Necrosis Kidney - Papillary Tip Necrosis Small vessel disease > ischemia >> >> Coagulative necrosis >> Repair/Fibrosis-scarring Can slough off and clog the ureter.

DM-Anatomic Findings: Pyelonephritis Neuropathy -> bladder dysfunction -> UTIs UTIs -> Pyelonephritis (also impaired response to infections) Pyelonephritis - acute Fibrinous necrosis/exudate Neutrophilic infiltrate Repair/Fibrosis-scarring Pyelonephritis - chronic (segmental scarring) Fibrosis: glomerular hyalinization/fibrosis; Medullary fibrosis Inflammatory infiltrate (lymphocytes) Atrophy of nephron unit Cortical Thinning/Medullary loss Kidney - Chronic Pyelonephritis

Test q: A 52y/o insulin-dependent diabetic develops chronic pyelonephritis. You would expect a renal biopsy to show: micronodular glomerulosclerosis, Test q: A renal biopsy was performed and micronodular glomerulosclerosis was found in the glomeruli. The person is most likely to have: Elevated Hemoglobin A1c. Test q: A 54y/o man w/type I diabetes mellitus had worsening renal function w/proteinuria and elevated BUN and creatinine. A renal biopsy was performed. Which of the following is the most likely finding? Micronodular glomerulosclerosis. Test q: A 50y/o female has declining renal function. She has repeated urinary tract infections. A biopsy shows micronodular glomerulosclerosis. The diagnosis is: Type I IDDM.

Lumpy, not finely granular. DM - Anatomic Findings: Large Vessels Other Infections Yeast (skin, bladder, throat) Cardiovascular System: Atherosclerosis Intimal thickening (hyperplasia) Increased growth factors (smooth muscle proliferation), increased ECM production, AGEs - protein trapping Media atheromas Increased LDL, oxidized lipids and monocyte activation Thrombosis (increased pro-coagulants) Ischemia/Infarcts Myocardial infarcts Cerebral infarcts Renal infarcts Bowel infarcts Ischemic skin ulcers/gangrene Extremity infarcts/gangrene - amputations Aneurysm/Rupture - Aorta Diabetes Treatment Treatment Insulin (injections or pumps) Oral hypoglycemics (Type II diabetics) Islet Cell or pancreas transplants Impact of Tight Control/Treatment Reduced renal disease Reduced cardiovascular disease Reduced retinopathy Reduced neuropathy

Diseases of Immunity Objectives: Review basic principles of cellular and humoral immunity Immune tolerance Four types of hypersensitivity reactions Transplant / Graft rejection

Fri. 09/03/10

Adaptive Immunity: Cellular and humoral immunology & tolerance T-cells CD4: MHC Class II CD8: MHC Class I B-cells produce antibodies and cytokines (regulate innate immune cells as well as other cell types) Antigen presenting cells (APCs) important in stimulating B cells Cytokines Macrophages / Eos / Basophils / PMNs innate immune cells but also important in adaptive immune response FIGURE 6-1 The principal classes of lymphocytes and their functions in adaptive immunity: Lymphocytes produce antibodies in response to antigenic challenge. Get B cell maturation and production of IgG, which is produced by plasma cells. The antibody then mediates antigen clearance. CD4 typically produce cytokines that act to orchestrate the activity of the other innate immune cells, as well as the adaptive immune cells (B cells). CD8 important in clearance of tumors and virus-infected cells.
Test q: What cells constitute the major immune defense mechanism against tumors? Cytotoxic T lymphocytes.

Key Cytokines Cytokines work to up-regulate or down-regulate immune responses. General proinflammatory: IL-1, TNF-a (affect not only immune cells but also other cells like liver acute phase reaction) General antiinflammatory/repair: TGF-b, IL-10 Proliferative: IL-2 Differentiation: IL-7 (B-/T-cell maturation); IL-4 (Th2); IL-12 (Th1); TGF-b & IL-23 (Th17) Th cell lineage specific: Th1: IFN-g/IL-2/IL-12 Th2: IL-4/5 Th17: IL-17/chemokines Cell mediated immunity effector cells (macrophages, NK cells): IFN-g Humoral & IgE mediated effector cells (MAST cells, eosinophils): IL-4, IL-5
Test q: A 9y/o boy has had a chronic cough and fever for the past month. A chest radiograph shows enlargement of hilar lymph nodes and bilateral pulmonary nodular interstitial infiltrates. A sputum sample contains acid-fast bacilli. (Once- said A sputum culture grows Mycobacterium tuberculosis.) A transbronchial biopsy specimen shows granulomatous inflammation within the lung, marked by the presence of Langhans giant cells. Which of the following mediators is most likely to contribute to giant cell formation and granulomas? Interferon-. REPEATED x3!! Test q: A 50y/o male moved to Indy two years ago. A chest x-ray for suspected pneumonia reveals a coin lesion in the left upper lung lobe. Biopsy reveals caseous necrosis w/giant cells and granuloma. The microscopic appearance of his lung biopsy is consistent w/activation of: IFN- . Test q: All of the following are true about cytokine function EXCEPT: Interleukins 2&7 (IL-2 and IL-7) are potent inhibitors of lymphocyte proliferation. (Other choices: IL-1 and TNFa are produced by various cell types and have diverse activities on both immune and non-immune cells leading to a generalized inflammatory state. IL-10 and TGF-b are produced by a number of cells, including Treg cells, and play an important role in inhibiting the inflammatory response and stimulating tissue repair processes. IL-4 and IL-5 are important regulators of Th2-mediated cellular responses. IFN-g is a potent regulator of cell mediated function and Th1-mediated cellular responses.)

CD4+ Th Peripheral Development: Th1 cells are stimulated by and also produce IFN-. Th1mediated responses tend to be involved in Type IV hypersensitivity reactions (which are cell-mediated and involve macrophages, monocytes). Th2 important in Type 1 hypersensitivity and atopic reactions (allergies). These individuals have a Th2polarized cytokine profile have more IL-4 and IL-5 as compared to others like IFN-. Th17 often produced in Type IV hypersensitivity reactions. Tregulatory (Treg cells) help to inhibit inflammatory reactions. Produce anti-inflam cytokines (IL-10, TGF-) FoxP3 is transcription factor need to make Treg cells. Xlinked autoimmune disease occurs when -/- for FoxP3.
Test q: All of the following are true about hypersensitivity reactions EXCEPT: Individuals w/atopy (predisposition to allergic disorders) frequently have elevated levels of Th1 cytokines (Th1 polarized phenotype). (Other choices: Type I HS reactions are initiated by antigen (allergen) binding to IgE-coated mast cells resulting in IgEmediated mast cell activation. A patient w/type A blood was mistakenly tranfused w/type B blood. The resulting hemolytic transfusion reaction is an example of a type II HS reaction. Using anti-immunoglobulin fluorescent antibodies, a granular appearing pattern of immunofluorescence (Direct IF) in the glomeruli of a patient with a skin infection positive for Streptococcus would be consistent w/a type III HS reaction.)

Initiation of Adaptive Immune Response by Professional APCs: Dendritic Cells Activated APC: Migrates to LN for presentation of Ag to CD4 & CD8 cells Migration and homing (e.g to High Endothelial Venules): Requires Specific Chemokines (e.g. CCL19/21) & Adhesion Receptors (e.g. ICAM-1) IMMUNE TOLERANCE: Central T-cell Tolerance Central tolerance: Clonal deletion of self-reactive T cells in thymus. First: Positive selection for T-cells recognizing MHC molecules (cells lacking recognition are deleted). Second: Negative selection for cells with high affinity interaction with self antigens (cells deleted). Some T-cells that recognize self Ag are converted to Treg cells. AIRE (Autoimmune regulator) gene: Transcriptional regulator induces peripheral Ag expression in thymic cells (Mutated in autoimmune polyendocrinopathy) T-cell peripheral tolerance: CD80/86 (B7.1/7.2) co-stimulation Activation vs. Tolerance: Dependent on maturation state of APC cells. Immature APC cells: low MHC Class I & II and CD80/86 (B7.1/7.2) co-stimulatory molecules Mature APC cells: Activation by microbial molecules or proteins; released from necrotic cells: Increased MHC & CD80/86 T-cells also express inhibitory B7 receptor, CTLA-4 ((-/-) mice assoc. with AI disease) Polymorphisms assoc. with human AI disease.

Peripheral tolerance: We do not develop immunity to eyes, testes, etc. because they express high levels of FasL any lymphocyte that tries to get into those tissues is apoptosed. Tumors surround themselves w/a cloak of Treg cells.
Test q: What ligand is overexpressed in human eyes and testes: FasL. Test q: All of the following are key mechanisms for peripheral T-cell tolerance EXCEPT: Activation of toll-like receptor-2 on T-cells induces marked anergy to subsequent antigen activation of the T-cells. (Other choices: Secretion of IL-10 and TGF- by regulatory T-cells (Treg). Fas ligand expression on testicular epithelium induces T-cell apoptosis. Immature antigen presenting cells fail to activate the co-stimulatory molecule CD28 on T-cells during antigen presentation. T-cells expressing the CTLA-4 receptor fail to respond to antigen presentation.) Test q: All of the following are mechanisms for peripheral T cell tolerance EXCEPT: The autoimmune regulatory element (AIRE) transcription factor induces expression of extrathymic self antigens within medullary thymic epithelial cells, resulting in clonal deletion of immature T cells that recognize these antigens. (Other choices: An immature APC that lacks B7.1/7.2 expression presents antigen to a Th cell, resulting in T cell anergy. T cells express an inhibitory receptor, CTLA4, that acts to suppress T cell function. Regulatory T helper cells (Treg) release cytokines that act to suppress immune cell function. Repeated stimulation of T cells induces expression of Fas, which can induce apoptosis in the T cell when it binds to Fas ligand (FasL) on other cells.)

B-cell Activation: Peripheral Tolerance

B-cell Tolerance Central (BM): Immature B-cells exposed to high levels of circulating self antigens undergo receptor editing (BCR rearrangements in hypervariable region that do not recognize self). If not successful, undergo apoptosis Peripheral: Requires T-cells. Ag must also be recognized by Th cells to stimulate B-cell development. (Remember CD40:CD40L interaction?) T-cell independent antigens: (e.g. Pokeweed mitogen & Carbohydrate antigens) induce low affinity IgM and no memory cells Hyper IgM immunodeficiency Syndrome: Absence of CD40L/CD40
Test q: What statement best characterizes hyper IgM syndrome: the disease is caused by a mutation or loss of the CD40 ligand on T cells. Test q: Hyper IgM syndrome is a primary immunodeficiency syndrome associated w/production of IgM only. Since IfM is typically a low-affinity antibody with weak activity against most microbes, patients are prone to recurring infections w/pyogenic bacteria. This syndrome is characterized by loss of function of what cellular protein? A mutation resulting in loss of the CD40 ligand on T cells that mediates binding to the CD40 receptor on B cells.

FOUR TYPES OF HYPERSENSITIVITY REACTIONS: Type I: IgE mediated allergic response Type II: Hypersensitivity: Antibody-Mediated Reactions Type III: Immune Complex Type IV: Hypersensitivity: Cell mediated hypersensitivity

Type I: IgE mediated allergic response

Comparisons of Type I-IV Hypersensitivity Reactions:

Test q: A 22y/o man has had a urethral discharge for the past week. A culture of the exudates from the urethra grows Neisseria gonorrhoeae. He is treated w/penicillin G, but within minutes after injection, he develops itching and erythema of the skin. This is quickly followed by severe respiratory difficulty w/wheezing and stridor. Which of the following immunoglobulins has most likely become attached to the penicillin G and mast cells to produce these symptoms? IgE.

Primary Mediators (preformed granules) Histamine (Antihistamines): Chemotactic factors: (LTB4, Eosinophil chemotactic factor) Secondary Mediators (Lipid mediators) Cysteinal Leukotrienes (C4, D4), PGD2, PAF CysLT1 receptor antagonists: zafirlukast (Accolate), montelukast (Singulair). 5-LO inhibitors: Zileuton (Zyflow) Secondary Mediators (Cytokines) TNF IL-4, IL-5 Early response: Wheel and flare / bronchospasm (LKT C4/D4, PAF) and mucous hypersecretion: vasodilation (histamine, PAF); vascular permeability (histamine, PAF, LKT C4/D4). Late-phase response: Recruitment of eosinophils, basophils, neutrophils, CD4+ (TH2). Tissue remodeling. Extreme Early Response: Anaphylaxis The Acute Mast Cell Degranulation Response is followed by a delayed response mediated by circulating Eos/Baso/CD4+ T-cells/PMNs:

Fatal acute laryngeal edema during an anaphylactic reaction to penicillin. Over time, the eosinophilic infiltrate can result in tissue remodeling. Asthma: long-term remodeling of the bronchial tree

Asthma: Tissue Remodeling

Test q: A protein found in peanuts, calls Ara h 1, is found to be the major antigenic determinate that generates an IgE response in susceptible individuals. Subsequent exposure to this antigen results in mast cell degranulation, resulting in rapid vasodilation, vascular permeability, and smooth muscle contraction. What type of HS? Type I Hypersensitivity reaction. Test q: Microscopic features of the tissue remodeling characteristic of asthma include all of the following EXCEPT: smooth muscle atrophy. (Other choices: mucous gland hyperplasia, mucus plugs, basement membrane thickening, and inflammation w/eosinophils)

Type 2 Hypersensitivity: Antibody-Mediated Reactions In all cases: Immune reaction against Ab/complement bound to cell or tissue. Type II: Cytotoxic Antibody Mediated Ab binding to Ag on cell results in complement fixation and opsonization, resulting in lysis or phagocytosis (e.g. transfusion reaction, AI hemolytic anemia, AI thrombocytopenia)
Test q: Lab tests are ordered for 2 hospitalized patients. During the phlebotomy procedure, the Vacutainer tubes drawn from these patients are mislabeled. One of the patients receives a blood transfusion later that day. Within 1 hour after the tranfusion of RBCs begins, the patient becomes tachycardic and hypotensive and passes pink-colored urine. Which of the following statements best describes how this reaction is mediated? Complement-mediated lysis of RBCs. REPEATED TWICE

Type II: Antibody Dependent Cellular Cytotoxicity Type II: Antireceptor antibodies:

Above: Ab binding to cells activates effector cells: NK cells & also Monocytes, PMNs, Eosinophils, that lyse Ag-coated cells via cytotoxic mechanisms (e.g. granzyme/perforin) (No phagocytosis). Eos use this mechanism to destroy parasites.

Above: Antibody has natural ligand activity wants TSH receptor (Graves Disease)

Above: Antibody has natural ligand blocking activity (Myasthenia gravis)

Diseases associated with Type II antibody-mediated reactions: Goodpastures Syndrome -Type IV collagen Bullous pemphigoid -skin basement membrane Pernicious anemia -intrinsic factor Acute rheumatic fever -antibodies against streptococcus cross-react with heart Erythroblastosis fetalis -Rh D antigen Transfusion reactions -ABO and minor antigens Aschoff Bodies: Hallmark of Rheumatic Fever: Granulomatous lesions that follow necrosis/degenerative phase and precede fibrosis. (Found in only a small subset of patients) Are actually type IV reaction (part of repair process that occurs later).

Test q: Lesions found in the myocardium at autopsy are the result of type __ HS. II. Test q: In acute rheumatic fever, the immunopathologic reaction is triggered by human antibody directed against: Streptococcus pyogenes. REPEATED TWICE Test q: A 29-y/o female dies during surgery to repair aortic and mitral valves and relieve chronic heart failure. As a child she experienced a severe upper respiratory infection w/subsequent pericarditis. The etiology for both the acute illness and valve malfunction is most likely: a bacterial infection.

Test q: The classic histopathologic lesion seen in the myocardium in rheumatic heart disease is: Granulomas.

Type III: Immune Complex Phase 1: Formation of immune complexes in the circulation Phase 2: Deposition of the immune complexes in various tissues (vasculitis, glomerulonephrits, or arthritis) Phase 3: An inflammatory reaction provoked by activation of PMNs and macrophages by Fc or C3b receptors.

Immune Complex Vasculitis: Fibrinoid necrosis & infiltrates

Type III Prototype: Acute post-streptococcal glomerulonephritis The reaction in the tissue is characterized by necrosis, with deposits of fibrinoid and an infiltrate of PMNs.

Key difference from Type II Reaction: Reaction is to immune complex deposition (bystander effect) rather than to Ab bound to specific Ag on Tissue/cell.

Direct IF: Detects Ab/Immune Complex Directly in biopsy Granular pattern Type III IC Acute poststreptococcal GN / SLE

Smooth pattern Type II Ab Goodpasture Syndrome Anti-Glomerular BM (collagen IV)

Type 4 Hypersensitivity: Cell mediated hypersensitivity No Antibodies CD4+ Th1/Th17: Delayed type hypersensitivity (Granuloma, tuberculin reaction) Activated macrophages / histiocytes CD8+ CTL: Direct cell cytotoxity (graft rejection & virus infection): Perforin/granzyme & FAS/FASL T cellmediated (type IV) hypersensitivity reactions: Unlike other hypersentivity reactions, this reaction takes days CD4+ TH1 cells (and sometimes CD8+ T cells, not shown) respond to tissue antigens by secreting cytokines that stimulate inflammation and activate phagocytes, leading to tissue injury. CD4+ TH17 cells contribute to inflammation by recruiting neutrophils (and, to a lesser extent, monocytes).

Classic Tuberculin Reaction (PPD): Reddening and induration peaking at 2472 hrs. Perivascular infiltration of T cells (CD4+) and mononuclear phagocytes perivascular cuffing.

CD8+ cytotoxic T lymphocytes (CTLs) directly kill tissue cells. The Good: Likely important in clearing of virally infected cells / intracellular pathogens & tumor immune surveillance. The Bad: Important in mediating graft rejection (Direct Pathway) and some autoimmune diseases

Test q: A person clearing brush in their back yard develops a blistering rash that causes extreme discomfort due to itching. A physician diagnoses this as a case of poison ivy, or contact dermatitis. Histologically this would appear as an epidermal blister with dermal and epidermal mononuclear infiltrate. What type of HS reaction? Type IV.

Chronic Type IV reaction: Granuloma

Chronic Type IV reactions to persistant or nondegradable Ags (TB and foreign bodies (e.g. sutures) lead to granulomatous inflammation.

Transplant/Graft Rejection: REJECTION PROCESSES Hyperacute: Preformed antidonor antibody (ABO incompatibility). RARE Characterized by Ab: Complement in endothelium Gross: Pale/cyanotic non-functioning organ Histology: Rapid thrombotic vasculitis w/ PMN perivascular infiltrates/ischemic necrosis Acute & Chronic Rejection

Test q: A 41y/o man receives a renal transplant because of end-stage renal disease resulting from acute glomerulonephritis as a juvenile. Just after the graft is anastomosed, the kidney becomes cyanotic, mottled, and flaccid. A few drops of bloody urine are excreted. A renal biopsy is taken, and there is neutrophil accumulation within the arterioles, glomeruli, and peritubular capillaries. Which of the following is responsible for this condition? Preformed antidonor antibodies.

In direct pathway, recipient CD8/CD4 cells are stimulated by APCs within the graft so the donors graft APCs within that organ are the ones presenting Ag to the recipient T-cells. Indirect recipients APCs. The difference is that the only pathway activated is CD4.

Acute Humoral Rejection: Type II Ab-mediated (Acute rejection vasculitis) Can range from Necrotizing vasculitis (more acute-Ab mediated activation of phagocytes) to less acute: Intimal thickening with inflammatory cells, foamy macrophages and smooth muscle proliferation. Need B-cell depleting therapy: (e.g. Rituximab (Rituxan), humanized monoclonal anti-CD20, a B lymphocyte-specific antigen).

Acute Cellular Rejection: Acute tubulointerstitial mononuclear infiltrate. Type IV Hypersensitivity response

Chronic Rejection: Obliterative intimal fibrosis Graft ateriosclerosis

Test q: Graft rejection is characterized by which of the following? Acute graft rejection is generally mediated by both type II antibody-mediated and type IV cell-mediated hypersensitivity reactions.

Graft arteriosclerosis. The vascular lumen is replaced by an accumulation of smooth muscle cells and connective tissue (fibrosis) in the vessel intima. Chronic repetitive repair process.
Test q: The classic histopathologic changes observed in the chronic rejection of a transplanted kidney (allograft) are: interstitial fibrosis and tubular atrophy. Test q: A 45y/o man w/chronic renal failure received a kidney transplant from his brother 36mo ago. For the next 30 months, he had only minor episodes of rejection that were controlled with immunosuppressive therapy. However, in the past 6mo, he has had increasing serum creatinine and urea nitrogen levels. Microscopic exam of a urinalysis specimen shows no WBCs. CT scan of the pelvis shows that the allograft is reduced in size. Which of the following immunologic processes most likely account for these findings? Vascular intimal and interstitial fibrosis. REPEATED x3 (Once, had two changes. It included On physical exam, he is afebrile and the answer was only vascular intimal fibrosis.) Test q: Which of the following is true regarding hyperacute graft rejection? Hyperacute graft rejection has been markedly curtailed by treating the donated tissue w/anti-thymocyte antibodies that remove the donor T-cells that recognize the recipients tissue as foreign, and thus mount an attack on the hosts tissue. Same question another year, different answer: The incidence of hyperacute rejection has been dramatically reduced by performing cross-matches to determine if the recipients serum contains preformed immunoglobulin against donor HLA or minor histocompatibility antigens. This prevents the rapid type II HS reaction that results in thrombotic necrotizing vasculitis in the donor tissue. Test q: Graft vs. host response is most likely to occur in patients receiving which type of transplant? Bone marrow (Other choices were kidney, heart, liver, and lung)

Chronic Rejection: Interstitial fibrosis and tubular atrophy

Graft vs. Host Disease Bone Marrow Transplants: Transplanted T-cells recognize recipient as foreign and mount rejection response. Treatment with High dose steroids and anti-thymocyte globulins. High mortality rate: sepsis T-cell Depletion (anti-T cell antibodies) Besides the icterus (yellow color, jaundice) in this skin there is a fine scaling rash in this patient following bone marrow transplantation with a 5 out of 6 antigen match. This is an example of graft versus host disease in which donor lymphocytes attack host tissues. Apoptosis and lymphocytic infiltrates in epidermal/dermal junction

GVHD Cholestasis (brown bile) & lymphocytic infiltrate

Conference: Lab Testing 1

09/03/10

Electrolyte Panel (reference range): Sodium: the major extracellular cation (135-150 mmol/L) Decreased sodium: fluid overload, SIADH, Addisons disease Increased sodium: cranial injuries, severe dehydration Potassium: the major intracellular cation (3.5 to 5.5 mmol/L) Decreased potassium: Diuretics, fluid overload Increased potassium: Diabetic acidosis, burns, renal failure Both increased and decreased potassium are associated with EKG changes Chloride: the major extracellular anion (92 to 102 mmol/L) Chloride changes typically accompany sodium changes, electroneutrality Bicarbonate: 21 -29 mmol/L The bicarbonate buffer system keeps the blood pH=7.4 Test q: All of the following lab tests would typically be ordered in the evaluation of Decreased bicarbonate: sepsis, metabolic acidosis syndrome of inappropriate ADH EXCEPT: Increased bicarbonate: COPD, metabolic alkalosis A. Plasma sodium Anion gap: Na [Cl + HCO2] >15 An unmeasured anion is present Renal failure Multiple myeloma CHF Metabolic toxins: methanol, ethylene glycol Limited Metabolic Profile: The four electrolyte results plus: Glucose (fasting <110 mg/dL) Increased : diabetes mellitus, Cushings Decreased: newborns, insulin overdose Urea (blood urea nitrogen, BUN) (<20 mg/dL) Creatinine (<1.6 mg/dL) Both are elevated in renal failure Only BUN increased in GI bleeding, CHF Calcium (8.6 -10.5 mg/dL) Decreased: post parathyroidectomy surgery Increased: parathyroid adenoma, malignancy, multiple myeloma, sarcoidosis Comprehensive Metabolic Profile: The eight analytes in the limited metabolic profile plus:
B. C. D. E. Plasma potassium Plasma osmolarity Urine sodium Urine osmolarity

Test q: You think that your patient has a paraneoplastic syndrome due to small cell lung cancer. Which of the following groups of tests would be most useful in the evaluation of a patient for suspected syndrome or inappropriate antidiuretic hormone (SIADH): Serum and urine sodium and osmolality. Test q: Ingestion of which of the following may result in a profound metabolic acidosis? Methanol. Test q: Which of the following casts may be found in normal urine? Hyaline cast. (Other choices WBC cast, RBC cast, and waxy cast) Didnt know where to put this q...maybe will be covered in Lab Testing 2? Test q: Lab results for your patient are: Total bilirubin: 8.4 <1.0 Direct bilirubin: 5.0 <0.1 Indirect bilirubin: 3.4 <1.0 Alkaline phosphatase: 473 <115 Alanine aminotransferase: 50 <35 The most likely cause of jaundice is: A. Hemolytic jaundice B. Hepatic jaundice C. Obstructive jaundice D. Hemochromatosis E. Iron deficiency

Albumin (>3.5 g/dL) Decreased: Protein loss, hepatic failure Total protein (6.4 8.3 g/dL) Decreased: protein loss Increased: multiple myeloma ^ REPEATED TWICE Total bilirubin (<1.0 mg/dL) Bilirubin is a end product of hemoglobin metabolism Alkaline phosphatase (<125 U/L) Elevated in bone growth, Padgets bone disease; biliary tract obstruction Asparate aminotransferase (AST) and Alanine aminotransferase (ALT) (<35U/L) Increased in hepatic diseases Lipid Profile Total cholesterol (<200 mg/dL) HDL cholesterol (>40 mg/dL, >60 desirable) Triglycerides (<150 mg/dL) LDL is calculated using the following formula: (<110 mg/dL) LDL = TC HDLC Triglycerides 5

Complete Blood Count and Differential WBC (5 -10,000) Types of WBCs (differential) - Neutrophils: (55 65%) - Lymphocytes (20 30%) - Monocytes (3 9%) - Eosinophils (1 5%) - Basophils (0 2%) Hemoglobin concentration (Men: 14-18 g/dL; Women: 12-16 g/dL) Hematocrit or packed cell volume: (Men: 39-49%; Women: 35-45 %) Platelet (150,000 450,000)

Test q: All of the following are Medicare approved lab panels EXCEPT: A. Electrolyte panel B. Basic metabolic profile C. Comprehensive metabolic profile D. Complete blood count E. Liver function tests

RBC morphology: presence of erythrocyte abnormalities RBC indices - MCV: mean corpuscular volume (80-100fL) - MCH: mean corpuscular hemoglobin - MCHC: mean corpuscular hemoglobin concentration Both MCH and MCHC are rarely used The CK-MB returns to normal 2-3 days post MI Because CK-MB may be of myocardial or skeletal muscle origin, most physicians prefer the CK Index (<5%) CK-Index = CKMB Total CK Troponins Cardiac Troponin I (TpnI) and Troponin T (TpnT) are proteins associated with the myocardial contractile complex Both TpnI and TpnT are fairly specific for myocardial injuries Post MI the TpnI and TpnT remain elevated for 5-7 days Problem 1 Statistics - New test for gastric CA - 180/200 pts with gastric CA have a (+) test - 40/160 pts w/o gastric CA have a (+) test - In your clinical practice 1/200 pts have gastric CA (this is the prevalence) - Calculate: sensitivity, specificity, PV+ - Sensitivity and specificity are unique to the test, but PPV changes with the population. - How do you improve PV+? What is sensitivity? - The percent of patient with gastric CA that have a positive test; a highly sensitive test misses few pts with disease - What is a true positive? A person who has the disease who tests positive. - What is the formula for sensitivity? - TP= gastric CA and + test - Sensitivity% = TP/TP+FN x100 - FN= gastric CA but a (-) test 180/180+20= 90% How about specificity? How often does it call a (-) a (+)? TN- pts without gastric CA and a (-) test FP- pts without gastric CA and a (+) test

Test q: A new test for prostate cancer is developed. 80% of men w/prostate cancer test positive. 90% of men without the disease test negative. In a population of 1000 men, 30% have the disease (300 men). Prevalence = 30%. What is the sensitivity of this test? 80%. What is predictive value of a positive test? 77%. Test q: You have developed a new serum test for gastric carcinoma. You run this test on 200 patients w/histologically confirmed gastric cancer and get 180 positive results. You run this same test on a group of 160 patients that have either non-gastric malignancies and benign diseases of the GI tract and get 40 positive results. You are very proud of this test and run it on all your patients. Gastric carcinoma has a prevalence of 1/200 patients in your practice setting. What would be the sensitivity of this new test? 90%. Test q: The prevalence of a disease in your practice is 33% and you find this distribution of results: TP = 90 TN = 100 FP = 100 FN = 10 The sensitivity of the test is: 90%. The specificity is: 50%. Test q: The percent of positive results that are true positives is known as the: predictive value. Test q: In a setting where the prevalence of a disease is 50% and a test has a sensitivity of 100% and a specificity of 50%, what would the predictive value of a positive result be? 67%.

Specificity - TN/TN+FP - TN= 120 - FP= 40 Specificity% = 120/(120+40) x100 Specificity= 75%

Predictive Value o What % of (+) tests are really positive o What % of (-) tests are really negative o PV(+)% = TP/TP+FP x100 o PV(-)% = TN/TN+FN x100 o Prevalence and Incidence o Prevalence- # of people with disease currently o Cumulative (total) o Diabetes has high prevalence and low incidence o Incidence- annual # of people who get disease o Annual (new cases) o Common cold has high incidence and low prevalence Prevalence: o # pts in the study with the disease/total population studied o TP+FN/TP+FP+TN+FN o In this population it is 1/200 o If you see 10,000 pts and run the test, 9,950 do not have gastric CA and 50 have gastric CA TP= 90% x 50= 45 FN= 10% x 50= 5 TN= 75% x 9,950= 7,462 FP= 25% x 9,950= 2,488 Prevalence and Tests A higher prevalence increases the PV+ A lower prevalence decreases the PV+ Case 1 68-y.o.male with wt loss, anorexia, nausea and constipation. Mucous membranes and sclera are icteric. A 5 cm-mass is palpated in the RUQ. Bili (tot)= 7.1 mg/dL (<1.0) Bili(d)= 3.4 mg/dL (<0.2) AST= 102 U/L (<40U/L) ALT= 88 U/L (<40 U/L) AlkPhos= 506 U/L (115 U/L) gammaGT= 258 U/L (35 U/L) d/tot Bili= >4 Transaminases are slightly elevated Alk phos and gamma GT are markedly elevated, suggesting obstruction Pt had CA of the head of the pancreas PV+% = TP/TP+FP x100 = 45/45+2,488 x 100 = 1.8% If the prevalence is 10% what is the PPV+? 1000 of 10,000 have the disease 1000/1000+2488 x 100 About 28%

Case 2 Test q: 6 y.o. male with fever, anorexia, vomiting and recent onset of abdominal pain and lassitude. Sclera were icteric and the spleen was palpable. Bili (tot)= 6.8 mg/dL Hemolysis suspected- d/tot bili< .2 Bili (d)= 0.6 mg/dL Plasma HB is elevated and haptoglobin is decreased, Plasma-Free Hb= 26 mg/dL (<10 mg/dL) both c/w hemolysis Haptoglobin= 0 mg/dL (35-200 mg/dL) Pt had hereditary eliptocytosis o o o The blue section above was a test q, the answer was: hemolytic anemia. Why was the spleen palpable? The spleen is large because when the eliptocytes get destroyed, they get destroyed in the spleen. How would a case of hepatitis present? d/totBili? Most of the bili would be direct because the problem is in the liver. Transaminases? Very high Alk Phos? Minimally elevated Plasma free-Hb and Haptoglobin? Normal

Case 3 53-y.o. male presents to the ER (ED) with chest pain for 16 hours. EKG c/w MI Myo 358 (<110ng/mL) CK-MB 12.9 (<5 ng/mL)

TnI 32.8 (<0.5ng/mL) Case 3 is an acute MI Myoglobin positive at 2 hrs post infarct; it offers the least specificity TnI positive at 4 hrs (3-6) post infarct CK-MB positive at 6 hrs (3-6) post infarct Would this patient be a candidate for clot busters or angioplasty? Probably yes, but at this point, 16hr after MI onset, its debatable. Most places will go ahead and do it because youre not exactly sure when the MI started. But youd like to get it done within 4 hours, and definitely by 12 hr. Case 4 75-y.o. male presented to the ER (ED) after MVA with chest pain. EKG is nondiagnostic. MI vs contusion 6 hrs: Myo= >1000; CK-MB= 2.1; TnI= 0.3 16 hrs: Myo= >1000; CK-MB= 0.9; TnI= 0.3 Myoglobin elevation due to skeletal muscle injury