Interpretation of Laboratory Tests: A Case-Oriented Review of Clinical Laboratory Diagnosis

1. Interpretation of Laboratory Tests:A Case-Oriented Review of Clinical Laboratory Diagnosis Roger L. Bertholf, Ph.D. Associate Professor of Pathology University of Florida Health Science Center/Jacksonville

2. Case 1: Oliguria and hematuria

3. Case 1: Oliguria and hematuria • A 7-year-old boy was brought to the pediatrician because of vomiting and malaise. On physical examination he was slightly flushed, and had some noticeable swelling of his hands and feet. The patient was uncomfortable, and complained of pain “in his tummy”. He had a slight fever. Heart was normal and lungs were clear. His past medical history did not include any chronic diseases. The mother noted that he had a severe sore throat “about two weeks ago”, but that it had cleared up on its own. The child was not taking any medications. There were no masses in the abdomen, and lymphadenopathy was not present. The child had some difficulty producing a urine specimen, but finally was able to produce a small amount of urine, which was dipstick-positive for blood and protein. American Society of Clinical Pathologists

4. Questions. . . • What is the differential diagnosis in this case? • What laboratory tests might be helpful in establishing the diagnosis? American Society of Clinical Pathologists

5. What do the kidneys do? • Regulate body fluid osmolality and volume • Regulate electrolyte balance • Regulate acid-base balance • Excrete metabolic products and foreign substances • Produce and excrete hormones American Society of Clinical Pathologists

6. The kidneys as regulatory organs “The kidney presents in the highest degree the phenomenon of sensibility, the power of reacting to various stimuli in a direction which is appropriate for the survival of the organism; a power of adaptation which almost gives one the idea that its component parts must be endowed with intelligence.” E. Starling (1909) American Society of Clinical Pathologists

7. Review of Renal Anatomy and Physiology • The kidneys are a pair of fist-sized organs that are located on either side of the spinal column just behind the lower abdomen (L1-3). • A kidney consists of an outer layer (renal cortex) and an inner region (renal medulla). • The functional unit of the kidney is the nephron; each kidney has approximately 106 nephrons. American Society of Clinical Pathologists

8. Renal anatomy Cortex Pelvis Capsule Medulla To the bladder American Society of Clinical Pathologists

9. Proximal tubule Afferent arteriole Distal tubule Glomerulus Bowman’s capsule Collecting duct Renal artery Henle’s Loop The Nephron American Society of Clinical Pathologists

10. Mean capillary blood pressure = 50 mm Hg BC pressure = 10 mm Hg Onc. pressure = 30 mm Hg Net hydrostatic = 10 mm Hg Glomerular filtration Glomerlular capillary membrane Vascular space Bowman’s space  2,000 Liters per day (25% of cardiac output)  200 Liters per day GFR  130 mL/min American Society of Clinical Pathologists

11. What gets filtered in the glomerulus? • Some filtered • 2-microglobulin • RBP • 1-microglobulin • Albumin • None filtered • Immunoglobulins • Ferritin • Cells • Freely filtered • H2O • Na+, K+, Cl-, HCO3-, Ca++, Mg+, PO4, etc. • Glucose • Urea • Creatinine • Insulin American Society of Clinical Pathologists

12. Then what happens? • If 200 liters of filtrate enter the nephrons each day, but only 1-2 liters of urine result, then obviously most of the filtrate (99+ %) is reabsorbed. • Reabsorption can be active or passive, and occurs in virtually all segments of the nephron. American Society of Clinical Pathologists

13. Reabsorption from glomerular filtrate American Society of Clinical Pathologists

14. How does water get reabsorbed? • Reabsorption of water is passive, in response to osmotic gradients and renal tubular permeability. • The osmotic gradient is generated primarily by active sodium transport • The permeability of renal tubules is under the control of the renin-angiotensin-aldosterone system. • The driving force for water reabsorption, the osmotic gradient, is generated by the Loop of Henle. American Society of Clinical Pathologists

15. Na+ 300 mOsm/Kg H2O Na+ Increasing osmolality Na+ Na+ 1200 mOsm/Kg The Loop of Henle Proximal tubule Distal tubule Renal Cortex Descending loop Ascending loop Renal Medulla American Society of Clinical Pathologists

16. Angiotensinogen  Na+ Renin  BP Angiotensin I Angiotensin II vasoconstriction Angiotensin III Adrenal cortex Aldosterone Na+ Na+ Regulation of distal tubule Na+ permeability JGA American Society of Clinical Pathologists

17. Plasma hyperosmolality H2O H2O ADH (vasopressin) Regulation of H2O reabsorption Pituitary Renal Medulla (osmolality 1200 mOsm/Kg) American Society of Clinical Pathologists

18. Summary of renal physiology TRPF (Filtered and secreted) Filtration - Reabsorption + Secretion = Elimination GFR (Filtered but not reabsorbed or secreted) American Society of Clinical Pathologists

19. Measurement of GFR Cu = Concentration in urine Vu(24h) = 24-hour urine volume Cp = Concentration in plasma 0.694 = 1000 mL/1440 min American Society of Clinical Pathologists

20. Compounds used to measure GFR • Should not be metabolized, or alter GFR • Should be freely filtered in the glomeruli, but neither reabsorbed nor secreted • Inulin (a polysaccharide) is ideal • Creatinine is most popular • There is some exchange of creatinine in the tubules • As a result, creatinine clearance overestimates GFR by about 10% (But. . .) • Urea can be used, but about 40% is (passively) reabsorbed American Society of Clinical Pathologists

21. Relationship between creatinine and GFR 6 5 4 3 Plasma creatinine 2 1 0 0 20 40 60 80 100 120 140 GFR (mL/min) American Society of Clinical Pathologists

22. Measurement of TRPF • Para-aminohippurate (PAH) is freely filtered in the glomeruli and actively secreted in the tubules. • PAH clearance gives an estimate of the total amount of plasma from which a constituent can be removed. American Society of Clinical Pathologists

23. Creatinine Creatine Creatinine 1-2% of creatine is hydrolyzed to creatinine each day American Society of Clinical Pathologists

24. Jaffe method for creatinine Janovsky Complex max = 490-500 nm Max Eduard Jaffe (1841-1911), German physiologic chemist American Society of Clinical Pathologists

25. Modifications of the Jaffe method • Fuller’s Earth (aluminum silicate, Lloyd’s reagent) • adsorbs creatinine to eliminate protein interference • Acid blanking • after color development; dissociates Janovsky complex • Pre-oxidation • addition of ferricyanide oxidizes bilirubin • Kinetic methods American Society of Clinical Pathologists

26. A t 20 80 Kinetic Jaffe method Slow-reacting (protein) Fast-reacting (pyruvate, glucose, ascorbate) Absorbance ( = 520 nm) creatinine (and -keto acids) 0 Time (sec)  American Society of Clinical Pathologists

27. Enzymatic creatinine methods • Creatininase • creatininecreatineCKADPPKLD • Creatinase • creatininecreatinesarcosinesarcosine oxidaseperoxideperoxidase reaction • Creatinine deaminase (iminohydrolase) • most common American Society of Clinical Pathologists

28. Creatinine iminohydrolase ATP N-Methylhydantoin + H2O NMH amidohydrolase NCS amidohydrolase Sarcosine N-Carbamoylsarcosine ADP - NH3, CO2 Sarcosine oxidase Formaldehyde + glycine + O2 H2O H2O2 H2O Oxygen receptor Colored product Peroxidase Creatinine deaminase method Creatinine American Society of Clinical Pathologists

29. Measurement of urine protein • Specimen • Timed 24-h is best • Urine protein/creatinine ratio can be used with random specimen • Normal protein excretion is <150 mg/24h • 50-60% albumin • Smaller proteins (1-, 2-microglobulins) • Tamm-Horsfall (uromucoid, secreted by tubules) • IgA, tubular epithelial enzymes, and other non-filtered components American Society of Clinical Pathologists

30. Dipstick method for urine protein • Method is based on protein association with pH indicator • Test pad contains dye tetrabromphenol blue at pH=3 • If protein binds to the pH indicator, H+ is displaced and the color changes from yellow to green (or blue) • Most sensitive to albumin (poor method for detecting tubular proteinuria) American Society of Clinical Pathologists

31. What causes excess urinary protein? • Overload proteinuria • Bence-Jones (multiple myeloma) • Myoglobin (crush injury, rhabdomyolysis) • Hemoglobin • Tubular proteinuria • Mostly low MW proteins (not albumin) • Fanconi’s, Wilson’s, pyelonephritis, cystinosis • Glomerular proteinuria • Mostly albumin at first, but larger proteins appear as glomerular membrane selectivity is lost. American Society of Clinical Pathologists

32. Classification of proteinuria: Minimal • <1 gram of protein per day • Chronic pyelonephritis • Mild glomerular disease • Nephrosclerosis (usually due to hypertension) • Chronic interstitial nephritis (usually analgesic-related) • Renal tubular disease American Society of Clinical Pathologists

33. Classification of proteinuria: Moderate • 1.0 - 4.0 grams of protein per day • Usually associated with glomerular disease • Overflow proteinuria from multiple myeloma • Toxic nephropathies American Society of Clinical Pathologists

34. Classification of proteinuria: Severe • >4 grams of protein per day • Nephrotic syndrome (GBM permeability) • Sx: edema, proteinuria, hypoalbuminemia, hyperlipidemia • In adults, usually 2 to systemic disease (SLE, diabetes) • In children, cause is usually primary renal disease • Minimal Change Disease (Lipoid Nephrosis) • Most common cause of NS in children • Relatively benign (cause unknown, not autoimmune) American Society of Clinical Pathologists

35. Proteinuria due to glomerulonephritis • Acute, rapidly progressive, or chronic GN can result in severe proteinuria • Often the result of immune reaction (Circulating Immune-Complex Nephritis) • Antigen can be endogenous (SLE) or exogeneous • Glomerular damage is mostly complement-mediated • If antigen is continuously presented, GN can become chronic American Society of Clinical Pathologists

36. How do red blood cells get in urine? • Hematuria can result from bleeding anywhere in the kidneys or urinary tract • Disease, trauma, toxicity • Hemoglobinuria can result from intravascular hemolysis • Disease, trauma, toxicity American Society of Clinical Pathologists

37. Heme H2O2 + chromogen* Oxidized chromogen + H2O Peroxidase Dipstick method for hemoglobin • Ascorbic acid inhibits the reaction, causing a false negative test • Depends on RBC lysis (may not occur in urine with high specific gravity) • Detection limit approximately 10 RBC/L *tetramethylbenzidine; oxidized form is green American Society of Clinical Pathologists

38. Microscopic examination of urine sediment American Society of Clinical Pathologists

39. Significance of RBC casts in urine • Indicative of blood crossing the GBM • Casts form in the distal tubules • Stasis produces brown, granular casts • RBC casts almost always reflect glomerular disease American Society of Clinical Pathologists

40. Bright’s Disease (acute glomerulonephritis) • Characterized by oliguria, proteinuria, and hematuria • Most common cause is immune-related Richard Bright (1789-1858) American Society of Clinical Pathologists

41. Primary Glomerulonephritis • Proliferative GN • Acute Post-infectious GN • Idiopathic or Crescentic GN • -GBM disease • Membranoproliferative GN • Focal GN • IgA nephropathy American Society of Clinical Pathologists

42. Primary Glomerulonephritis, cont. • Idiopathic membranous GN • Histological diagnosis, probably immune complex • Chronic GN • Clinical Dx; many potential causes • Lipoid Nephrosis • Histological findings normal; “Nephrosis” • Focal Glomerular Sclerosis • Probably immune (IgM) related American Society of Clinical Pathologists

43. Secondary Glomerulonephritis • Systemic Lupus Erythematosus • Renal failure accounts for 50% of SLE deaths • Polyarteritis (inflammatory vasculitis) • Wegener’s Granulomatosis (lung and URT) • Henoch-Schönlein Syndrome • Lacks edema assoc. with post-streptococcal GN • Goodpasture’s Syndrome (pulmonary hemorrhage) • Hemolytic-Uremic Syndrome • Progressive Systemic Sclerosis (blood vessels) American Society of Clinical Pathologists

44. Case 3: Chest Pain

45. Case 3: Chest Pain A 63 year old male was brought to the emergency department after complaining of severe chest pain that had lasted for two hours. He had been mowing his lawn when the pain developed, and he became concerned when the pain did not subside after he stopped the activity. He had no previous history of heart disease. On presentation he was moderately overweight, dia- phoretic, and in obvious discomfort. He described his chest pain as “beginning in the center of my chest, then my arms, neck, and jaw began to ache too.” Diagnostic procedures were performed. American Society of Clinical Pathologists

46. Questions • What is the most important consideration in the triage of this patient? • What tests should be ordered? American Society of Clinical Pathologists

47. Chest pain • One of the most common reasons for seeking medical attention • Characteristics of cardiogenic chest pain (angina) • induced by exercise • described as “pressure” • radiates to extremities • MI not relieved by rest or vasodilatory drugs (NG) • Only 25% of patients presenting with chest pain as the primary complaint will ultimately be diagnosed as MI (specificity=25%; sensitivity=80%) American Society of Clinical Pathologists

48. Aorta Pulmonary arteries Superior vena cava LA RA LV RV The Heart American Society of Clinical Pathologists

49. Aorta Superior vena cava Pulmonary arteries Pulmonary veins Inferior vena cava The Heart (posterior view) American Society of Clinical Pathologists

50. Cardiac physiology American Society of Clinical Pathologists