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1 Interpretation of Laboratory Tests: A Case-Oriented Review of Clinical Laboratory Diagnosis Roger L. Bertholf, Ph.D. Associate Professor of Pathology.

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Presentation on theme: "1 Interpretation of Laboratory Tests: A Case-Oriented Review of Clinical Laboratory Diagnosis Roger L. Bertholf, Ph.D. Associate Professor of Pathology."— Presentation transcript:

1 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 2 Case 1: Oliguria and hematuria

3 R. BertholfAmerican Society of Clinical Pathologists3 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.

4 R. BertholfAmerican Society of Clinical Pathologists4 Questions... What is the differential diagnosis in this case? What laboratory tests might be helpful in establishing the diagnosis?

5 R. BertholfAmerican Society of Clinical Pathologists5 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

6 R. BertholfAmerican Society of Clinical Pathologists6 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)

7 R. BertholfAmerican Society of Clinical Pathologists7 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 10 6 nephrons.

8 R. BertholfAmerican Society of Clinical Pathologists8 Renal anatomy Cortex Medulla Pelvis To the bladder Capsule

9 R. BertholfAmerican Society of Clinical Pathologists9 The Nephron Renal artery Glomerulus Bowman’s capsule Proximal tubule Distal tubule Collecting duct Henle’s Loop Afferent arteriole

10 R. BertholfAmerican Society of Clinical Pathologists10 Glomerular filtration Glomerlular capillary membrane Vascular space Bowman’s space Mean capillary blood pressure = 50 mm Hg BC pressure = 10 mm Hg Onc. pressure = 30 mm Hg Net hydrostatic = 10 mm Hg  2,000 Liters per day (25% of cardiac output)  200 Liters per day GFR  130 mL/min

11 R. BertholfAmerican Society of Clinical Pathologists11 What gets filtered in the glomerulus? Freely filtered –H2O–H2O –Na +, K +, Cl -, HCO 3 -, Ca ++, Mg +, PO 4, etc. –Glucose –Urea –Creatinine –Insulin Some filtered –  2 -microglobulin –RBP –  1 -microglobulin –Albumin None filtered –Immunoglobulins –Ferritin –Cells

12 R. BertholfAmerican Society of Clinical Pathologists12 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.

13 R. BertholfAmerican Society of Clinical Pathologists13 Reabsorption from glomerular filtrate

14 R. BertholfAmerican Society of Clinical Pathologists14 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.

15 R. BertholfAmerican Society of Clinical Pathologists15 The Loop of Henle Proximal tubuleDistal tubule Descending loop Ascending loop Increasing osmolality Renal Cortex Renal Medulla Na + H 2 O Na mOsm/Kg 300 mOsm/Kg

16 R. BertholfAmerican Society of Clinical Pathologists16 Regulation of distal tubule Na + permeability JGA Renin  Na +  BP Angiotensinogen Angiotensin I Angiotensin II Angiotensin III vasoconstriction Aldosterone Adrenal cortex Na +

17 R. BertholfAmerican Society of Clinical Pathologists17 Regulation of H 2 O reabsorption Pituitary ADH (vasopressin) Plasma hyperosmolality H2OH2OH2OH2O Renal Medulla (osmolality  1200 mOsm/Kg)

18 R. BertholfAmerican Society of Clinical Pathologists18 Summary of renal physiology Filtration - Reabsorption + Secretion = Elimination GFR (Filtered but not reabsorbed or secreted) TRPF (Filtered and secreted)

19 R. BertholfAmerican Society of Clinical Pathologists19 Measurement of GFR C u = Concentration in urine V u(24h) = 24-hour urine volume C p = Concentration in plasma = 1000 mL/1440 min

20 R. BertholfAmerican Society of Clinical Pathologists20 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

21 R. BertholfAmerican Society of Clinical Pathologists21 Relationship between creatinine and GFR Plasma creatinine GFR (mL/min)

22 R. BertholfAmerican Society of Clinical Pathologists22 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.

23 R. BertholfAmerican Society of Clinical Pathologists23 Creatinine CreatineCreatinine 1-2% of creatine is hydrolyzed to creatinine each day

24 R. BertholfAmerican Society of Clinical Pathologists24 Jaffe method for creatinine Janovsky Complex max = nm Max Eduard Jaffe ( ), German physiologic chemist

25 R. BertholfAmerican Society of Clinical Pathologists25 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

26 R. BertholfAmerican Society of Clinical Pathologists26 Kinetic Jaffe method Absorbance ( = 520 nm) Time (sec)  Fast-reacting (pyruvate, glucose, ascorbate) Slow-reacting (protein) tt AA creatinine (and  -keto acids)

27 R. BertholfAmerican Society of Clinical Pathologists27 Enzymatic creatinine methods Creatininase –creatinine  creatine  CK  ADP  PK  LD Creatinase –creatinine  creatine  sarcosine  sarcosine oxidase  peroxide  peroxidase reaction Creatinine deaminase (iminohydrolase) –most common

28 R. BertholfAmerican Society of Clinical Pathologists28 Creatinine deaminase method Creatinine iminohydrolase + H 2 O N-Methylhydantoin ATP ADP NMH amidohydrolase N-Carbamoylsarcosine H2OH2O Peroxidase Oxygen receptorColored product Sarcosine NCS amidohydrolase - NH 3, CO 2 + O 2 Sarcosine oxidase H2OH2OH2O2H2O2 Formaldehyde + glycine

29 R. BertholfAmerican Society of Clinical Pathologists29 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

30 R. BertholfAmerican Society of Clinical Pathologists30 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)

31 R. BertholfAmerican Society of Clinical Pathologists31 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.

32 R. BertholfAmerican Society of Clinical Pathologists32 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

33 R. BertholfAmerican Society of Clinical Pathologists33 Classification of proteinuria: Moderate grams of protein per day Usually associated with glomerular disease Overflow proteinuria from multiple myeloma Toxic nephropathies

34 R. BertholfAmerican Society of Clinical Pathologists34 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)

35 R. BertholfAmerican Society of Clinical Pathologists35 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

36 R. BertholfAmerican Society of Clinical Pathologists36 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

37 R. BertholfAmerican Society of Clinical Pathologists37 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 H 2 O 2 + chromogen*Oxidized chromogen + H 2 O Heme Peroxidase *tetramethylbenzidine; oxidized form is green

38 R. BertholfAmerican Society of Clinical Pathologists38 Microscopic examination of urine sediment

39 R. BertholfAmerican Society of Clinical Pathologists39 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

40 R. BertholfAmerican Society of Clinical Pathologists40 Bright’s Disease (acute glomerulonephritis) Characterized by oliguria, proteinuria, and hematuria Most common cause is immune-related Richard Bright ( )

41 R. BertholfAmerican Society of Clinical Pathologists41 Primary Glomerulonephritis Proliferative GN –Acute Post-infectious GN –Idiopathic or Crescentic GN –  -GBM disease –Membranoproliferative GN Focal GN –IgA nephropathy

42 R. BertholfAmerican Society of Clinical Pathologists42 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

43 R. BertholfAmerican Society of Clinical Pathologists43 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)

44 44 Case 3: Chest Pain

45 R. BertholfAmerican Society of Clinical Pathologists45 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.

46 R. BertholfAmerican Society of Clinical Pathologists46 Questions What is the most important consideration in the triage of this patient? What tests should be ordered?

47 R. BertholfAmerican Society of Clinical Pathologists47 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%)

48 R. BertholfAmerican Society of Clinical Pathologists48 The Heart Aorta Superior vena cava RA LA RV LV Pulmonary arteries

49 R. BertholfAmerican Society of Clinical Pathologists49 The Heart (posterior view) Aorta Superior vena cava Inferior vena cava Pulmonary veins Pulmonary arteries

50 R. BertholfAmerican Society of Clinical Pathologists50 Cardiac physiology

51 R. BertholfAmerican Society of Clinical Pathologists51 Cardiac conduction system Sinoatrial (SA) node Atrioventral (AV) node His bundle Right bundle branch Left bundle branch

52 R. BertholfAmerican Society of Clinical Pathologists52 Normal Electrocardiogram P Q R S T U

53 R. BertholfAmerican Society of Clinical Pathologists53 Myocardial infarction Right coronary artery Left coronary artery Anterior left ventricle

54 R. BertholfAmerican Society of Clinical Pathologists54 ECG changes in myocardial infarction S P R T QQ S-T elevation

55 R. BertholfAmerican Society of Clinical Pathologists55 Diagnostic value of ECG ECG changes depend on the location and severity of myocardial necrosis Virtually 100% of patients with characteristic Q- wave and S-T segment changes are diagnosed with myocardial infarction (100% specificity) However, as many as 50% of myocardial infarctions do not produce characteristic ECG changes (sensitivity  50%) ECG may be insensitive for detecting prognostically significant ischemia

56 R. BertholfAmerican Society of Clinical Pathologists56 History of cardiac markers 1975: Galen describes the use of CK, LD, and isoenzymes in the diagnosis of myocardial infarction. 1980: Automated methods for CK-MB (activity) and LD-1 become available. 1985: CK-MB isoforms are introduced. 1989: Heterogeneous immunoassays for CK-MB (mass) become available. 1991: Troponin T immunoassay is introduced. 1992: Troponin I immunoassay is introduced.

57 R. BertholfAmerican Society of Clinical Pathologists57 Enzyme markers Aspartate transaminase (AST; SGOT) 2-Hydroxybutyrate dehydrogenase Lactate dehydrogenase –Five isoenzymes, composed of combinations of H (heart) and M (muscle) subunits Creatine kinase –Three isoenzymes, composed of combinations of M (muscle) and B (brain) subunits

58 R. BertholfAmerican Society of Clinical Pathologists58 Lactate dehydrogenase (LD) Pyruvate LD activity is measured by monitoring absorbance at = 340 nm (NADH) Methods can be P  L or L  P –But...reference range is different Total LD activity has poor specificity Lactate LD NAD + NADH

59 R. BertholfAmerican Society of Clinical Pathologists59 Tissue specificity of LD isoenzymes

60 R. BertholfAmerican Society of Clinical Pathologists60 LD isoenzyme electrophoresis (normal) LD-1 LD-2 LD-3 LD-4 LD-5 LD-2 > LD-1 > LD-3 > LD-4 > LD-5 Cathode (-)Anode (+)

61 R. BertholfAmerican Society of Clinical Pathologists61 LD isoenzyme electrophoresis (abnormal) LD-1 LD-2 LD-3 LD-4 LD-5 LD-1 > LD-2 Cathode (-)Anode (+)

62 R. BertholfAmerican Society of Clinical Pathologists62 Direct measurement of LD-1 Electrophoresis is time-consuming and only semi- quantitative Antibodies to the M subunit can be used to precipitate LD-2, 3, 5, and 5, leaving only LD-1 –Method can be automated –Normal LD-1/LD total ratio is less than 40%

63 R. BertholfAmerican Society of Clinical Pathologists63 Sensitivity and specificity of LD-1 Sensitivity and specificity of the LD 1:2 “flip”, or LD-1 > 40% of total, are 90+% within 24 hours of MI, but... –May be normal for 12 or more hours after symptoms appear (peak in hours) –May not detect minor infarctions Elevations persist for up to 10 days Even slight hemolysis can cause non-diagnostic elevations in LD-1

64 R. BertholfAmerican Society of Clinical Pathologists64 Creatine Kinase (CK) Phosphocreatine ADP HK GlucoseGlucose-6-phosphate NADPH =340 nm NADP + GPD 6-Phosphogluconate Oliver and Rosalki method (1967) Creatine CK ADPATP Mg ++

65 R. BertholfAmerican Society of Clinical Pathologists65 Tissue specificities of CK isoenzymes

66 R. BertholfAmerican Society of Clinical Pathologists66 Measurement of CK isoenzymes Electrophoresis (not used anymore) Immunoinhibition/precipitation –Antibody to M subunit –Multiply results by 2 –Interference from CK-1 (BB) Most modern methods use two-site (“sandwich”) heterogeneous immunoassay –Measures CK-MB mass, rather than activity –Gives rise to a pseudo-percentage, often called the “CK-MB index”

67 R. BertholfAmerican Society of Clinical Pathologists67 Sensitivity/specificity of CK-MB Sensitivity and specificity of CK-MB for myocardial infarction are >90% within 7-18 hours; peak concentrations occur within 24 hours CK is a relatively small enzyme (MW = 86K), so it is filtered and cleared by the kidneys; levels return to normal after 2-3 days Sensitivity is poor when total CK is very high, and specificity is poor when total CK is low Presence of macro-CK results in false elevations

68 R. BertholfAmerican Society of Clinical Pathologists68 CK isoforms C-terminal lysine is removed from the M subunit-- therefore, there are three isoforms of CK-3 (MM) t ½ : CK-MB 1 > CK-MB 2 Ratio of CK-MB 2 to CK-MB 1 exceeds 1.5 within six hours of the onset of symptoms Only method currently available is electrophoresis CK-MB 2 (tissue)CK-MB 1 (circulating) C-terminal lysine Plasma carboxypeptidase

69 R. BertholfAmerican Society of Clinical Pathologists69 Myoglobin O 2 -binding cytosolic protein found in all muscle tissue (functional and structural analog of hemoglobin) Low molecular weight (17,800 daltons) Elevations detected within 1-4 hours after symptoms; returns to normal after 12 hours Nonspecific but sensitive marker--primarily used for negative predictive value Usually measured by sandwich, nephelometric, turbidimetric, or fluorescence immunoassay

70 R. BertholfAmerican Society of Clinical Pathologists70 Temporal changes in myoglobin and CK-MB

71 R. BertholfAmerican Society of Clinical Pathologists71 Troponin Thick Filament Myosin TropomyosinActin TnC TnT (42 Kd) TnI (23 Kd)

72 R. BertholfAmerican Society of Clinical Pathologists72 Tissue specificity of Troponin subunits Troponin C is the same in all muscle tissue Troponins I and T have cardiac-specific forms, cTnI and cTnT Circulating concentrations of cTnI and cTnT are very low cTnI and cTnT remain elevated for several days Hence, Troponins would seem to have the specificity of CK-MB (or better), and the long-term sensitivity of LD-1

73 R. BertholfAmerican Society of Clinical Pathologists73 Is cTnI more sensitive than CK/CK-MB? 79 y/o female with Hx of HTN, CHF, CRI, Type II diabetes

74 R. BertholfAmerican Society of Clinical Pathologists74 Measurement of cTnI and cTnT All methods are immunochemical (ELISA, MEIA, CIA, ECIA) Roche Diagnostics (formerly BMC) is the sole manufacturer of cTnT assays –First generation assay may have had some cross- reactivity with skeletal muscle TnT –Second generation assay is cTnT-specific –Also available in qualitative POC method Many diagnostics companies have cTnI methods

75 R. BertholfAmerican Society of Clinical Pathologists75 W.H.O. has a Myocardial Infarction? A clinical history of ischemic-type chest discomfort Changes on serially obtained ECG tracings A rise and fall in serum cardiac markers A patient presenting with any two of the following: Source JACC 28;1996:

76 R. BertholfAmerican Society of Clinical Pathologists76 Sensitivity/Specificity of WHO Criteria

77 R. BertholfAmerican Society of Clinical Pathologists77 What Cardiac Markers do Labs Offer?


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