INTRODUCTION

1. Basis of Course and Objectives
2. Requesting Tests, the Rationale for Selection

I. SPECIMEN COLLECTIONS AND NON-DISEASE VARIABLES

1. Sample Identification
2. Whole Blood
3. Plasma and Serum
4. Urine
5. Other Physiological Samples
6. Conditions Modifying Sample Results
        (Hospital Laboratory Tour Video)

II. ELECTROLYTES, INORGANICS AND BLOOD GASES

1. Oxygen and Carbon Dioxide
2. Hydrogen Ion
3. Bicarbonate Ion
4. Sodium and Potassium Ions
5. Chloride Ion
6. Calcium and Phosphate Ions
7. Magnesium Ion
8. Zinc and Iron
        (Laboratory Videos on Cl^- and Li⁺ Determinations)

III. AMINO ACIDS, PROTEINS AND OTHER NITROGENOUS SUBSTANCES

IV. ENZYMES

1. Introduction

2. Aminotransferases (AST, ALT)

a. Clinical Significance b. Methods c. Reference Ranges

3. Creatine Kinase (CK)

a. Properties and Clinical Significance

b. Methods for CK Level Determination

c. Reference Ranges

d. Methodology for CK Isozyme Separation

e. CK Isozyme Level Ranges

4. Aldolase (ALD)

a. Properties and Clinical Significance

b. Normal Serum Levels

5. Lactic Dehydrogenase (LDH)

a. Properties

b. Myocardial Infarction and Other Clinically Significant Measurements of LDH

c. Methods for LDH d. Reference Ranges

6. Alkaline Phosphatase (ALP)

a. Properties

b. Clinical Significance

c. Methods for ALP d. Reference Ranges

7. Gamma Glutamyl Transpeptidase (GGT)

a. Properties

b. Clinical Significance

c. Methods for GGT

d. Reference Ranges

8. Pancreatic Digestive Enzymes

1) Properties
2) Clinical Significance
3) Methods
4) Reference Ranges

9. Lipase

a. Properties

b. Clinical Significance

c. Methods

d. Reference Range

10. Trypsin and Chymotrypsin

a. Properties

b. Clinical Significance

c. Methods

d. Reference Range

11. Cholinesterase (SChE)

a. Properties

b. Clinical Significance

c. Methods

d. Reference Ranges

12. Acid Phosphatases (ACP)

a. Properties

b. Clinical Significance

c. Specimens

d. Methods

e. Reference Ranges

13. Terminal Deoxynucleotide Transferase (TdT)

a. Clinical Significance

14. Lysosomal Enzyme Deficiencies

a. Tay-Sach's Disease

b. N-Acetyl-ß-hexosaminidase A (ß-NAGS A)

15. Disorders of Erythrocyte Metabolism

a. Glucose-6-phosphate Dehydrogenase (G-6-PD)

1) Specimens and Methods
2) Reference Ranges

b. Erythrocyte Glutathione

1) Clinical Significance and Methods
2) Reference Ranges

(Clinical Case - Acute Myocardial Infarction)

V. PEPTIDE HORMONES AND IMMUNOLOGICAL METHODS

1. Introduction

2. Immunoassays

a. Basis of Immunoassays

b. Monoclonal Antibodies

c. Radioimmunoassay (RIA)

d. Immunoradiometric-assay (IRMA)

e. Enzyme Multiplied Immunoassay Technique (EMIT)

f. Enzyme-Linked Immunosorbent Assay (ELISA)

g. Fluorescence Polarization Immunoassay (FPIA)

h. Substrate-Laballed Fluorescent Immunoassay (SLFIA)

i. Immunodiffusion Techniques

1) Single Immunodiffusion (Radial Immunodiffusion (RID)
2) Double Immunodiffusion

j. Immunoelectrophoresis Techniques

1) Immunoelectrophoresis (IEP)
2) Crossed Immunoelectrophoresis (CRIE)
3) Immunofixation (IF)
4) Western Blotting
5) Electro-immunoassay (EIA)

k. Quantitative Immunological Rate Assays by Turbidimetry or Nephelometry

3. The Peptide Hormones

a. Insulin

1) Clinical Significance
2) Methods
3) Reference Ranges
4) Specimens

b. Peptide Hormones of the Anterior Pituitary

1) Growth Hormone (GH)

a) Properties
b) Clinical Significance
c) Methods for GH and Somatomedins

2) Prolactin (PRL)

a) Properties
b) Clinical Significance
c) Methods
d) Reference Ranges

c. Corticotropin (ACTH) and Related Peptides

1) Properties
2) Clinical Significance
3) Specimens
4) Methods
5) Reference Ranges

d. Gonadotropins (LH, FSH, ICSH and hCG)

1) Properties
2) Clinical Significance
3) Specimens
4) Methods
5) Reference Ranges

e. Thyrotropin Releasing Hormone (TRH), T₃, T₄, rT₃, TBG, TSH and hTSI

1) Properties
2) Clinical Significance
3) Specimens
4) Methods
5) Reference Ranges

f. Posterior Pituitary Hormones, Oxytoin and Vasopressin (ADH)

1) Properties
2) Clinical Significance
3) Specimens
4) Methods
5) Reference Ranges

g. Renin and Angiotensin

1) Properties
2) Clinical Significance
3) Specimens
4) Methods
5) Reference Ranges

h. Parathyroid Hormone (PTH) and Calcitonin (CT)

1) Properties
2) Clinical Significance
3) Specimens
4) Methods
5) Reference Ranges

i. Gastrin and Secretin

1) Properties
2) Clinical Significance
3) Specimens
4) Methods
5) Reference Ranges

4. Catecholamine Neurotransmitters and Hormones

a. Clinical Significance

b. Methods

1) Urinary Metanephrine
2) Urinary Vanillyl Mandelic Acid (VMA)
3) Urinary 3-Methoxy-4-hydroxyphenylglycol (MHPG)
4) Urinary Homovanillic Acid (HVA)
5) Urinary Free Catecholamines
6) Catecholamines in Plasma

a) Radioenzymatic Methods

5. Serotonin (5HT) and 5-Hydroxyindoleacetic Acid (5HIAA)

a. Clinical Significance

b. Methods

(Clinical Case - Hypothyroidism) (Laboratory - Theophylline by EMIT)

VI. STEROIDS

1. Introduction

2. The Steroid Hormones - General Methods

a. Specimens for Steroid Hormones

b. Hydrolysis, Extraction and Separation of Steroid Hormones

c. Quantitation

1) Colorimetric
2) Fluorometric
3) Gas Chromatography (GC)
4) High Performance Liquid Chromatography (HPLC)
5) Radioimmunoassay (RIA)
6) Competitive Protein Binding (CPBA) and Radioreceptor Assays (RRA)

3. Cortisol

a. Properties

b. Clinical Significance

c. Physiologic Tests for Differential Diagnosis of Addison's Disease and Cushing's Syndrome Involving Cortisol Measurements

d. Methods for Cortisol in Blood and Urine

e. Reference Ranges for Cortisol in Plasma and Urine

f. Determination of 11-Deoxycortisol in Plasma

g. Reference Ranges for 11-Deoxycortisol in Plasma

4. Aldosterone

a. Clinical Significance

b. Saline and Fludrocortisone Suppression Tests for Primary Aldosteronism

c. Methods for Determination of Aldosterone in Plasma and Urine (RIA)

d. Reference Ranges for Plasma and Urinary Aldosterone by RIA

5. 17-Hydroxyprogesterone

a. Clinical Significance

b. Determination of 17-Hydroxyprogesterone in Plasma

c. Reference Ranges for Plasma 17-Hydroxyprogesterone

6. Dehydroepiandrosterone Sulfate (DHEAS)

a. Clinical Significance

b. Methods for DHEAS in Plasma

c. Reference Ranges for Plasma DHEAS

7. Urinary 17-Hydroxycorticosteroids (17-HOCS), 17-Ketosteroids (17-KS) and 17-Ketogenic Steroids

a. Clinical Significance

b. Determination of 17-HOCS in Urine by Porter-Silber Method

c. Reference Ranges of 17-HOCS in Urine

d. Determination of Total 17-HOCS (Ketogenic Steroids) in Urine

e. Reference Ranges of Total Ketogenic Steroids in Urine

f. Determination of 17-Ketosteroids in Urine

g. Reference Ranges of 17-Ketosteroids in Urine

8. Testosterone

a. Blood Distribution

b. Clinical Significance

c. Determination of Free (Non-Protein Bound) Testosterone in Plasma

d. Reference Ranges of Free Plasma Testosterone

e. Determination of Plasma Testosterone by RIA

f. Reference Ranges of Plasma Testosterone

9. Progesterone

a. Properties

b. Clinical Significance

c. Methods for Progesterone in Plasma (RIA)

d. Reference Ranges for Progesterone in Plasma

10. Pregnanediol

a. Clinical Significance

b. Reference Ranges for pregnanediol in Urine

11. Estrogens

a. Properties

b. Clinical Significance

c. Methods for Determination of Estrogens in Plasma

1) Determination of Estradiol by RIA
2) Reference Ranges for Estradiol in Plasma

d. Methods for Determination of Estrogens in Urine

1) Kober Color Reaction
2) Reference Ranges for Total Urinary Estrogens

e. Estriol

1) Clinical Significance
2) Methods for Estriol

a) Estriol in Plasma
b) Estriol Plasma Levels
c) Estriol in Urine
d) Estriol Urine Levels

f. Estrogens and Breast Cancer

1) Relationship Between Estrogens and Breast Cancer
2) Measurements of Estrogen and Progesterone Receptors in Breast Cancer

12. Vitamin D

a. Properties

b. Clinical Significance of Changes in Vitamin D Metabolites

c. Methods for Vitamin D Metabolites

1) Total 25-(OH)D (D₂ + D₃) in Plasma or Serum by Competitive Protein Binding Assay
2) 5-(OH)D₂, 25-(OH)D₃, Vitamins D₂ and D₃ by HPLC and Ultraviolet Detection
3) Determination of Total 1,25(OH)₂D in Plasma by a Competitive Protein Binding Assay

d. Reference Ranges for Vitamin D and Metabolites in Serum

13. Bile Acids

a. Biochemistry

b. Clinical Significance

c. Methods for Bile Acids in Serum

d. Specimens for Bile Acids in Serum

e. Reference Ranges for Serum Bile Acids by GLC (Total Bile Acids) and RIA (Individual Bile Acids)

VII. LIPIDS AND LIPOPROTEINS

1. Introduction

2. Total Cholesterol

a. Specimens for Plasma Cholesterol

b. Reference Values for Total Cholesterol and LDL-Cholesterol (Table 7-2)

c. Enzymatic Method for Total Plasma Cholesterol (TC)

d. Reference Ranges for Total Plasma Cholesterol (TC)

3. Triglycerides (TG)

a. Determination of Triglycerides (TG) by Enzymatic Methods

b. Reference Ranges for Plasma Triglycerides

c. Reference Values for Triglycerides (mg/dL) (Table 7-3)

d. Plasma Appearance

1) Electrophoretic Migration in Hyperlipoproteinemias (Figure 7-1)

4. High Density Lipoprotein-Cholesterol (HDL-C) and Low Density Lipoprotein-Cholesterol (LDL-C)

a. Method for HDL-C

b. Reference Ranges for HDL-C

1) Reference Values for HDL-C (Table 7-4)

c. Methods for Determining LDL-2

d. Reference Ranges for LDL-C

e. Coronary Heart Disease Risk Based on LDL-C/HDL-C (Table 7-5)

5. Further Tests for Lipoprotein Disorders

a. HDL-Subfractionation

1) Major and Minor Apolipoproteins (Table 7-6)
2) Apolipoprotein A
3) Apolipoprotein B
4) Apolipoprotein C
5) Apolipoprotein E
6) Immunoassay Methods for Apolipoproteins

a) Radial Immunodiffusion (RID)
b) Electroimmunoassay (EIA)
c) Immunonephelometry (INA)
d) Enzyme Linked Immunoassay (ELISA)
e) Fluorescent Immunoassay (FIA)
f) Radioimmunoassay (RIA)

7) Reference Ranges for Plasma Apolipoprotein Levels

b. Drug Induced Effects on Hyperlipoproteinemia

(Clinical Case - Hyperlipoproteinemia)

VIII. GLUCOSE AND HOME TESTING METHODS

1. Glucose Testing for Diabetes

a. Self Diagnostic Tests (Table 8-1)

2. Types of Diabetes

a. Type I b. Type II c. Other Types

3. Normal Blood Clucose Levels

4. Diagnostic Criteria for Diabetes

5. Specimens for Glucose Tests

6. Methods for Glucose

a. Hexokinase Methods

b. Glucose Oxidase Methods

c. Ortho-Toluidine Methods

7. Glycosylated Hemoglobin (HbA₁)

a. Function of HbA₁

b. Methods for Glycosylated Hemoglobins

1) Ion Exchange Microcolumn
2) HPLC
3) Colorimetry
4) Radioimmunoassay
5) Electrophoresis
6) Isoelectric Focussing
7) Affinity Chromatography

8. Home Glucose Monitoring

a. Urine Versus Blood Glucose (Figure 8-1)

b. Urine Glucose Testing

1) Clintest
2) Clinistix
3) Diastic
4) Tes-Tape
5) Chemstrip uG
6) Kyotest uGk

9. Practical Aspects of Urine Glucose Testing

a. When to Test

b. Recordkeeping

c. Common Errors in Urine Testing

10. Urine Testing for Ketone Bodies

a. Physiology

b. Patient Selection

c. Products for Urine Testing

11. Home Blood Glucose Monitoring

a. Rationale

b. Patient Selection

c. Recordkeeping

d. Accuracy of Home Capillary Glucose Monitoring

e. Adverse Effects of Home Capillary

f. Efficiency of Home Capillary Glucose Maintenance

g. Blood Glucose Testing Strip

1) Dextrostix
2) Dextrostix "Look-alikes"
3) Chemstrips bG
4) Trendstrips
5) Glucostix

h. Visual Confirmation - Strips for Instruments

1) Reflectance Meters

i. Use of Information Derived From Home Glucose Monitoring

1) Assessment of Maximum Therapy Response
2) Intensive Insulin Regimens
3) Assessment of Minimum Therapy Response

j. Conclusion

12. Systems for Clinical Chemistries Outside the Hospital Laboratories

a. Clinical Chemistry Instruments

1) Processing Mode
2) Reagent Type
3) Features of Chemistry Analyzer

a) Analyst
b) DT-60
c) Reflotron
d) Seralyzer
e) Vision
f) Other Semi-Automated Systems
g) Fully-Automated Systems
h) Electrolyte Systems

4) Quality Assurance
5) Conclusions

(Video - Clinical Chemistries by Reflectance Spectrophotometry)

(Clinical Case - Diabetes)

IX. HEMATOLOGY

1. Introduction

2. Hemoglobin

a. Specimens for Hemoglobin

b. Methods for Hemoglobin

c. Absorbance Maxima for Hemoglobins (Figure 9-1, Table 9-1)

d. Reference Ranges for Blood Hemoglobin

e. Decreased Levels of Hemoglobin

f. Elevated Levels of Hemoglobin

g. Abnormal Hemoglobins

h. Methods for Characterizing Hemoglobin Variants (Figure 9-2)

3. Hematocrit

a. Specimens for Hematocrit

b. Reference Ranges for Hematocrit

c. Increased Hematocrit

d. Decreased Hematocrit

4. Red Blood Cell Indices (Wintrobe Indices)

a. Red Blood Cell Counts

1) Specimens for RBC Counts
2) Reference Ranges for RBC Counts

b. Mean Corpuscular Volume (MCV)

1) Reference Ranges for MCV

c. Mean Corpuscular Hemoglobin (MCH)

1) Reference Ranges for MCH

d. Mean Corpuscular Hemoglobin Concentration (MCHC)

1) Reference Ranges for MCHC

e. Additional Diseases Diagnosed by MCV, MCH and MCHC

5. Peripheral Blood Smear

6. Reticulocyte Count in Blood

a. Specimens for Reticulocyte Count

b. Decreased Reticulocytes

c. Increased Reticulocytes

7. White Blood Cell Counts

a. Reference Ranges for WBC Counts

b. Normal Constitution of Leucocytes of Varying Developmental Stages

c. Decreased WBC Levels

d. Increased WBC Levels

8. Platelet Counts (Thrombocytes)

a. Reference Ranges for Platelet Counts

b. Decreased Platelet Counts (Thrombocytopenia)

c. Increased Platelet Counts (Thrombocytosis or Thrombocythemia)

9. Deficiency Anemias

a. Iron Deficiency Anemia

b. Vitamin B12 Deficiency Anemia

1) Methods for Serum Vitamin B 12 (cyanocobalamin)
2) Reference Ranges for Vitamin B12
3) Schilling Test for Vitamin B12 Absorption

c. Folate Deficiency Anemia

1) Methods for Folic Acid in Serum
2) Reference Ranges for Serum Folate

10. Additional Tests for Hemolytic Disease

a. Lactic Dehydrogenase

b. Serum Haptoglobin

c. Direct Coombs Test

11. Immunohematology

a. Antigen

b. Antibody

c. Agglutinogen

d. Agglutinin

e. Hemolysin

f. Isoantibodies

g. Autoantibodies

h. Natural Antibodies

i. Complete (Bivalent) Antibodies

j. Incomplete (Univalent) Antibodies

k. Warm Antibody

l. Cold Antibody

12. Drug Induced Hemolytic Anemia

a. Mechanism I (Drug Adsorption)

b. Mechanism II (Modified RBC Antigen)

c. Mechanism III (Innocent Bystander)

d. Partial List of Drugs Associated with Both an Acquired Positive Direct Coomb's Test and Hemolytic Anemia (Table 9-2)

13. Congenital Anemias

14. Leukemias, Lymphomas and Myeloproliferative Syndromes

15. Blood Coagulation

a. Bleeding Time

b. Prothrombin Time (PT)

1) Specimens for PT
2) PT Test
3) Decreased PT
4) Increased PT

c. Activated Partial Thromboplastin Time (APPT) and Partial Thromboplastin Time (PPT)

1) Specimens for PPT and APPT
2) APPT Test
3) Decreased APPT
4) Increased APPT

16. Erythrocyte Sedimentation Rate (ESR) (SED Rate)

a. Specimens for ESR

b. Methods for ESR

c. Reference Ranges for ESR (SED Rate)

d. Decreased ESR

e. Increased ESR

f. Blood Sedimentation Tubes (Figure 9-4)

(Video - Iron and Hemoglobin by Visible Spectrophotometry)

X. THERAPEUTIC DRUG MONITORING AND TOXICOLOGY

1. Introduction

2. Therapeutic Drug Monitoring (TDM)

a. Specimens for TDM

1) Request Form for TDM or CT (Figure 10-1)

b. General Methods for TDM c. Anticonvulsants

1) Phenobarbital
2) Primidone
3) Phenytoin (Diphenylhydantoin)
4) Ethosuximide
5) Clonazepam
6) Carbamazepine
7) Valproic Acid
8) Methods for Anticonvulsants (Figure 10-2)

d. Cardiovascular Agents

1) Digoxin
2) Quinidine
3) Lidocaine
4) Procainamide
5) Disopyramide
6) Verapamil
7) Propranolol
8) Methods for Cardiovascular Agents

e. Antipsychotic Drugs (Cyclic Antidepressants and Lithium)

1) Imipramine
2) Desimpramine
3) Amitriptyline
4) Doxepin
5) Maprotiline
6) Amoxapine
7) Trazodone
8) Methods for Cyclic Antidepressants
9) Lithium
10) Methods of Lithium

f. Antibiotics-Aminoglycosides, Vancomycin and Chloramphenicol

1) Gentamicin
2) Kanamycin
3) Tobramycin
4) Vancomycin
5) Chloramphenicol
6) Methods for Antibiotics

g. Antineoplastic Drugs

1) Methotrexate (MTX)
2) Methods for MTX

h. Immunosuppressants

1) Cyclosporine
2) Methods for Cyclosporine

i. Bronchodilators

1) Theophylline
2) Caffeine
3) Methods of Theophylline

j. Commentary on TDM

3. Clinical Toxicology (CT)

a. Screening Procedures (Figure 10-8)

1) Spot Tests
2) Thin-Layer Chromatography (TLC)
3) Gas-Liquid Chromatography (GLC)
4) High Performance Liquid Chromatography (HPLC)
5) Mass Spectrometry (MS)
6) Nuclear Magnetic Resonance (NMR) Spectroscopy and Fourier Transform Infra-Red (FT-IR) Spectroscopy

b. Separating Toxic Substances From Biological Matrices

1) Volatile Substances (Table 10-2)
2) Non-Volatile Substances (Figures 10-9 and 10-10)

c. Selected Toxic Agents

1) Ethanol and Methanol
2) Salicylates (Figure 10-11)
3) Acetaminophen and Phenacetin (Figure 10-12)
4) Amphetamines
5) Benzodiazepines
6) Barbiturates (Table 10-3, Figure 10-13)
7) Narcotics
8) Metals
9) Fluoride and Bromide
10) Other Toxic Agents

d. Commentary on Clinical Toxicology