Clinical Laboratory Techniques and Analysis

Introduction to Clinical Laboratory Techniques and Analysis

• Introduction to Clinical Laboratory Techniques and Analysis by Ming Jin, PhD, Department of Pathology & Laboratory Medicine, University of California Irvine, April 3, 2025.

Laboratory Medicine at UCI

• Clinical Chemistry

• Molecular pathology

• Microbiology

• Hematology

• Coagulation

• Cytogenetics

• Blood bank (transfusion medicine)

Testing Areas in Clinical Chemistry Lab

• General chemistry: electrolytes, cholesterol, glucose, albumin, bilirubin, calcium, amylase, endocrine testing, cardiac markers, tumor markers, etc.

• Therapeutic drug monitoring (TDM) and Toxicology: gentamicin, urine drug screening for cocaine, etc.

• Special chemistry: protein electrophoresis, etc.

• Urine analysis: Dipstick method for protein, glucose, etc.

• Blood gases: $PO<em>2$, $PCO</em>2$, pH, etc.

Basic Metabolic Panel

• Sodium (Na)

  • Normal range: 135-145 mEq/L

  • Mnemonic: 135-145 is normal

• Potassium (K)

  • Normal range: 3.5-5 mEq/L

  • Mnemonic: 3-5 Bananas/Bunch

• Blood Urea Nitrogen (BUN)

  • Normal range: 5-20 mg/dL

  • Mnemonic: BUNion: 5 digits/limb, 20 total

• Creatinine

  • Normal range: 0.6-1.2 mg/dL

  • Mnemonic: 0.9 (nine) right in middle

• Glucose

  • Normal range: 70-100 mg/dL

  • Mnemonic: Energy: Low at 70-100 yrs old

• Calcium (Ca)

  • Normal range: 8.5-10.5 mg/dL

  • Mnemonic: Milk: Weighs 8.5 pounds +2%

• Chloride (Cl)

  • Normal range: 95-105 mEq/L

  • Mnemonic: Hot Tub: 95-105 degrees F

• Bicarbonate (HCO3)

  • Normal range: 23-29 mEq/L

  • Mnemonic: Carbonation: (2)12 soda = 24

Chemistry Testing Panel

• Analytes and Panels

  • Electrolyte Panel: Carbon Dioxide, Chloride, Potassium, Sodium

  • Basic Metabolic Panel: Carbon Dioxide, Chloride, Potassium, Sodium, Calcium, Creatinine, Urea Nitrogen (BUN), Glucose

  • Renal Function Panel: Carbon Dioxide, Chloride, Potassium, Sodium, Calcium, Creatinine, Urea Nitrogen (BUN), Glucose, Phosphorus, Albumin

Drug Abuse Test at UCI

Common Specimen Used in Clinical Chemistry

• Blood: whole blood, serum, or plasma

  • Venous Blood

  • Arterial Blood (for blood gas analysis)

• Urine

• Spinal fluid

• Body fluid

  • Depends on the test (serum or plasma)

Phases of Testing

• Pre-analytical (52%)

  • Test Selection (pre-preanalytical)

  • Ordering

  • Collection

  • Identification

  • Transport

  • Sample Preparation

• Analytical (33%)

  • Sample Measurement

  • Instrument Calibration & Maintenance

• Post-analytical (15%)

  • Reporting

  • Interpretation & Action (post-postanalytical)

Pre-analytical

• Pre-analysis refers to all steps before specimen analysis.

• Factors altering test results before the test:

  • Physiological variation

  • Specimen collection and handling

  • Exercise

  • Circadian variation

  • Diet

  • Posture

Pre-analytical Variables: Exercise-Related Changes

• Recent strenuous exercise increases:

  • Alanine Aminotransferase (ALT)

  • Creatinine

  • Creatine Kinase (CK), etc.

• Recent strenuous exercise decreases:

  • Cholesterol

  • Triglycerides, etc.

Pre-analytical Variables: Circadian Variation

• ACTH-pituitary -> Adrenal gland -> Big difference at breakfast time vs. Midnight

Pre-Analytical Variables: Diet Effects

• After eating a meal, glucose and triglyceride levels are higher.

• High meat or protein-rich diets increase serum urea, ammonia, and urate.

• Some fruits and vegetables (bananas, pineapples) are rich in serotonin; may observe 5’hydroxyindoleacetic acid in urine.

Pre-analytical Variables: Postural Effect

• Changing from supine to sitting causes fluid shift from intravascular to interstitial space.

• Sitting blood collection (compared to supine) can increase macromolecular analytes by ~10%:

  • Albumin

  • Lipids (cholesterol or triglyceride)

  • Enzymes

  • Immunoglobulin

Pre-analytical Variables: Variability by Sample Collection

• Poor phlebotomy technique

• Improper centrifuging of the specimen

• Improper tube selection

• Improper storage of the specimen

• Effects of blood sample hemolysis:

  • Potassium

  • Magnesium

  • Phosphorous

  • Lactate dehydrogenase (LDH)

• Improper labeling of the specimen causes pre-analytical errors.

Collection Tubes

• Collection tubes may contain anticoagulants.

Collection Tubes (cont.)

Collection Tube Additives

• Heparin Plasma: for general chemistry tests

• EDTA Plasma: for blood cell count, some special tests, ex. BNP

• Citrate Plasma: for coagulation tests

• Sodium Fluoride: an antiglycolytic agent for glucose test

• Serum separator: using polymer gel for the separation of serum (or plasma) from blood cell. Certain analyses and therapeutic drugs may bind to the gel.

• Clot Activator: tubes contain a silica clot activator attached to the wall with a silicone surfactant for enhance clot formation in vitro.

BD Vacutainer® Order of Draw for Multiple Tube Collections

Specimen Collection of TDM

• Time of specimen collection:

  • Random

  • Trough

  • Peak

• Optimal test tube of specimen collection:

  • Red top tube

    • No anticoagulant

    • No gel for serum separation

• If not mentioned trough or peak, it will be random.

Specimen Collection of Drug of Abuse Tests

• Urine

• Blood

Common Methods Used in the Clinical Chemistry Lab

• Spectrophotometry

• Immunoassay

• Laboratory Automation

• Chromatography/Mass Spectrometry

Spectrophotometry

• Components of a single-beam spectrophotometer:

  • Exciter lamp

  • Entrance slit

  • Monochromator

  • Exit slit

  • Cuvette

  • Photodetector

  • Light-emitting diode (LED) display

Spectrophotometry Beer-Lambert Law

• $A = \epsilon C L$

• Add color.

• Show.

• We Calibrate to Identity.

Common Interferences of Spectrophotometry Method

• Bilirubin

• Hemolysis

• Lipemia

Interferences of Albumin Assay by Beckman Spectrophotometry Method

• at 30 my below color evident Use

• Use that to Find articles?

Immunoassay

• Radioimmunoassay (RIA)

• Enzyme immunoassay (EIA, ELISA)

• Fluorescence immunoassay (FIA)

• Chemiluminescence immunoassay (CIA)

• Electro-chemiluminescence immunoassay (ECIA)

Immunoassay

• Heterogeneous assay

  • Example, “Sandwich” method

• Homogenous assay

  • Enzyme multiplied immunoassay technique (EMIT)

• Large molecule -> smaller molecular such as cocaine or morphine

Immunoassay

• Heterogeneous assay

  • Example, “Sandwich” method

• Homogenous assay

  • Enzyme multiplied immunoassay technique (EMIT)