Clinical Laboratory Fundamentals: Instruments, Assays, Values

Clinical Laboratory Equipment Essentials

Water Bath

Used to incubate samples at a desired temperature.

Centrifuge

An equipment used to separate substances that differ significantly in their masses by using centrifugal force.

Spectrophotometer

Measures substances in the ultraviolet (200 – 400 nm) as well as in the visible range (400 – 700 nm) of the electromagnetic spectrum of light.

Micropipettes

Used to dispense micro volumes of samples or reagents.

Enzyme Kinetics and Inhibition

Competitive vs. Non-Competitive Inhibitors

Relationship Between Km and Substrate Affinity

• An enzyme with a high Km has a low affinity for its substrate and requires a greater concentration of substrate to achieve Vmax.

Blood Collection Tubes: Types and Uses

Grey Stopper Tube

The grey stopper tube is specifically used for glucose determinations. It contains Sodium fluoride, which inhibits glycolysis, and Oxalate, which acts as an anticoagulant, preserving glucose levels.

Clinical Chemistry Assay Principles

Calcium Determination Method

The method is based on the specific binding of cresolphthalein complexone (OCC), a metallochromic indicator, and calcium at alkaline pH.

OCC + Calcium pH 10.7 → OCC-Calcium Complex

Phosphate Determination Method

In an acid medium, inorganic phosphate reacts with Ammonium molybdate, forming a yellow phosphomolybdic complex.

PO43- + H+ + (NH4)6 Mo7O24 → Phosphomolybdic Complex

Glucose Determination Method

In the presence of glucose oxidase (GOD), glucose is oxidized to gluconic acid and hydrogen peroxide. The hydrogen peroxide then reacts, in the presence of peroxidases, with phenol and 4-aminoantipyrine to form a quinonimine dye.

Glucose + O2 GOD → Gluconic Acid + H2O2

2H2O2 + 4-Aminoantipyrine + Phenol → Quinoneimine + 4H2O

Clinical Reference Ranges and Associated Conditions

Glucose Levels

• Normal Value: 70-110 mg/dL
• Hyperglycemia (High Glucose):
  • Diabetes Mellitus
  • Hyperactivity of thyroid, pituitary, or adrenal gland
  • Surgical removal of pancreas
• Hypoglycemia (Low Glucose):
  • Overdosage of insulin in treatment of Diabetes Mellitus
  • Hyperactivity of thyroid or pituitary gland
  • Starvation
  • Severe exercise

Phosphate Levels

• Normal Value: 3-4 mg/dL
• Hyperphosphatemia (High Phosphate):
  • Hyperparathyroidism
  • Renal failure
• Hypophosphatemia (Low Phosphate):
  • Rickets
  • Osteomalacia
  • Hyperparathyroidism

Calcium Levels

• Normal Value: 9-11 mg/dL

Calcium Concentration Calculation:

Absorbance of Test

-------------------- x Standard Concentration

Absorbance of Standard

• Hypercalcemia (High Calcium):
  • Hyperparathyroidism
  • Vitamin D poisoning
  • Multiple Myeloma
  • Renal failure
  • Sarcoidosis
• Hypocalcemia (Low Calcium):
  • Hypoparathyroidism
  • Vitamin D deficiency
  • Pseudohypoparathyroidism
  • Certain kidney diseases
  • Low protein levels