HUMAN DISORDERS AND BIOCHEMICAL TESTS

General Principles of Clinical Chemistry Analysis.

Definitions of Terms   

·         Human disorder is an illness caused by abnormalities in genes or chromosomes especially a condition that is present from before birth. Eg Phenylketonuria,

·         Clinical chemistry is the branch of science which deals with investigation of chemical constituents of the body.

·         An anticoagulant is a substance that prevents coagulation (clotting) of blood.

·         Preservative these are chemical substances which are used to keep or prevent deterioration urine constituents for a long period.

·         Biochemistry is the chemical composition in the living matter.

·         Qualitative analysis is the procedures or steps performed to determine the presence or absence of substance in the body or specimen.

·         Quantitative analysis is the procedures or steps performed to determine the exact amount of substance present in the body or specimen

Other definitions:

·         Reducing substances are substances found in specimens that reduce blue copper II sulphate to red orange cuprous oxide.

·         Fundamental Laws of Chemistry: These are important elementary Laws of Chemistry which include, Law of Conservation of Mass, Law of Constant Composition and the Gas Laws.

·         Blank (Zero Solution) is a solution which does not contain an analyte but contains all reagents /chemicals used in the measurement.Usually used to zero or calibrate the machine. 

·         Standard Solution is solution with a known concentration used for the calculation of concentrations of unknown (test samples).

·         Control Solution is a solution or material solely used for the purpose of quality control and not for calibration.

·         Wavelength is the characteristic of electromagnetic radiation; the distance between two wave crests.

·         Analytical procedures are diagnostic methods carried on specimens.

Describe common Analysis (measurements) in Clinical Chemistry

Measurement of serum or plasma;

·         Creatinine, Urea, Uric acid

·         Blood/plasma Glucose

·         Bilirubin

·         Protein

·         Alkaline Phosphatase

·         Alanine Aminotrasferase (ALT)

·         Aspartate   Aminotransferase (AST)

·         Cholesterol

 Measurement of urine;

·         Protein

·         Glucose

·         Bilirubin

·         Urobilinogen

·         Specific gravity

Measurement of CSF;

·         Glucose

·         Protein

Measurement of Faeces:

·         Occult Blood

Discuss the underlying principles of Spectrophotometry 

·         Spectrophotometry is underlined by measurement of the amount of light that a sample absorbs at a particular wavelength and then used to determine the concentration of the sample by comparison with an appropriate standards or reference data, based on the Beer-Lambert Law.

Explanation;

·         When a white light passed through the coloured solution, the light rays of coloured solution are transmitted and absorbed.

·         Light rays of one colour  of that solution is more reabsorbed strongly than others, the deeper the colour the more the light rays of the same colour are reabsorbed.

·         The amount of light absorbed increases with an increase in their concentration of coloured compound, the intensity of colour obtained is directly proportional to the concentration of coloured compound of solution in chosen wavelength or filter.

·         Note: For the proper function of Spectrophotometer the following must be adhered; Wavelength selection, use of blank solution, use of standard solution and use of control solution. It is based on the formula:  A = a.b.c

     

A = Absorbance;  light that was not transmitted after passage of light through a solution;

a  = absorptivity coefficient (L. cm/g)

b  =  cuvette path length (1 cm)

c =  concentration

A is calculated from % Transmittance which is detected in the colourimeter:  A = 2 - log₁₀ %T 

Explanation;

·         When a white light passed through a monochromator it selects one wavelength of light that when directed through the coloured solution, the light rays of coloured solution are transmitted and absorbed.

·         Light rays of one colour  of that solution is more absorbed strongly than others, the deeper the colourthe more the concentrated the molecules and the more light rays of the same colour are reabsorbed.

·         The amount of light absorbed increases with an increase in their concentration of coloured compound, the intensity of colour obtained is directly proportional to the concentration of coloured compound of solution in chosen wavelength or filter.

Concentration versus Absorbance Curve

·         When Absorbance of standard solutions (known concentration) are analysed, the relationship between Absorbance and Concentration is plotted.

·         When patient samples and other unknowns are analysed in the same way, their absorbance readings can be used to determine concentration from this plot. To do this with a manual procedure , determine the absorbance of a set of known standards and plot the standard ABS on the Y axis versus standard concentration on the x axis using linear graph paper.   The points should fall in a straight line and angle up to the right (positive slope).

 

Measure absorbance of the patient sample and read the concentration by following the absorbance reading horizontally until it intersects with the line and then following that point vertically down to the concentration.

Calculating Concentration from Absorbance

•      A single standard (known concentration) can be used to determine concentration of an unknown standard.  First measure the Absorbance of the standard and then measure Absorbance of the patient sample. Determine patient concentration from its measured Abs with this formula:

ConcStd/ Abs Std = Conc patient/ Abs patient

Example:   Glucose 10 mmol/L standard gave Abs 0.500 and patient Abs was 0.225.  What is patient concentration?    

Conc patient = Abs patient (Conc std/ Abs std) 

? = 0.225 (10/0.5) =   4.5 mmol/L

  

Problem #1 Calculations using Beer’s Law

A standard with molar absorptivity of 1.25  L/g. cm was analysed in a spectrophotometer with cuvette pathlength of 1 cm. and gave an absorbance reading of 0.125.

•      What is the a?

•      What is the b?

•      What is the A

•      Calculate the concentration in g/L

Answer: (not to be listed on student handout)

A = a.b.c

a = 1.25

b = 1

A = 0.125

c = A / (a x b)  = 0.125 / 1.25 x 1  = 0.1 g/L

Problem #2

Albumin Determined from the Standard Concentration versus Abs curve

 

Using this graph, determine the concentration in g/L of albumin with absorbance reading of 0.25

Answer (not to be listed on student handout):by following the absorbance reading of around 0.25 horizontally to where it meets the graph and tracing vertically down, you get 40 g/L.

Problem #3

An albumin  30gl/L standard gave Abs 0.250 and the patient Abs when analysed for albumin was 0.125.  What is patient concentration for albumin?    

Answer (not to be listed on student handout): Conc patient = Abs patient (Concstd/ Abs std) 

? = 0.125 (30/0.250) =   15 g/L

 Explain anticoagulants and preservatives used in clinical chemistry 

·         Anticoagulants-These are substances that prevent coagulation (clotting) of blood.

·         The ratio of anticoagulant/preservative and specimen used in Clinical chemistry should be observed.

Common Anticoagulants used in Clinical Chemistry:

·         Ethylamine Disodium Tetra Acetic acid (EDTA)

·         Heparin

·         Trisodium Citrate

·         Potassium or Sodium Oxalate

·         Ammonium Oxalate/Potassium Oxalate (Heller and Paul anticoagulant)

Ethylamine Disodium Tetra Acetic acid (EDTA)

·         Concentration used is 1.2mg/ml of blood.

Mode of action:

·         These chemical prevent clotting by binding with calcium.

·         It inhibits conversion of fibrinogen to fibrin.

·         It is also used for haematological investigation.

·         Not suitable for coagulation purposes, PCV decrease, and cause platelets fragmentation.

·         It cannot be used when electrolyte are to be measured

Heparin

·         It is very expensive.

·         It inhibits formation of thrombin.

·         Concentration used is 2mg/10ml of blood.

·         It does not interfere with the most chemical reactions.

·         It helps to minimize haemolysis.

Ammonium Oxalate/Potassium Oxalate (Heller and Paul anticoagulant)

·         Combine with calcium to form insoluble calcium oxalate. Cannot used for blood transfusion. (It is poisoning).

·         Convert Calcium iron to insoluble oxalate.

·         Not suitable for determination of Urea or Ammonia.

Sodium Fluoride.

·         It inhibits red cells metabolism (Glycolysis) or bacteria action.

·         Usually mixed with potassium oxalate 3 parts in 1 part of fluoride.

·         It is not suitable in enzyme estimation because it is poison

PRESERVATIVES

·         Preservative these are chemical substances which are used to keep or prevent deterioration urine constituents for a long period.

Purpose:

·         To prevent decomposition of urea to ammonia.

·         Inhibit oxidation of urobilinogen.

·         It inhibits bacteria growth.

Common preservatives are:

·         2.0M Hydrochloric acid used for quantitative Protein, Amino acid, in Organic phosphate and Calcium.

·         Thymol crystal used for Sodium, Creatinine, Potassium, Chloride, Urea, Protein, Amylase, Reducing substances.

·         Boric acid powder or crystals.

·         Chloroform.

·         Toluene

·         Sodium Azide

·         Refrigeration at 2 – 8^oC not more than two hours