LABORATORY ASPECT OF PCR, IMMUNOASSAY ELECTROPHORESIS

Dr.Budi Dermawan Lubis, SpPK

Protein Electrophoresis   Electrophoresis separates protein based on their electric

charge densities.

  At pH > isoelectric point protein negatively charged and moved to anode on agarose or cellulose acetate gel.

  Sample: serum, urine, CSF, EDTA for Hb electrophoresis

Protein Electrophoresis

  Absolute protein fraction: percentage x TTP

  Caution: plasma contain fibrinogen can be mistake as paraprotein, intravascular hemolyticà Hb-haptoglobin à ↑ α2 band, light chain myeloma à no M spike detected on SPE

Immunoassay •  Sample collection and processing is a complex à

no simple system that will meet the requirements for all analyte, it is important to determine the type of specimen required before begins to obtains the specimen

•  Immmunoassay : 1ml blood for 3- 4 tests •  Haemolysis : in vivo or in vitro, >300mg/dl à red color of serum, increase intracellular constituents (K, LDH), decrease

extracellular constituent, interference with analytical assay •  Lipaemic : caused by elevated lipoprotein , plasma turbidity at TG concentrations > 300 mg/dl, interference with photometric & electrophoretic analysis •  Icteric : Interference with spectral & chemical (oxidase / peroxidase •  Centrifuge sample 1200g 10minute for kompleks AG-AB with turbidimetry method

Laboratory Criteria For Unacceptable Samples

1.  Inadequate sample identification

2.  Inadequate volume of blood collected into an

additive tube

3.  Use of an improper collection tube

4.  Hemolysis

5.  Improper transportation & storage : sample for

blood gases transported on ice

6.  Contamination suspected

7.  Unknown time delay

Immunoassay

Polymerase Chain Reaction

•  Rapid and highly-specific amplification of DNA fragments

•  Components underlie the PCR process:thermostable DNA polymerases and the ability to specific oligonucleotide primers

•  PCR consists of three stages, multiply repeated: denaturing, annealing, and elongation. After the reaction is completed amplicon can be visualized by gel electrophoresis.

•  Advantage: rapid, sensitive, specific, robust. Disadvantage: expensive, extraneous DNA may contaminate reaction, mutation gen have to design primer.

•  Indication : Dx malignancy, bacteria, viruses, thallasemia.

Temp Time Σ Cycle

Denaturation Annealing Elongation

94°C 50-65°C

72°C

15-30 s 30-60 s

45 s – 3 min

25 - 30

PCR