Effects of heparin on polymerase chain reaction for blood white cells.
Author(s): Yokota M, Tatsumi N, Nathalang O, Yamada T, Tsuda I
Publication: J Clin Lab Anal, 1999, Vol. 13, Page 133-40
PubMed ID: 10323479 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate if the inhibitory effect of heparin on PCR amplification is affected by heparin concentration, cell count, template amount or Taq polymerase type.
Conclusion of Paper
Although, DNA yield and purity were unaffected by heparin concentration in blood, increasing concentrations of heparin necessitated higher leukocyte counts for human leukocyte antigen (HLA) typing. However, the effect depended on the type of Taq polymerase with the highest success rate for heparinized specimens obtained using Ex Taq.
Studies
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Study Purpose
The purpose of this study was to compare HLA type determination in EDTA and heparin anticoagulated specimens and to determine if the inhibitory effect of heparin was dependent on heparin concentration, cell count, template amount or Taq polymerase type. Sodium heparin (16, 48 or 96 U/mL blood) and K2EDTA (1.5 mg/mL) anticoagulated blood specimens were obtained from 10 healthy donors. Leukocytes were obtained by double centrifugation, and concentrations were adjusted by adding plasma. DNA was extracted using phenol-chloroform.
Summary of Findings:
HLA type was successfully determined for all 10 EDTA blood specimens, but failed for 2 of the 10 heparinized (16 U/mL) specimens. Both specimens that failed to amplify also had low leukocyte counts (<3000 leukocytes/mL). DNA yield and purity, as determined by spectrophotometer, were unaffected by heparin concentration in blood, but as heparin concentration increased, more leukocytes were necessary in order to obtain sufficient amplification for HLA typing. Further, amplification depended on Taq polymerase type, with the highest success rate obtained using Ex Taq. Accordingly, higher concentrations of heparin were necessary to inhibit amplification with Ex Taq than with the other Taq polymerases.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA PCR DNA Spectrophotometry Cell count/volume Hematology/ auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Potassium EDTA
Sodium heparin
Biospecimen Aliquots and Components Biospecimen components 2,700 leukocytes/mL blood
12,000 leukocytes/mL blood
PCR Specific Template/input amount 100 ng
50 ng
33 ng
16 ng
PCR Specific Nucleic acid amplification Takara Taq
Ex Taq
PE Taq
Ph Taq
Wako Taq
Qia Taq