Fresh frozen neoplastic tissues from 70 infiltrating ductal breast carcinomas were analysed for cytosolic oestrogen receptor (ER) protein content using a solid phase enzyme immunoassay (EIA) method based on a "sandwich" principle (Abbott ER-EIA monoclonal). Formalin-fixed, paraffin-embedded sections from the same carcinomas were examined for nuclear immunoreactivity against a monoclonal antibody for ER protein (Dako) using the standard avidin-biotin complex immunoperoxidase (IP) method after microwave antigen retrieval. The degree of ER positivity by IP was also scored according to a visual estimation of the percentage of cells expressing immunopositivity and the intensity of staining. Twenty-eight (40%) of the carcinomas were ER-positive by EIA and 34 (48.6%) were positive by IP. Twenty-five (35.7%) were ER-positive and 33 (47.1%) were ER-negative by both methods. Nine (12.9%) were ER-negative by EIA but were positive by IP, this discrepancy being ascribed to sampling inadequacy for EIA. However, 3 (4.3%) tumours were ER-positive by EIA and negative by IP. This discrepancy may be variously due to inadequate antigen retrieval, faulty technique and the possibility that the two methods do not measure identical ER proteins. IP appears to have an advantage over EIA in that it has a higher pick-up rate, does not require fresh tissue and can be applied to archival material. However, to reduce false negative estimations, it may be necessary to run IP staining using more than one ER antibody. Standardisation of the IP method for ER is desirable before this method is to be widely adopted in Malaysian laboratories. Quantitation of ER positivity by IP scoring correlated poorly with actual cytosolic levels. Caution should be exercised in attaching patient management value to visual IP scoring.
Matched MeSH terms: Receptors, Cytoplasmic and Nuclear/analysis*
The polymerase chain reaction (PCR) is useful for amplifying specific mRNAs, particularly those present in low copy numbers. However, due to the exponential nature of the amplification process, PCR cannot readily be used to quantify gene expression. A competitive PCR technique was developed to address this shortcoming. An internal standard that is 100% homologous to, but shorter than, the target gene was constructed. The practicality of the method was demonstrated by determining the expression levels of a human transcription factor, peroxisome proliferator-activated receptor gamma 1 (hPPARgamma1) which is normally present in low copy numbers in selected cells. A mock system was used to test the accuracy and sensitivity of the method, which was subsequently used to determine the expression of this receptor in lipopolysaccharide (LPS)-activated monocytes, which are known to express hPPARgamma1 differentially during cellular activation. Densitometric analysis showed that the competitive PCR method reliably estimated the expression levels of hPPARgamma1 at the attomole (10(-18)) level in monocytes.
Matched MeSH terms: Receptors, Cytoplasmic and Nuclear/analysis