Development of a simple method for rapid isolation of polymorphonuclear leukocytes from human blood.

Polymorphonuclear leukocytes (PMNs; commonly known as neutrophils) play essential roles in innate immunity and inflammation. Although there are standardized methods for the isolation of human neutrophils, they are time consuming and demand considerable technical expertise, making them unfeasible for many clinical applications. Here, we describe a simple and time-efficient technique for the isolation of human neutrophils, which adapts a readily available commercial cell preparation tube (CPT) currently in use for isolation of peripheral blood mononuclear cells (PBMC) and plasma and is now adapted to also yield neutrophils. The total time required for neutrophil isolation was less than 1 hr. Neutrophils isolated by this method were highly purified (> or =97%) as assessed by surface expression of the neutrophil specific marker, CD66b. Neutrophils isolated by this method were functional as demonstrated by their ability to secrete interleukin-1 receptor antagonist (IL-1RA). Neutrophils isolated using this new technique secreted significant amounts of soluble IL-1RA (929.3+/-197 pg/10(6)cells/mL) in response to lipopolysaccharide (LPS). Use of this adapted CPT method allows simultaneous isolation of functional human neutrophils as well as PBMC and plasma. Adoption of this new method will allow the conduct of different neutrophil assays at any clinical site without requiring trained laboratory personnel or a large staff time commitment.

Detection of the soluble heat shock protein 27 (hsp27) in human serum by an ELISA.

Increased levels of autoantibodies against heat shock protein 27 (hsp27) in patients with breast, ovarian, or endometrial cancer strongly suggest the presence and increased levels of hsp27 in their circulation. Therefore, we have developed a sensitive and reproducible ELISA for quantification of soluble hsp27 levels in biological fluid such as serum. The assay is highly specific for hsp27. The limit of detection of the ELISA is about 0.5 ng/mL. The mean intra- and inter-coefficients of variation were 7.45 and 8.18, respectively. The recovery of the recombinant protein was nearly 100%. The assay could detect soluble hsp27 levels in normal human serum when the level was >0.5 ng/mL. Out of 28 serum samples we tested, 10 samples were not detected for any hsp27 level in our ELISA. However, hsp27 levels could be detected in the other 18 samples. The median serum hsp27 level was 3.27 ng/mL when all the 28 normal control samples were included. Low levels of hsp27 in normal human serum may be useful to distinguish the hsp27 levels in breast or other cancer patients during the progression of the disease. Therefore, the use of hsp27 ELISA could be extremely useful in evaluating the role of soluble hsp27 in breast or other cancers.