Yasumasa Akai

A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum

We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2-3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages.

Combination Analysis of a Whole Lymph Node by One-Step Nucleic Acid Amplification and Histology for Intraoperative Detection of Micrometastasis

Objective: The aim was to develop a more efficient molecular detection system than histological examination (HE) for lymph node (LN) metastasis. Methods: Cytokeratin (CK) 19 mRNA copy numbers of 5 colon carcinoma cell lines (Lovo, DLD1, WiDr, Colo201 and Colo320) were calculated and compared by one-step nucleic acid amplification (OSNA) and conventional real-time reverse-transcription polymerase chain reaction (RT-PCR). Then, 91 LN submitted for HE from 6 patients with advanced colorectal adenocarcinoma and 64 LN submitted for frozen diagnosis from 47 patients with different malignancies were examined by OSNA and HE. Results: CK19 mRNA copy numbers of all but Colo320 cells detected by OSNA were within double of those detected by RT-PCR. The least cell count of Lovo cells detected at one reaction (2 µl) by OSNA was calculated as 0.8 cells. Carcinoma metastasis showing either HE+ or OSNA+ was detected in 7.9% of the LN from advanced colorectal adenocarcinomas and in 30.0% of the LN for frozen diagnosis from different malignancies; HE–/OSNA+ metastasis was detected in 4.8 and 4.0%, respectively. OSNA analysis of 1 LN could be completed within 40 min. Conclusion: A combined analysis of LN by HE and OSNA could increase the sensitivity for detecting micrometastasis during surgery.