Can cytoplasmic nucleophosmin be detected by immunocytochemical staining of cell smears in acute myeloid leukemia?

Mutations in the C-terminal region of nucleophosmin in acute myeloid leukemia (AML) result in aberrant cytoplasmic nucleophosmin (cNPM) in leukemic blast cells which is detectable by immunocytochemistry in bone marrow trephine (BMT) biopsy sections. We tested whether cNPM is detectable by immunocytochemistry in air-dried smears of AML with nucleophosmin1 (NPM1) mutations. An immunoalkaline phosphatase method was developed using the OCI-AML3 cell line, known to have mutated NPM1, and assessed on blood and marrow smears of 60 AML cases. NPM was detectable in all blast cell nucleoli and cNPM in 21 of 31 of NPM1 mutated and 15 of 29 wild-type cases. Paired air-dried smears and BMT biopsies from the same case (mutated and wild-type) gave discrepancies in cNPM expression and there was no correlation in 10 of 22 cases. Due to the high false positive and negative rates for cNPM in cell smears, this method should not be used as a surrogate for NPM1 mutations in AML.

Labeling of multiple cell markers and mRNA using automated apparatus.

Double immunoenzymatic labeling of 2 different molecules in tissue sections is a widely used technique. However, it is time consuming since the 2 immunoenzymatic procedures are carried out in sequence, and they must also be optimally performed to avoid unwanted background labeling. In this paper, we report that double immunoenzymatic staining performed using automated immunostaining apparatus considerably reduces the requirements in terms of time and is also highly reproducible and free of background. Three tissue markers can also be visualized by performing (after immunoperoxidase labeling) 2 sequential immuno-alkaline phosphatase procedures using different substrates. Furthermore, single or double detection of mRNA by in situ hybridization can be combined with immunoenzymatic labeling. Finally, automated labeling could also be performed on peripheral blood and bone marrow smears, opening the possibility of using this procedure in the analysis of hematologic/cytology samples.

Detection of genetic alterations by immunoFISH analysis of whole cells extracted from routine biopsy material.

The detection of genetic abnormalities (eg, translocations, amplifications) in paraffin-embedded samples by the fluorescence in situ hybridization (FISH) technique is usually performed on tissue sections. FISH analysis of nuclei extracted from paraffin-embedded samples is also possible, but the technique is not widely used, principally because of the extra labor involved and the loss of information on tissue architecture. In this article, we report that nuclei extracted from paraffin-embedded tissue often retain at least part of the surrounding cytoplasm. Consequently, immunocytochemical labeling for a range of cellular markers (eg, of lineage or proliferation) can be performed in combination with FISH labeling, allowing specific cell populations to be analyzed for genetic abnormalities. These cell preparations are largely free of the problems associated with tissue sections (eg, truncation artifact, signals in different focal planes) so that interpretation is easy and numerical chromosomal abnormalities are readily assessed. Cells isolated from paraffin sections can be stored in suspension so that arrays can be created as and when needed from a range of neoplasms for investigation by the immunoFISH technique (for example, for studying a new genetic abnormality). This procedure represents a novel methodology, which in some settings offers clear advantages over analysis of tissue sections.