Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

The application of Fluorescence In Situ Hybridization called FISH to genetic risk stratification in multiple myeloma is essential. The critical part of FISH reports is not all nucleated cells, but clonal plasma cells specifically purified or identified by sorting with anti-CD138 magnetic beads or marking with cytoplasmic kappa or Lambda light chain immunoglobulin with FISH testing called cIg FISH. Interphase FISH after plasma cell sorting or cIg FISH turns out to be significant in the diagnosis of multiple myeloma, due to relatively lower proportion of plasma cells in the bone marrow.

However, these methods have some shortcomings including complex processes, high specimen demands and high experimental costs. Plasma cells can be quickly located in a bone marrow smear. Based on this, we use bone marrow smear FISH for the detection of multiple myeloma cells.

This study was conducted according to the principles of the Helsinki Declaration and approved by the Ethics Committee of Zhongnan Hospital of Wuhan University. The specimens were collected from a multiple myeloma patient in the Department of Hematology, Zhongnan Hospital of Wuhan University of China. Place the first 0.2 milliliter of bone marrow solution on a clean disposable glass slide, spread the bone marrow smears to uniform thickness and sharp tails by quickly removing the bone marrow then dry it naturally.

Cover the bone marrow films with one milliliter of Wright-Giemsa solution for approximately 10 seconds at room temperature. Add one milliliter of phosphate buffer to the slides, care was required to prevent the dye solution from drying or flowing off the slides. Mix the dye solution gently and maintain it for 15 minutes at room temperature.

Rinse the slides with clean water and dry it in the air at room temperature. Use forward light microscopy to detect malignant plasma cells, the plasma cells should be well dispersed. Now what we see in the field is the plasma cells which are fairly dispersed.

This is a typical Binuclear nuclear plasma cell. Cover the hybridized area with fixative solution for 10 minutes to discolor and fix plasma cells. Rinse the slide with deionized water and dry it in air at room temperature.

Preheat a jar containing two times SSC buffer to 56 degrees Celsius in a water bath. Wash the prepared bone marrow smear in the preheated two times SSC buffer for 10 minutes followed by dipping into deionized water at room temperature. Dehydrate the smear in an ethanol gradient 70%85%and 100%ethanol each for one minute.

Dry the smear in air for 10 minutes. First add TP53 on centromere of chromosome 17 probe mixture to the hybridization area and cover it with a cover slip press gently the fine pointed tweezers to remove air bubbles from underneath. Seal all sides of cover slip with rubber cement to ensure good tightness.

After solidification of the rubber cement the slide was placed on an automatic FISH machine denaturation was performed at 78 degrees Celsius for five minutes followed by hybridization at 37 degrees Celsius overnight. Pick up the slide from the FISH machine. Use a fine pointed tweezers to remove the rubber cement gently.

Immerse the slide in two times SSC at room temperature for approximately one or two minutes to wash the cover slip off. Wash the slide in 68 degrees Celsius preheating buffer in a water bath for two minutes. Wash the slides with two times SSC in a 37 degree Celsius water bath for one minute.

Set up right in the dark to dry thoroughly for 10 minutes at 10 microliter of DAPI to the hybridized area, cover with a cover slip. View hybridized slide using a suitable filter set on a fluorescence microscope, use monochromatic blue fluorophore optical filter to observe fluorescence intensity of the cell nucleus. Take cell nucleus images under a DAPI filter set.

CEP17 was labeled with green fluorescence. Use spectrum green fluorophore optical filter to observe the location of CEP17. Take CEP17 signals image under green filter set.

The TP53 gene was labeled with orange fluorescence. Use spectrum orange fluorophore optical filter to observe TP53, take TP53 signals image under the set, merge these three images into an entirety and examine for numerical abnormalities. In a normal interphase cell two orange and two green signals are observed inside a nucleus with blue fluorescence indicating one pair of normal chromosomes 17.

The results showed the morphology and genetics of bone marrow in a multiple myeloma patient. Figure A showed that 15%of plasma cells in a bone marrow smear were have found to have larger and darker and nuclear along with large amounts of cytoplasm than normal. Figure B showed a gray scale image of representative by a nucleated plasma cell in a bone marrow smear.

Figure C showed four orange and four green signals in one interphase nucleus of plasma cell suggesting that four copies of chromosome 17 were localized in a nucleus. Figure D showed that the abnormal chromosome karyogram of this patient confirmed two pairs of chromosomes 17 in one nucleus. Bone marrow smears are much easier to obtain than anticoagulant bone marrow specimens because obtaining anticoagulated bone marrow requires complex procedures to obtain the nucleus.

Accordingly, we established a convincing method for bone marrow smear FISH for the detection of multiple myeloma cells.