This project was supported by the Innovation Fund of WNLO 2018WNLOKF023.

This study was conducted according to the principles of the Helsinki Declaration and approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (No. 2019065). The specimens were collected from a MM patient in the Department of Hematology, Zhongnan Hospital of Wuhan University (China).
1. Preparation of the BM smears
<ol>
	<li>Place the first 0.2 mL of BM solution on a clean disposable glass slide.</li>
	<li>Spread the BM smears to uniform thickness with sharp ...

<ol>
	<li>Sonneveld, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+multiple+myeloma+with+high-risk+cytogenetics:+a+consensus+of+the+International+Myeloma+Working+Group.">Treatment of multiple myeloma with high-risk cytogenetics: a consensus of the International Myeloma Working Group.</a> Blood. 127, 2955-2962 (2016).</li><li>Radbruch, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competence+and+competition:+the+challenge+of+becoming+a+long-lived+plasma+cell.">Competence and competition: the challenge of becoming a long-lived plasma cell.</a> Nature Reviews Immunology. 6, 741-750 (2006).</li><li>Lai, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+cytogenetics+in+multiple+myeloma:+a+study+of+151+patients+including+117+patients+at+diagnosis.">Improved cytogenetics in multiple myeloma: a study of 151 patients including 117 patients at diagnosis.</a> Blood. 85 (9), 2490-2497 (1995).</li><li>Hallek, M., Bergsagel, P. L., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+myeloma:+increasing+evidence+for+a+multistep+transformation+process.">Multiple myeloma: increasing evidence for a multistep transformation process.</a> Blood. 91, 3-21 (1998).</li><li>Munsh, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+recommendations+for+risk+stratification+in+multiple+myeloma:+report+of+the+International+Myeloma+Workshop+Consensus+Panel+2.">Consensus recommendations for risk stratification in multiple myeloma: report of the International Myeloma Workshop Consensus Panel 2.</a> Blood. 117, 4696-4700 (2011).</li><li>Gole, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cIg-FISH+On+Patients+with+Multiple+Myeloma+-+A+Modified+and+Simple+Technique+Easily+Incorporated+Into+Routine+Clinical+Service.">cIg-FISH On Patients with Multiple Myeloma - A Modified and Simple Technique Easily Incorporated Into Routine Clinical Service.</a> Blood. 120, 4792-4792 (2012).</li><li>Lee, S. H., Erber, W., Porwit, A., Tomonaga, M., Peterson, L. I. C. S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematology,+ICSH+guidelines+for+the+standardization+of+bone+marrow+specimens+and+reports.">Hematology, ICSH guidelines for the standardization of bone marrow specimens and reports.</a> International Journal of Laboratory Hematology. 30, 349-364 (2008).</li><li>&#352;tifter, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+evaluation+of+bone+marrow+aspirate+and+biopsy+is+superior+in+the+prognosis+of+multiple+myeloma.">Combined evaluation of bone marrow aspirate and biopsy is superior in the prognosis of multiple myeloma.</a> Diagnostic Pathology. 5, 30 (2010).</li><li>Huegel, A., Coyle, L., McNeil, R., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+interphase+fluorescence+in+situ+hybridization+on+direct+hematological+bone+marrow+smears.">Evaluation of interphase fluorescence in situ hybridization on direct hematological bone marrow smears.</a> Pathology. 27, 86-90 (1995).</li><li>Jacobsson, B., Bernell, P., Arvidsson, I., Hast, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+morphology,+esterase+cytochemistry,+and+interphase+cytogenetics+of+peripheral+blood+and+bone+marrow+smears.">Classical morphology, esterase cytochemistry, and interphase cytogenetics of peripheral blood and bone marrow smears.</a> The Journal of Histochemistry and Cytochemistry. 44, 1303-1309 (1996).</li><li>Dunning, K., Safo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ultimate+Wright-Giemsa+stain:+60+years+in+the+making.">The ultimate Wright-Giemsa stain: 60 years in the making.</a> Biotechnic &amp; Histochemistry. 86, 69-75 (2011).</li><li>Woronzoff-Dashkoff, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wright-giemsa+stain.+Secrets+revealed.">The wright-giemsa stain. Secrets revealed.</a> Clinics in Laboratory Medicine. 22, 15-23 (2002).</li><li>McPherson, R. A., Pincus, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Henry's+Clinical+diagnosis+and+Management+by+Laboratory+Methods+E-book.">Henry's Clinical diagnosis and Management by Laboratory Methods E-book.</a> Elsevier Health Sciences. , (2017).</li><li>Ross, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+from+the+European+Myeloma+Network+on+interphase+FISH+in+multiple+myeloma+and+related+disorders.">Report from the European Myeloma Network on interphase FISH in multiple myeloma and related disorders.</a> Haematologica. 97, 1272-1277 (2012).</li></ol>

The application of FISH to genetic risk stratification in MM is essential. The critical part of FISH reports is not all nucleated cells, but clonal PCs specifically purified or identified by sorting with anti-CD138 magnetic beads or marking with cytoplasmic immunoglobulin light chain &#954; or &#955;. Interphase FISH after PC sorting or cIg-FISH was found to be significant in the diagnosis of MM due to the relatively lower proportion of PCs in the BM. However, these methods have some shortcomings, including complex proce...

Multiple myeloma (MM) is a malignant plasma cell (PC) disease with strong biological heterogeneity and large individual differences in clinical efficacy, with survival periods ranging from months to decades. Cytogenetic characteristics are important prognostic indicators of MM. The risk stratification system and individualized treatments based on genetic characteristics have become topics of intense interest in clinical research on MM1. The aberrations of PCs tested in a fluorescence in situ hybridization (FISH) panel of bone marrow (BM) include del 13q14 (RB1), del 17p13 (TP53), t(4;14) (IGH/FGFR3), t(11;14) (IGH/MYEOV), t(14;16) (IGH/MAF), t(14:20) (IGH/MAFB), 1q21 (CKS1B) gain/amplification, and 1p (CDKN2C) deletion.
Standard metaphase cytogenetics should be included in the initial assessment of MM patients. Although conventional G-banded karyotyping offers the benefit of a whole-chromosome analysis, the low yield of this method leads to many false negative results2. Traditionally, PCs have been viewed as largely incapable of splitting because they are the end-stage products of B lymphocyte differentiation. This makes it difficult to obtain split images. Improved cytogenetic analysis in MM with long-term cultures (6 days) and stimulation of cultures by cytokines may be a promising method for identifying cytogenetic abnormalities in newly diagnosed MM patients3. Even when the culture time was prolonged to 6 days, however, cytogenetic abnormalities were accurately reported in 30%-50% of MM patients4. Furthermore, MM is characterized by complex cytogenetic aberrations that reflect its prognostic heterogeneity. However, the resolution of traditional chromosome banding technology is low, which may easily lead to missed detection of chromosomal abnormalities in MM.
Interphase FISH, preferably after CD138-positive magnetic bead-based PC sorting or marking with cytoplasmic immunoglobulin light chain &#954;/&#955;, is indispensable in the analysis of MM5,6. A CD138-positive selected sample is strongly recommended for the optimized yield of tumor cells. However, accurate quantitation of cytogenetic aberrations requires at least 4 mL of anticoagulated BM for PC sorting. Additionally, the combinations of either CD138 immunomagnetic bead sorting with FISH or cytoplasmic &#954;/&#955; light chain immunoglobulin with FISH testing (cIg-FISH) increase numerous experimental costs and are time-consuming.
Usually, the PC ratio is first assessed from the morphological examination of stained BM smears or BM biopsy sections7,8. BM specimens of the highest quality (first marrow aspirate samples) are used for morphological testing, whereas those sent for FISH or other detection are often the secondary aspirate samples with high ratios of dilution with peripheral blood.
As early as the 1990s, multiple studies showed that BM smears can be directly used for interstitial FISH examinations, which has proven to be a reliable and repeatable method9. An elegant study based on cell morphology and esterase cytochemistry combined with FISH of peripheral blood and BM smears confirmed its great clinical significance for elucidating chromosomal abnormalities in patients with granulocytic and lymphocytic leukemia10.
Herein, we provide a novel method for improving the success of interphase FISH detection in MM patients.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>automatic FISH machine</td>
			<td>Leica&#160; Corporation</td>
			<td>S500-24</td>
			<td>FINAL Assy Thermobrite 240V</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Abbott Molecular Inc.</td>
			<td>06J49-001</td>
			<td>DAPI Counterstain</td>
		</tr>
		<tr>
			<td>FISH Analysis Software</td>
			<td>IMSTAR&#160; Corporation</td>
			<td>IMSTAR</td>
			<td>FISH Analysis Software</td>
		</tr>
		<tr>
			<td>FISH Probe</td>
			<td>Abbott Molecular Inc.</td>
			<td>05N56-020</td>
			<td>Vysis Locus Specifc Identifer TP53 / CEP 17 FISH Probe Kit</td>
		</tr>
		<tr>
			<td>Fixed volume pipette</td>
			<td>Eppendorf China Ltd.</td>
			<td>M33768H</td>
			<td>10&#160; microliter</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Olympus Corporation</td>
			<td>BX53</td>
			<td>Forward Fluorescence Microscope</td>
		</tr>
		<tr>
			<td>Karyotype Analysis Software</td>
			<td>IMSTAR&#160; Corporation</td>
			<td>IMSTAR</td>
			<td>Karyotype Analysis Software</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Olympus&#160; Corporation</td>
			<td>BX41</td>
			<td>Forward Light Microscope</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Abbott Molecular Inc.</td>
			<td>07J05-001</td>
			<td>NP-40</td>
		</tr>
		<tr>
			<td>Plastic staining dyeing rack</td>
			<td>Guangzhou Kaixiu Trading Co., Ltd.</td>
			<td>RSJ-501</td>
			<td>&#160;24 slides</td>
		</tr>
		<tr>
			<td>Plastic staining dyeing tank</td>
			<td>Guangzhou Kaixiu Trading Co., Ltd.</td>
			<td>RSJ-516</td>
			<td>&#160;24 slides</td>
		</tr>
		<tr>
			<td>Rubber Cement</td>
			<td>Marabu&#160;GmbH &#38; Co. KG</td>
			<td>FixoGum&#160;</td>
			<td>Rubber Cement</td>
		</tr>
		<tr>
			<td>SSC</td>
			<td>Abbott Molecular Inc.</td>
			<td>02J10-032</td>
			<td>20&#215;SSC</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Shanghai Boxun Medical Bio-Instrument Co., Ltd.</td>
			<td>DK-8D</td>
			<td>Multiple Temperature Water bath</td>
		</tr>
	</tbody>
</table>

interphase fluorescence in situ hybridization of bone marrow smears of multiple myeloma

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of the most common hematological malignancies. The identifying characteristic of MM is accumulated malignant plasma cells in bone marrow. FISH reports for MM mainly focus on purified or identified clonal plasma cells, rather than all nucleated cells, by sorting with anti-CD138 magnetic beads or marking with cytoplasmic immunoglobulin light chain &#954; or &#955;. Bone marrow interphase nuclei are usually obtained from fresh bone marrow cells. However, satisfactory enrichment of plasma cell specimens requires large amounts of fresh heparin anti-coagulated bone marrow, which cannot be obtained in the case of difficult bone marrow extraction or a bone marrow dry tap. Herein, we establish a novel method to improve the success of FISH detection on stained or unstained bone marrow smears. Bone marrow smears are easier to obtain than anticoagulated bone marrow specimens.

In the initial morphological assessment of a newly diagnosed MM patient, 15% of PCs in a BM smear were found to have larger and darker nuclei along with larger amounts of cytoplasm than normal PCs (Figure 2A). The first tube of heparin anti-coagulated BM detected by the immunophenotype technique revealed only 2.3% of the monoclonal aberrant PCs. CD138 immunomagnetic bead sorting in combination with FISH or the cIg-FISH technique is essential for obtaining an accurate FISH result. However, as...

Watch this Scientific Journal Video about Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma at JoVE.com

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

Here, we present a protocol for improving the success of interphase fluorescence in situ hybridization detection on bone marrow smears from multiple myeloma patients.

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

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of ...

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.

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4.0K visualizações.  Zhongnan Hospital of Wuhan University. A aplicação da Fluorescência In Situ Hibridização chamada FISH à estratificação de risco genético em mieloma múltiplo é essencial. A parte crítica dos relatórios fish não são todas as células nucleadas, mas as células plasmáticas clonais especificamente purificadas ou identificadas por triagem com contas magnéticas anti-CD138 ou marcação com kappa citoplasmica ou imunoglobulina da cadeia de luz Lambda com teste de PEIXE chamado cIg FISH. Peixe interfáltico após a triage...