Name: interphase fluorescence in situ hybridization of bone marrow smears of multiple myeloma
Uploaded: 2022-04-15T14:00:00
Description: Watch this Scientific Journal Video about Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma at JoVE.com

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>
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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. 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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 ...

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