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

Summary

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

Abstract

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 κ or λ. 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.

Introduction

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)....

Protocol

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

1. Place the first 0.2 mL of BM solution on a clean disposable glass slide.
2. Spread the BM smears to uniform thickness with sharp .......

Discussion

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 κ or λ. 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.......

Materials

Name	Company	Catalog Number	Comments
automatic FISH machine	Leica  Corporation	S500-24	FINAL Assy Thermobrite 240V
DAPI	Abbott Molecular Inc.	06J49-001	DAPI Counterstain
FISH Analysis Software	IMSTAR  Corporation	IMSTAR	FISH Analysis Software
FISH Probe	Abbott Molecular Inc.	05N56-020	Vysis Locus Specifc Identifer TP53 / CEP 17 FISH Probe Kit
Fixed volume pipette	Eppendorf China Ltd.	M33768H	10  microliter
Fluorescence Microscope	Olympus Corporation	BX53	Forward Fluorescence Microscope
Karyotype Analysis Software	IMSTAR  Corporation	IMSTAR	Karyotype Analysis Software
Light Microscope	Olympus  Corporation	BX41	Forward Light Microscope
NP-40	Abbott Molecular Inc.	07J05-001	NP-40
Plastic staining dyeing rack	Guangzhou Kaixiu Trading Co., Ltd.	RSJ-501	24 slides
Plastic staining dyeing tank	Guangzhou Kaixiu Trading Co., Ltd.	RSJ-516	24 slides
Rubber Cement	Marabu GmbH & Co. KG	FixoGum	Rubber Cement
SSC	Abbott Molecular Inc.	02J10-032	20×SSC
Water bath	Shanghai Boxun Medical Bio-Instrument Co., Ltd.	DK-8D	Multiple Temperature Water bath