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) (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 κ/λ, is indispensable in the analysis of MM^5,6. 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 κ/λ 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 sections^7,8. 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.

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 tails by quickly removing the BM. Then, dry naturally.
3. Cover the BM films with 1 mL of Wright-Giemsa solution for approximately 10 s at room temperature.
4. Add 1 mL of phosphate buffer to the slides.
   NOTE: Take care to prevent the dye solution from drying or flowing off the slides.
5. Mix the dye solution gently and maintain it for 15 min at room temperature.
6. Rinse the slides with clean water and dry them in air at room temperature.
7. Use forward light microscopy to detect malignant PCs. The PCs should be well dispersed.

2. FISH pre-processing of the BM smears

1. Cover the hybridized area with fixative solution for 10 min to discolor and fix the PCs.
   1. To prepare fresh fixative solution, mix methanol with glacial acetic acid in a volume ratio of 3:1.
2. Rinse the slide with deionized water (dH₂O) and dry it in air at room temperature.
3. Preheat a jar containing 2x SSC buffer to 56 °C in a water bath. Freshly dilute the buffer to 20x SSC before use.
   1. To prepare 20x SSC, thoroughly mix 176 g of sodium chloride and 88 g of sodium citrate with 800 mL of dH₂O. Measure the pH and adjust to pH 5.3 ± 0.2. Then, add dH₂O to bring the final volume to 1 L.
4. Wash the prepared BM smear in the preheated 2x SSC buffer for 10 min, followed by dipping it into deionized water at room temperature.
5. Dehydrate the smear in an ethanol gradient (70%, 85%, and 100% ethanol, each for 1 min). Air-dry the smear for 10 min.

3. BM smear FISH and washing

NOTE: All steps were performed in a dark room to prevent fluorescence quenching.

1. Add the TP53/centromere of chromosome 17 (CEP17) probe mixture to the hybridization area and cover it with a coverslip. Press gently with fine-pointed tweezers to remove air bubbles from underneath.
2. Seal all sides of the coverslip with rubber cement to ensure good tightness.
3. After solidification of the rubber cement, place the slide on an automatic FISH machine. Perform denaturation at 78 °C for 5 min, followed by hybridization at 37 °C overnight.
4. Pick up the slide from the FISH machine. Use fine-pointed tweezers to remove the rubber cement gently.
5. Immerse the slide in 2x SSC at room temperature for approximately 1 or 2 min to wash the coverslip off.
6. Wash the slide in 68 °C preheated 0.4x SSC/0.3% NP-40 buffer in a water bath for 2 min.
   1. Prepare 0.4x SSC/0.3% NP-40 solution by thoroughly mixing 20 mL of 20x SSC (pH 5.3) with 950 mL of dH₂O. Then, add 3 mL of NP-40 and mix thoroughly until completely dissolved. Measure the pH and adjust to 7.0-7.5 with NaOH. Then, add dH₂O to bring the final volume of the total solution to 1 L.
7. Wash the slides with 2x SSC in a 37 °C water bath for 1 min. Set upright in the dark to dry thoroughly for 10 min.
8. Add 10 µL of DAPI to the hybridized area and cover with a coverslip.

4. FISH analysis and imaging

1. View the hybridized slide using a suitable filter set on a fluorescence microscope.
2. Use a monochromatic blue fluorophore optical filter to observe the fluorescence intensity of the cell nucleus. Take cell nucleus images under a DAPI filter set.
3. Use a chromosome centromere probe to show how many chromosomes a cell contains. Label CEP17 with green fluorescence. Use a spectrum green fluorophore optical filter to observe the location of CEP17. Take a CEP17 signals image under the green filter set.
4. Label the TP53 gene with orange fluorescence. Use a spectrum orange fluorophore optical filter to observe TP53. Take a TP53 signals image under this set.
5. Merge these three images and examine them for numerical abnormalities.
   NOTE: In a normal interphase cell, two orange and two green signals are observed inside a nucleus with blue fluorescence, indicating one pair of normal chromosome 17 (Figure 1).

Representative Results

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, aspirated BM is limited in the event of it being a dry tap. BM smears were used to test interphase FISH. A representative binucleated PC in BM film was localized by a fluorescence microscope stage Vernier caliper (Figure 2B). FISH was applied to the BM smears to test for TP53 and CEP17. The FISH results showed four orange and four green signals in one interphase PC nucleus (Figure 2C), suggesting that four copies of chromosome 17 were localized in the metaphase PC nuclei. Karyotyping results were obtained by extending the culture time up to 3 days and further analyzed using FISH analytic software (Figure 2D). The accuracy of the interphase FISH results on the BM smears was further verified.

Figure 1: Normal signals of the TP53/CEP17 FISH probe for interphase cells (1000x). The fluorescence intensity of the cell nucleus was blue, the TP53 was orange, and the CEP17 was green. In those 3 normal interphase cells, both two orange and two green signals appeared inside the blue-fluorescing nucleus, indicating two pairs of the normal chromosome 17. Please click here to view a larger version of this figure.

Figure 2: Morphology and genetic images of bone marrow (BM) in multiple myeloma (MM) patients. (A) Myeloma cells with clustered distribution indicated by the arrows () after Wright-Giemsa staining of the BM smear (1000×). (B) Binucleate plasmacytoid cells indicated by arrows () on a grayscale image taken by a black and white camera on a fluorescence microscope (1000×). (C) TP53/ centromere of chromosome 17 (CEP17) fluorescence in situ hybridization (FISH) probe test for the BM smear. FISH reveals four TP53 and four CEP17 signals in each nucleus (→) (1000×). (D) Abnormal chromosome karyogram shows two pairs of chromosome 17 in one nucleus (). Please click here to view a larger version of this figure.

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 processes, high specimen demands, and high experimental costs. Fifty PCs can be quickly located by a microscope-stage Vernier caliper. BM smears are much easier to obtain than anticoagulant BM specimens because obtaining anti-coagulated BM requires complex procedures to obtain the nucleus. Accordingly, we established a convincing method for BM smear FISH for the detection of MM cells.

First, BM smears should be made by an experienced morphological analyst. To spread the BM into smears, the spreader is placed in front of the aspirate at an angle of 30°-45° and pulled back to make contact with the fluid¹¹. Then, the spreader is moved forward uniformly in a steady motion. The density of the BM smear must be tailored so that the length is approximately three-fourths of the slide¹². The BM film must be narrower than the slide to guarantee that cells on all edges can be detected¹³. The selection of the hybridization area and the localization of PCs was critical in our protocol, and this requires an experienced morphological analyst. For BM smears containing fewer PCs, the PCs should be localized by using a forward fluorescence microscope oil immersion objective and Vernier calipers on the objective stage, and their images can be taken by monochrome shots. For example, if the PCs accounted for only 3% of the total nucleated cells in a BM film by morphological examination, a well-dispersed hybrid region of 8 mm x 8 mm should contain no less than 50 PCs for a FISH test. After FISH hybridization, fluorescence signals in at least 50 PCs must be counted by an experienced analyst¹⁴.

However, a limitation of this technique is the requirement for fresh BM smears. If the BM smears are stored for over 2 years, the resulting FISH images might be obscure, resulting in misinterpretation.

Taken together, the abovementioned BM smear FISH can be extensively used for patients with hematological malignancies when BM extraction is difficult or BM dry tap occurs, even in hypo-proliferative diseases with fewer nucleated cells. We hope that more patients with hematological malignancies can benefit from this method.

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