To Propose a Reliable means of Identifying Plasma
Cells (PCs) from Haematological Bone Marrow Smears
(BMSs), prior to fluorescent in situ hybridisation
(FISH) studies, for patients referred for
Multiple Myeloma (MM).
LSI 13 (13q14.3) (Locus Specific Indicator) and IgH
(Immunoglobulin heavy chain) Break Apart
Translocation Probes were then used to Analyse these
PCs for Chromosome Abnormalities associated with
MM.
By Layla Nadine Al-Bustani
Nottingham University Hospitals (NUH), City Hospital Campus

• Aim of project
• Introduction
• What is MM?
• Prevalence
• What are PCs and their morphology?
• Clinical features associated with MM
• Strategies
• Methods and Materials
•
Section 1: To test the technique: Is it possible to capture PCs, remove MGG stain and carryout FISH twice, using the
LSI 13q14.3 and IgH break apart probe on the same slide.
• Section 2: Making bone marrow smears
• Section 3: Analysis of Mature Plasma Cells (MPCs) from 5 patients, referred for Myeloma
• Section 4: Analysis of Immature Plasma Cells (IPCs) from 5 patients, referred for Myeloma
• Discussion
• Conclusion
• Summary
• References
• Acknowledgements

• To devise a method where by PC identification could be performed, prior to fluorescent in situ hybridisation (FISH) studies using the standard VYSIS LSI 13 (Locus Specific
Indicator) (13q14.3) and VYSIS IgH (Immunoglobulin heavy chain) break apart translocation probe.
• The LSI 13 D13S319 probe was selected, because this region is known to be deleted in patients with MM.
• The IgH break apart translocation probe was selected because patients with MM are known to have rearrangements involving the IgH region.

What is Multiple Myeloma (MM)?
• A clonal B cell neoplasia. The term neoplasia is used to describe the process in which a genetically altered precursor cell (in this case haematopoietic stem cells) gives rise to an abnormal clone of cells
PCs, which show abnormality in proliferation and maturation.
• MM is characterised by the expansion of a malignant PC population within the bone marrow with a low proliferative index.
Prevalence of MM
• MM accounts for 10% of haematological malignancies and it is found to be more common in males than females.
• The risk of MM is said to increase with age.

• PCs are the most mature cells in the B-lymphocyte lineage. They have a unique ability of secreting antigen-specific immunoglobulin (paraprotein: IgA, IgD, IgE, IgG, IgM).
• Due to the elevated number of malignant PCs in patients with
MM, excessive quantities as well as abnormal forms of a paraprotein are secreted.
• PCs are rarely found in peripheral blood.
• PCs of normal individuals are located in the bone marrow, where their levels range between 0.2% - 2.8% of the white blood cells.

Mature plasma cells
Small-sized, with dense chromatin and prominent cytoplasm
Intermediate Plasma cells
Medium or large-sized, with less dense chromatin but still prominent cytoplasm .
Immature Plasma cells
These have a pronounced nucleolus and fine chromatin. The immature plasma cells are associated with proliferation and poorer prognosis .
Plasmablasts (immature plasma cell).
These are much larger than mature plasma cells and have no perinuclear zone.

• Bone damage is often the most significant feature.
• Bone damage (caused by an altered balance between bone production and destruction) leads to areas of thinning of the bone. The “holes” in the bone, which are produced, are called osteolytic lesions.
• The presence of Bence-Jones proteins . (dimers of immunoglobulin κ, or λ, light chains, that are found in the urine).
• High levels of paraprotein. IgG (normal levels: 5.3-16.5g/L, abnormal levels: 20.5g/L).
• High creatinine, (abnormal levels 540 μmol/L, normal levels 60-
125μmol/L).

•
To recognise and capture PCs, according to their morphology, from BM smeared slides, which have been stained with May
Grunwald Giemsa (MGG).
•
To illustrate the ability to recognize PCs from BM smeared slides, by using the CD138 marker specific to the cytoplasm of the PCs.
To remove the MGG stain from these BM smeared slides
•
To perform FISH on the identified PCs, using the LSI 13 probe and then the LSI IGH dual colour, break apart translocation probe, using the same slide.
•
To try and make our own BM smeared slides.
•
To run through diagnostic cases referred for MM.
• To identify Immature, Intermediate, Plasmablasts and Mature
PCs from BM smeared slides. Then carry out FISH analysis using the same two probes. A comparison between the abnormalities found in Immature, Intermediate PCs and Plasmablasts to Mature
PCs was then made.

To capture PCs
• BMSs referred for MM, which were stained with MGG, were obtained from the Haematology department at NCH.
• After liaising with our haematology department, I was taught how to identify
PC according to their morphology
• We identified and captured PC images according to their morphology, using
MetaSystems.
• This was done prior to any FISH, because the FISH protocol destroys the cytoplasm, which is a crucial characteristic, in identifying PCs.
• Metasystems stored the location of every image captured, which in turn allowed the relocation of the PCs after FISH.
• A marker pen and later a diamond pen was used to mark out the area containing PCs on the slide.
• This area indicated where the probe was placed.

• The image below represents May Grunwald Giemsa stained plasma cell
(on the left), which has been identified by its morphology.
• And the CD138 marker specific to the cytoplasm of plasma cells (on the right), which confirms the CORRECT identification and therefore validation of the ability to recognise plasma cells.
PC
PC

To remove MGG stain
• Initially, 2 slides were put through an ethanol series (ETOHs), (each ethanol %, for 4 minutes).
• The slides were then left to air dry before FISH was carried out.
FISH protocol
• Slide denaturation: 1min 45sec in 70% formamide at 73ºC and probe denaturation: 5 minute at 73ºC. (water bath).
• The appropriate volume of probe was then added to each slide which was cover slipped and placed in a hybridisation chamber overnight in an incubator at 37 ºC.
Post hybridisation washes
• Vysis wash 1 at 73ºC for 2 minutes and Vysis wash 2 (2 lots of
30secs) at room temperature (RT).
• The slides were then mounted
• The slides were viewed using a fluorescence microscope.

4 minutes in each ethanol %.
PC
PC
PC
PC
PC
PC
Trouble shooting
• The volume of probe was insufficient, which resulted in the absence of signals in some PCs.
• A consistent problem was the presence of a green haze ( green arrow ) that was observed and became more apparent, the longer the slide was under the fluorescence microscope.
• This impeded the visualisation of the LSI 13 signals.
Actions taken
• The probe volume was increased.
• Various ways were used to try and reduce or remove the green haze.

Slide immersed into fix for 40 seconds Formamide
8 minutes in each Ethanol % (Slide a)
From the images it was deduced that the most effective way to remove the MGG was when the slide was placed in each
Ethanol % for 8 minutes.

• Slide a was placed in 4xSSC for 10 minutes to remove the IgH probe.
• FISH was then carried out on this slide again except this time the LSI 13 (q14.3) was used
• The FISH protocol used was identical to the first FISH carried out, except that the slide was only denature for 1 minute in
70% formamide at 73ºC.
• This is because the chromosomes were fragile from the first
FISH run.

PC
Image of
MGG stained
PCs , prior to
FISH studies
PC
Primary FISH using the LSI 13 (D13S319) probe
Secondary FISH using the LSI IgH breakapart translocation probe
PC
PC
PC
PC

• Attempts to make BM smeared slides from samples referred to cytogenetics was unsuccessful.
• The Clinical Cytogenetics department in lithium heparin or RPMI 1640 medium plus heparin.
• After contacting Dr Fiona Ross as well as the haematology department at NCH, I discovered that the distorted morphology and inconsistent staining of the BMs was due the medium that it was received in.
• I was also informed that the first few drops of BM are taken and put onto slides (and sent to haematology for morphology studies) at the same time the BM samples are taken.
• This is because; it is the first few drops that contain the highest proportion of PCs cells.
• From this information, unfixed, unstained BM smeared slides referred for MM were henceforth collected from the haematology department at
NUH, City hospital campus.

• Unfixed, unstained BMSs were obtained from the Haematology dept.
• Slides were fixed for 5 minutes in 100% ETOH, then stained for 5 minutes in MG stain (Aldrich)
• Slides were then washed with water and stained for a further 5 minutes in Giemsa (BDH, British Drug House ) stain
• This stain was also washed off with water and slides were left to dry on a hot plate.
• These patients were then FISHed using the standard LSI 13q14.3 and
IgH probe and analysed for LSI 13 deletions and IgH rearrangements.

•
IgH breakapart probe: The red and green signal had to be at least 1 signal width apart to be considered a split signal.
•
LSI 13 probe: a 10% cut off level was used

The % of mature PCs with 1 and 2, LSI 13 signals, as well as the % of mature PCs with different IgH rearrangements, from 5 patients referred for Myeloma
Table 1
PATIENT NUMBER
3
4
5
1
2
Number of LSI 13 signals
1 2
3%
12%
93%
87%
86%
22%
24%
13%
61%
68%
IgHBA signal pattern
2F 1F 1R 1G 1F
70%
76%
0%
4%
13%
4%
43 %
27%
28%
0 %
33 %
0%
50%
11 %
50%
KEY
Figures in blue indicate a normal FISH result
Figures in red indicate an abnormal FISH result

• Metasystems was used to identify and capture immature,
Intermediate and Plasmablasts (categorised as the IPCs).
• Slides were fixed, stained and destained in the usual manner.
• FISH was then performed using the standard LSI 13(q14.3) and IgH break apart translocation probe.
• The results can be seen in the following tables and graphs.

Table 2: FISH results obtained from 5 patients referred for Myeloma. In these cases Mature and
IPCs were analysed for LSI 13 deletions.
PATIENT NUMBER
6
7
8
9
10
Number of LSI 13 signals
Mature Plasma Cells Immature Plasma Cells
1
16%
14%
84%
0%
90%
2
66%
67%
8%
57%
10%
1
18%
9%
100%
20%
80%
2
68%
90%
0%
80%
20%
Table 3: FISH results obtained from the same patients as in table 2 (patients 6-10), referred for
Myeloma. MPCs and IPCs were analysed for IgH rearrangements.
PATIENT
NUMBER
9
10
6
7
8
2F
3%
10%
5%
6%
44%
1F
0%
50%
23%
31%
0%
Mature Plasma cells
1R 1G 1F 1R 2G 1F
38%
0%
0%
6%
22%
22%
0%
11%
0%
5%
IgHBA signal pattern
1G 1F
3%
15%
35%
37%
5%
1F
0%
36%
11%
28%
20%
Immature Plasma cells
1R 1G 1F 2R 2G
46%
0%
0%
0%
20%
20%
0%
0%
0%
0%
1G 1F
0%
18%
66%
57%
20%
Patient 10 (DOTH) failed to produce a conclusive FISH result for immature plasma cells using the IgH break apart translocation probe
KEY
Figures in blue indicate a normal FISH result
Figures in red indicate the abnormality present in the highest proportion (%) of plasma cells
Figures in green indicate the second highest abnormality present in the plasma cells

Bar Graph 1: Represents the IgH Rearrangements observed in the IMMATURE Plasma Cells of Four
Patients referred for Multiple Myeloma
9
8
7
6
9
8
7
6
0
0 10 20 30
% IPCs
40 50 60
Bar Graph 2: The results obtained from four patients who have displayed IgH Rearrangements in their MATURE Plasma Cells
10 20 30
% MPCs
40 50
70
60
KEY
1R 1G 1F
2R 2G
1F
1G 1F
KEY
1R 1G 1F
1R 2G 1F
1F
1G 1F

• From the ten patients analysed in this project, 3 patients (3,8 and 10) illustrated evidence of a deletion involving the region 13q14.3.
• From tables 1 and 3, analysis of MPCs using the IgH break apart translocation probe, showed that 7 patients had an abnormal FISH result.
• It is important to identify the partner involved in an IgH rearrangement
• Patients that have the t(11;14) (q13;q32) are said to have a neutral prognosis unlike patients with the t(4;14)(p16;q32) which is associated with an unfavourable prognosis.
• From table 2, patients 6, 7 and 9 have normal (i.e. 2 copies) of LSI 13
D13S319 in their IPCs and MPCs
• Unlike patients 8 and 10 who have only 1 copy of the LSI 13 D13S319 probe in their IPCs and MPC.
• From these five patients it can be deduced that, if an abnormality is present in the IPCs (i.e at the early stages of B cell differentiation) it is more likely for these abnormalities to be carried through, until the B cell has differentiated into a MPC.

• Cytogeneticists can identify PCs according to their morphology.
• Unfixed, unstained BMSs can be fixed (100% ETOH) and stained using MGG.
• The MGG stain can be removed successfully
• FISH can be performed successfully on BMSs.
• PCs from patients referred for MM can be analysed for chromosome abnormalities.

• The Association of Clinical Cytogenetics, 2007, Best Practice guidelines state that, “ In the absence of a reliable means of identifying plasma cells, totally normal FISH results shall be qualified, explaining that the possibility of a false negative result is much higher than might be anticipated from assessment of the morphology smears” .
• This project illustrates that identifying PCs (by morphology) is “Reliable” and selecting only the PCs, ensures that the correct population of cells are being analysed
• Selecting PCs from BMSs, which will then be FISHed, is an accurate way of identifying specific chromosome abnormalities associated with MM
• The analysis would result in much higher proportion of abnormal PCs being scored. This can be clearly be seen from the results obtained from the patients in this project
• This technique is currently being implemented into the Cytogenetics laboratory at NUH, City hospital campus

• The probes that have been selected are the Kreatech 13(q14.3)/ 17p13 (p53) dual set probes and Kreatech t(4;14)(p16;q32) dual fusion probes
• That is, patients who are +ve for a deletion and/or translocation would be given a certain type of treatment
• In the case of a patient with a del(13)(q14.3) or a t(4;14)(p16;q32) they would follow the Velcade/PAD intensification protocol, after 3 courses of
CTD (Cyclophosphamide, pharmion, Thalidomide and Dexamethasone) chemotherapy.
• A publication in 2006 by Chiecchio et al suggested that, patients who show a deletion of 13q either by cytogenetics or interphase FISH, have a poor prognosis.
• However, when cases with a deleted 13 detectable by both cytogenetics and interphase FISH, were separated from those by interphase FISH, only the poor prognosis of interphase FISH detectable deleted 13, disappeared.
• It was therefore deduced that observing the deleted 13 in metaphase preparations is a critical prognostic factor in myeloma.
• From this information, cytogenetics would be performed on patients who are
+ve for a 13q deletion on interphase FISH.

•
•
•
•
•
•
•
•
•
•
•
Human Cytogenetics: malignancy and acquired abnormality 3rd edition by
D.E.Rooney practical approach pp 279.
Chromosomal analysis in multiple myeloma: cytogenetic evidence of two different diseases. Leukemia 1998 12 , pp960-969 N-V Smadja, C Fruchart, F Isnard, C
Louvet, J-L Dutel, N Cheron, M-J Grange, M Monconduit and C Bastard.
Hypodiploidy is a major prognostic factor in multiple myeloma by Nicole
Véronique Smadja , Christian Bastard , Christophe Brigaudeau , Dominique Leroux , and Christophe Fruchart Blood , 2001, volume 98, number 7, pp. 2229-2238.
http://www.multiplemyeloma.org/about_myeloma/
Textbook of Malignant Haematology edited by Laurent Degos, David C Linch and
Bob Lowenberg. Published in 1999 by Martin Dunitz. Pages 37-45
Multiple Myeloma booklet. Leukemia Research http://web.cc.yamaguchi-u.ac.jp/~plasma/msg/nextpage/plasma01.html
Immature Plasma Cells, Obtained from a Malignant Pleural Effusion
Jon Fukumoto, M.D., Yu Wong, M.D., Ph.D., and Steven Coutre, M.D. http://ashimagebank.hematologylibrary.org/cgi/content/full/ashimgbank;2006/110
8/6-00027 http://igitur-archive.library.uu.nl/dissertations/2006-1005-202204/c2.pdf Different aspects of thalidomide treatment and stem cell transplantation in multiple myeloma patients by Adriana Margaretha Willemina van Marion. Doctoral thesis
Oxford Handbook of Clinical Haematology by D.Provan et al, pg 206-210.

Haematology department at NUH, City hospital campus
• Dr Krishnamurthy who taught me how to recognise plasma cells
• Dr Wadelin
• Dr Byrne
• Mr Alan Grey
• Ms Elaine Smith
• Mr Ken Morrison
For providing the bone marrow smeared slides and help whilst making our own BMSs
Clinical cytogenetics department at NUH, City hospital campus
• Mrs Kate Martin
• Mrs Adele Calvert
• Mr Nigel Smith for their complete, undivided help and support throughout my project.