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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 44  |  Issue : 4  |  Page : 227-231

Sensitivity of flow cytometry immunophenotyping compared with bone marrow morphology in diagnosis of multiple myeloma


1 Demonstrator at Clinical Pathology Department of South Egypt Cancer Institute, Assiut University, Assiut, Egypt
2 Lecturer at Clinical Pathology Department of South Egypt Cancer Institute, Assiut University, Assiut, Egypt
3 Professor at Clinical Pathology Department of the Faculty of Medicine, Assiut University, Assiut, Egypt
4 Internal Medicine, Faculty of Medicine, Assiut University, Assiut, Egypt
5 Professor at Clinical Pathology Department of South Egypt Cancer Institute, Assiut University, Egypt

Date of Submission05-Oct-2019
Date of Acceptance21-Oct-2019
Date of Web Publication20-Jul-2020

Correspondence Address:
Eman H Ahmed
As. Professor Clinical Pathology/South Egypt Cancer Institute
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_42_19

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  Abstract 


Background The diagnosis of multiple myeloma (MM) is challenging, as it is based on several factors, including serum and urine protein electrophoresis, immunofixation, bone marrow (BM) aspirate, and biopsy, in addition to immunophenotyping. Moreover, quantification of BM plasma cells (PCs) by conventional morphology is a mandatory test for the diagnosis and response assessment in MM. One alternative would be to use multiparameter flow cytometry immunophenotyping to quantify BMPCs, although this is currently still limited to research studies and the differential diagnosis of unusual cases.
Aim The aim was to determine the diagnostic sensitivity of flow cytometry in the detection of abnormal PCs in BM specimens of MM compared with BM morphology.
Patients and methods We retrospectively analyzed bone marrow aspiration (BMA), bone marrow biopsy (BMB), and immunophenotypic reports of 50 newly diagnosed patients with MM. Patients’ data were collected and analyzed.
Results BMA of 43 (86%) patients showed PCs greater than or equal to 10%; BMB of 35 of them showed PCs greater than or equal to 10%, and five patients were with poor BMB quality. The sensitivity of BMB in detection of PCs was 90% and 86% for BMA. Concerning immunophenotyping, the gate of mononuclear cells was in the range of 4–88%; those gated cells were 100% positive for CD138 and CD38. CD56 was positive in 84% whereas CD19 was negative in 96%. Overall, 46% were κ restricted and 54% were λ restricted, with 100% sensitivity to malignant PCs.
Conclusion Unlike morphologic and histologic studies, FC is not dependable for providing quantitative information about MM but provides qualitative information for assessing the immunophenotype and light chain clonality of PCs.

Keywords: biopsy, bone marrow aspirate, flow cytometry, immunophenotyping, multiple myeloma


How to cite this article:
Yousof EA, Ahmed EH, Mansor SG, Mohamed HO, Thabet AF, Sayed DM. Sensitivity of flow cytometry immunophenotyping compared with bone marrow morphology in diagnosis of multiple myeloma. Egypt J Haematol 2019;44:227-31

How to cite this URL:
Yousof EA, Ahmed EH, Mansor SG, Mohamed HO, Thabet AF, Sayed DM. Sensitivity of flow cytometry immunophenotyping compared with bone marrow morphology in diagnosis of multiple myeloma. Egypt J Haematol [serial online] 2019 [cited 2020 Aug 8];44:227-31. Available from: http://www.ehj.eg.net/text.asp?2019/44/4/227/290234




  Background Top


Multiple myeloma (MM) is a clonal B-cell disorder in which malignant plasma cells (PCs) accumulate in the bone marrow (BM), producing lytic lesions, excessive amounts of monoclonal protein in the serum or urine, and evidence of end-organ damage [hypercalcemia, renal insufficiency, anemia, or bone lesions ‘CRAB’ (hypercalcemia Renal insufficiency Anemia Binelesion) criteria] [1].

A conventional diagnosis in MM is based on a variety of laboratory results: morphologic features, analysis of M component, hematologic features, biochemical parameters, immunophenotyping, cytogenetics, and labeling index–proliferative activity of PCs [2].

The updated criteria for the diagnosis of myeloma represent a paradigm shift in the approach to myeloma and have considerable effect on the management of the disease [3].

Incorporation of immunophenotypic studies in the management of patients with PC disorders is still far from being routinely established in many diagnostic flow cytometry laboratories and also, immunophenotypic analysis is restricted to research purposes and to differential diagnosis of unusual atypical cases. For clonal PC disorders, multiparameter flow cytometry is of clear and established clinical relevance in: first, the differential diagnosis between MM and other PC-related disorders; second, the identification of high-risk monoclonal gammopathy of undetermined significance and smoldering MM; third, minimal residual disease investigation after therapy; and additionally it may also be useful for fourth, the definition of prognosis-associated antigenic profiles and fifth, the identification of new therapeutic targets [4].

The aim of this study was to determine diagnostic sensitivity of flow cytometry in the detection of abnormal PCs in BM specimens of MM compared with BM morphology.


  Patients and methods Top


This study was done on 50 patients with MM. Those patients were presented to South Egypt Cancer Institute and Assiut University hospitals. The initial diagnostic workup that was carried out for all patients before any chemotherapy was history taking and clinical examination, with careful assessment of clinical signs relevant to MM.

Complete blood picture (CELL-DYE CD-3500CS; Abbott Diagnostics, USA), serum chemistry (creatinine, calcium, albumin, and β2MG) (All these tests were performed on Cobas Integra 400 Plus, Swiss, serial number: 500558), and serum protein electrophoresis (Pretty Interlab device, Rome, Italy, serial number: 38405301) were performed for all patients included in our study. Bone marrow aspiration and biopsy (BMA and BMB) and immunophenotyping data were also obtained. The patients were diagnosed as MM according to WHO criteria 2016. This study was carried out after the approval of Ethical Committee, Assiut University.

Immunophenotyping was performed with a panel of flourochromes including the following:
  1. CD38 by fluorescence isothiocyanate conjugated anti CD38 (clone A07778; Beckman Coulter, Germany).
  2. CD138 by Phycoerythrin (clone DL-101; BD Bioscience).
  3. CD56 Allophycocyanin (clone N901;Beckman Coulter).
  4. CD19 peridinin–chlorophyll–protein complex (Per CP PerCP-Cy5.5, clone SJ25C1; BD Bioscience.
  5. Cytoplasmic κ, (Ig κ Light Chain, fluorescence isothiocyanate, clone TB28-2; BD Bioscience).
  6. Cytoplasmic Lambda (Ig λ Light Chain, Phycoerythrin, clone 1-155-2; BD Bioscience).


Statistical analysis was done using SPSS (Statistical Package for the Social Sciences, version 20; IBM, Armonk, New York, USA). Continuous data were expressed in form of mean (range), whereas nominal data were expressed in the form of frequency (percentage).


  Results Top


Mean age of enrolled patients was 52.42±9.28 years, with range between 32 and 75 years. Majority (52%) of patients were males, and 48% were females. Other laboratory investigations are demonstrated in [Table 1].
Table 1 Some laboratory investigations of patients with multiple myeloma

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Morphologic and hematological results of the cases

The patients were divided into two groups based on BMA pcs% (≥10% or <10%), and each group was further subdivided into two groups based BMB PCs% (≥10% or <10%). In the present study 43 (86%) patients were with BMA PCs% greater than or equal to 10%, 35 of them were BMB PCs greater than or equal to 10%, and five patients were with poor BMB quality, with sensitivity of BMB of 90% ([Figure 1]).
Figure 1 Morghological findings of multiple myeloma.

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The other group of BMA PCs% less than 10% were seven patients, two of them with inadequate BMA, which may be diluted, but immunophenotyping (IPT) results demonstrated that one sample has 2% malignant PCs, and the other sample has 6% malignant PCs. All seven cases were with BMB PCs greater than or equal to 10%. Regarding the pattern of BMB infiltration by PCs, 16% of cases were focal in distribution.

Regarding the five (10%) cases with poor quality BMB, two (4%) of them were with normal serum protein electrophoresis, another two (4%) were with polyclonal band, whereas one case was with M band. They were diagnosed as MM based on BMA PCs% and positive CRAB criteria. Moreover, those five cases with poor BMB quality demonstrated more than 10% malignant monoclonal PCs by flow cytometry.

Analysis of flow cytometric data and marker expression

PCs could be sufficiently identified through back CD138 gating on mononuclear cells. Regarding gated mononuclear cells percentage by immunophenotyping, the gate was in the range of 4–88%, with the mean of 35.65±24.1%; from this gate, the percentage of strictly malignant PCs was in the range of 2–80%, and the mean was 20.54±19.1%.

The overall positive expression rate of CD138 was 100% (50/50). The expression of CD38 was 100% (50/50), CD56 was 84% (42/50), and CD19 was 4% (2/50) in neoplastic myeloma cells. Regarding monoclonality, 23 (46%) patients were κ restricted and 27 (54%) patients were λ restricted; as a quantity of PCs, 44 (88%) of cases had greater than or equal to 10% myeloma cells and the other six (12%) cases had a percentage less than 10 myeloma cells, and their immunophenotypic profile is in [Table 2].
Table 2 The immunophenotyping of patients with multiple myeloma

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Comparison between sensitivity of FCMI, SPEP, and BM morphology in diagnosis of multiple myeloma

In 43/50 cases, BMA helped in the diagnosis of MM in combination with other WHO criteria (86% sensitivity). In 45/50 cases, BMB helped in the diagnosis of MM in combination with other criteria (90% sensitivity). The SPEP showed monoclonal M protein and helped in diagnosis of MM only in 34 (68%) cases. Regarding FCMI, all cases had positive immunophenotping criteria that helped in the diagnosis of MM, with 100% senstivity ([Figure 2]).
Figure 2 Sensitivity of both bone marrow biopsy and IPT and electrophoresis.

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  Discussion Top


Accurate quantification of PCs in BM is critical for diagnosis and assessment of treatment response in patients with MM [5].

Moreover, our study demonstrated that BMA PCs% was lower than BMB PCs percentage, and these results were in agreement with Lee et al. [6]. These findings support the importance of evaluating BMA and BMB slides in the diagnosis and monitoring of the course of MM, as suggested by other authors [5]. In addition, our study results suggest that combined analysis of BMA and BMB is useful as a routine procedure to achieve more accurate and informative diagnostic data, and this in agreement with Stifter et al. [7]. Regarding BMB infiltration by PCs, in our study, 16% of cases were focal in distribution, which is slightly lower than results confirmed by Štifter et al. [7] (20% focal), and it may have some effect on the accuracy and reliability of the PC percentage assessment in BMA. As demonstrated elsewhere, there are notable discrepancies and a relatively poor correlation in determining the percentage of BM PCs between BMA and BMB [8]. Aasen and Mckenna [9] discussed the limited sensitivity of BMA and BMB in determination of PC clonality and should do immunohistochemistry to improve morphologic impression and categorize disease.

Flow cytometry slightly underestimated PC compared with aspirate and trephine microscopy. The 10% diagnostic cutoff for MM on aspirate microscopy diagnosed only 86% of cases. However, in all cases, FC demonstrated a monoclonal population of malignant PCs. MM assessment by trephine microscopy (BMA and BMB) and FC agreed in 44/50 cases (88%), but all six discordant cases were monoclonal by FC, but their PC percentage was less than 10%. Our results were in agreement with Lee et al. [6] and Paiva et al. [4], who also discussed that FCI is more important than morphology in diagnosis of MM. In contrast, unlike morphologic and histologic studies, FC is not dependable for providing quantitative information about MM, but it provides qualitative information for assessing the immunophenotype and light chain clonality of PCs, as all cases were reported as monoclonal PCs. Our result was in agreement with Flores-Montero et al. [10]. Regarding SPEP, it diagnosed only 34 (68%) cases as monoclonal M protein, which indicates low sensitivity of SPEP in determination of monoclonality, and this was discussed by Uddin et al. [11], who demonstrated that SPEP has low sensitivity regarding monoclonality of PCs.

In the current study, the sensitivity of BMA and BMB was 86 and 90%, respectively, whereas it was 100% in FCMI in quantitation of malignant PCs in BM. Our results is in agreement with Ajise et al. [12], whose results demonstrated that FC was the highest sensitive tool in assessment of clonality [96% (79/82)], whereas morphology was positive in 84% (71/82) of cases. Moreover, their results showed that marrow morphology is a sensitive method for assessment of BM PCN burden, whereas FC is the most sensitive method to establish the clonal nature of the PCN.

The expression of some markers in MM cases

The B-lineage-associated antigen CD19 is a marker acquired early during B-cell differentiation, expressed during B-cell maturation, and still present in most normal PCs; however, it is retained in a small proportion of patients with PC myeloma. In our study, CD19 was positive in 4% of the patients, which was markedly lower than that reported by Boshnak and Hashem [13], and this may be referred to lower number of cases. Moreover, our figure was lower than that reported by Shin et al. (8.8%) [14], but similar to that reported by Mateo et al. (4%) [15] and higher than Lin et al. (1%) [16].

CD56 is an adhesion molecule that is involved in anchoring PCs to the stromal structure of the BM. The absence of CD56 has been related to malignancy in PCs, and its downregulation showed high proliferation and spreading of malignant PCs [17]. In contrast, in another study, the presence of CD56 correlates with the aggressiveness of disease in patients with myeloma [18].

The current study showed that CD56 was positive in 43 (84%) patients, which was much higher than that observed by Boshnak and Hashem (41.9% of patients) [13]. In contrast, our results were relatively similar to that observed by Shin et al. (66%) [14], Aasen and McKenna [9] (70%), and Pan et al. (71%) [17]. Such discrepancies may be attributed to differences in the sample size.


  Conclusion Top


Unlike morphologic and histologic studies, FC is not dependable for providing quantitative information about MM but provides qualitative information for assessing the immunophenotype and light chain clonality of PCs.

Both BM morphology and FC immunophenotyping are essential for diagnosis of MM. Taken together, these results support the incorporation of multiparameter flow cytometry immunophenotyping into the routine evaluation of all patients with MM at diagnosis.

Large-scale studies with larger number of cases and long-term follow up are also recommended.

Acknowledgements

The authors thank the SECI flow cytometry laboratory team for their excellent job and commitment throughout the whole period of the research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Rajkumar SV. Multiple myeloma: 2016 update on diagnosis, risk‐stratification, and management. Am J Hematol 2016; 91:719–734.  Back to cited text no. 3
    
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Paiva B, Vidriales M-B, Perez JJ, Mateo G, Montalbán MA, Mateos MV et al. Multiparameter flow cytometry quantification of bone marrow cells at diagnosis provides more prognostic information than morphological assessment in myeloma patients. Haematologica 2009; 94:1599–1602.  Back to cited text no. 4
    
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Lee N, Moon S, Lee J, Park H, Kong S, Bang S et al. Discrepancies between the percentage of plasma cells in bone marrow aspiration and BM biopsy: impact on the revised IMWG diagnostic criteria of multiple myeloma. Blood Cancer J 2017; 7:e530.  Back to cited text no. 6
    
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Štifter S, Babarović E, Valković T, Seili-Bekafigo I, Štemberger C, Načinović A et al. Combined evaluation of bone marrow aspirate and biopsy is superior in the prognosis of multiple myeloma. Diagn Pathol 2010; 5:30.  Back to cited text no. 7
    
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Uddin MM, Rahman MM, Sultana SA, Saha D. Superiority of serum immunofixation electrophoresis over serum protein electrophoresis in the diagnosis of multiple myeloma. J Bangladesh Coll Physicians Surg 2018; 36:95–100.  Back to cited text no. 11
    
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Ajise OE, Roshal M, Wang L, Sukhram GN, Smith KM, Maslak P, Dogan A. Clinical utility of morphology, immunohistochemistry, flow cytometry, and FISH analysis in monitoring of plasma cell neoplasms in the bone marrow. J Hematopathol 2016; 9:9–18.  Back to cited text no. 12
    
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Boshnak NH, Hashem AE. Association between immunophenotypic markers and cytogenetic aberrations in Egyptian patients with plasma cell myeloma. Egypt J Haematol 2017; 42:1.  Back to cited text no. 13
    
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Shin SY, Lee ST, Kim HJ, Kim SJ, Kim K, Kang ES, Kim SH. Antigen expression patterns of plasma cell myeloma: an association of cytogenetic abnormality and International Staging System (ISS) for myeloma. J Clin Lab Anal 2015; 29:505–510.  Back to cited text no. 14
    
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Mateo G, Montalban MA, Vidriales MB, Lahuerta JJ, Mateos MV, Gutiérrez N et al. Prognostic value of immunophenotyping in MM: a study by the PETHAMA/GEM cooperative study groups on patients uniformly treated with high dose theraphy. J Clin Oncol 2008; 26:2737–2743.  Back to cited text no. 15
    
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