An alternative processing approach to increase CD138 intensity in flow cytometric analysis of plasma cells.

BACKGROUND: Surface median immunofluorescence intensity (MFI) of plasma cells antigens, particularly CD138, by flow cytometry underestimates plasma cell populations when compared with that estimated by morphological assessment on Wright's-stained slides. CD138 MFI using traditional sample preparation methods for flow cytometric analysis is often dim and difficult to interpret due to multiple factors. This becomes critical when diagnosing and accurately classifying plasma cell dyscrasias. METHODS: In this study, we analyzed 280 flow cytometric results collected from 2016 to 2022 for CD38 and CD138 MFI on bone marrow aspirates performed by two different methods of sample processing-traditional method of lyse-wash and the alternative method of lyse-no-wash. RESULTS: Visual examination of histograms showed a clear advantage to CD138 expression intensity with the no-wash method. Although no significant difference was observed in CD38 MFI between the two techniques (p = 0.3), considerable improvement was observed in CD138 MFI with the lyse-no-wash technique of sample processing compared with the conventional method (p = 0.003). CONCLUSIONS: We concluded that the method of lyse-no-wash is superior to traditional methods especially when it comes to handling bone marrow aspirate samples for plasma cell immunophenotyping. This alternate technique increases the sensitivity of flow cytometry to detect plasma cells resulting in bright and crisp signal intensity for surface CD138. This technique may be particularly advantageous when analyzing low tumor burden such as minimal residual disease.

Differential Tissue Metabolic Signatures in IgG4-Related Ophthalmic Disease and Orbital Mucosa-Associated Lymphoid Tissue Lymphoma.

Purpose: To identify tissue metabolomic profiles in biopsy specimens with IgG4-related ophthalmic disease (IgG4-ROD) and mucosa-associated lymphoid tissue (MALT) lymphoma and investigate their potential implication in the disease pathogenesis and biomarkers. Methods: We conducted a comprehensive analysis of the metabolomes and lipidomes of biopsy-proven IgG4-ROD (n = 22) and orbital MALT lymphoma (n = 21) specimens and matched adjacent microscopically normal adipose tissues using liquid chromatography time-of-flight mass spectrometry. The altered metabolomic profiles were visualized by heat map and principal component analysis. Metabolic pathway analysis was performed by Metabo Analyst 4.0 using differentially expressed metabolites. The diagnostic performance of the metabolic markers was evaluated using receiver operating characteristic curves. Machine learning algorithms were implemented by random forest using the R environment. Finally, an independent set of 18 IgG4-ROD and 17 orbital MALT lymphoma specimens were used to validate the identified biomarkers. Results: The principal component analysis showed a significant difference of both IgG4-ROD and orbital MALT lymphoma for biopsy specimens and controls. Interestingly, lesions in IgG4-ROD were uniquely enriched in arachidonic metabolism, whereas those in orbital MALT lymphoma were enriched in tricarboxylic acid cycle metabolism. We identified spermine as the best discriminator between IgG4-ROD and orbital MALT lymphoma, and the area under the receiver operating characteristic curve of the spermine to discriminate between the two diseases was 0.89 (95% confidence interval, 0.803-0.984). A random forest model incorporating a panel of five metabolites showed a high area under the receiver operating characteristic curve value of 0.983 (95% confidence interval, 0.981-0.984). The results of validation revealed that four tissue metabolites: N1,N12-diacetylspermine, spermine, malate, and glycolate, had statistically significant differences between IgG4-ROD and orbital MALT lymphoma with receiver operating characteristic values from 0.708 to 0.863. Conclusions: These data revealed the characteristic differences in metabolomic profiles between IgG4-ROD and orbital MALT lymphoma, which may be useful for developing new diagnostic biomarkers and elucidating the pathogenic mechanisms of these common orbital lymphoproliferative disorders.

Aberrant Expression of Nodal and Paranodal Molecules in Neuropathy Associated With IgM Monoclonal Gammopathy With Anti-Myelin-Associated Glycoprotein Antibodies.

To clarify the pathogenesis of anti-myelin-associated glycoprotein (MAG) antibody neuropathy associated with IgM monoclonal gammopathy (anti-MAG neuropathy), sural nerve biopsy specimens from 15 patients were investigated. Sodium channels, potassium channels, contactin-associated protein 1 (Caspr1), contactin 1, and neurofascin were evaluated by immunofluorescence in teased-fiber preparations. Immunoreactivity to the pan-sodium channel in both anti-MAG neuropathy patients and in normal controls was concentrated at the node of Ranvier unless there was demyelination, which was defined as the widening of the node of Ranvier. However, this immunoreactivity became weak or disappeared as demyelination progressed. In contrast, KCNQ2 immunostaining was nearly absent even in the absence of demyelination. The lengths of Caspr1, contactin 1, and pan-neurofascin immunostaining sites at the paranode were significantly increased compared with those of normal controls despite the absence of demyelination. The length of paranodal neurofascin staining correlated with the anti-MAG antibody titer, nerve conduction indices, the frequency of de/remyelination in teased-fiber preparations, and the frequency of widely spaced myelin (p < 0.05, p < 0.05, p < 0.01, and <0.05, respectively). These findings suggest that nodal and paranodal molecular alterations occur in early stages preceding the morphological changes associated with demyelination in anti-MAG neuropathy.

An update to the pathogenesis for monoclonal gammopathy of renal significance.

Monoclonal gammopathy of renal significance (MGRS) is characterized by the nephrotoxic monoclonal immunoglobulin secreted by an otherwise asymptomatic or indolent B cell or plasma cell clone, without hematologic criteria for treatment. These MGRS-associated diseases can involve one or more renal compartments, including glomeruli, tubules, and vessels. Hydrophobic residue replacement, N-glycosylated, increase in isoelectric point in monoclonal immunoglobulin (MIg) causes it to transform from soluble form to tissue deposition, and consequently resulting in glomerular damage. In addition to MIg deposition, complement deposition is also found in C3 glomerulopathy with monoclonal glomerulopathy, which is caused by an abnormality of the alternative pathway and may involve multiple factors including complement component 3 nephritic factor, anti-complement factor auto-antibodies, or MIg which directly cleaves C3. Furthermore, inflammatory factors, growth factors, and virus infection may also participate in the development of the diseases. In this review, for the first time, we discussed current highlights in the mechanism of MGRS-related lesions.

Sporadic late-onset nemaline myopathy: Clinical spectrum, survival, and treatment outcomes.

OBJECTIVE: To describe the clinical phenotype, long-term treatment outcome, and overall survival of sporadic late-onset nemaline myopathy (SLONM) with or without a monoclonal protein (MP). METHODS: We conducted a retrospective chart review of patients seen between September 2000 and June 2017 and collected clinical, laboratory, and survival data. Treatment response was classified as mild, moderate, or marked as adjudged by predefined criteria. RESULTS: We identified 28 patients with SLONM; 17 (61%) had an associated MP. Median age at symptom onset was 62 years. Diagnosis was often delayed by a median of 35 months from symptom onset. There was no difference in clinical or laboratory features between patients with or without MP. Although the majority of patients had proximal or axial weakness at onset, about 18% of patients had atypical presentations. A total of 7/9 (78%) patients receiving IV immunoglobulin (IVIg), 6/8 (75%) receiving hematologic therapy as either autologous stem cell transplant (ASCT) or chemotherapy, and 1/8 (13%) receiving immunosuppressive therapies responded to treatment (p = 0.001). All 3 patients with marked response were treated with IVIg; 2 of them had an MP. The 5-year and 10-year overall survival from symptom onset was 92% and 68%, respectively, with no difference between patients with or without MP. CONCLUSION: SLONM has a wide spectrum of clinical presentations. In this contemporary case series, overall survival of patients did not seem to be affected by the presence of an MP. Initial treatment with IVIg is reasonable in all patients, followed by ASCT or chemotherapy as second-line therapy in patients with an associated MP.

Generalized crystal-storing histiocytosis with diffuse large B-cell lymphoma and monoclonal gammopathy in a Chinese elderly woman: a case report.

BACKGROUND: Crystal-storing histiocytosis (CSH) is a rare lesion characterized by sheets of crystal-laden non-neoplastic histiocytes. CSH shows a prominent association with lymphoproliferative disorders that express monoclonal immunoglobulins, mainly multiple myeloma (MM), lymphoplasmacytic lymphoma (LPL) and monoclonal gammopathy of undetermined significance (MGUS). However, no aggressive B cell lymphoma has been reported to be associated with CSH. CASE PRESENTATION: A 74-year-old Chinese woman presented with multiple subcutaneous masses, abdominal pain, and fever. An IgM kappa type of monoclonal gammopathy (MG) was noted by immunofixation performed on the patient's serum. Computed tomographic (CT) scan revealed subcutaneous masses on the left upper arm and at the waist and multiple low-density lesions in the spleen. Microscopically, sections of subcutaneous masses revealed sheets of large polygonal and spindle cells with abundant eosinophilic cytoplasm, round to ovoid eccentric nuclei, reticulate chromatin, and median nucleoli. Massive needle-shaped crystals were confined to the cytoplasm. Immunohistochemically, these crystal-containing cells were positive for CD68/PGM1, CD163, IgM, and Igκ. Meanwhile, the splenic tumour was diagnosed as diffuse large B-cell lymphoma (DLBCL), non-germinal-centre B (non-GCB) subtype (Hans algorithm). Immunohistochemistry for IgM was positive in the cytoplasm of some neoplastic cells. Immunoglobulin heavy chain (IgH) gene rearrangement was detected by PCR analysis of the subcutaneous mass and the splenic tumour. CONCLUSION: To the best of our knowledge, this is the first case of generalized CSH with DLBCL and MG. Although the rarity of CSH and separate locations of CSH and lymphoma led to a diagnostic dilemma, the presence of MG was a clue to appreciate the relation between CSH and DLBCL. This case stressed a full investigation into the underlying lymphoproliferative disorder for integrated diagnosis and correct treatments.

Noninvasive assessment of corneal alterations associated with monoclonal gammopathy.

Aggregation of monoclonal immunoglobulin can lead to organ damage. However, the necessity of invasive examination such as biopsy has hampered better understanding of the pathophysiology. Corneal crystalline deposition is a rarely reported but known ocular manifestation of multiple myeloma. It is unclear whether the cornea is a common target of monoclonal immunoglobulin deposition. We conducted a prospective clinical case-control study to objectively quantify monoclonal gammopathy-associated corneal changes as well as any therapeutic response. Using an ophthalmic Scheimpflug camera imaging for noninvasive corneal assessments, we quantified densitometry values in 30 patients. Although none had crystalline keratopathy, corneal transparency in monoclonal gammopathy patients was significantly impaired compared to that in age-matched controls, based on noninvasive Scheimpflug camera imaging. Furthermore, treatment for multiple myeloma seemed to eradicate the diffuse aggregation of monoclonal proteins. Our results indicate that exposure to monoclonal immunoglobulin may induce the accumulation of monoclonal immunoglobulin in the cornea, and ophthalmic examinations such as corneal densitometry measurements with a Scheimpflug camera may be useful for noninvasive evaluation of monoclonal immunoglobulin deposition diseases.

Monoclonal Immunoglobulin Deposition Disease and Related Diseases.

Abnormal proliferation of plasma cells and some monoclonal B cells frequently cause the secretion of monoclonal immunoglobulins or immunoglobulin fragments into the serum, causing monoclonal gammopathy, which leads to various diseases including renal diseases. Therefore, monoclonal gammopathy is frequently associated with kidney diseases, including glomerular and tubulointerstitial diseases. Glomerular disease, with the deposition of monoclonal immunoglobulins or their components, includes monoclonal immunoglobulin deposition disease (MIDD), AL or AH amyloidosis, type I cryoglobulinemia, proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID), immunotactoid glomerulopathy, and fibrillary glomerulonephritis. In addition, tubulointerstitial diseases with the deposition of monoclonal immunoglobulins or their components are constituted by light chain (myeloma) cast nephropathy, light chain associated Fanconi's syndrome (light chain proximal [crystal] tubulopathy), and crystal-storing histiocytosis. In the present review article, we demonstrate the clinicopathological characteristics of MIDD, which is one of the representative diseases of plasma cell dyscrasias, and discuss various renal diseases with the deposition of monoclonal immunoglobulins or their components in glomeruli and the tubulointerstitium. We recommend that these renal diseases are arranged as one disease category, "renal diseases with deposition of monoclonal immunoglobulins or their components", in order to simplify the understanding of complicated diseases in plasma cell dysplasia.

Multiparameter Flow Cytometry Identification of Neoplastic Subclones: A New Biomarker in Monoclonal Gammopathy of Undetermined Significance and Multiple Myeloma.

Multiparameter flow cytometry (MFC)-based clonality assessment is a powerful method of diagnosis and follow-up in monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). However, the relevance of intraclonal heterogeneity in immunophenotypic studies remains poorly understood. The main objective of this work was to characterize the different immunophenotypic subclones in MGUS and MM patients and to investigate their correlation with disease stages. An 8-color MFC protocol with 17 markers was used to identify the subclones within the neoplastic compartment of 56 MGUS subjects, 151 newly diagnosed MM patients, 30 MM subjects in complete remission with detectable minimal residual disease, and 36 relapsed/refractory MM patients. Two or more clusters were observed in > 85% of MGUS subjects, 75% of stage I MM patients, and < 15% in stage III. Likewise, a significant correlation between the dominant subclone size, secondary cytogenetic features, and changes in the expression of CD27, CD44, and CD81 was detected. The loss of intraclonal equilibrium may be an important factor related with kinetics and risk of progression not well considered to date in MFC studies. The MFC strategy used in this work can provide useful biomarkers in MGUS and MM.

κ Light-chain monoclonal gammopathy and cast nephropathy in a horse with multiple myeloma.

CASE DESCRIPTION A 27-year-old Dutch Warmblood mare was evaluated because of a history of lethargy, reluctance to move, weight loss, persistent hyperproteinemia, and recurrent episodes of mild lameness. CLINICAL FINDINGS Hematologic evaluation revealed anemia (RBC concentration, 3.84 × 106 cells/µL), thrombocytopenia (47 × 103 thrombocytes/µL), and hyperproteinemia (total protein concentration, 11.2 g/dL) with hyperglobulinemia and hypoalbuminemia. Results of protein electrophoresis of serum and urine samples indicated a monoclonal gammopathy; the paraprotein was identified as a κ light chain. On abdominal ultrasonographic examination, hypoechoic nodules were visualized in the spleen. Results of cytologic examination of a splenic fine-needle aspirate and histologic examination of a bone marrow biopsy sample were consistent with plasma cell myeloma. TREATMENT AND OUTCOME Treatment was declined owing to the age of the horse and poor prognosis. The horse was discharged from the hospital, and the owner was given palliative care instructions. The horse was euthanized 2 weeks later because of recurrent episodes of lethargy, anorexia, and signs of colic. Necropsy confirmed the diagnosis of multiple (plasma cell) myeloma. Plasma cell aggregates in the liver, spleen, bone marrow, and kidney and the presence of cast nephropathy were identified on histologic examination. CLINICAL RELEVANCE Multiple myeloma is rarely reported in horses. A monoclonal peak on serum protein electrophoresis should raise the suspicion of neoplasia, specifically multiple myeloma. The findings for this patient confirmed the importance of considering neoplasia in horses with nonspecific clinical signs.

IL-10 inducible CD8+ regulatory T-cells are enriched in patients with multiple myeloma and impact the generation of antigen-specific T-cells.

Tumor-mediated immunosuppression via regulatory T-cells is a key player among the various immune-escape mechanisms in multiple myeloma. We analyzed the generation, distribution, function and immunophenotype of CD8+CD28- regulatory T-cells in patients with multiple myeloma. Functionality of CD8+CD28- T-cells was assessed by immunological assays using ex vivo generated antigen-specific T-cells from patients with plasma cell dyscrasias and healthy donors. Detailed analysis of distribution, immunophenotype and cytotoxic potential of CD8+CD28- T-cells was performed by flow cytometry and ELISA. We found that the amount of CD8+CD28- T-cells was directly correlated with the suppression of antigen-specific T-cell responses in patients with plasma cell dyscrasia. Analyzing the CD8+CD28- T-cells in detail, increased numbers of these cells were observed in the bone marrow (i.e., tumor microenvironment) of patients with plasma cell dyscrasia. Furthermore, we identified the expression of lymphocyte function-associated antigen 1 (LFA-1) as a marker of immunosuppression and defined the CD8+CD28-CD57+LFA-1high population as the relevant immunosuppressive compartment. These regulatory T-cells act as immunosuppressors via soluble factors and incubation with IL-10 augmented their immunosuppressive capacity. The immunosuppressive regulatory network of IL-10 and the CD8+CD28-CD57+LFA-1high regulatory T-cells show unique characteristics and contribute to the tumor immune escape mechanism in patients with multiple myeloma.

Paraproteinaemia in Primary Cutaneous Marginal Zone Lymphoma.

Primary cutaneous marginal zone lymphomas (PCMZL) frequently exhibit lymphoplasmacytoid/plasmacytic differentiation, implying the capacity to produce monoclonal immunoglobulins. As these paraproteins are secreted, and thus are measurable in blood and urine, they may correlate with disease burden and serve as tumour markers reflecting therapeutic response. This study retrospectively analysed the records of 23 patients with PCMZL. During treatment and follow-up, laboratory tests, including full blood count, lactate dehydrogenase, serum protein electrophoresis and turbidimetric analyses, were conducted. Thirty-nine percent of cases showed a suspicious serum protein electrophoresis in terms of paraproteinaemia. In 44% of cases the heavy and light chain restriction in tissue samples correlated with serological findings. Altogether, 89% of the PCMZL patients with paraproteinaemia eventually experienced a relapse, in contrast to 62% of the group without paraproteinaemia. This study analysed the incidence and clinical implications of paraproteinaemia in patients with PCMZL. A clear correlation was found between paraproteinaemia, tumour relapse and therapeutic intervention.

Immunochemical Diagnosis of Multiple Myeloma.

The diagnostic potentialities of complex immunochemical analysis of the serum and daily urine were evaluated in 118 patients with multiple myeloma. In 95 patients, we observed secretion of monoclonal intact immunoglobulins with heavy chains G (N=69), A (N=19), and M (N=4) and biclonal secretion of paraproteins G and A (N=3). Bence-Jones protein was detected in the sera and daily urine of 16 patients and Bence-Jones proteinuria alone was detected in 3 patients. The diagnostic sensitivity of serum immunoelectrophoresis in multiple myeloma is 94.1%. Analysis of paraproteinuria is particularly important in Bence-Jones myeloma, when paraprotein excretion may be not associated with paraproteinemia. Complex study by immunoelectrophoretic and immunoturbidimetric methods in multiple myeloma increases the diagnostic sensitivity to 99.2%.

A functional assay to identify amyloidogenic light chains.

INTRODUCTION: Multiple myeloma (MM) and light chain monoclonal gammopathy of undetermined significance (LCMGUS) are plasma cell disorders associated with the secretion of monoclonal free light-chain (LC) proteins. Due to the high concentrations of LC in circulation, both of these populations are at risk for developing LC-associated amyloidosis (AL) - a protein misfolding disease characterized by the deposition of LC protein fibrils in organs and tissues, leading to dysfunction and significant morbidity. At present, accurate identification of subjects at risk for developing amyloidosis is not possible, but with the advent of novel, amyloid-targeted therapies, identification of pre-symptomatic individuals is of clinical import. METHODS: To address this, a competition assay has been developed to discern LC proteins with enhanced amyloidogenic potential. Numerous factors that may influence the efficacy of the assay have been evaluated to yield optimal conditions. RESULTS: Using a panel of nine patient-derived LC, we have demonstrated that amyloid-associated LC inhibited the recruitment of a biotinyl-λ6 variable domain by homologous amyloid-like fibrils significantly more than MM LC (p < .01). CONCLUSION: The assay accurately discriminated AL from MM patient populations, suggesting that it may aid in the identification of patients with monoclonal gammopathies who have an increased risk of developing amyloidosis.

Evaluation of CD229 as a new alternative plasma cell gating marker in the flow cytometric immunophenotyping of monoclonal gammopathies.

BACKGROUND: Current flow-cytometric plasma cell (PC) gating is based on CD138, CD38, and CD45 expression. CD138 is known for variable expression and loss during storage and processing. Introduction of anti-CD38 and anti-CD138 monoclonal-antibody therapies has limited the use of these markers during follow-up. Hence, additional reliable PC-gating markers are required. Recently, CD229 has been claimed as an alternative PC-gating marker. However, these studies are limited to a small cohort of samples. We evaluated the utility of CD229 as a new PC-gating marker in routine laboratory practice. METHODS: Expression of CD229 was studied in 310 bone marrow (BM) samples (251 plasma-cell disorders and 59 controls) and compared with CD138 and CD38 expression. We also evaluated the effect of additional processing for cytoplasmic immunoglobulin-light-chains (CyIgL) staining on the quantitation of PC. RESULTS: Expression of CD229 was consistently stronger on PC than other hematopoietic-cells (p < 0.001). PC-percentages using CD229 in combination with CD38 or CD138 and CD45 revealed high correlation with a reference gating-strategy using CD138, CD38 and CD45 (r = 0.98, r = 0.99 r = 0.99 respectively) and r = 0.92 using CD229 and CD45 without CD38 or CD138. In contrast, CD138 expression showed significant variability (CV-MFI, 97.5) and loss from PC in 53% of samples. Quantitation of PC was found to be lower in 69.3% and higher in 30.7% samples processed for CyIgL-staining as compared to surface-staining. CONCLUSIONS: CD229 is a reliable new alternative PC-gating marker in routine laboratory practice. Quantitation of PC based on CD138 expression or from samples processed for CyIgL-staining should be used with caution. © 2018 International Clinical Cytometry Society.

Flow cytometric aberrancies in plasma cell myeloma and MGUS - correlation with laboratory parameters.

BACKGROUND: Multiparametric flow cytometry is a useful tool for diagnosis of plasma cell (PC) dyscrasias and assessment of minimal residual disease in plasma cell myeloma (PCM). However, the immunophenotypic differences between the clonal PCs of PCM and those of monoclonal gammopathy of undetermined significance (MGUS) as well as the correlation of these flow cytometric markers with pertinent laboratory parameters have not been evaluated. METHODS: We retrospectively identified all newly diagnosed treatment-naive PCM and MGUS patients between 09/2014 and 06/2015 who underwent 10-color flow-cytometric evaluation: CD45, CD38, CD138, cKappa, cLambda, CD19, CD27, CD28, CD56, CD117. FACSDiva analysis was used to identify antigenic aberrancies and associations with pertinent laboratory parameters were evaluated. RESULTS: All cases demonstrated at least two aberrancies. There was a trend toward a greater number of aberrancies in PCM, with 68% showing >/= 4 aberrancies compared with 44% in MGUS (P = 0.11). The only marker more frequently aberrant in one disease class was CD19, aberrant in 68% of PCM and 25% of MGUS (P < 0.01). In PCM, significant associations were found for CD56 non-aberrancy (P = 0.05) and the presence of amyloid and CD27 aberrancy and normal serum albumin (P = 0.05). In MGUS, CD117 expression was associated with normal hemoglobin (P = 0.03). CONCLUSIONS: The PCs of PCM show a trend toward more antigenic aberrancy than those of MGUS. There is significant association between the antigenic profiles of PCM/MGUS and clinical parameters including amyloidosis, albumin level, and hemoglobin. © 2018 International Clinical Cytometry Society.