Immunoglobulin Kappa Light Chain Research Articles

A clonality assay in canine B cell tumors targeting the immunoglobulin light chain lambda locus

Clonality assays for antigen receptor rearrangement have been used as adjunct examinations of lymphoproliferative diseases. These assays have been useful for differentiation between inflammation and clonal expansion of lymphocytes. Whereas the immunoglobulin heavy chain (IGH) and immunoglobulin light chain kappa (IGK) loci have been targeted in canine clonality assays previously, the immunoglobulin light chain lambda gene (IGL) locus has not yet been investigated. This study aimed to evaluate the usefulness of clonality assays in dogs using IGL. Canine diffuse large B cell lymphomas (DLBCL), cutaneous plasmacytomas, and pathologically diagnosed lymph nodes without lymphoma, were used in this study. Genomic DNA was extracted from formalin-fixed paraffin embedded sections. Sequences of IGLV and IGLJ were obtained from the ImMunoGeneTics database. Several primers against IGLVs and IGLJs were designed in the regions showing homology, by alignment of the gene segments. Products of polymerase chain reaction were analyzed on a capillary electrophoresis. In total, 20 of 23 cases of DLBCL showed clonality (87.0 %), whereas 8 of 30 cutaneous plasmacytomas were clonal (26.7 %). One of 23 lymph nodes without lymphoma showed clonality, thus the specificity was 95.7 %. These data indicate that the IGL locus could be a target for canine clonality assays and that the sensitivity of IGL-based clonality assays in cutaneous plasmacytomas was lower than that in DLBCL.

Cutaneous Ossifying Amyloidoma

Amyloidoma, otherwise known as tumoral amyloidosis, is a localized deposition of amyloid (AL-type or AA type) without systemic amyloidosis. It is the rarest form of tissue amyloid deposition, and up to 7% of amyloidomas develop systemic amyloidosis.Cutaneous AL-type amyloidoma is considered by many authors as an unusual variant of primary cutaneous marginal zone lymphoma. Although cutaneous amyloidoma can form calcifications, ossification is extremely unusual, with only 1 case previously published to date.We report the case of a 75-year-old woman with voluminous and strikingly ossifying AL-type amyloidoma in the left pretibial skin. Her medical history included excision of hepatic hydatidic cysts 25 years prior and diffuse large B-cell lymphoma of the left parotid gland 8 years prior treated with chemotherapy and radiotherapy with complete response. After the diagnosis of amyloidoma, an extension study with cervical, chest, abdominal, and pelvic TC was performed, with no additional lesions found. Serum and protein electrophoresis revealed elevations in kappa light chain and IgA immunoglobulin levels but did not reveal monoclonal bands. In situ hybridization for immunoglobulin light chains showed monotypic kappa expression in plasma cells infiltrating the amyloidoma.Extensive ossification in amyloidomas can make diagnosis difficult; therefore, we describe an interesting case of this histopathologically peculiar amyloidoma.

Clinical and pathologic features of primary membranous nephropathy in Turkey: a multicenter study by the Turkish Society of Nephrology Glomerular Diseases Working Group

Background We aimed to evaluate the features of primary membranous nephropathy (MNP) in Turkish people. Methods This is a retrospective analysis of patients with biopsy-proven primary MNP. We obtained the data collected between 2009 and 2019 in the primary glomerulonephritis registry of the Turkish Society of Nephrology Glomerular Diseases Study Group (TSN-GOLD). Patients with a secondary cause for MNP were excluded. Clinical, demographic, laboratory, and histopathological findings were analyzed. Results A total of 995 patients with primary MNP were included in the analyses. Males constituted the majority (58.8%). The mean age was 48.4 ± 13.9 years. The most common presentation was the presence of nephrotic syndrome (81.7%) and sub nephrotic proteinuria (10.3%). Microscopic hematuria was detected in one-third of patients. The median estimated glomerular filtration rate (eGFR) was 100.6 mL/min/1.73 m2 (IQR, 75.4–116.3), and median proteinuria was 6000 mg/d (IQR, 3656–9457). Serum C3 and C4 complement levels were decreased in 3.7 and 1.7% of patients, respectively. Twenty-four (2.4%) patients had glomerular crescents in their kidney biopsy samples. Basal membrane thickening was detected in 93.8% of cases under light microscopy. Mesangial proliferation and interstitial inflammation were evident in 32.8 and 55.9% of the patients, respectively. The most commonly detected depositions were IgG (93%), C3 complement (68.8%), and kappa and lambda immunoglobulin light chains (70%). Although renal functions were normal at presentation, vascular, interstitial, and glomerular findings were more prominent on biopsy in hypertensive patients. No significant effect of BMI on biopsy findings was observed. Conclusions Despite some atypical findings, the main features of primary MNP in Turkey were similar to the published literature. This is the largest MNP study to date conducted in Turkish people.

Multiple Myeloma Presenting with Multiple Thoracic Manifestations

Multiple myeloma is a malignant proliferation of plasma cells that affects mainly bone marrow but may also involve other organs as well. We report thoracic involvement in the form of left-sided pleural effusion, osseous lesions, bronchial infiltration, and mediastinal lymphadenopathy in a 61-year-old woman, non-smoker presented with chest pain, dyspnoea, cough and deterioration in general health over the preceding seven months. Immunoelectrophoresis and immunofixation showed raised kappa-light chain immunoglobulin G (IgG) in serum and pleural fluid. Bronchial and pleural biopsies documented myelomatous infiltration and bone marrow aspirate revealed extensive plasma cell infiltration. At eight months, following the fourth cycle of melphalan, endoxan and prednisone based chemotherapy, the patient died.

Differences between tumor clones of B lymphocytes with restriction of kappa or lambda immunoglobulin light chains in patients with chronic lymphocytic leukemia.

e19520 Background: The purpose of this study was to compare morphological and immunophenotypic characteristics of the tumor population of B-lymphocytes with varying restrictions of immunoglobulin light chains in patients with chronic lymphocytic leukemia (CLL). Methods: We examined 30 patients with CLL (median age 64.9±8.6 years). All patients underwent CBC+DIFF blood tests (Sysmex XE 2100, Japan), morphological examinations (BioVision; Micros, Austria), and immunophenotyping (IPT) by multicolor flow cytometry (Navios 10/3, Beckman Coulter, USA) of the bone marrow and venous blood. According to the IPT results, the patients were divided into: group 1 - 22 patients (73.3%) with tumor cells expressing kappa immunoglobulin light chains, and group 2 - 8 patients (26.7%) with lambda immunoglobulin light chains. Statistical processing of results was performed in the STATISTICA 13.0 program. Results: Morphological tests revealed differences between tumor populations of B lymphocytes. In group 1, the tumor population was represented by small cells of the same type with scanty, often unvisible cytoplasm and nuclei with a lumpy chromatin structure, without distinct nucleoli. In group 2, the sizes of cells varied from small to large, with abundant cytoplasm, round or folded nuclei, smoothed chromatin structure and 1-2 nucleoli. Prolymphocytes were observed among tumor cells (8.1±1.7% of WBC). IPT also revealed differences in tumor clones: morphological uniformity of tumor cells in group 1 was observed in the distribution of tumor cells reflected in low parameters of light scattering on the diagram: location to the left along the FSC axis - from 200 to 400 units and below along the SSC axis - from 10 to 160 units. In group 2, the lymphoid zone was heterogeneous and stretched on the plot: location to the right along the FSC axis - from 200 to 1000 units and higher along the SSC axis - from 10 to 400 units, closer to the monocyte zone, which indicated morphological polymorphism of tumor cells. The expression of CD45 also differed: in group 1, the expression was higher and tumor B lymphocytes were higher by fluorescence intensity on a dot plot on the CD45 / SSC scale in the second half of the third decade and in the fourth decade - to the right compared with group 2, where aberrant B lymphocytes were in the third decade and more to the left. The CD45 expression allowed defining differentiation of the tumor clone: the population in group 1 was represented by mature cells, and in group 2 by less mature and/or intermediate forms. There were no significant differences in the expression of other markers. The revealed differences were typical of both the peripheral blood and bone marrow. Conclusions: The study established morphological and immunophenotypic differences between tumor clones of B lymphocytes expressing either kappa or lambda immunoglobulin light chains in patients with CLL.

Clonality assessment and detection of clonal diversity in classic Hodgkin lymphoma by next-generation sequencing of immunoglobulin gene rearrangements

AbstractClonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.

Ultrasensitive RNA In Situ Hybridization for Kappa and Lambda Light Chains Assists in the Differential Diagnosis of Nodular Lymphocyte-predominant Hodgkin Lymphoma.

Establishing a diagnosis of nodular lymphocyte-predominant Hodgkin lymphoma (nLPHL) is often challenging as the differential diagnosis is broad, including classic Hodgkin lymphoma (cHL), progressive transformation of germinal centers (PTGC), and other lymphoproliferative disorders. In this study, we investigate the utility of a recently described ultrasensitive in situ hybridization assay for kappa and lambda immunoglobulin light chains in distinguishing nLPHL, cHL, and PTGC. A total of 72 cases were examined (21 nLPHL, 33 cHL, and 18 PTGC). In nLPHL, the large neoplastic cells were light chain restricted in 21/21 (100%) cases (16 kappa, 5 lambda). In contrast, Reed-Sternberg cells of cHL were negative for kappa and lambda in all cases (0/33, 0%; P <0.001). In PTGC, polytypic B cells were noted in mantle zones and germinal centers in all cases, with 1 case (5%) also showing focal collections of light chain restricted large B cells. Background monotypic small B cells were identified in 3 cases, including 1 nLPHL and 2 cHL (1 of which arose in chronic lymphocytic leukemia). Ultrasensitive in situ hybridization for kappa and lambda is a useful addition to a standard immunophenotyping panel for the evaluation of suspected nLPHL.

Physiology, Diagnosis and Treatment of Cardiac Light Chain Amyloidosis.

AL (light-chain) amyloidosis is a systemic disease in which amyloid fibers are formed from kappa or lambda immunoglobulin light chains, or fragments thereof, produced by a neoplastic clone of plasmocytes. The produced protein is deposited in tissues and organs in the form of extracellular deposits, which leads to impairment of their functions and, consequently, to death. Despite the development of research on pathogenesis and therapy, the mortality rate of patients with late diagnosed amyloidosis is 30%. The diagnosis is delayed due to the complex clinical picture and the slow progression of the disease. This is the type of amyloidosis that most often contributes to cardiac lesions and causes cardiac amyloidosis (CA). Early diagnosis and correct identification of the type of amyloid plays a crucial role in the planning and effectiveness of therapy. In addition to standard histological studies based on Congo red staining, diagnostics are enriched by tests to determine the degree of cardiac involvement. In this paper, we discuss current diagnostic methods used in cardiac light chain amyloidosis and the latest therapies that contribute to an improved patient prognosis.

Next-Generation Sequencing-Based Clonality Detection of Immunoglobulin Gene Rearrangements in B-Cell Lymphoma.

Immunoglobulin (IG) clonality assessment is a widely used supplementary test for the diagnosis of suspected lymphoid malignancies. The specific rearrangements of the immunoglobulin (IG) heavy and light chain genes act as a unique hallmark of a B-cell lymphoma, a feature that is used in clonality assessment. The widely used BIOMED-2/EuroClonality IG clonality assay, visualized by GeneScanning or heteroduplex analysis, has an unprecedented high detection rate because of the complementarity of this approach. However, the BIOMED-2/EuroClonality clonality assays have been developed for the assessment of specimens with optimal DNA quality. Further improvements for the assessment of samples with suboptimal DNA quality, such as from formalin-fixed paraffin-embedded (FFPE) specimens or specimens with a limited tumor burden, are required. The EuroClonality-NGS Working Group recently developed a next-generation sequencing (NGS)-based clonality assay for the detection of the IG heavy and kappa light chain rearrangements, using the same complementary approach as in the conventional assay. By employing next-generation sequencing, both the sensitivity and specificity of the clonality assay have increased, which not only is very useful for diagnostic clonality testing but also allows robust comparison of clonality patterns in a patient with multiple lymphoma's that have suboptimal DNA quality. Here, we describe the protocols for IG-NGS clonality assessment that are compatible for Ion Torrent and Illumina sequencing platforms including pre-analytical DNA isolation, the analytical phase, and the post-analytical data analysis.

Engineered mRNA-expressed bispecific antibody prevent intestinal cancer via lipid nanoparticle delivery

ABSTRACT The potential of antibodies, especially for the bispecific antibodies, are limited by high cost and complex technical process of development and manufacturing. A cost-effective and rapid platform for the endogenous antibodies expression via using the in vitro transcription (IVT) technique to produce nucleoside-modified mRNA and then encapsulated into lipid nanoparticle (LNP) may turn the body to a manufactory. Coinhibitory pathway of programmed death ligand 1 (PD-L1) and programmed cell death protein 1 receptor (PD-1) could suppress the T-cell mediated immunity. We hypothesized that the coblocking of PD-L1 and PD-1 via bispecific antibodies may achieve more potential antitumor efficacies compare with the monospecific ones. Here, we described the application of mRNA to encode a bispecific antibody with ablated Fc immune effector functions that targets both human PD-L1 and PD-1, termed XA-1, which was further assessed the in vitro functional activities and in vivo antitumor efficacies. The in vitro mRNA-encoded XA-1 held comparable abilities to fully block the PD-1/PD-L1 pathway as well as to enhance functional T cell activation compared to XA-1 protein from CHO cell source. Pharmacokinetic tests showed enhanced area under curve (AUC) of mRNA-encoded XA-1 compared with XA-1 at same dose. Chronic treatment of LNP-encapsulated XA-1 mRNA in the mouse tumor models which were reconstituted with human immune cells effectively induced promising antitumor efficacies compared to XA-1 protein. Current results collectively demonstrated that LNP-encapsulated mRNA represents the viable delivery platform for treating cancer and hold potential to be applied in the treatment of many diseases. Abbreviations: IVT: in vitro transcription; LNP: lipid nanoparticle; hPD-1: human PD-1; hPD-L1: human PD-L1; ITS-G: Insulin-Transferrin-Selenium; Pen/Strep: penicillin-streptomycin; FBS: fetal bovine serum; TGI: tumor growth inhibition; IE1: cytomegalovirus immediate early 1; SP: signal peptide; hIgLC: human immunoglobulin kappa light chain; hIgHC: human IgG1 heavy chain; AUC: area under the curve; Cl: serum clearance; Vss: steady-state distributed volume; MLR: mixed lymphocyte reaction.

The characteristics of patients with kidney light chain deposition disease concurrent with light chain amyloidosis

The type of monoclonal light chain nephropathy is thought to be largely a function of the structural and physiochemical properties of light chains; hence most affected patients have only one light chain kidney disease type. Here, we report the first series of kidney light chain deposition disease (LCDD) concomitant with light chain amyloidosis (LCDD+AL), with or without light chain cast nephropathy (LCCN). Our LCDD+AL cohort consisted of 37 patients (54% females, median age 70 years (range 40-86)). All cases showed Congo red-positive amyloid deposits staining for one light chain isotype on immunofluorescence (62% lambda), and LCDD with diffuse linear staining of glomerular and tubular basement membranes for one light chain isotype (97% same isotype as the amyloidogenic light chain) and ultrastructural non-fibrillar punctate deposits. Twelve of 37 cases (about 1/3 of patients) had concomitant LCCN of same light chain isotype. Proteomic analysis of amyloid and/or LCDD deposits in eight revealed a single light chain variable domain mutable subgroup in all cases (including three with separate microdissections of LCDD and amyloid light chain deposits). Clinical data on 21 patients showed proteinuria (100%), hematuria (75%), kidney insufficiency and nephrotic syndrome (55%). Extra-kidney involvement was present in 43% of the patients. Multiple myeloma occurred in 68% (about 2/3) of these patients; none had lymphoma. On follow up (median 16 months), 63% developed kidney failure and 56% died. The median kidney and patient survivals were 12 and 32 months, respectively. LCDD+AL mainly affected patients 60 years of age or older. Thus, LCDD+AL could be caused by two pathological light chains produced by subclones stemming from one immunoglobulin light chain lambda or kappa rearrangement, with a distinct mutated complementary determining region.

Pathological review of cardiac amyloidosis using autopsy cases in a single Japanese institution

AimAmyloidosis is a systemic or localized disease of protein deposition characterized by amorphous eosinophilic morphology and positivity of Congo Red staining. The typing of amyloidosis is becoming increasingly important because therapeutic agents for each amyloidosis type have been developed. Herein, the authors review the autopsy cases at an institution to reveal the putative Japanese characteristics of each amyloidosis type and evaluate the clinicopathological significance of each type. Materials and methods: A total of 131 autopsy cases of systemic and localized amyloidosis were retrieved for classification by immunohistochemistry. Immunohistochemistry for transthyretin, amyloid A (AA), immunoglobulin light-chain kappa and lambda, and β2-microglobulin was performed for all cases. ResultsThe 131 amyloidosis cases were classified as follows: 71 cases (54.2%) of transthyretin amyloidosis, 32 cases (24.4%) of AA amyloidosis, 8 cases (6.1%) of light-chain amyloidosis, and 5 cases (3.8%) of β2-microglobulin amyloidosis, along with 15 equivocal cases (11.5%). All cases showed myocardial involvement of amyloidosis. Histopathologically, the transthyretin type was significantly associated with the interstitial and nodular patterns, and with the absence of the perivascular and endocardial patterns. The AA type was significantly associated with the perivascular and endocardial patterns, and with the absence of the nodular pattern. ConclusionThe authors revealed the putative characteristics of cardiac amyloidosis in Japan by using autopsy cases. About 90% of amyloidosis cases were successfully classified using only commercially available antibodies.

Chimeric Antigen Receptor T Cell Therapy and Its Significance in Multiple Myeloma.

Multiple myeloma (MM) has a five-year prevalence worldwide of 230,000 people and is known as the second most common hematological malignancy within the United States. Extensive research has been conducted to gain a wide range of treatment strategies, providing hope to these patients. Combination therapy using chemotherapy, monoclonal antibodies, and immunomodulatory drugs are the current management of choice. After the introduction of chimeric antigen receptor (CAR) T cell therapy, promising results have been evidenced. In this therapy, T cells are derived from the patient and modified in-vitro to induce receptors that later target specific antigens when they are injected into patients. CAR T cells use three mechanisms to kill tumor cells: cytolytic pathways, cytokine release, and Fas/FasL axis. In this review, we highlight the different tumor markers targeted for therapy against multiple myeloma (MM). Target antigens for CAR T cell therapy include B-cell maturation antigen (BCMA), signaling lymphocyte activation molecule F7 (SLAMF7), CD38, CD138, CD19, immunoglobulin kappa light chain, orphan G protein-coupled receptor class C group 5 member D (GPRC5D). With the benefit of improving survival and prognosis, this therapy does carry a risk of some adverse events such as cytokine release syndrome, encephalopathy, infections, hypogammaglobulinemia, and tumor lysis syndrome.

Immunoglobulin gene rearrangement in Koreans with multiple myeloma: Clonality assessment and repertoire analysis using next-generation sequencing.

IntroductionWe assessed the applicability of next-generation sequencing (NGS)-based IGH/IGK clonality testing and analyzed the repertoire of immunoglobulin heavy chain (IGH) or immunoglobulin kappa light chain (IGK) gene usage in Korean patients with multiple myeloma (MM) for the first time.MethodsFifty-nine bone marrow samples from 57 Korean patients with MM were analyzed, and NGS-based clonality testing that targeted the IGH and IGK genes was performed using IGH FR1 and IGK primer sets.ResultsClonal IGH and IGK rearrangements were observed in 74.2% and 67.7% of samples from Korean patients with kappa-restricted MM, respectively (90.3% had one or both), and in 60.7% and 95.5% of samples from those with lambda-restricted MM, respectively (85.7% had one or both). In total, 88.1% of samples from Koreans with MM had clonal IGH and/or IGK rearrangement. Clonal rearrangement was not significantly associated with the bone marrow plasma cells as a proportion of all BM lymphoid cells. IGHV3-9 (11.63%) and IGHV4-31 (9.30%) were the most frequently reported IGHV genes and were more common in Koreans with MM than in Western counterparts. IGHD3-10 and IGHD3-3 (13.95% each) were the most frequent IGHD genes; IGHD3-3 was more common in Koreans with MM. No IGK rearrangement was particularly prevalent, but single IGKV-J rearrangements were less common in Koreans with kappa-restricted MM than in Western counterparts. IGKV4-1 was less frequent in Koreans regardless of light chain type. Otherwise, the usages of the IGH V, D, and J genes and of the IGK gene were like those observed in previous Western studies.ConclusionNGS-based IGH/IGK clonality testing ought to be applicable to most Koreans with MM. The overrepresentation of IGHV3-9, IGHV4-31, and IGHD3-3 along with the underrepresentation of IGKV4-1 and the differences in IGK gene rearrangement types suggest the existence of ethnicity-specific variations in this disease.

Issue Highlights — May 2021

Issue Highlights — May 2021

IgG4-positive plasma cells are more often detected in chronic periapical lesions arising from permanent rather than primary teeth.

To characterize plasma cell subsets in chronic periapical lesions affecting permanent and primary teeth. Only chronic periapical lesions without root canal treatment were selected. Twenty-one radicular cysts and 7 periapical granulomas affecting permanent teeth and 19 radicular cysts and 4 periapical granulomas affecting primary teeth were assessed for immunoglobulin (Ig) light chain (kappa and lambda), Ig heavy chain (IgG, IgG4, IgA, IgM and IgD) and plasma cell immunohistochemical markers (MUM1/IRF4, EMA and CD138). The data acquired were analysed by Student's t test, Mann-Whitney U, Friedman test followed by Dunn's multiple comparison test and Spearman's rank correlation. All cases were polyclonal (having similar kappa/lambda light chain ratios). IgG was most abundant compared to other Ig heavy chains (all, P<0.001); like Ig light chains, but unlike IgA, there was greater expression of IgG in the primary compared to the permanent dentition, for both radicular cysts (P<0.001) and periapical granulomas (P=0.53). Notably, IgG4 expression was greater in the permanent than the primary dentition, for both radicular cyst (P<0.05) and periapical granuloma (P=0.65). IgM and IgD expression was scarce and variable, whereas plasma cell populations were detected efficiently through EMA, CD138 and MUM1/IRF4 markers, the latter being more sensitive in both dentitions. There were slight variations in the Ig light and heavy chain profiles in chronic periapical lesions when comparing the permanent and primary dentitions. The ability of IgG4+ plasma cell infiltration to modulate inflammatory responses in chronic periapical lesions arising from permanent as opposed to primary teeth should be considered in future studies.

Autopsy findings in a patient with primary systemic AL (kappa light chain) amyloidosis.

ABSTRACTFirst described by Rokitansky in 1842, and further characterized by Virchow in 1854, amyloidosis is a disorder caused by amyloid deposition, a fibrillary insoluble protein. The clinical spectrum of amyloidosis is broad, as the amyloid deposition may virtually occur in all tissues. Herein, we report the case of a 66-year-old man with a long-lasting emaciating disease, diagnosed, at autopsy, with primary systemic amyloidosis. Amyloid protein deposition was found in many tissues and organs. The involvement of the vessels’ wall rendered ischemic injury most prominent in the intestinal loops causing mesenteric ischemia. Despite the thorough organic involvement, the immediate cause of death was aspiration bronchopneumonia. Massive amyloid deposition was found in virtually all major organs, such as the heart, liver, kidneys, spleen, pancreas, adrenals, prostate, skin, and thyroid: the latter, a complication of the amyloidosis known as amyloid goiter. Post-mortem review of the deceased’s laboratory workup showed a slightly abnormal kappa:lambda ratio in the blood; however, no clonal lymphoplasmacytic disorder was confirmed in the bone marrow and other lymphoreticular system organs either by the microscopic examination and immunohistochemical staining. Laser-capture microdissection and tandem mass spectrometry of the splenic tissue detected a peptide profile consistent with an immunoglobulin Kappa light chain. The presence of amyloid purpura favors the diagnosis of primary systemic amyloidosis.

Heart damage in the combined types of systemic amyloidosis

To identify and determine the clinical and morphological characteristics of combined types of SA in patients with biventricular chronic heart failure (CHF). Eighty autopsy protocols for biventricular CHF deaths were retrospectively analyzed. Immunohistochemistry and confocal laser scanning microscopy (CLCM) with antibodies to amyloid A (AA), serum amyloid-P (SAP), prealbumin, and immunoglobulin kappa (κ) and lambda (λ) light chains were performed. The myocardium showed a combination of different types of SA in 6 (7.5%) cases, including Alλ-CA+ATTR-CA, ALκ-CA+ATTR-CA, and AA-CA+ATTR-CA in 4, 1, and 1 cases, respectively. Macroscopically, the heart mass averaged 470±20 g; the thickness of the left and right ventricular myocardium was 1.5±0.2 and 0.4±0.1 cm, respectively; the interventricular septum averaged 1.2±0.2 cm; and the cardiac index was 0.008. The myocardium was dense, dark red with diffuse layers of whitish dense fibrous connective tissue; the heart cavities were enlarged. Microscopically, in 25% of cases, all heart parts had ALλ-CA that was characterized by massive amyloid deposits localized predominantly in the intramyocardial vessel wall, intermuscular connective tissue, and perivascularly. The myocardium also displayed small amyloid deposits of ALλ-CA and ATTR-CA in the intermuscular connective tissue and intramyocardial vessel wall. Amyloid deposits were located in different parts of the myocardium; there were also areas of co-localization of ALλ-CA+ATTR-CA. The combined types of SA occurred under the guise of coronary heart disease and the dilated cardiomyopathy phenotype. The combined amyloid AL-CA and ATTR-CA was generally localized in the interstitium and myocardial vessels. There were also small areas of co-localization of amyloid deposits, which were found mainly in the intramyocardial vessels.

Isotyping and Semi-Quantitation of Monkey Anti-Drug Antibodies by Immunocapture Liquid Chromatography-Mass Spectrometry

There is an urgent demand to develop new technologies to characterize immunogenicity to biotherapeutics. Here, we developed an immunocapture LC-MS assay to isotype and semi-quantify monkey anti-drug antibodies (ADAs) to fully human monoclonal antibody (mAb) drugs. ADAs were isolated from serum samples using an immunocapture step with the Fab of the full-length mAb cross-linked to magnetic beads to minimize matrix interference. A positive monoclonal antibody control against the human immunoglobulin kappa light chain was used as a calibration standard for ADA quantitation. The final LC-MS method contains 17 multiple reaction monitoring (MRM) transitions and an optimized 15-min LC method. The results suggested that IgG1 was the most abundant isotype in ADA-positive samples. IgG2 and IgG4 were identified at lower levels, whereas IgG3 and IgA levels were only observed at very minor levels. In addition, levels of total ADA measured by the LC-MS assay were comparable to results obtained using a traditional ligand binding assay (LBA). The LC-MS ADA assay enabled rapid immunogenicity assessment with additional isotype information that LBAs cannot provide.

Importance of Flow Cytometry in the Differential Diagnosis of Hairy Cell Leukemia in the State of Rio Grande Do Norte, Brazil

Introduction:Hairy Cell Leukemia (HCL) is a B-cell non-Hodgkin's Lymphoma (B-NHL) representing about 2% of chronic leukemias, is manifested in adults with an average age of 55 years old or more and the ratio of male: female is 5:1, being more common among white people. It is characterized by the presence of neoplastic lymphocytes with cytoplasmic projections (villous cells), a characteristic commonly observed in other DLPCs such as variant HCL (HCL-v) and splenic villous cell lymphoma (SVCL), being the immunophenotyping by flow cytometry determinant in the differential diagnosis of these neoplasms. HCL is characterized by splenomegaly, hepatomegaly, pancytopenia in peripheral blood (PB) with leukopenia, anemia, neutropenia, monocytopenia, and thrombocytopenia. It has a low number of circulating tumor cells, spleen, liver, and bone marrow (BM) infiltration.Objective:To investigate, by flow cytometry, patients with lymphocytosis and presence of villous lymphocytes in the characterization of HCL and HCV-v and SMZL.Methodology:Were investigated samples of peripheral blood (SP) and bone marrow (MO) from 27 patients previously diagnosed with DLPC and presence of villous lymphocytes which were by flow cytometry with a panel of monoclonal antibodies (MoAb) conjugated to fluorochromes and targeted to T-lymphocytes: CD1a, CD2, CD3, CD5, CD7, subpopulation T-helper (CD3/CD4) and T-cytotoxic (CD3/CD8), in addition to TCR a/b and TCR g/d; Natural Killer cells: CD16-56; B-lymphocytes: CD19, CD20, CD21, CD22, CD23, CD79b, CD200, IgM, IgG, IgD, anti-kappa and anti-lambda, in addition to CD10, TdT (Terminal deoxynucleotidyl Transferase), CD103, CD123, CD11c, CD25, CD38, CD138, CD45 and CD14. At the same time, a complete blood count with differential white blood cell count and investigation of clinical and demographic data such as age, sex and ethnicity / race were also performed.Results:The distribution of patients according to ethnicity and gender, there was a predominance of white individuals and males. The age group most affected was in patients older than 60 years. All patients expressed pan-B antigens on leukemic cells with expression of CD19, CD22 / CD20 (Forte), sIgH, associated with clonal restriction for immunoglobulin light chain (kappa= 20 and lambda= 7), associated with FMC7 expression, HLADR, CD38 and CD45 strong and negativity to CD10, CD138, CD200, CD23, CD5, TdT and related T antigens. Sixteen cases were categorized as HCL, six HCLv and five SVCL. The immunophenotyping of HCL cases was positive for CD103, CD25, CD123 and CD11c. HCLv was negative for CD103 in three cases and CD25 and SVCL negative for CD103, CD123 and CD11c and CD25 in all cases.Conclusions:The precise diagnosis of HCL has fundamental importance because each NHL-B has a specific treatment, besides emphasizing the sensitivity and speed of the IFC regarding diagnosis and follow-up. Disclosures No relevant conflicts of interest to declare.