A case of immunofluorescence-negative light chain deposition disease diagnosed by mass spectrometry

Abstract

A 53-year-old man was referred to our department for the evaluation of kidney dysfunction and edema. Kidney biopsy revealed nodular glomerular lesions; however, immunofluorescence showed no specific deposition of immunoglobulins or light chains, and a definitive diagnosis could not be confirmed. Serum and urine immunofixation electrophoresis detected κ-type Bence Jones protein, raising suspicion of monoclonal immunoglobulin-related kidney disease. Bone marrow examination led to a diagnosis of multiple myeloma. Electron microscopy revealed band-like, powdery, electron-dense deposits extending from the glomerular basement membrane to the subendothelial area as well as deposits along the tubular basement membranes, suggestive of monoclonal immunoglobulin deposition disease. However, additional evaluation using immunofluorescence on paraffin-embedded tissues and immunohistochemical staining failed to demonstrate light-chain deposition, and a pathological relationship between the plasma cell disorder and kidney lesion could not be established. Laser microdissection followed by liquid chromatography–tandem mass spectrometry identified κ light chain deposition, leading to a definitive diagnosis of light chain deposition disease and providing pathological evidence linking the plasma cell disorder to the kidney lesion. Plasma cell–directed therapy was subsequently initiated, resulting in improved kidney function and the avoidance of dialysis. This case highlights the potential utility of mass spectrometry for establishing a definitive diagnosis and guiding therapeutic decision-making when light-chain deposition cannot be demonstrated by conventional or additional immunostaining.

Introduction

Light chain deposition disease (LCDD) is a clonal plasma cell disorder characterized by the deposition of non-amyloid monoclonal light chains in several organs. It is a relatively rare condition that remains undiagnosed during the asymptomatic phase. LCDD is categorized as a monoclonal immunoglobulin deposition disease (MIDD) [1]. MIDD is included within the spectrum of monoclonal gammopathy of renal significance and may lead to progressive kidney dysfunction. Therefore, early diagnosis and appropriate therapeutic interventions are essential. A kidney biopsy is indispensable for diagnosis and demonstration of monoclonal immunoglobulin deposition using immunofluorescence (IF) is considered particularly important. However, cases with IF negativity cannot be diagnosed properly, making the diagnosis challenging.

In recent years, monoclonal immunoglobulin-related kidney diseases have been recognized to exhibit diverse pathological features, and advanced diagnostic techniques, including electron microscopy and mass spectrometry, have become increasingly important [2].

Proteomic analysis using laser microdissection combined with liquid chromatography–tandem mass spectrometry (LMD-LC–MS/MS) enables direct identification of deposited proteins, and its usefulness has been reported even in IF-negative cases. Here, we report a case in which light chain deposition could not be identified using IF on kidney biopsy, making the diagnosis challenging. However, it was eventually confirmed as LCDD by LMD-LC–MS/MS. Additionally, we present a review of the relevant literature.

Case report

A 53-year-old man with a history of hypertension was followed up by a general practitioner. In February of year X, the patient developed uncontrolled hypertension and edema of the extremities. Urinalysis revealed hematuria (2 +) and proteinuria (2 +), and laboratory tests showed kidney dysfunction (serum creatinine [Cr] 2.64 mg/dL, estimated glomerular filtration rate [eGFR] 21.5 mL/min/1.73m²) and anemia. The patient was referred to our department for further evaluation and treatment. At the initial visit, a urine protein/creatinine ratio was 1.0 g/gCr, and urinary sediment showed 10–19 red blood cells per high-power field (HPF), indicating mild proteinuria and hematuria. The initial screening tests did not reveal any other abnormalities. Because the kidneys were mildly enlarged, a percutaneous kidney biopsy was planned to investigate the underlying cause, and the patient was admitted to our department in March of the same year.

Upon admission, his blood pressure increased to 180/90 mmHg. Physical examination revealed conjunctival pallor. Heart and lung sounds were within normal limits. Bilateral lower extremity edema was present, and no skin rash was observed. Laboratory findings revealed anemia with a hemoglobin level of 9.3 g/dL. Serum biochemical tests revealed no abnormalities in the electrolyte levels or liver function. Kidney dysfunction persisted, with a serum creatinine level of 2.72 mg/dL and eGFR 20.8 mL/min/1.73m². Serum immunoglobulin levels (IgG, IgA, and IgM) decreased. Complement levels were within the normal range, and antineutrophil cytoplasmic antibodies and anti–glomerular basement membrane antibodies tested negative. Urinalysis revealed hematuria (10–19 red blood cells/HPF) and proteinuria (1.0 g/day). Although no M-protein peak was detected on serum or urine protein electrophoresis, immunofixation electrophoresis revealed κ-type Bence Jones protein in both serum and urine. The serum free light chain κ/λ ratio was 173.0 (Table 1). Chest radiography did not reveal pleural effusion or cardiomegaly. Abdominal computed tomography revealed bilateral kidney enlargement without hydronephrosis. Based on the kidney dysfunction and urinary abnormalities, glomerulonephritis was suspected, and a kidney biopsy was performed on hospital day 5.

Table 1.

Laboratory data

Urinary test			Biochemistry			Immunological findings
proteinuria	2+		TP	6.3	g/dL	IgG	416	mg/dL
hematuria	2+		Alb	4.2	g/dL	IgA	43	mg/dL
RBC	10–19	/HPF	AST	12	IU/L	IgM	14	mg/dL
U-TP/Cr	1.0	g/gCr	ALT	18	IU/L	CH50	78.5	U/mL
			LDH	170	IU/L	C3	113.1	mg/dL
Blood cell count			BUN	24.5	mg/dL	C4	28.7	mg/dL
WBC	3300	/μL	Cr	2.72	mg/dL	MPO-ANCA	–
Seg	53.0	%	eGFR	20.8	mL/min/1.73m2	PR3-ANCA	–
Lymph	35.0	%	Na	147	mEq/L	Anti-GBM	–
Mono	5.0	%	K	3.8	mEq/L	κ	3130	mg/L
Eosino	6.0	%	Cl	112	mEq/L	λ	18.1	mg/L
Baso	1.0	%	Ca	9.2	mg/dL	κ/λ	173.0
RBC	2.99×106	/μL	P	3.5	mg/dL	Immunofixation electrophoresis
Hb	9.3	g/dL	CRP	0.11	mg/dL	Serum IFE	BJP(κ)
PLT	171×103	/μL				Urine IFE	BJP(κ)

WBC white blood cell, Seg segmented neutrophils, Lymph lymphocyte, Mono monocyte, Eosino eosinophil, Baso basophil, RBC red blood cell, Hb hemoglobin, PLT platelet, TP total protein, Alb albumin, AST aspartate aminotransferase, ALT alanine aminotransferase, LDH lactate dehydrogenase, BUN blood urea nitrogen, Cr creatinine, Na sodium, K potassium, Cl chloride, Ca calcium, P phosphorus, CRP C-reactive protein

Light microscopy revealed nodular glomerular lesions and tubular basement membrane thickening (Fig. 1). No abnormalities were observed in the vessels or tubulointerstitial area, except for focal tubular atrophy. Congo red staining results were negative. Although serum and urine immunofixation electrophoresis detected a κ-type Bence Jones protein, suggesting a hematologic disorder-related kidney disease, IF showed no specific staining for immunoglobulins or κ/λ light chains (Fig. 2).

Fig. 1.

Light microscopy findings of the kidney biopsy specimen. Nodular glomerulosclerosis is observed in the glomeruli. Thickening of the tubular basement membranes is observed (PAS×200 PAM×100). Focal tubular atrophy is observed (PAS×100)

Fig. 2.

Immunofluorescence shows no specific staining

Bone marrow aspiration revealed approximately 10% plasma cells, and together with the immunohistochemical staining results, multiple myeloma was diagnosed. Mild anemia was observed; however, neither hypercalcemia nor osteolytic lesions were present and kidney impairment was the predominant clinical manifestation. Despite the diagnosis of multiple myeloma, a pathological relationship between the plasma cell disorder and kidney lesion could not be established, making treatment decisions challenging. Kidney function progressively deteriorated, with serum creatinine level rising to 6.0 mg/dL and proteinuria worsening to a urinary protein/creatinine ratio of 5.0–7.0 g/gCr. Considering the risk of progression to end-stage kidney disease, treatment for multiple myeloma was considered. However, treatment was withheld because a pathological relationship between the plasma cell disorder and kidney lesion had not been established, and there was concern about potential treatment-related adverse effects.

Subsequent electron microscopy revealed band-like, powdery, electron-dense deposits extending from the glomerular basement membrane to the subendothelial area as well as deposits along the tubular basement membranes, suggestive of MIDD (Fig. 3). Based on the clinical course, monoclonal proliferation of κ-type Bence Jones protein due to multiple myeloma with deposition of κ light chains in the glomeruli was suspected, consistent with LCDD. However, repeated immunohistochemical staining, including immunoenzymatic methods and pronase-digested paraffin tissue IF, failed to demonstrate specific deposition of the light chain.

Fig. 3.

Electron microscopy findings. Electron microscopy demonstrated band-like, powdery, electron-dense deposits extending from the glomerular basement membrane to the subendothelial area (a), (b), and finely granular electron-dense deposits along the tubular basement membranes (c) ((a)×2500, (b)×10000, (c)×4000)

Given the diagnostic difficulty in determining the cause of kidney lesions, mass spectrometry was performed. In the mass spectrometry analysis, κ light chain was among the most abundant proteins detected, whereas proteins associated with other deposition diseases were not among the most abundant proteins, supporting the interpretation that the deposits were predominantly composed of κ light chain. (Fig. 4). Following the diagnosis, bortezomib and dexamethasone therapy was initiated on hospital day 193. After treatment, proteinuria decreased to <1.0 g/gCr, and the κ/λ ratio improved from 173.0 to 28.2. Kidney function was stabilized, with serum creatinine levels ranging from 3.0 to 4.0 mg/dL, and dialysis initiation was avoided. Figure 5 shows the pathological approach and a diagram of the clinical course for this case.

Fig. 4.

LMD-LC–MS/MS shows monotypic κ light chain deposition

Fig. 5.

Pathological work up and clinical course IF, immunofluorescence; IHC,immunohistochemistry; Cr, creatinine; UTP/UCr, urinary total protein/urinary creatinine; PSL, prednisolone; Weekly BD, weekly bortezomib anddexamethasone therapy 　

Discussion

Although multiple myeloma was diagnosed in this case, a definitive diagnosis was challenging because light chain deposition was not detected by IF or immunostaining for light chains on kidney biopsy.

Monoclonal immunoglobulins are produced by clonal plasma or B cells. Monoclonal immunoglobulins can cause kidney injury even in the absence of overt hematologic malignancy. Monoclonal immunoglobulin–associated kidney disease is heterogeneous. Because accurate classification is closely related to the treatment strategy and prognosis, precise pathological diagnosis is essential [2]. LCDD is categorized as MIDD, a group of multisystem disorders characterized by the deposition of abnormally truncated monoclonal immunoglobulins in various organs, particularly the kidneys [3]. The deposits are typically Congo red–negative, show a single light or heavy chain on IF, and appear as powdery deposits on electron microscopy. MIDD includes LCDD, the most prevalent disorder constituting approximately 80% of MIDD, heavy chain deposition disease, and light and heavy chain deposition disease, which are extremely rare [4, 5]. LCDD is usually reported in the course of plasma cell dyscrasias, including multiple myeloma (11–65%), monoclonal gammopathy of undetermined significance (32–86%), macroglobulinemia (2%), and other lymphoproliferative disorders (2–3%) [6, 7].

Patients with MIDD who achieve remission with treatment have a favorable kidney prognosis, with a 3-year kidney survival rate of 86%; therefore, early diagnosis and therapeutic intervention are crucial [8]. MIDD, particularly LCDD, is characterized by the deposition of monoclonal immunoglobulins along the glomerular and tubular basement membranes, and IF is considered essential for its diagnosis. However, in some cases, deposits may not be identified using IF [5]. Possible explanations for the negative findings of frozen section IF include the loss of deposited proteins during pretreatment or washing procedures, resulting in levels below the detection threshold, structural abnormalities of antigenic epitopes, and technical factors related to specimen handling [9]. Even when IF results are negative, a diagnosis of LCDD has been established in previous cases based on electron microscopy findings. Therefore, MIDD cannot be excluded solely based on negative IF results [10].

In the present case, a definitive diagnosis could not be established by light microscopy or conventional IF alone, and it was necessary to await the electron microscopy findings. Nodular lesions and nonfibrillar electron-dense deposits observed by electron microscopy strongly suggested MIDD. Therefore, additional evaluations using IF on paraffin-embedded tissue and immunohistochemical staining were performed; however, light chain deposition could not be identified. Consequently, a clear pathological relationship between the plasma cell disorder and kidney lesion could not be established. Considering the potential adverse events associated with chemotherapy, treatment was not initiated at that time due to the absence of sufficient evidence.

Proteomic analysis using mass spectrometry enables the direct identification of deposited proteins. Therefore, even when deposits cannot be identified by IF, a diagnosis may be achieved through the identification of deposited proteins. Several cases in which mass spectrometry was performed based on the suspicion of MIDD have been reported, including cases with nodular lesions and negative IF findings that were ultimately diagnosed as LCDD [11–15] (Table 2). In the present case, LMD-LC-MS/MS identified κ light chain deposition, leading to a definitive diagnosis of LCDD. This provided pathological evidence linking the plasma cell disorder to the kidney lesion. Chemotherapy was subsequently initiated, resulting in improved kidney function and avoidance of dialysis.

Table 2.

Cases in which mass spectrometry contributed to the diagnosis

Reference	Age/Sex	Cr (mg/dL)	UPro/Cr (g/gCr)	IF findings	EM findings	Diagnosis
Qian Q, 2010[11]	57/M	2.5	1.04	Negative	Fibrillar structures	HLCDD
Royal V, 2015[12]	77/M	2.2	5.3	Negative	Fibrillar structures	HCDD
Tomomichi Kasagi, 2017[13]	42/M	2.36	3.94	Negative	Fibrillar structures	LCDD
Best Rocha A, 2019[14]	68/M	2.3	0.53	Negative	Powdery electron-dense deposits	HCDD
Tsushima T, 2021[15]	64/F	5.6	1.8	κ(+) λ(−)	Powdery electron-dense deposits	LCDD
This case	53/M	2.72	1.0	Negative	Powdery electron-dense deposits	LCDD

Cr creatinine, Upro urinary protein, IF immunofluorescence, EM electron microscopy, HLCDD heavy and light chain deposition disease, HCDD heavy chain deposition disease, LCDD light chain deposition disease

Mass spectrometry is available only in a limited number of institutions and is not readily accessible in all cases. Therefore, when kidney involvement associated with a hematological disorder is clinically suspected but a diagnosis cannot be established by conventional IF, additional studies, such as IF on paraffin-embedded tissue and immunohistochemical staining, may facilitate earlier diagnosis. Furthermore, when diagnosis remains difficult despite these additional evaluations, the identification of deposited proteins by mass spectrometry may contribute to establishing a definitive diagnosis and enabling appropriate therapeutic intervention.

Declarations

Conflict of interest

The authors have declared that no conflict of interest exists.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Written informed consent was obtained from the legal representatives of the patient for the publication of information and images.