Diagnostic utility of immunofixation and free light chain assay in AL amyloidosis: a systematic review and meta-analysis

Abstract

In this systematic review, we aimed to assess the sensitivity of serum immunofixation (SIFE), urine immunofixation (UIFE), and serum free light chain (sFLC) in detecting plasma cell dyscrasia associated with light chain (AL) amyloidosis. We searched the literature using PubMed, Embase, and CENTRAL databases since inception through March 2024. We included studies reporting the diagnostic accuracy of immunofixation and/or sFLC in patients with AL amyloidosis. We used the GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) approach to assess the certainty of the evidence and QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies-2) to assess the risk of bias. After deduplication, 29 237 records were screened, and 44 studies (n = 18 071) were included. For the meta-analysis, 28 studies reported sensitivity for SIFE (n = 3390), 24 studies for UIFE (n = 2830), 19 studies for SIFE and UIFE (n = 2819), 21 studies for sFLC ratio (n = 3678), 6 studies for SIFE and sFLC ratio (n = 372), 3 studies for UIFE and sFLC ratio (n = 242), and 10 studies for all 3 tests (n = 1430). Pooled sensitivities were as follows: SIFE, 0.68 (95% confidence interval [CI], 0.62-0.73); UIFE, 0.77 (95% CI, 0.72-0.81); SIFE and UIFE, 0.89 (95% CI, 0.84-0.93); and sFLC ratio, 0.82 (95% CI, 0.78-0.86). When SIFE, UIFE, and sFLC ratio were combined, the sensitivity reached 0.99 (95% CI, 0.96-1.00). The high sensitivity of combining SIFE, UIFE, and sFLC ratio supports their use as a reliable initial testing strategy to raise suspicion for AL amyloidosis and guide decisions regarding future diagnostic modalities.

Introduction

Amyloidosis is a group of heterogeneous diseases that develop as a consequence of the deposition of insoluble fibrils in various organs and tissues.1, 2, 3 Light chain (AL) amyloidosis results from a population of clonal immunoglobulin-secreting cells that produce κ or λ type monoclonal light chains. Rarely, patients with AL amyloidosis may produce heavy chains in diseases such as lymphoplasmacytic lymphoma.⁴ The clinical manifestations of AL amyloidosis differ depending on which organ is affected, with the kidneys, heart, gastrointestinal tract including liver, and nerves being the most commonly involved.

Early diagnosis and prompt treatment are important factors that allow for improved patient survival. Unfortunately, most initial symptoms of AL amyloidosis are nonspecific, with pathognomonic symptoms such as periorbital purpura and macroglossia occurring sporadically and/or at advanced stages, which may lead to delayed diagnosis and progression of organ damage.^5,6 Hester et al showed that the median time between the earliest symptoms and the final diagnosis is 2.3 years.⁷ Diagnosis of amyloidosis is based on the detection of monoclonal light chains, along with histological confirmation of light chain deposition within affected tissues. Serum immunofixation (SIFE), urine immunofixation (UIFE), and serum free light chain (sFLC) assays are typically used as initial tests because they are widely accessible and easily performed, which may facilitate an earlier diagnosis. Additionally, sFLC detection has been demonstrated to precede the presentation of symptoms in individuals with AL amyloidosis.⁸

Considering the importance of establishing an early diagnosis of AL amyloidosis combined with the role of immunofixation and free light chain in the diagnostic pathway, this systematic review aims to evaluate the diagnostic test accuracy (DTA) of SIFE, UIFE, and sFLC to diagnose plasma cell dyscrasia (PCD) associated with AL amyloidosis.

Methods

This review, which focused on the DTA of SIFE, UIFE, and sFLC ratio, was done to inform 2 PICO (population, index test, comparator, and outcome) questions in the 2026 American Society of Hematology AL amyloidosis diagnostic guideline.⁹ The guideline effort included a systematic review team of 12 members, and a clinical panel consisting of 20 members (that included 12 hematologists, 3 cardiologists, a neurologist, a nephrologist, 2 internal medicine specialists, and a pathologist with expertise in amyloidosis, as well as a patient representative). We conducted the systematic review based on the Cochrane handbook for systematic reviews of DTA and reported according to the preferred reporting items for a systematic review and meta-analysis of DTA studies: the PRISMA(Preferred Reporting Items for Systematic reviews and Meta-Analyses)-DTA Statement.^10,11 We used the PICO framework. We registered the protocol in PROSPERO (International Prospective Register of Systematic Reviews) (identifier CRD42024527372).

Data source and search

We performed a comprehensive umbrella systematic search in PubMed, Embase, and the Cochrane Central Register of Controlled Trials from inception till March 2024. The search was designed and constructed by an experienced librarian, with input from the systematic review team and the clinical panel members. The full search strategy is available in Supplemental table 2.

Eligibility criteria

Teams of 2 reviewers independently and in duplicate screened the title and abstract of the studies using Covidence systematic review software (Australia) and assessed the eligibility of full-text reports on LASER AI (Poland). In case of disagreement between decisions, a third reviewer (M.A., H.K., and R.A.M.) assessed the study, or the study was presented to the clinical panel to resolve the conflict and reach a consensus.

We included studies that report on DTA for SIFE, UIFE, sFLC, or any combination of the 3 tests with an acceptable reference standard in adults (aged >18 years). We included studies reporting on patients with suspected or confirmed AL amyloidosis. We excluded studies reporting on cohorts with only secondary amyloidosis, transthyretin amyloidosis (ATTR), familial amyloid angiopathy, localized amyloidosis, amyloid deposition in the brain, amyloidosis (unspecified) or other types of amyloidosis, including amyloidosis associated with lymphoplasmacytic lymphoma, heavy chain amyloidosis, and B-cell lymphoproliferative disorders. We included studies with the following design: cohort, case control, or cross-sectional. We excluded case reports, case series with <10 patients, conference abstracts published before 2021, protocols, narrative reviews, and systematic reviews. We included studies reporting on ≥1 of the following diagnostic measures: sensitivity, specificity, positive predictive value, and negative predictive value.

To minimize the risk of including overlapping populations, we evaluated all eligible studies for potential duplication of participants through comparing study author lists, recruitment centers, and enrollment periods. When multiple reports from a single cohort were identified that both address the same analysis, we included the most comprehensive or recent publication with the largest sample size or most complete outcome reporting.

For the calculation of diagnostic test measures, SIFE, UIFE, sFLC, or any combination of the 3 tests, which are the index test (test of interest that DTA measures will be calculated for) and a reference test (test used as the best estimate of the actual disease state) needed to be performed in the same individual. For this review, we did not limit to specific acceptable reference tests, and we accepted each study’s diagnosis of AL amyloidosis at face validity.

Data extraction

Teams of 2 reviewers independently extracted relevant data using a standard piloted Excel form. We designed the form to systematically capture study characteristics and relevant outcome data required to calculate the sensitivity. Collected data included: study design, amyloidosis type, organ involvement, index test, cutoff, reference test, and diagnostic test measures (true positive, true negative, false positive, false negative, sensitivity, specificity, positive predictive value, negative predictive value).

Statistical analysis

For the meta-analysis, we used OpenMeta[analyst] (Providence, RI). We used the random model when we had ≥5 studies and reported the sensitivity as a range when we had <5 studies. Furthermore, we planned on subgroup analyses based on studies reporting on renal adjustment as well as the method of urine collection.

Quality assessment

We evaluated risk of bias using QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies-2).¹² We used the GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) approach to assess the certainty of the evidence.^13,14 Two reviewers (H.K. and M.A.) familiar with the GRADEpro software (https://gradepro.org) constructed a summary of findings table for each research question. The reviewers considered study design, risk of bias, inconsistency, indirectness, imprecision, and publication bias for the assessment of the quality of evidence for each outcome.^15,16 The certainty of the evidence is represented using symbols as follows: ⨁◯◯◯ indicates very low certainty, ⨁⨁◯◯ indicates low certainty, ⨁⨁⨁◯ indicates moderate certainty, and ⨁⨁⨁⨁ indicates high certainty.

Results

After deduplication, 29 237 records were screened, from which 44 studies were included with a total of 18 071 patients. For the meta-analysis, we included 28 studies that reported the sensitivity for SIFE (n = 3390); 24 studies that reported on UIFE (n = 2830); 19 studies that reported on SIFE and UIFE (n = 2819); 21 studies that reported on sFLC ratio (n = 3678); 6 studies that reported on SIFE and sFLC ratio (n = 372); and 3 studies that reported on UIFE and sFLC ratio (n = 242). Lastly, 10 studies reported on the sensitivity for all 3 tests with a total of 1430 patients. The PRISMA flowchart is shown in Figure 1.

Figure 1.

PRISMA flowchart.

Characteristics of included studies

Tables 1 and 2 show the characteristics of included studies in the systematic review. Most studies were based in North America (48%), followed by Europe (34%). Nearly half (43%) of all studies were published between 2009 and 2016. Regarding study design, nearly two-thirds (61.4%) of the studies were case control, followed by cohort designs (32%). The most used reference for SIFE, UIFE, or sFLC was organ biopsy (multiple types of organ biopsies), with 47.7% of the studies using it as the reference.

Table 1.

Characteristics of included studies

Author (year), country	Population	Study design	Index test	Cutoff for sFLC	Reference test	Summary of ROB (QUADAS-2)
Halushka et al17 (2015), United States	N = 85 Mean age, 68.2 y (SD 8.7) Inclusion: patients with AL amyloidosis	Case-control	κ:λ FLC ratio	<0.5 or >5.0	EMB	High
Mahmood et al18 (2016), United Kingdom	N = 94 Median age, 64 y (Range 55.2-72.2) Inclusion: consecutive patients with systemic AL amyloidosis seen at the National Amyloidosis Centre	Case-control	κ:λ FLC ratio, SIFE, UIFE	NA	Organ Bx (unspecified)	High
Palladini et al19 (2016), Italy	N = 982 Median age, 65 y (Range 56-71) Inclusion: patients diagnosed with AL amyloidosis at the Pavia Amyloidosis Research and Treatment Center between 2004 and 2014	Case-control	κ:λ FLC ratio	<0.26 or >1.65	Organ Bx (unspecified)	High
Chang et al20 (2019), United States	N = 1173 Mean age, 64 y (SD 15) Inclusion: consecutive newly diagnosed patients with AL amyloidosis	Cohort	κ:λ FLC ratio	<0.26 or >1.65	NA	High
Morris et al21 (2007), Australia	N = 31 Median age, 61 y Inclusion: patients with documented AL amyloidosis referred to 4 affiliated hospitals between April 2002 and February 2006 were retrospectively identified	Case-control	κ:λ FLC ratio	<0.26 or >1.65	Organ Bx	High
Xu et al22 (2017), China	N = 1340 Median age, 58 y (Range 14-94) Inclusion: 4786 cases with a complete set of myeloma detection in preliminary diagnosis at Wuhan Tongji Hospital from September 2012 to June 2016	Cohort	κ:λ FLC ratio	<0.26 or >1.65	NA	High
Liu et al23 (2022), China	N = 38 Median age, 62 y (Range 52-68) Inclusion: all patients at the National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing, China, from 2009 to 2019	Cohort	κ:λ FLC ratio	<0.26 or >1.65	Kidney/BM Bx	High
Shimojima et al24 (2005), Japan	N = 17 Median age, 59.9 y (Range 44-71) Inclusion: 17 patients with primary AL amyloidosis who were referred to our hospital between April 2000 and October 2003	Case-control	κ:λ FLC ratio, SIFE, UIFE	NA	Organ Bx (unspecified)	High
Arbustini et al25 (2009), Italy	N = 15 Median age, 60 y (Range 50-72) Inclusion: 369 patients evaluated at the Amyloid Center in Pavia from December 1986 to December 2000	Case-control	SIFE, UIFE	NA	Fat pad Bx	High
Traynor et al26 (1991), United States	N = 6 Median age, 59.5 y (Range 50-72) Inclusion: 6 patients for whom cranial neuropathy was a major manifestation of primary amyloidosis	Case series	SIFE, UIFE	NA	Organ Bx	High
Kyle et al27 (1983), United States	N = 229 Median age, 65 y Inclusion: all Mayo Clinic records reporting a histologic diagnosis of amyloidosis from 1 January1970 to 31 December 1980	Case-control	SIFE, UIFE	NA	Organ Bx	High
Wolf et al28 (1986), United States	N = 45 Age, NA Inclusion: 54 cases of Bx-proven systemic amyloidosis in which BM Bxs and/or particle sections	Cohort	SIFE, UIFE	NA	BM Bx	High
Muchtar et al29 (2019), United States	N = 592 Median age, 63 y (Range 56-71) Inclusion: 592 patients with Bx-proven, MS-confirmed AL amyloidosis diagnosed from 1 January 2008 through 31 August 2015 were included	Case-control	κ:λ FLC ratio, SIFE, UIFE	NA	Organ Bx (unspecified)	High
Lee et al30 (2023), South Korea	N = 98 Median age, 62 y (Range 40-82) Inclusion: patients with newly diagnosed amyloidosis by pathologic examination between 2005 and 2015	Case-control	κ:λ FLC ratio, SIFE, UIFE	NA	BM Bx	High
Rauf et al31 (2023), United Kingdom	N = 3354 Median age, 78 y (Range 70-83) Inclusion: 3354 patients with suspected or proven cardiac amyloidosis who were referred for evaluation	Cohort	κ:λ FLC ratio, SIFE, UIFE	<0.26 or >1.65	EMB/extracardiac Bx	High
Palladini et al32 (2009), Italy	N = 121 Age, NA Inclusion: 121 patients with Bx-proven systemic AL amyloidosis were consecutively enrolled between January 2004 and May 2005; none of the patients was previously treated (AL)	Case-control	κ:λ FLC ratio, SIFE, UIFE	NA	NA	High
Katzmann et al33 (2005), United States	N = 1020 Age, NA Inclusion: all Mayo Clinic patients who were tested for serum k and 1 FLCs from 1 January 2003 to 31 December 2003	Cohort	κ:λ FLC ratio, SIFE, UIFE	<0.26 or >1.65	Bx (unspecified)	High
Cermakova et al34 (2013), Czech Republic	N = 281 Age range, 30-88 y Inclusion: 281 patients who had been diagnosed with malignant monoclonal gammopathy in the outpatient department of the Haemato-Oncology Clinic of the University Hospital Brno	Cohort	κ:λ FLC ratio, SIFE, UIFE	NA	NA	High
Hernández-Reyes et al35 (2012), Mexico	N = 23 Median age, 57 y (Range 39-98) Inclusion: all patients in whom AL was diagnosed at the Centro de Hematología y Medicina In-terna de Puebla in Puebla, Mexico	Case-control	SIFE	NA	Organ Bx (unspecified)	High
Katzmann et al36 (2002), United States	N = 127 Age range, = 21-90 y Inclusion: 127 healthy donors aged 21-62 years (68 women [54%] and 59 men [46%]) were obtained from a pool of donors who had typical FLC values (Mayo Clinic) (AL)	Case-control	SIFE, UIFE	NA	NA	High
Real de Asúa et al37 (2013), Spain	N = 54 Mean age, 64 y (SD 13) Inclusion: patient with diagnoses with AL amyloidosis	Case-control	SIFE, UIFE	NA	Organ Bx	High
Akar et al38 (2005), United States	N = 189 Age range, 32-88 y Inclusion: 169 patients with systemic AL amyloidosis and 20 controls; all patients with AL amyloidosis had a positive tissue Bx by Congo red staining	Case-control	κ:λ FLC ratio, SIFE, UIFE	NA	Organ Bx (unspecified)	High
Krsnik et al39 (2015), Spain	N = 32 Median age, 63 y (Range 39-87) Inclusion: 32 consecutive patients treated at the Department of Hematology of the University Hospital Puerta de Hierro, Majadahonda (Madrid, Spain), between 2005 and 2013 who had an unequivocal histological diagnosis of AL amyloidosis	Case series	SIFE, UIFE	NA	Organ Bx	High
Bochtler et al40 (2008), Germany	N = 133 Median age, 63 y (Range 40-81) Inclusion: 133 consecutive untreated patients	Case series	κ:λ FLC ratio, SIFE, UIFE	<0.26 or >1.65	Organ Bx (unspecified)	High
Gerth et al41 (2012), Germany	N = 209 Mean age, 64.19 y Inclusion: all patients with the histological diagnosis of AL or MIDD were included	Cohort	κ:λ FLC ratio, SIFE, UIFE	NA	Renal Bx	High
Prokaeva et al42 (2016), United States	N = 199 Median age, 59.4 y (Range 29.2-80.4) Inclusion: 199 untreated patients with Bx-proven AL amyloidosis, evaluated in the Amyloidosis Center at Boston University School of Medicine and Boston Medical Center, from May 1995 to June 2009	Case-control	κ:λ FLC ratio, SIFE, UIFE	<0.26 or >1.65	Bx (not specified)	High
Miyazaki et al43 (2016), Japan	N = 50 Median age, 62.5 y (Range 40-84) Inclusion: patient with diagnoses with AL amyloidosis	Case-control	κ:λ FLC ratio, SIFE	NA	Organ Bx (unspecified)	High
Parepalli et al44 (2022), India	N = 41 Mean age, 56 y (SD 9.6) Inclusion: all diagnosed cases of monoclonal gammopathy with renal involvement	Cohort	SIFE	NA	Renal Bx	High
Lisenko et al45 (2016), Germany	N = 63 Median age, 62 y (Range 40-77) Inclusion: 63 patients who were newly diagnosed with systemic AL amyloidosis from May 2014 to May 2015 at our Amyloidosis Center at the University Hospital in Heidelberg, Germany	Case-control	SIFE, UIFE	NA	Organ Bx	High
Zhou et al46 (2013), China	N = 625 Mean age, 55.4 y (Range 40-83) Inclusion: 625 consecutive patients were recruited	Cohort	SIFE, UIFE	NA	Renal Bx	High
Matsuda et al47 (2014), Japan	N = 230 Mean age, 58.7 y (SD 9.5) Inclusion: 230 Japanese patients (127 men and 103 women; age range, 15-81 y; mean, 58.1 y) who had visited our hospital and been diagnosed with AL amyloidosis between April 2001 and December 2012	Case-control	SIFE, UIFE	NA	Organ Bx	High
Staron et al48 (2020), United States	N = 673 Age, NA Inclusion: 673 patients with AL amyloidosis who presented between 2010 and 2019 (AL)	Case-control	SIFE, UIFE	NA	Organ Bx (unspecified)	High
Perfetti et al,49 1995, Italy	N = 23 Age, NA Inclusion: 16 patients with Bx-proven amyloidosis were studied; primary amyloidosis was suspected on the basis of clinical histories and presentation	Case-control	SIFE	NA	Organ Bx	High
Katzmann et al50 (2009), United States	N = 1877 Age, NA Inclusion: all Mayo Clinic patients with a monoclonal gammopathy who also had serum PEL, IFE, and FLC, and urine PEL and IFE, performed within 30 days of diagnosis	Cohort	SIFE	NA	NA	High
Sarosiek et al51 (2021), United States	N = 36 Age, NA Inclusion: 45 newly diagnosed patients with suspected AL amyloidosis	Case-control	SIFE, UIFE	NA	BM Bx	High
Maleszewski et al52 (2013), United States	N = 143 Mean age, 67.5 y Inclusion: all cases of cardiac Bx-proven amyloidosis that had mass spectrometry testing	Case-control	κ:λ FLC ratio, SIFE, UIFE	NA	EMB	High
Milani et al53 (2017), United States	N = 1069 Mean ag,: 65.19 y Inclusion: consecutive, newly diagnosed patients with AL amyloidosis evaluated between 2004 and 2015	Case-control	κ:λ FLC ratio, SIFE, UIFE	<0.26 or >1.65	Organ Bx (unspecified)	High
Nasr et al54 (2013), United States	N = 218 Median age, 63 y Inclusion: 16 Mayo Clinic patients with a diagnosis of renal AH (n = 5) or AHL (n = 11) and 202 Mayo Clinic patients with a diagnosis of AL (AL-2 in 165 and AL-κ in 37) were identified by retrospective review of all native renal Bxs evaluated in the Renal Pathology Laboratory at Mayo Clinic, Rochester, from 2007 to 2011	Case-control	κ:λ FLC ratio, SIFE	NA	Renal Bx	High
Russo et al55 (2017), Italy	N = 75 Age, NR Inclusion: patients with AL amyloidosis	Case-control	UIFE	NA	NA	High
Park et al56 (2003), United States	N = 68 Age, NA Inclusion: 68 patients with AL amyloidosis, in whom uMPs were detected by high-resolution IFE electrophoresis	Case-control	SIFE, UIFE	NA	Liver Bx	High
Kyle et al1 (1992), United States	N = 21 Median age: 75 y (Range 47-92) Inclusion: patients with a diagnosis of amyloidosis, including primary, secondary, senile, localized, and familial amyloidosis, were obtained, all patients with amyloidosis from a secondary cause, those with a positive family history of amyloid, or those with amyloid localized to an organ were excluded	Case-control	SIFE, UIFE	NA	Organ Bx	High
Gillmore et al57 (2016) United States	N = 1498 Age, NA Inclusion: patients with suspected or histologically proven amyloidosis who had been referred	Cohort	κ:λ FLC ratio, SIFE, UIFE	<0.26 or >1.65	Organ Bx (unspecified)	High
Abroud et al58 (2022) France	N = 144 Mean age, 73 y Inclusion: patients suspected of having cardiac amyloidosis who were referred to the Referral Center for Cardiac Amyloidoisis in Henri Mondor Hospital (Créteil, France)	Case-control	κ:λ FLC ratio, SIFE, UIFE	0.37-1.44	Organ Bx (unspecified)	High
Kumar et al59 (2010), United States	N = 730 Median age, 63.3 y (Range 32-90) Inclusion: patients with Bx-proven AL seen at Mayo Clinic between 1980 and 2006, who had FLC measurements within 90 days of diagnosis performed as part of clinical evaluation or subsequently or stored serum	Cohort	κ:λ FLC ratio	<0.26 or >1.65	Organ Bx (unspecified)	High

AH, heavy chain amyloidosis; AHL, heavy and light chain amyloidosis; BM, bone marrow; Bx, biopsy; EMB, endomyocardial biopsy; MIDD, monoclonal immunoglobulin deposition disease; MS, mass spectrometry; NA, not applicable; NR, not reported; PEL, serum protein electrophoresis; ROB, risk of bias; uMP, urinary monoclonal proteins.

Table 2.

Summary of the study characteristics

Category	Variable	No. of studies (N = 44) n (%)
Study region	North America	21 (47.7)
	Europe	15 (34.1)
	Asia	7 (15.9)
	Oceania	1 (2.3)
Year of publication	Before 2000	5 (1.4)
	2000-2008	8 (18.2)
	2009-2016	19 (43.3)
	2017-2023	12 (27.3)
Study design	Cohort	14 (31.8)
	Case-control	27 (61.4)
	Case series	3 (6.8)
Reference test	Organ biopsy	21 (47.7)
	Surrogate biopsy	4 (9)
	Biopsy (organ or surrogate)	6 (13.6)
	Renal biopsy	3 (6.8)
	Endomyocardial biopsy	2 (4.5)
	Liver biopsy	1 (2.2)
	Unspecified	7 (15.9)

Sensitivity of immunofixation

A total of 28 studies (n = 3390) reported sensitivity for SIFE in patients with AL amyloidosis.24, 25, 26, 27, 28, 29, 30, 32, 34, 35, 36, 37, 38, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 54, 56, 58, 60 The pooled sensitivity was 0.68 (95% confidence interval [CI], 0.62-0.73; Figure 2A). A total of 24 studies reported sensitivity for UIFE (n = 2830) with a pooled sensitivity of 0.77 (95% CI, 0.72-0.81; Figure 2B).24, 25, 26, 27, 28, 29^,32, 33, 34^,36, 37, 38^,40,41,45, 46, 47, 48^{,51,55,56,58,60} We found limited evidence on studies reporting on DTA for UIFE using 24-hour collection vs spot urine, therefore a subgroup analysis was not feasible. When performing both SIFE and UIFE, considering the test positive when either is positive, we included 19 studies with 2819 patients.^18,24,25,28, 29, 30^,32, 33, 32^,38, 39, 40, 24, 41, 42^,46, 47, 48^,53,56 The pooled sensitivity for both tests done in combination was 0.89 (95% CI, 0.84-0.93; Figure 2C). For the sensitivity of SIFE, UIFE, and SIFE plus UIFE, we judged the certainty of the evidence to be low (⨁⨁◯◯) due to risk of bias concerns, because of the case-control design of the studies and lack of information on timing between index and reference test, as well as inconsistency in the accuracy measures.

Figure 2.

Sensitivity of immunofixation for detecting plasma cell dyscrasia in AL amyloidosis. Forrest plots for SIFE (A), UIFE (B), and SIFE and UIFE in combination (C). FN, false negative; TP, true positive.

Sensitivity of sFLC ratio

For the sFLC ratio, we included 21 studies (n = 3678).^12,17, 18, 19, 20, 21, 22, 23, 24^,29,32, 33, 34^,38,40, 41, 42^,52,54,58,59 The pooled sensitivity was 0.82 (95% CI, 0.78-0.86; Figure 3A). Fifteen studies (n = 3388) reported the sensitivity of sFLC ratio alone with a cutoff <0.26 or >1.65, for which the pooled sensitivity was 0.84 (95% CI, 0.80-0.88). One study (Halushka et al) reported the sensitivity for sFLC ratio using a cutoff of <0.5 or >5.0, for which they reported the sensitivity to be 1.¹⁷ Another study (Nasr et al) used a higher cutoff of <0.125 or >8, for which the sensitivity was reported to be 0.53.⁵⁴ We performed a sensitivity analysis removing the studies with an inclusive cutoff (supplemental Figure 1), and the sensitivity was 0.82 (95% CI, 0.77-0.86). For the sFLC ratio, we judged the certainty of the evidence to be low (⨁⨁◯◯) due to risk of bias, because of the case-control design of the studies and lack of information on timing between the index and reference test, as well as inconsistency in the accuracy measures. Limited evidence on studies reporting on DTA with and without renal adjustment did not allow for subgroup analyses.

Figure 3.

Sensitivity of serum free light chains for detecting plasma cell dyscrasia in AL amyloidosis. Forrest plot for sFlC alone (A), SIFE and sFLC in combination (B), and SIFE, UIFE, and sFLC in combination (C). FN, false negative; TP, true positive.

As for combining the sFLC ratio with immunofixation, 6 studies reported on SIFE and sFLC (n = 372); the pooled sensitivity was 0.96 (95% CI, 0.93-0.98; Figure 3B).^24,32, 33, 34^,43,58 We included 3 studies (n = 242) that reported on UIFE and sFLC, the sensitivities ranged from 0.91 to 1.32, 33, 34 For SIFE and sFLC, we judged the certainty of evidence to be moderate (⨁⨁⨁◯) due to risk of bias concerns, whereas for UIFE and sFLC we judged it to be low (⨁⨁◯◯) due to risk of bias concerns and imprecision, due to the small number of included patients.

Sensitivity for the combination of SIFE, UIFE, and sFLC ratio

For the combination of SIFE, UIFE, and sFLC ratio, in which a positive result on any individual test constituted overall positivity, we included 10 studies with 1430 patients.^24,29, 30, 31, 32, 33, 34^,52,57,58 The pooled sensitivity was 0.99 (95% CI, 0.96-1.00; Figure 3C). We judged the certainty of the evidence to be moderate (⨁⨁⨁◯) due to risk of bias concerns, because of the case-control design of the studies and lack of information on timing between index and reference test (Table 3). Figure 4 illustrates the pooled sensitivity for each of the tests and the combinations of those tests.

Table 3.

Evidence profile for the use of a combination of SIFE, UIFE, and sFLC ratio

Sensitivity	0.99 (95% CI, 0.97-1.00)	Prevalences	1%	10%
Specificity	0.24 (95% CI, 0.21-0.28)

Outcome	No. of studies (No. of patients)	Study design	Factors that may decrease certainty of the evidence					Effect per 1000 patients tested		Test accuracy CoE
			ROB	Indirectness	Inconsistency	Imprecision	Publication bias	Pretest probability of 1%	Pretest probability of 10%
True positives (patients with suspicion of AL amyloidosis)	10 studies 1926 patients	Cohort and case-control type studies	Serious∗	Not serious	Not serious	Not serious	None	10 (10-10)	99 (97-100)	⨁⨁⨁◯ Moderate∗
False negatives (patients incorrectly classified as not having suspicion of AL amyloidosis)								0 (0-0)	1 (0-3)
True negatives (patients without suspicion of AL amyloidosis)	1 study 576 patients	Cohort and case-control type studies	Serious∗	Serious†	Not serious	Not serious	None	242 (208-277)	220 (189-252)	⨁⨁◯◯ Low∗,†
False positives (patients incorrectly classified as having suspicion of AL amyloidosis)								748 (713-782)	680 (648-711)

CoE, certainty of evidence; ROB, risk of bias.

^∗We assessed ROB for all the studies using QUADAS-2 tool. We downgraded for ROB because we have some concerns for ROB. In some studies, not all the patients underwent the same reference test, in all the studies included the duration between the index and reference test was not reported (selection domain and flow).

^†For indirectness the study reports data on the performance of a single part of the index test and does not report on the remaining components. The specificity will not play a major role in decision-making and hence is unlikely to affect the recommendation.

Figure 4.

Bar chart showing the sensitivity of different index tests.

Discussion

This is, to our knowledge, the first comprehensive systematic review comparing the sensitivity of SIFE, UIFE, and sFLC for diagnosing PCD associated with AL amyloidosis. This question was prioritized as part of the AL amyloidosis diagnostic guideline effort because of its clinical importance in raising suspicion for AL amyloidosis and informing subsequent diagnostic evaluation. Our results showed that the sensitivity increased from 67% when using SIFE alone up to 99% when performing all 3 tests (SIFE, UIFE, and sFLC), with a positive result being a single positive test among any of the 3. The high sensitivity of all tests combined decreases the chance of having a false negative result, which in this case would be potential patients with AL amyloidosis being misdiagnosed. Relying on SIFE alone or UIFE alone would result in 33% and 24% of patients being missed, respectively. Furthermore, deferring UIFE, which is considered a simple test, and only performing SIFE and sFLC leads to an additional 4% of patients being missed. Missing patients would delay correct treatments, worsen the prognosis of the disease, and may even increase mortality.⁶¹ The combination of sFLC, SIFE, and UIFE is an excellent initial testing paradigm to increase the suspicion of AL amyloidosis, especially in patients with cardiac symptoms or asymptomatic proteinuria of unknown cause. The high sensitivity of the 3 tests combined allows for their use as an initial testing group to increase suspicion of AL amyloidosis and can guide further testing. Additionally, the usefulness of these tests also lies in differentiating between AL and ATTR amyloidosis, which a recently published scoping review highlighted as having overlapping symptomology between both diseases.⁶ Nevertheless, in Phull et al⁶² it is estimated that nearly 40% of patients in the ATTR cohort had a concomitant monoclonal gammopathy of undetermined significance. In these patients, differentiating between AL and ATTR amyloidosis would usually require further work-up, including a biopsy with mass spectrometry–based typing.

When using the cutoff of <0.26 or >1.65 for sFLC the sensitivity was 84.2% and decreases to 53% when using <0.125 or >8, and increases to 100% when using a cutoff of <0.5 or >5.0. Although not analyzed in this review, using the more inclusive cutoff would have a tradeoff on the specificity of the test. Patients with a falsely positive test could lead to a work-up to diagnose other differentials such as myeloma. It is important to note that the monoclonal sFLC interpretation is subject to analytical issues leading to inconsistency in linear responses and imprecision due to reagent lot-to-lot variation resulting in a clear drift toward higher sFLC ratios over the years. Since 2022, laboratories worldwide have been unable to reproduce the defined sFLC ranges in healthy controls. Laboratories may need to update their practices and reference ranges to reflect changes in sFLC distribution and ensure the validity and reliability of sFLC testing. To add to this, N-latex assays and assays from the Binding Site are 2 common approaches for the measurement of sFLCs. Numerous studies have shown that the analytical platform can dramatically affect sFLC results and warrants the introduction of instrument-specific reference ranges. These variations may explain the inconsistencies found in the different sensitivity measures between studies. We did not formally evaluate the effect different assays have on the sensitivity of tests.

This review focuses on the sensitivity of SIFE, UIFE, and sFLC, as the specificity of these tests depend on the starting population used in cohort studies, or the comparator population in case-control studies. Therefore, the specificity in 1 clinical scenario may not necessarily reflect the comparison addressed. Patients with false positive results may undergo unnecessary additional and possibly invasive testing, and there might be a resulting increase in disease-related anxiety. This review included only patients with AL amyloidosis. Restricting the population to AL amyloidosis enhances the accuracy of our estimates, as other conditions, such as lymphoplasmacytic lymphomas, may produce monoclonal proteins that yield false positive results on immunofixation testing.

For the limitations, first, the certainty of evidence was low to moderate because of concerns related to risk of bias, in which studies do not report the timing between the SIFE, UIFE, and sFLC and the reference test. Additionally, for some analyses, we had inconsistency in the accuracy measures as well as imprecision due to a small number of patients. Second, most included studies were case-control studies with the participants referred to tertiary centers. The case-control design and tertiary setting of these studies, in which DTA is calculated based on patients with known disease and a control group without the condition, may exaggerate diagnostic accuracy as they introduce spectrum bias.¹² This made it possible to accurately calculate the sensitivity (true positive [TP]/TP + false negative [FN]), but difficult to calculate the specificity (true negative [TN]/TN + false positive [FP]). Even when it was possible to calculate the specificity, it would be inaccurate as the specificity would vary according to the population. Xu et al²² conducted a case-control study that reported the performance of sFLC alone in patients with PCD including patients with AL amyloidosis (1.1% of study participants). The study yielded a specificity of 24% and a sensitivity of 88% (including 17 cases and 576 controls). This low specificity reflects many false positives or individuals without AL amyloidosis. Third, both planned subgroup analyses for renal adjustment of sFLC and method of urine collection for UIFE were not feasible due to limited reporting on these subgroups in the included studies. We acknowledge that renal function can influence sFLC due to reduced renal clearance, leading to higher circulating light chains and altered κ:λ ratios and that kidney-adjusted cutoffs may therefore be clinically relevant; however, we were unable to evaluate or compare different cutoff thresholds separately and instead relied on the cutoffs as defined in the individual studies, accepting them at face value. Future comparative studies evaluating the test accuracy, comparing studies reporting on renal adjustments, as well as studies comparing urine collection method would allow for a better understanding of the impact each collection method has on the accuracy of the test. Furthermore, in our analysis, we do not assess the impact of sFLC assay variability, reference range drift, and platform differences on test accuracy. Lastly, we did not limit to a specific reference standard and were inclusive for all references. Some studies specified the reference standard used (ie, biopsy, fat pad biopsy, organ biopsy), whereas other studies do not specify exactly how patients were diagnosed. If no proper method was used to reach a diagnosis of AL amyloidosis, this may have led patients to be misclassified and ultimately impact the DTA estimates. Ideally, a diagnosis of amyloidosis would have been made through a Congo red–positive biopsy with mass spectrometry–based typing.

The high sensitivity of SIFE, UIFE, and sFLC together at 0.99 make them a reliable test to increase the suspicion of AL amyloidosis. The results of these tests would help to discuss the benefits and harms of more invasive confirmatory tests such as biopsy with patients and guide further testing requirements.

Conflict-of-interest disclosure: All authors were members of the guideline panel, members of the systematic review team, or both. As such, they completed a disclosure of interest form, which was reviewed by the American Society of Hematology and is available in the supplemental Material 1.