Common causes are investigated first unless there is remarkable evidence concerning one or more LSDs, and further details are discussed in the sections below. Among these common causes of HSM are metabolic (Hemochromatosis, Wilson’s disease), congestive (heart failure, thrombosis), neoplastic (leukemia, lymphoma, hepatoblastoma, hepatocellular carcinoma and secondary metastasis to liver), hematologic (thalassemia, sickle cell anemia), infectious (cytomegalovirus, toxoplasmosis, hepatitis virus), inflammatory (sarcoidosis, systemic lupus erythematosus), toxic (acetaminophen overdose), and infiltrative (amyloidosis) causes [19,20].

LSDs usually present with non-tender hepatomegaly and/or splenomegaly, as well as smooth liver and spleen borders [21], but it is important to emphasize that an unsmooth liver/spleen does not exclude LSDs. HSM is more common in the severe and early-onset forms of LSDs. Table 1 presents a comprehensive list of all the LSDs with HSM as a clinical manifestation, grouped into classes and coupled with their respective genes. Disorders of lysosome-related organelles, such as Chediak–Higashi and Hermansky–Pudlak syndromes, are excluded from this review, even though they could potentially lead to HSM in the setting of hemophagocytic lymphohistiocytosis [22].

HSM is a common feature of mucopolysaccharidoses (MPSs) type I-VII, ASMD, lysosomal acid lipase deficiency (Wolman disease in children or CESD in adults), GM1 gangliosidosis type I, mucolipidosis type II, galactosialidosis, saposin C deficiency, NPC, and Gaucher disease. It is rare in other LSDs such as aspartylglucosaminuria and Sandhoff, whereas, it is absent in metachromatic leukodystrophy, among others.

Other genetic diseases can present with splenomegaly, like sickle cell disease or hepatomegaly in the case of alpha-1-antitrypsin deficiency and Beckwith–Wiedemann syndrome [21,52]. Aside from LSDs, other IMDs that lead to HSM are urea cycle disorders, classical galactosemia, glycogen storage disorders, hereditary fructose intolerance, tyrosinemia type I, prolidase deficiency, mitochondrial diseases, fatty acid oxidation defects, congenital disorders of glycosylation, peroxisomal disorders, among others [19]. Thus, genetic disorders and IMDs are important to include in a differential diagnosis for HSM.

Clinical manifestations of LSDs: MPSs, oligosaccharidoses and mucolipidoses have findings of a particular form of skeletal dysplasia called “dysostosis multiplex”. Coarse facial features that become more prominent with age may be an important diagnostic sign [39,60]. Coarse facial features involve the presence of frontal bossing, a depressed nasal bridge, an enlarged tongue, prominent supraorbital ridges, rounded cheeks, thick lips, and wide spaced teeth. Additionally, progressive macrocephaly is noticed in children with GM1 gangliosidosis, Sandhoff diseases and GM2 gangliosidosis due to GM2 activator protein deficiency [61]. Contrary to childhood-onset LSDs, attenuated forms of these conditions may present subtly without any striking facial or bony features that are usually characteristic for LSDs. Other systems that may be affected include dermatological findings. Angiokeratomas are present in certain oligosaccharidoses [39] and abundant dermal melanocytosis is common in children with MPSs [62,63]. However, other skin disorders have also been observed in adults with MPS disorders [64]. Farber disease has a classic triad that encompasses a hoarse voice, subcutaneous nodules, and joint deformities. Attenuated forms, often mimicking juvenile arthritis, have previously been described [26]. Individuals with Sialidosis and Galactosialidosis can develop nephrotic syndrome, while renal Fanconi syndrome is common in cystinosis [65,66,67].

In the presence of HSM, further evaluations recommended an ophthalmological exam that may reveal a cherry-red spot concurrent with severe early-onset forms of LSDs, whilst corneal clouding and corneal cystine crystals can be present in children or adults [67,68,69]. On the other hand, vertical supranuclear gaze palsy is a key clinical feature of NPC, and the slowing of horizontal ocular saccades is characteristic of neuronopathic Gaucher disease [70,71].

Imaging of the liver and spleen can be helpful in ruling out benign and malignant neoplasms along with etiologies that produce cysts [20] or gaucheromas [72]. Abdominal imaging may also detect disease-specific pathologies, such as adrenal calcifications in Wolman disease [73].

The application of imaging modalities other than MRI or ultrasound of the liver has been evaluated in LSDs. As an example, elastography has been shown to be useful in the assessment of liver fibrosis in Gaucher disease [74]. The additional value of the fibroscan is the non-invasive evaluation of controlled attenuation parameters and liver stiffness [75].

The skeletal survey can identify dysostosis multiplex that includes findings like J-shaped sella, the thickening of diploic spaces, short and thick clavicles, broad oar-shaped ribs, dysplastic vertebral bodies, scoliosis, inferior pelvic tapering, rounded iliac wings, hip dysplasia, shortened long bones with hypoplastic epiphyses, short and wide metacarpals with thin cortices and proximal pointing, irregular and hypoplastic carpal and tarsal bones. Dysostosis multiplex is characteristic of MPSs, oligosaccharidoses and mucolipidoses [39,60,64]. Bone infarction, arthropathy, and lytic lesions are classical features of Gaucher disease [76], while reduced bone mineral density is a feature of Gaucher disease and ASMD, especially in adult patients [77].

First-line laboratories involve a liberal workup which is required to determine additional liver problems such as hepatocellular damage, cholestasis, and cirrhosis. Most of the LSDs that present with organomegaly have normal liver function test results; nonetheless, the absence of such laboratory abnormalities does not rule out these conditions [24]. Cholestasis is commonly observed in early-onset NPC; liver failure can be evidenced in ASMD and NPC; elevated liver function test results can be expected in Farber and Pompe disease; rare cases of cirrhosis have been reported in Gaucher disease [78], as well as MPS type I and II [9]. CESD is suspected in individuals with high cholesterol, low-density lipoprotein, and triglycerides [79], who may also have persistent mildly elevated liver function tests. The childhood-onset form, Wolman disease, manifests with failure to thrive and very abnormal biochemical and hematological tests.

Apart from biochemical blood and urine tests, hematological investigations are important. Chronic anemia, mild to severe thrombocytopenia and abnormal coagulation tests are often seen in pediatric and adult-onset (attenuated) cases of Gaucher disease. Mild but persistent thrombocytopenia of around 110,000 platelets per microliter was reported as the only laboratory investigation in some attenuated Gaucher disease patients, which led to further investigations and diagnosis at the age of 35 (personal observations of KMS). A range of cytological abnormalities have been described in some LSDs, especially vacuolated lymphocytes [80] and azurophilic inclusions in lymphocytes [81].

Certain non-LSD metabolic disorders may have similar hepatic manifestations and should be considered as outlined above in Section 2.3. Wilson disease and alpha-1-antitrypsin deficiency are likely if cirrhosis is also present. Hepatomegaly in conjunction with hypoglycemia and seizures can be associated with hepatic glycogen storage diseases or gluconeogenesis defects; tyrosinemia type I or Fanconi–Bickel disease should be considered with co-existing renal tubulopathy; and hereditary fructose intolerance often demonstrates abnormal liver function. These disorders are diagnosed mainly in children, but should also be included in the differential diagnoses in adulthood, especially if temporally related to a catabolic stressor like fasting, pregnancy, or steroid use [7].

First line basic and metabolic investigations for HSM are:

Second-line laboratory investigations aiming at the diagnosis of LSDs include analyses of enzyme function, glycosaminoglycans (GAGs), oligosaccharides, and other specific and non-specific biomarkers. These analyses are performed in specialized biochemical laboratories and are not readily available in general commercial labs.

Genetic testing is a useful non-invasive (blood, saliva and buccal swab samples are commonly used) diagnostic approach in centers with and without limited access to the specialized LSD biochemical diagnostic services because it can capture genetic variants (formerly known as mutations) associated with LSDs, as well as other genetic diseases concurrently. Pre-and post-test genetic counseling is highly encouraged. Molecular testing can be diagnostic when likely pathogenic or pathogenic variants are identified. Nevertheless, findings like variants of uncertain clinical significance can lead to more targeted/specific biochemical tests including enzymology or biomarker evaluations that eventually confirm the diagnosis and may upgrade variant classification. Variants of uncertain clinical significance are non-diagnostic and are reported more commonly in populations underrepresented in the genomic databases. Genetic testing frequently occurs in tandem with or after the laboratories discussed below: biochemical urine tests, enzymatic assays, and biomarker evaluation.

Today, Sanger sequencing for single gene testing is rarely used. In contrary, short read next-generation sequencing has become widely available and more affordable, facilitating different gene testing approaches ranging from gene panels to exome or genome sequencing [82]. Gene panels simultaneously investigative multiple genes associated with a specific clinical presentation or group of diseases. For instance, gene panels are commercially available for LSDs or HSM [83]. It is important to check whether or not the genes of interest are included in the ordered genetic testing and the estimated turnaround time (about 3–4 weeks for most commercial laboratories). In the outpatient setting, gene panels or exome sequencing are the preferred option. Rapid exome or genome sequencing can deliver quicker results in emergency cases and the critical care setting within 1 week [84].

Likewise, some limitations must be considered. Depending on the type of genetic testing, some variants cannot be easily identified. As an example, gene panels based exclusively on short reads detected by massive-parallel sequencing might miss the deletion of a partial exon [85]. Moreover, pseudogenes can hinder the sequencing of specific genes, such as GBA and IDS, which are associated with Gaucher disease and MPS II, respectively [82].

Enzymatic analyses on plasma, leukocytes or fibroblasts often can provide a definite biochemical diagnosis of LSDs. However, enzymatic testing is not available for disorders of integral membrane proteins. About 60% of lysosomal disorders are associated with a deficiency of a specific lysosomal hydrolase, and these account for approximately 85–90% of patients diagnosed [10]. Some laboratories routinely offer large panels while others are more selective. It is important for the clinical team to understand the diagnostic strategy of the referral laboratory to ensure that the requested panel includes the suspected disorders. Enzyme activities are routinely measured in plasma and leukocytes isolated from peripheral blood. The use of dried blood spots as an initial sample source has increasingly been developed for some enzymes [86].

Historically, enzyme activity was measured using cultured skin fibroblasts as a sample source, which have largely been replaced by using peripheral blood, but can still be required to establish a diagnosis in some atypical cases. The disadvantage of using fibroblasts is that it requires a skin biopsy, an invasive procedure. There is also a time delay for the fibroblast cultures to establish and grow. However, once the cultures have been established, they can be cryopreserved and recovered if further investigations are required. Cultured cells (usually fibroblasts) are the gold standard sample type to measure neuraminidase for the diagnosis of sailidosis and galactosialidosis, and they can be used for filipin staining in suspected cases of NPC [57,87,88].

When a potential biochemical diagnosis is identified by the deficiency of a sulfatase enzyme, further investigations are required to establish or exclude the differential diagnosis of multiple sulfatase deficiency (MSD). It has been found that not all sulfatase enzymes are deficient in MSD, particularly in attenuated patients. It is therefore recommended that at least three different sulfatase enzymes are measured [30,89]. Mucolipidoses are characterized by an increased enzymatic level of hydrolases in the plasma contrasted with the decreased level in the fibroblasts [45].

A widely described pitfall of lysosomal enzymatic analysis is pseudodeficiency. The enzyme activity is reduced in vitro, but individuals do not have the typical expected clinical manifestations of associated LSD [45]. Pseudodeficiency is explained by decreased specificity toward an artificial substrate used in assays due to the presence of pseudodeficiency variants. Enzyme activity can be reduced to levels that overlap with those seen in affected individuals. Additional biomarkers and genetic analysis may be required to clarify a potential diagnosis. Another rare complication of enzyme analysis can be normal enzyme activity towards artificial substrates, giving a normal result in an affected patient. This has been reported as an issue in some patients affected by ASMD with a specific variant SMPD1, p.Q294K (hg38) [90].

Importantly, the enzyme activity may differ in early-onset versus adult-onset cases. Although the correlation is not perfect, severe and early-onset LSDs correspond to absent or minimal enzyme activity, while attenuated and late-onset patients show residual enzyme activity, reflecting the underlying variants in the relevant gene. The residual enzyme activity may not be detected in leucocytes or dried blood spots but can often be measured in cultured skin fibroblasts. In some cases, particularly with adult patients, the enzyme activity may be just below the reference range and a highly suspicious clinical diagnosis may need further support from enzyme analysis in multiple sample types, as well as genetic and biomarker analysis.

Biochemical urine tests for LSDs include GAGs, oligosaccharides, and sialic acid analyses, which should be considered when HSM is present. A full and complete investigation usually entails quantitative GAG analysis; GAG extraction; and two-dimensional low-voltage electrophoresis and thin layer chromatography (TLC) for oligosaccharides and sialic acids. As there are various qualitative (one- or two-dimensional electrophoresis) and quantitative methods available for the measurement of GAGs, all laboratories may not offer the same testing strategy and therefore clinicians should be aware of which disorders are being screened for at their referral laboratory of choice. For instance, some laboratories analyze quantitative GAGs and perform GAG extraction (Supplemental Figure S1) only when the initial test is positive. Therefore, clinicians should be aware of what is being offered at their referral center.

Increased amounts of quantitative GAGs in urine, as a screening method, may indicate MPS. However, it is known that attenuated MPS patients may not show this increase. Also, GAGs naturally decrease with age. Therefore, it is imperative for a laboratory to report against their own in-house validated age-related reference ranges.

Excess oligosaccharides and sialic acid in urine can be detected using TLC. Again, there is variance across laboratories in which analyses are performed. For instance, the Willink laboratory performs TLC for oligosaccharides and sialic acids as standard on all urine samples arriving at the laboratory with a clinical suspicion of MPS. This strategy is used due to overlapping phenotypes for a variety of LSDs. Abnormal amounts of these compounds can give an indication of certain metabolic disorders such as Alpha mannosidosis, aspartylglucosaminuria, galactosialidosis, GM1 gangliosidosis, M\ucolipidosis II/III, neuraminidase deficiency and free sialic acid storage disease (Supplemental Figure S2). Similarly to GAGs, levels of intensity and detection of abnormal oligosaccharides and sialic acids decline with age. Results should always be confirmed by means of relevant enzymology and/or gene analysis of the appropriate indicated gene or genes.

In recent years, some laboratories have used liquid chromatography tandem mass spectrometry for the analysis of GAGs and oligosaccharides. These new methods, such as non-reducing end, appear to have a better performance with a higher sensitivity and specificity compared with those previously mentioned, although they are not widely available and are costly [91,92].

The LSD biomarkers currently available and emerging are listed in Table 2. Many novel biomarkers have been reported to have close to 100% sensitivity and specificity during initial evaluation, yet subsequently demonstrate limitations once used routinely. Studies reporting non-specific increases in LSD biomarkers are listed in Table 3; expanding collective understanding of these limitations is of high importance as the biomarker approach to laboratory diagnosis further proliferates.

The use of biomarkers in the LSD field is ever expanding and is frequently driven by the desire for high-throughput applications (e.g., newborn screening) or to overcome specific diagnostic challenges. To date, LSD biomarkers have found the highest value in situations where traditional enzymology cannot be used effectively (e.g., NPC, Cystinosis). Biomarkers can also be used to support more traditional enzymatic approaches to diagnosis of LSDs. For instance, equivocal leukocyte arylsulfatase A activity may be supported by the analysis of sulfatides; this may be of particular benefit to distinguish deficiencies (affected MLD) from pseudodeficiencies.

In some cases, analysis of a panel of biomarkers selected on the basis of the diagnostic performance and/or likely clinical overlap can improve specificity compared with analysis of an isolated biomarker. For example, N-palmitoyl-O-phosphocholineserine in combination with sphingosylphosphorylcholine and hexosyl-sphingosine were used for the differentiation of NPC, ASMD and Gaucher disease. Appropriate follow-up by means of enzymology can also be critical to achieving a definitive diagnosis via the exclusion of alternative causes of a biomarker pattern (e.g., measurement of lysosomal acid lipase activity in response to an increase in plasma oxysterol and/or N-palmitoyl-O-phosphocholineserine).

Biomarkers could prove to be helpful for the reclassification of variants of uncertain clinical significance, but this requires a holistic approach and careful consideration of all relevant biochemical, genetic and clinical factors.

Pathology requires invasive procedures. Thus, biopsies should be avoided unless molecular testing or enzyme analysis are non-diagnostic or unavailable. Biopsies can be obtained from different tissues depending on the suspected LSDs. Skin biopsy is a minimally invasive source of fibroblasts which after being cultured can facilitate the diagnosis of certain LSDs based on enzyme levels or biomarkers. It can also be used to detect deposits in fibroblast and in other annex structures (glands, erector pili muscle, end terminal nerves). Patterns of filipin staining suggest NPC [134]. Sialic acid is elevated in the fibroblasts of patients with free sialic acid storage disease [135]. Biopsy of the subcutaneous nodules can show the typical “Farber bodies” [136].

Liver and muscle biopsies carry a higher risk of complications [57]. However, certain findings can help to narrow the differential, such as a positive PAS muscle biopsy, which is indicative of Pompe disease. Some findings in the liver biopsy are typical in lysosomal acid lipase deficiency like “sea-blue” histiocytes, vacuolated Kupffer cells, and cholesterol crystals [137]. ASMD presents with foamy cells and fibrosis [138]. “Gaucher cells” can be observed in liver, spleen or bone marrow biopsies [94].

Muscle biopsy may show secondary mitochondrial changes in LSDs [139]. Bone marrow biopsy is a test performed by hematologists when malignancy is suspected and is not thoroughly discussed in this review.