Hematological Findings in Lysosomal Storage Disorders: A Perspective from the Medical Laboratory

Lysosomal storage disorders (LSDs) are a group of rare and genetic diseases produced by mutations in genes coding for proteins involved in lysosome functioning. Protein defect leads to the lysosomal accumulation of undegraded macromolecules including glycoproteins, glycosaminoglycans, lipids, and glycogen. Depending on the stored substrate, several pathogenic cascades may be activated leading to multisystemic and progressive disorders affecting the brain, eye, ear, lungs, heart, liver, spleen, kidney, skin, or bone. In addition, for some of these disorders, hematological findings have been also reported. In this paper, we review the major hematological alterations in LSDs based on 56 case reports published between 2010 and 2020. Hematological alterations were reported in sphingolipidosis, mucopolysaccharidoses, mucolipidoses, neuronal ceroid lipofuscinosis, glycogenosis, glycoproteinosis, cystinosis, and cholesteryl ester storage disease. They were reported alterations in red cell linage and leukocytes, such as anemia and morphology changes in eosinophils, neutrophils, monocytes, and lymphocytes. In addition, changes in platelet counts (thrombocytopenia) and leukocyte abnormalities on non-peripheral blood samples were also reported for some LSDs. Although in most of the cases these hematological alterations are not pathognomonic of a specific disease or group of LSDs, since they can be easily identified in general clinical laboratories, their identification may contribute to the diagnosis of these disorders. In this sense, we hope that this review contributes to the awareness of the importance of hematological alterations in the diagnosis of LSDs.


A R T I C L E I N F O A B S T R A C T
Lysosomal storage disorders (LSDs) are a group of rare and genetic diseases produced by mutations in genes coding for proteins involved in lysosome functioning. Protein defect leads to the lysosomal accumulation of undegraded macromolecules including glycoproteins, glycosaminoglycans, lipids, and glycogen. Depending on the stored substrate, several pathogenic cascades may be activated leading to multisystemic and progressive disorders affecting the brain, eye, ear, lungs, heart, liver, spleen, kidney, skin, or bone. In addition, for some of these disorders, hematological findings have been also reported. In this paper, we review the major hematological alterations in LSDs based on 56 case reports published between 2010 and 2020. Hematological alterations were reported in sphingolipidosis, mucopolysaccharidoses, mucolipidoses, neuronal ceroid

INTRODUCTION
Lysosomal storage disorders (LSDs) are a group of monogenic metabolic diseases produced by mutations in genes encoding for proteins involved in the lysosomal function. These mutations lead to the synthesis of proteins with none or reduced activity, producing the progressive accumulation of partially degraded substrates into the lysosome [1]. Although the clinical, diagnostics, and pathophysiology can be heterogeneous, the LSDs can be classified into 7 groups according to the accumulated general substrate ( Table 1).
The diagnosis of a LSDs is based on clinical evaluation of the patient and laboratory tests such as the evaluation of the enzymatic activity [2], or the primary stored substrate (e.g., glycosaminoglycans, oligosaccharides, sphingolipids, and glycogen, among others) [3,4]. Molecular diagnosis is still difficult considering the lack of complete understanding of the clinical implications of novel variants [5][6][7]. Nevertheless, molecular diagnosis should be considered during the diagnosis of some LSDs, such as neuronal ceroid lipofuscinosis [8]. Moreover, lyso-Gb3 for Fabry disease (FD) or macrophage inflammatory protein 1-alpha (MIP-1α) in Gaucher disease (GD) have been suggested as novel biomarkers of these LSDs [5]. Chitotriosidase has also been proposed as a biomarker for some LSDs such as GD [9], cystinosis [10], GM1 gangliosidoses [11], and Niemann-Pick type C [12]. Particularly in GD, some hematological alterations on macrophages are frequently found in both smear blood and biologic fluids (i.e., Bronchoalveolar lavage-BAL), which may contribute to the diagnosis. Nevertheless, leukocyte abnormalities also can be found in other LSDs. In this review, we described the major hematological alterations in LSDs found in 56 case reports published between 2010 and 2020. Reference values of all hematological findings were adjusted to the guidelines on standard operating procedures for hematology of the World Health Organization (WHO).

LSD AND GENERAL DIAGNOSIS
Lysosomes were first described in the 1950s by Christian De Duve, who recognized the role of this organelle in the degradation and recycling of intracellular and extracellular macromolecules [15]. During the last decade, they have been recognized other lysosomal functions due to the fundamental role as a metabolic hub, influencing and sensing diverse nutrient processes, secretion, gene regulation, plasmatic membrane repair, ionic homeostasis, immune response, and cholesterol transport, among others [16]. The lysosomal biogenesis is triggered by the transcription factor EB (TFEB), which is a master regulator of the lysosomal biogenesis and influences the interaction of the lysosome with other organelles and the cellular homeostasis [16].
LSDs are a group of about 70 monogenic metabolic disorders caused by the deficiency of a specific protein (i.e., enzyme, transporter, or cofactor) involved in the lysosomal function. This deficiency leads to the lysosomal accumulation of partially degraded substrates that generates and spectrum of clinical manifestations depending of the stored substrate and the affected tissues [17]. LSDs have an overall prevalence between 1 in 4,000 and 7,000 live births. Nevertheless, the exact prevalence of LSDs is difficult to calculate due to their wide heterogeneity, which sometimes leads to misdiagnosis [5]. An important group of LSDs are associated with central nervous system (CNS) impairment, while others are mainly characterized by their effect on peripheral organs. The reader is referred to recent review for more details about LSDs [18].

DIAGNOSIS
Given the enormous heterogeneity found in the LSDs, the diagnosis is always a challenge, and it requires a common effort between clinicians, biochemists, and medical laboratory scientists to achieve an adequate and timely diagnosis. In this sense, clinical suspicion should be accompanied by accurate routine and specialized laboratories.

Clinical approach
LSDs clinical manifestation mainly depends on the stored substrate and the affected tissues. Usually, the storage substrate occurs in the organs where the substrate is synthesized (i.e., liver, spleen, bone, brain, muscle). In this sense, the accumulation of the substrate will generate a disruption of the cellular homeostasis, inducing cellular damage, mitochondrial stress, apoptosis, dysregulation of redox processes, and disruption of the lysosome in/out transport [19][20][21]. Phenotypic features of patients with LSDs vary depending of the stored substrate and may include coarse face, short stature, skeletal abnormalities, hepatosplenomegaly, cardiac and lung disease, eye and ear impairment, and central nervous system involvement [5,18].

Laboratory approach
As previously noted, the diagnostic confirmation of a LSD requires a specialized laboratory including the identification of biomarkers (e.g., stored substrate quantitation), determination of enzymatic activities, and molecular diagnosis. In these scenarios, important advances have been reached. For instance, the measure of GAGs by several qualitative and quantitative methods such as alcian blue, toluidine blue, paper, and thin-layer chromatography, high-pressure chromatography, gas chromatography, mass spectrometry is used in MPS diagnosis and follow up [22]. Collagen type II, β-galactosidase, nidogen-1, and fatty acid-binding protein are also used in MPS [23]. For FD, it has been proposed the globotriaosylsphingosine determination, whereas the measure of chitotriosidase is used for GD. Other biomarkers for GD include glucosylsphingosine, macrophages protein 1-alpha and 1-beta, cathepsin K and osteopontin [23]. For sphingolipidoses such as Krabbe, the psychosine has been described as a biomarker, which may determine the progression of the disease [23], whereas the cholestane-3β, 5α, 6β-triol (C-triol), and 7-ketoholesterol seem to be a sensible biomarker in Niemann-Pick type C (NPC) [24,25]. In glycogenoses, as in Pompe disease, the most known biomarker is tetrasaccharide glucose (Glc4), which correlates with therapy response. Other two biomarkers, myostatin and insulin-growth factor I (IGF-I), can also be used in Pompe disease [23,26]. These biomarkers could be useful in pseudo-deficiencies, as reported for MPS I, MPS VII, and GD [27,28], for which the traditional biochemical tests not always lead to a specific diagnosis. The enzyme activity determination is the gold standard for the diagnosis of LSDs produced by the impairment of an enzyme. This activity can be assayed in a wide range of biological samples such as plasma, serum, leukocytes, cultured fibroblasts, chorionic villi, amniotic fluid, cultured amniocytes, and dried blood spots [29]. Finally, the molecular diagnosis can helpful to establish the diagnosis and genetic counseling interventions [3], and is the gold standard for other conditions as neuronal ceroid lipofuscinosis [8]. Nevertheless, biomarker-, enzyme activity-, or molecular-based diagnosis of LSDs requires highly specialized facilities and trained personnel. In contrast, the analysis of the hematological abnormalities, which can help in the diagnosis of some LSDs, can be performed in low and high-complexity laboratories. In this sense, in the next sections, we will review the complexity of the hematopoietic system and the major alterations reported in some LSDs. and indicated that the majority of blood cells originate from very few/unique multipotent HSCs capable of proliferation, differentiation, and self-renewal [30]. These HSC-independent hematopoietic cells originate in the embryo and persist in the adult hematopoietic system [31,32]. Whereas thrombopoietin, erythropoietin, colony-stimulating factor, and some interleukins promote the HSCs proliferation; several cytokines promote the HSC differentiation into multipotential colony-forming progenitor units (CFU) including: CFU-GEMM (Granulocytes, Ery throcytes, Monocytes, Megakaryocytes) for the myeloid lineage and CFU-Li (T, B or NK cells) for the lymphoid lineage ( Figure 1) [33,34].

OVERVIEW OF THE HEMATOPOIETIC SYSTEM
Since mature blood cells derived from HSC contain all the eukaryotic organelles, including lysosome, substrate accumulation observed in LSDs  [35,36]. Upon intrinsic and extrinsic signals (cytokines, growth factors, and transcription factors), HSCs can give origin to GFU-GEMM and CFU-Li to myeloid and lymphoid precursors, respectively [37]. can affect not only the hematopoietic precursors but also the most differentiated cells as red blood cells, leukocytes, and thrombocytes or even platelets [38,39]. In Table 2, we summarize the normal values for the blood count and in the next sections, we describe the normal findings of the blood cells as well as their alteration in the LDS context.
The age for male patients was between 2 weeks and 68 years; whereas females ranged from newborn to 69 years. We focused on the hematological parameters before the establishment of any clinical intervention to identify baseline values and morphological changes in blood cells that could be associated with the disease. All data is summarized in Table 3.

Red cell lineage
Anemia was reported in 30.3% of the cases (17/56), with GD having the highest number of reports (64.7%), followed by cystinosis, CESD, NPC, ML IV, MPS I, and aspartylglucosaminuria, with 5.9% each one. Based on the MCV, 3 cases of microcytic anemia (MCV <80fL) were identified, all of them corresponding to GD. Although mild anemia was the main finding, one case reported a severe microcytic hypochromic anemia (Hb: 50 g/L; MCV: 66 fL, MCH: 19.4 pg) in a 1-year-old man diagnosed with GD type 1 with slight anisocytosis and poikilocytosis [41]. Interestingly, a 69 years-old Japanese female with GD type 1 and gastric cancer had a reduction in the erythrocytes count (3.98x10 6 /µL) and hematocrit levels (34,6%), with marked reticulocytosis (20%) [42]. In this patient, a novel mutation c.587A>G (p.K157R) in the GBA gene was reported; however, the impact of this mutation on the β-glucocerebrosidase was not evaluated.

Leukocytes
As previously described, LSDs are a group of metabolic disorders characterized by substrate accumulation into the lysosome [1]. In this sense, it could be expected that the major hematological findings are related to the leukocyte morphology due to the presence of organelles in these cells [43]. The reader is referred to some of the reviewed publications for representative images of leukocytes alterations [44][45][46][47][48].

Granulocytes
Basophil abnormalities were not reported on any of the revised cases. On the other hand, eosinophils with enlarged and sparse granules in the blood smear were reported in three cases of GM1 gangliosidoses [44,49,50]. Although these alterations have been recognized since early reports as frequent alterations of GM1 gangliosidoses [51], they are not routinely investigated as part of suspicion of GM1 gangliosidoses unless the absolute leukocyte count is altered. Eosinophils with deep pink-blue granules were reported in a MPS type VI female patient, as well as abnormal lobulation on neutrophils [52]. These findings were observed after an alteration in the automated analysis, which led to a suspicion that was later confirmed as pseudobasophilia. On the other hand, the Alder-Reilly anomaly was identified on neutrophils from MPS I, IV, and VI patients [45,[52][53][54][55], representing a potential finding for this LSD group.
Although we did not find the Alder-Reilly anomaly in the reports for other LSDs, it is not possible to discard its presence in entities different than MPS. In addition, Alder-Reilly inclusions must not be confused with toxic granulations that are restricted to neutrophils in a transitory form during an inflammatory process [43].

Monocytes
Despite the crucial role of monocytic cells, morphology alterations were not frequently  [54,55], which are metachromatic after toluidine staining [46]. Nevertheless, the most common findings on monocytes seem to be restricted to the phagocytic activity of the macrophages from BM, liver, and spleen. These changes will be discussed later.

MPS IIIB [59], MPS IV [54] and MPS VII [59].
In ML type II (I-cell disease) vacuole-like inclusions are frequently observed on lymphocytes, in which accumulation of HLA class II molecules have been observed, suggesting a role of N-acetylglucosamine-1-phosphotransferase in the immune system [60]. In this sense, the study of the chemical nature of the inclusions may increase our knowledge about the physiopathology of the disease and lead to the identification of novel therapeutic targets. On the other hand, atypical and reactive states of lymphocytes were reported in one case of GD [61] and aspartylglucosaminuria [62], respectively. Also, electronic microscopy of peripheral blood samples allowed the identification of cytoplasmatic vacuoles containing electrodense periodic structures in lymphocytes from NCL patients [63][64][65].
In Pompe disease, it has been reported that vacuolation of lymphocytes [66] is a consequence of the lysosomal glycogen accumulation [67]. Electronic microscopy of peripheral blood cells, allowed the identification of inclusions in samples from Pompe disease patients [68]. Despite vacuolization is being suggestive of lysosomal storage, the use of a specific glycogen staining (i.e., periodic acid-Schiff, PAS), allows a more accurate diagnostic impression. This was validated by Hagemans et al., through the analysis of PAS-stained blood films obtained from 65 patients with classical infantile and adult forms of Pompe disease, with a sensitivity and specificity of 100% and 98%, respectively [69].

Leukocyte abnormalities on non-peripheral blood cells
The major goal of this review article was to describe the more common hematological findings in LSDs with a particular focus on the blood smear due to its routine use in medical laboratories. Nevertheless, interesting changes of leukocytes, particularly in monocyte-derived lineage, have been registered in several biological samples which are summarized in Table 4. For instance, for GD it has been described the presence of Gaucher cells in BM [80,86]. Gaucher cells are very large cells, usually macrophages, with a diameter between 20 and 80 µm with a small and eccentrically placed nucleus as well as a cytoplasm with wrinkles or striations [87].   Likewise, in Niemann Pick disease type B and C foamy macrophages have been observed in BM, bronchoalveolar lavage, and liver biopsy [88][89][90]. Histiocytes with refractile crystal in BM [76], and macrophages with intracytoplasmic granules in cerebrospinal fluid [91], were found in cystinosis and MPS I, respectively. Finally, azurophilic inclusions in plasma cells were found in BM from MPS IIIA patients [47].

CONCLUSIONS AND PERSPECTIVES
In LSDs, the accumulation of undegraded macromolecules into the lysosome triggers complex pathogenetic cascades leading to the clinical manifestations of these diseases. Impaired metabolism of lysosomal substrates may affect the autophagic-lysosomal system, the ubiquitinproteosome system, the lysosome membrane permeability, and promotes inclusion body formation, dysregulation of the signaling pathway, inflammation, calcium homeostasis abnormalities, mitochondrial dysfunction, and oxidative stress [18].
These cellular changes may affect several tissues leading to different clinical manifestations, including hematological alterations. Although these hematological alterations may not be common findings in LSDs, they may contribute to the diagnosis of the disease, especially considering that the tests to evaluate these hematological abnormalities are available in most clinical laboratories. Nevertheless, health care professionals need to be aware of hematological alterations and correlate them with patient symptoms and other clinical laboratory results.
In addition, it is necessary to extend the study of hematological alterations to other LSDs to explore the potential of these abnormalities as a biomarker for diagnosis or treatment follow-up.