Red blood cell deformability in multiple myeloma 1

1 Introduction

Multiple myeloma (MM) is a neoplasm of the plasma cells that covers a clinical spectrum from asymptomatic to aggressive forms referable to the deposition of abnormal immunoglobulin chains in different tissues.

The MM evolves from clinically silent pre-malignant stages denominated monoclonal gammopathy of undetermined significance (MGUS) and a middle clinical phenotype defined smoldering multiple myeloma [20].

Symptomatic myeloma is defined by the presence of end-organ damage (CRAB: hypercalcemia, renal insufficiency, anemia, bone lesions) in patients with a M-protein component and clonal bone marrow plasma cells. In many MM patients there is a cluster of clinical, laboratory, radiological and pathological findings. A M-protein is observed in the serum or urine in about 97% of patients (IgG 50%, IgA 20%, light chain 20%, IgD, IgE, IgM and biclonal <10%); ∼3% of cases are non-secretory. In 90% of MM patients there is a decrease in polyclonal Ig (<50% of normal). Other laboratory findings include hypercalcemia (20%), elevated creatinine (20–30%), hyperuricemia (>50%) and hypoalbuminemia (∼15%). Radiographic studies show at initial diagnosis lytic lesions, osteoporosis or fractures in 70% of cases.

The M-protein component in serum and urine is detected and evaluated by specific analysis (serum and urine electrophoresis and immunofixation). Furthermore, serum free light chain assay has a high sensitivity [9].

For the evaluation of the bone marrow plasma cell infiltration, bone marrow biopsy is proposed to identify quantitative and/or qualitative abnormalities of plasma cells. The diagnosis of multiple myeloma requires 10% or more clonal plasma cells. Bone marrow studies should include chromosome analysis by conventional karyotyping (cytogenetics) and fluorescence in situ hybridization (FISH).

The presence of bone lesions is generally investigated with a full skeleton radiographic survey, even if MRI, CT, or PET/CT are more sensitive tests [16].

In MM, the high concentration of monoclonal plasma proteins increases plasma viscosity; in the clinical practice there is a positive relationship between the paraprotein concentration and the values of plasma viscosity. Nevertheless the level of this relationship depends on the shape and size of these molecules (IgM>IgA>IgG); moreover, some immunoglobulins, such as IgA and IgG3, have a predisposition to polymerize [14, 29].

To date, several papers have examined the hemorheological pattern in MM patients observing an increase in plasma and in serum viscosity and in erythrocyte aggregation, associated with a hematocrit decrease; this hemorheological alteration may be associated with ocular abnormalities, bleeding diathesis and neurological symptoms, such as stroke [17–19] related to the cerebral ischemia and high output cardiac failure [27].

In the last years, some authors have described an altered lipid profile in the erythrocyte membrane [6] and in plasma of MM patients [7]. Others have suggested instead that the increasing fatty acid synthase (FAS) found in these patients should be the focus for pharmacological treatment [32]. Moreover, it has been observed how the proliferation of human myeloma cells may be contained with the inhibition of the fatty acid metabolism using etomoxir and orlistat [26]. Using the atomic force microscopy (AFM), that examines the morphological properties of red blood cells at nanometer scales, other authors observed marked differences between controls and MM patients [8, 34].

Recently, we published a paper regarding the hemorheological profile in MM patients [4], however the purpose of this research was to focus on the behaviour of the erythrocyte deformability in this clinical disorder.

2 Subjects

We enrolled 29 patients (11 women and 18 men; mean age 67.9±10.6 years) with MM. The group included 9 IgA, 16 IgG, 1 IgM and 3 non-secretory MM. Sixteen patients were recently diagnosed and at the initial stage of therapy, 8 were on consolidation/conservation therapy, whereas 5 patients had achieved a complete remission. The principal laboratory findings in this group of patients were: Hb (g/dl) 10.97±1.75 (range 7.6–15.0), RDW (%) 15.86±2.40 (range 13.2–22.4), creatinine (mg/dl) 1.14±0.61 (range 0.53–2.95), beta2-microglobulin (μg/ml) 4.098±2.698 (range 1.50–13.50), calcium (mg/dl) 9.153±0.741 (range 8.01–11.90), albumin (g/L) 36.41±5.16 (range 26.50–45.10), fibrinogen (mg/dl) 354.4±120.2 (range 207–817), IgG (mg/dl) 1343±1450 (range 161–5101), IgA (mg/dl) 517±1004 (range 6–3890), IgM (mg/dl) 35.1±30.3 (range 5–101), M-protein (g/dl) 1.226±1.207 (range 0–3.800). The control group included 31 subjects (18 women and 13 men; age range 23–65 years) free of diseases on the basis of clinical history, physical examination, electrocardiography, routine hematological and urine analysis.

3 Methods

Venous blood samples were collected in the morning by venous puncture from the antecubital vein of fasting subjects and immediately transferred to anticoagulated glass tubes for the evaluation of the following parameter.

– Erythrocyte deformability: to evaluate this hemorheological parameter, a 30μl of anticoagulated blood with 2 ml of dextran solution at a viscosity of 24 mPa was mixed. The measurement was obtained by using the diffractometer Rheodyn SSD of Myrenne, which measures the diffraction pattern of a laser beam passing through erythrocytes suspended in a viscous medium and deformed by a force with defined shear stress. The shear stress employed was 6, 12, 30 and 60 Pa. The erythrocyte deformation was expressed as elongation index (EI) = (l – w/l + w) x100, where l = length and w = width of the erythrocytes.

4 Statistical analysis

Data were expressed as means +/– S.D. The Student’s t test for unpaired data was used to compare normal controls and MM patients. The correlations between erythrocyte deformability and immunoglobulin levels in MM patients were examined using the linear regression.

5 Results

In MM patients a significant decrease in erythrocyte deformability, especially at low shear stresses, was observed (Fig. 1). EI at the shear stress of 60 Pa (EI 60) was 45.19±2.52 in control subjects and 42.34±4.65 in MM patients (p < 0.01); EI 30 was 41.67±2.20 in control subjects and 36.43±5.07 in MM patients (p < 0.001); EI 12 was 33.29±2.18 in control subjects and 25.54±5.31 in MM patients (p < 0.001); EI 6 was 24.94±2.32 in control subjects and 16.25±4.87 in MM patients (p < 0.001). We also assessed the correlation between all the values of the elongation index and the immunoglobulin levels; no statistical correlation between these parameters was found (data not shown).

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Fig.1

Erythrocyte deformability at different shear stresses in control subjects and multiple myeloma patients.

There is an evident decrease of the erythrocyte deformability in MM patients. Physiologically, the decrease of red cell deformability may depend on a reduced surface/volume ratio, an increased cytosolic viscosity, or an alteration of the membrane dynamic properties; the latter refers in particular to the qualitative and/or quantitative alteration of membrane lipids and proteins. Erythrocyte membrane lipids are represented by cholesterol (28%), phospholipids (62%), and glycolipids (5–10%); phospholipids are distributed asymmetrically: phosphatidylcoline (30%) and sphyngomyelin (25%) are neutral molecules localized in the external leaflet, while phosphatidylethanolamine (27%) and phosphatidylserine (14%) -negatively charged- are localized in the internal leaflet. This asymmetry is preserved by the activity of some enzymes, such as flippases and floppases that translocate lipids against gradient with an energy-dependent mechanism, while the scramblases move phospholipids in both directions dissipating the transmembrane gradient via an ATP independent mechanism. Erythrocyte membrane proteins are subdivided in integral and in peripheral proteins. The integral proteins are the band 3, the glycophorins and the glycosylated phosphatidylinositol-anchored proteins, while the peripheral proteins may be subdivided into anchoring (ankirin, protein 4.1, protein 4.2, adducin etc) and cytoskeletal (α and β-spectrin and actin).

In patients with MM, an alteration of the red cell membrane fatty acid profile has been found: an increase in satured fatty acids and in total polyunsatured fatty acids (n-6), and a decrease in monounsatured fatty acids, in total polyunsatured fatty acids (n-3), in total trans fatty acids, and in the ratio n-3/n-6 [6]. The plasma fatty acid profile in MM patients shows an increase in satured fatty acids, in monounsatured fatty acids and in total polyunsatured fatty acid (n-6) and a decrease in polyunsatured fatty acids (n-3), in total trans fatty acids, and in the ratio n-3/n-6. These alterations are ascribable to the functional alteration of the desaturase and of the elongase, which have a specific role in the maintenance of the lipid network in biological membranes [15, 31].

The increase in fatty acid synthase (FAS) observed in human myeloma cell lines has suggested that FAS should be a target of the pharmacological treatment in MM [32]. In fact, in that research the authors firstly demonstrate an increased FAS expression in bone marrow samples obtained from MM patients and in two human MM cell lines (U266 and RPMI8226). Thereafter, they treated in vitro U266 cells with cerulenin, a native inhibitor of FAS; this treatment reduced the metabolic activity/cell proliferation of the cell lines and activated significantly the apoptosis. Further considerations [26] could be made by bearing in mind the inhibition of fatty acid metabolism obtained with some drugs (etomoxir or orlistat) in human myeloma cells. These molecules inhibited significantly the beta-oxidation and “de novo“ acid synthesis without changes in the glucose metabolism; each molecule reduced by 40–70% the myeloma cell proliferation while the use of both molecules produced an additive inhibitory action.

By employing the atomic force microscopy (AFM), Zhang et al. [34] have demonstrated that the surface topographic image, the height profile and the surface ultrastructure of the erythrocytes distinguish the erythrocyte of healthy subjects from those of MM patients. Liu et al. [8] observed in MM erythrocytes also a marked irregularity of the outline of the histograms of the particle size extracted from the surface ultrastructure.

The altered membrane lipid profile, as well as the marked anomalies observed using the AFM, may explain the decrease of erythrocyte deformability observed in MM.

An alternative hypothesis might be the presence of a paroxysmal nocturnal hemoglobinuria-like defect in the red cell membrane of MM patients [5, 28]. This defect is characterized by an alterated synthesis of the glycosylphosphatidylinositol, which is essential for the binding of some surface proteins, such as CD55 and CD59, to protect the red cells from intravascular lysis. Up to now, PNH-like clones have been observed in different haematological diseases, such as aplastic anaemia, myelodysplastic syndrome and myeloproliferative disorders [13], but also in lymphoproliferative syndrome [10] and in acute leukemia [12]. PNH-like defect has been observed in patients with rheumatic diseases [2] while in diabetes mellitus [1] there was a reduction of the expression of CD55 and CD59 in endothelial cells incubated with high glucose concentration and normalized after co-incubation with verapamil.

Another aspect to underline is the possible role played by the altered erythrocyte deformability in the pathogenesis of anaemia, found in two thirds of MM patients. This clinical manifestation seems to be the consequence of an increased IL-6 production, of the macrophage inflammatory protein-1α secretion by myeloma cells, of the apoptosis of erythrocyte precursors (immature erythroblastes) and of low erythropoietin level (due partly to renal impairment) [23, 24]. Moreover, it is possible to argue that the alteration of the red cell membrane observed in this clinical condition, altering its rheology, may help to remove them.

7 Conclusions

In conclusion, from a rheological perspective, MM is a hematological neoplasm impairing the microcirculation because of the increase in plasma viscosity and associated with a decrease in red cell deformability, which acts together with the plasma viscosity in the microcirculatory area influencing the delivery of oxygen to the tissue [3, 33].

Conflict of interest

The authors declare that they have no conflict of interest.