Erythrocyte deformability and hemorheological profile in multiple myeloma

Abstract

The hemorheological profile in multiple myeloma (MM) has been extensively studied. Our investigation regarded the behavior of whole-blood viscosity, plasma viscosity and erythrocyte deformability in MM. We enrolled 24 MM patients; 13 of them had been recently diagnosed and were at the initial stage of therapy, 6 were on consolidation/conservation therapy and 5 had achieved a complete remission. On fasting venous blood we evaluated whole-blood and plasma viscosity at high and low shear rates, haematocrit, the ratios between whole-blood viscosity (at high and low shear rate) and haematocrit×100, the ratio between plasma viscosity at low and high shear rate and the erythrocyte deformability examined by using laser diffractometry and expressed as elongation index. A significant increase in plasma viscosity at low shear rate and a marked decrease in haematocrit were observed in MM patients compared with normal controls. Also the ratio between the high shear rate whole-blood viscosity and haematocrit ×100 and the ratio between the low and high shear rate plasma viscosity were significantly increased in MM patients. A significant decrease in erythrocyte deformability, especially at low shear stresses, was found. We discuss some hypotheses that might explain the behavior of red blood cell deformability in MM, considering that its impairment, in addition to the increase of plasma viscosity, can alter the microcirculatory flow in these patients.

Keywords

Multiple myeloma plasma viscosity erythrocyte deformability

1 Introduction

Multiple myeloma (MM) is a neoplasm of plasma cells that accumulate in bone marrow leading to bone destruction and marrow failure, with a monoclonal protein (M-protein) in serum and/or urine. The disease spans a clinical spectrum from asymptomatic to aggressive forms referable to deposition of abnormal immunoglobulin chains in different tissues.

The average age of MM patients is 62 years for men (75% >70 year old) and 61 years for women (79% >70 year old). 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 [40].

Symptomatic myeloma is defined by the presence of end-organ damage (CRAB: hypercalcemia, renal insufficiency, anemia, bone lesions) in a patients with a M-protein component and clonal bone marrow plasma cells. In several patients there is a cluster of clinical, laboratory, radiological and pathological findings. A M-protein is found 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 lytic lesions, osteoporosis or fractures in 70% of cases at initial diagnosis.

The medical history of the patient and the physical examination should be included in the initial diagnosis, associated with a complete blood cell count with differential leucocyte and platelet count, blood urea nitrogen, serum creatinine and electrolytes, serum calcium as well as albumin, lactate dehydrogenase (LDH) and beta-2 microglobulin. 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 [25].

For the evaluation of the bone marrow plasma cell infiltration, bone marrow aspiration and 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 at initial diagnosis should include chromosome analysis by conventional karyotyping (cytogenetics) and fluorescence in situ hybridization (FISH).

A full skeleton radiographic survey is advisable to evaluate bone lesions, however additional more sensitive tests to consider are MRI, CT, or PET/CT [30].

Multiple myeloma staging is performed by the Durie and Salmon system that considers the M-protein levels, bone lesions, hemoglobin levels and calcium concentration. Additional indicators of higher risk patients include elevated serum beta-2 microglobulin, low serum albumin, elevated LDH, high C-reactive protein, increased plasma cell proliferative activity, high degree of bone marrow replacement, plasmablastic morphology and genetics. An international staging system (ISS) for plasma cell myeloma provides highly significant prognostic correlations, using a combination of serum beta-2 microglobulin and albumin level to define the three stages.

According to recent guidelines, the therapeutic program is subdivided into three phases: induction, consolidation, and maintenance; the approach to each phase is individualized based on the features of the disease, age and comorbidities. These features allow to distinguish patients eligible for transplantation from those who are not eligible. In both cases the survival rate has clearly improved in the last years due to an extensive spectrum of new agents available for treatment [39].

MM may also occur in other different forms, such as micromolecular myeloma in which the plasma cells produce only immunoglobulin light chains; non-secretory myeloma in which the plasma cells do not produce immunoglobulin, although an excessive number of them is demonstrable; solitary plasmacytoma, a cancer that has a single location in a bone or extramedullary site and plasma cell leukemia with the plasma cells present in large numbers even in blood.

In MM the high concentration of monoclonal plasma proteins increseas plasma viscosity (PV), that is a major component of whole-blood viscosity (WBV). A plasma hyperviscosity condition can result from the presence of immunoglobulins with high molecular weight. There is generally a positive relationship between the paraprotein concentration and the values of PV. Nevertheless, the development of plasma hyperviscosity also depends on the shape and size of the molecules; in fact, IgM are greater than IgA, which are greater than IgG. Moreover some immunoglobulins, such as IgA and IgG3, show an inclination to polymerize [27 , 55].

The protein content is a pivotal factor for increasing PV and blood flow resistance, which regulates the vascular tone and maintains a functional capillary density [3 , 53]. It also has a specific impact on the rheological behavior of red blood cells, inducing the rouleaux phenomenon that is especially observed at low shear rates.

To date, several papers [1 , 54] have examined whole-blood, plasma and serum viscosity in MM patients, observing an increase in PV, serum viscosity and red cell aggregation, often associated with a low haematocrit (Ht). In a minority of MM patients (2–6%), a symptomatic hyperviscosity syndrome develops, with ocular, neurological and cardiovascular manifestations [1 , 59]; the condition is more frequent (10–30%) in Waldenstrom’s macroglobulinemia than in MM, due to the characteristics of IgM molecules.

In the last years some authors have demonstrated an altered lipid composition in the erythrocyte membrane [13] and plasma of MM patients [14], and the fatty acid metabolism has been suggested as a possible therapeutic target in MM [50, 58]. Moreover, significant differences between the erythrocytes of normal controls and those of MM patients have been observed using the atomic force microscopy (AFM), that investigates the morphological properties of cells on a nanometer scale [21, 61].

Taking into consideration the above studies, the purpose of this preliminary research was to examine the hemorheological profile, including the erythrocyte deformability measured with a diffractometer method, in a group of MM patients.

2 Subjects

We examined 24 patients (11 women and 13 men; mean age 66.7±10.9 years) with MM. The group included 8 IgA, 13 IgG, 1 IgM and 2 non-secretory MM. Thirteen patients were recently diagnosed and at the initial stage of therapy, 6 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) 11.17±1.69 (range 8.3–15.0), RDW (%) 15.59±2.19 (range 13.2–20.7), creatinine (mg/dl) 1.19±0.64 (range 0.53–2.95), beta2-microglobulin (μg/ml) 4.406±2.869 (range 1.50–13.50), calcium (mg/dl) 9.174±0.793 (range 8.01–11.90), albumin (g/L) 36.03±5.35 (range 26.50–45.10), fibrinogen (mg/dl) 351.4±129.6 (range 207–817), IgG (mg/dl) 1208±1295 (range 161–4688), IgA (mg/dl) 410.1±785.8 (range 6–3139), IgM (mg/dl) 34.3±29.6 (range 5–101), M-protein (g/dl) 0.952±1.047 (range 0–3.100). The control group included 21 subjects (13 men and 8 woman; age range 23–63 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 parameters:

WBV at the shear rate of 450 sec^–1, by using the cone-on-plate viscometer Well-Brookfield 1/2 LVT (Middleboro, MA, USA), at 37°C;

WBV at the shear rate of 0.51 sec^–1 employing the viscometer Contraves LS30 (proRheo GmbH, Althengstett, Germany), at 37°C;

PV at the shear rate of 450 sec^–1, by using the cone-on-plate viscometer Wells-Brookfield 1/2 LVT, at 37°C;

PV at the shear rates of 20.4 and 0.51 sec^–1 employing the viscometer Contraves, at 37°C;

Ht, obtained by using a micromethod;

WBV at 450 sec^–1/ Ht×100;

WBW at 0.51 sec^–1 / Ht×100;

PV at 0.51 sec^–1/ PV at 450 sec^–1;

Erythrocyte deformability; to evaluate this parameter, 30μl of anticoagulated blood were mixed with 2 ml of dextran solution with a viscosity of 24 mPa·s. 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) ×100, where l = length and w = width of the erythrocytes.

4 Statistical analysis

Data were expressed as means±S.D. and ranges. The Student’s t test for unpaired data was used to compare normal controls and MM patients. The study of the correlations between hemorheological parameters was carried out using linear regression. The difference in the hemorheological profile between MM patients subgroups subdivided according to the clinical stage (remission or active disease), or according to the median value of the red blood cell distribution width (RDW), were examined employing the Student’s t test for unpaired data.

5 Results

By comparing normal controls and MM patients, a significant increase in PV at low shear rates and a marked decrease in Ht were observed in MM subjects (Table 1). A correlation between WBV at high and low shear rates and Ht was evident in MM patients and normal controls (Fig. 1), although in MM patients, in particular at high shear rate, the degree of significance was lower. No correlations between PV at high and low shear rates and immunoglobulin levels were found (Fig. 2), nor was PV at any shear rate correlated with fibrinogen or M-protein concentration (data not shown). We also evaluated the correlation between high shear rate WBV and high shear rate PV and between low shear rate WBV and low shear rate PV in normal controls and in MM patients. From this analysis (data not shown) a significant correlation between high shear rate WBV and high shear rate PV was evident in both groups. The ratio between high and low shear rate WBV and Ht×100 and the ratio between PV at low and high shear rate were evaluated (Table 2). The ratio high shear rate WBV/Ht×100 and the ratio low shear rate PV/ high shear rate PV were significantly increased in MM patients. The MM patients were subdivided according to the clinical stage and according to the median value of RDW (15.45%). No difference in the hemorheological pattern was evident according to the clinical stage (data not shown); in the subgroup with higher RDW values a significant decrease of Ht (p < 0.05) and a marked increase in high shear rate WBV/Ht ratio×100 (p < 0.01) were observed. In MM patients a significant decrease in erythrocyte deformability, especially at low shear stresses, was also observed (Table 3). Considering this unexpected datum, a further evaluation was conducted on the possible correlation between EI and PV and between EI and fibrinogen, immunoglobulin and M-protein levels, but no significant correlation was found (data not shown).

Fig.1

Correlations between whole-blood viscosity (WBV) and haematocrit in control subjects (A-B) and in multiple myeloma patients (C-D).

Fig.2

Correlations between plasma viscosity (PV) and Ig concentrations in multiple myeloma patients.

Table 1

Means±S.D. and ranges of the hemorheological determinants in control subjects and multiple myeloma patients

	Control subjects	Multiple myeloma patients
WBV 450 sec^–1 (mPa · s)	2.934±0.393	2.886±0.435
	(2.410–3.780)	(2.100–3.750)
WBV 0.51 sec^–1 (mPa · s)	24.18±4.40	21.47±6.77
	(17.57–32.70)	(10.20–34.97)
PV 450 sec^–1 (mPa·s)	1.210±0.065	1.175±0.286
	(1.100–1.310)	(0.610–1.570)
PV 20.4 sec^–1 (mPa·s)	1.318±0.070	1.540±0.207^***
	(1.150–1.500)	(1.310–2.080)
PV 0.51 sec^–1 (mPa·s)	2.657±0.659	5.640±0.827^***
	(1.910–5.100)	(3.820–7.650)
Ht (%)	41.67±3.51	35.17±4.61^***
	(35.0–47.0)	(26.0–47.0)

^***p < 0.001 vs Control Subjects (Student’s t test for unpaired data). WBV = Whole-Blood Viscosity; PV = Plasma Viscosity; Ht = Haematocrit; mPa = milliPascal.

Table 2

Means±S.D. and ranges of the hemorheological indexes in control subjects and multiple myeloma patients

	Control subjects	Multiple myeloma patients
WBV 450/Ht×100	7.031±0.598	8.261±1.150^***
	(5.980–8.240)	(6.360–10.71)
WBV 0.51/Ht×100	57.74±7.07	60.23±14.84
	(48.81–74.32)	(37.78–87.43)
PV 0.51/PV 450	2.20±0.53	5.12±1.63^***
	(1.62–4.18)	(2.73–10.44)

^***p < 0.001 vs Control Subjects (Student’s t test for unpaired data). WBV = Whole-Blood Viscosity; PV = Plasma Viscosity; Ht = Haematocrit.

Table 3

Means±S.D. and ranges of erythrocyte deformability in control subjects and multiple myeloma patients

	Control subjects	Multiple myeloma patients
EI 60	45.13±2.67	42.75±4.61^*
	(40.11–50.08)	(30.25–53.00)
EI 30	41.84±2.42	37.01±4.94^***
	(37.50–46.80)	(24.90–47.68)
EI 12	33.37±2.35	26.33±5.04^***
	(29.10–38.18)	(15.05–35.86)
EI 6	25.05±2.39	17.01±4.59^***
	(20.73–29.78)	(7.91–26.32)

^*p < 0.05; ^***p < 0.001 vs Control Subjects (Student’s t test for unpaired data). EI = Elongation Index.

6 Discussion

This study must be considered preliminary because of several limitations, regarding both the enrolled patients and the methods we employed. The group of MM patients was small and heterogeneous, including subjects at different clinical stages and receiving different medical treatments. Moreover, the techniques we used to assess the hemorheological pattern are in some aspects questionable.

A first point to be made refers to the behavior of WBV at high and low shear rates. WBV is determined by Ht, PV, erythrocyte deformability and aggregation. In this research WBV did not discriminate normal controls from MM patients at any shear rate, since the low values of Ht counterbalanced any trend towards an increase of PV.

As regards the evaluation of PV, we observed increased values of it in MM patients only at low shear rates, and there was no significant correlation between plasma viscosity and every single plasma protein component (fibrinogen, Ig, M-protein). This is undoubtedly surprising, but may be due, at least in part, to of the heterogeneity of the MM patient group, who showed a rather wide range of fibrinogen concentrations but especially an extremely wide range of immunoglobulin levels; some of them had high plasma concentrations of the M-protein, in others it was absent.

PV has a remarkable intra-individual stability [12], although it changes significantly in several clinical conditions. Plasma is generally considered a newtonian fluid, so its viscosity is assumed not to be dependent on shear rate. High shear rate PV is considered the ‘true’ newtonian PV, but when PV is measured by rotational viscometers at various shear rates, an apparent non-newtonian behaviour emerges [6]. In our study MM patients showed a very different behaviour of low shear rate PV in comparison with controls, but these results must be put in the context of the particular technical conditions in which they were obtained. A measurement of PV by a capillary viscometer would be more accurate and, in general, a more in-depth analysis of the rheological blood behaviour in these MM patients is to be planned.

As expected on the basis of the relatively low values of high shear rate PV, no patient in our study experienced the symptoms of a hyperviscosity syndrome. Apart from the direct clinical relevance of extreme plasma hyperviscosity, a less pronounced increase in PV seems to be an independent risk factor for cardiovascular diseases [4 , 33].

The more interesting finding of this research, in our opinion, is the behavior of the erythrocyte deformability. EI was reduced in MM patients at all the tested shear stresses. The values showed higher variability in MM patients, and as shear stress decreased, a decreasing overlap between ranges in control subjects and MM patients was observed. Erythrocyte deformability can be influenced by age and gender, but there was no correlation between EI and age in MM patients nor was there a significant difference in EI between men and women at any shear stress. The lack of correlation between EI and PV or Ig levels does not suggest a direct influence of plasma proteins on erythrocyte deformability, which is more likely to depend on a cellular alteration.

The physiological factors able to reduce erythrocyte deformability are a reduced surface/volume ratio, an increased cytosolic viscosity and an alteration of the membrane dynamic properties. Other authors have observed a decreased deformability of erythrocytes in MM, mainly by filtration techniques [41, 49]. However, filterability is less reliable than diffractometry for the assessment of red cell deformability, since it is considerably influenced by many other factors including plasma protein pattern.

An intrinsic structural abnormality of erythrocytes in MM has been recently demonstrated by means of the atomic force microscopy [21, 61]. Marked irregularities in the cell shape and in the surface ultrastructure distinguished the erythrocytes of healthy subjects from those of MM patients. A possible cause of a red cell structural abnormality is a qualitative and/or quantitative alteration of membrane lipids and proteins. In MM patients a significant modification of the fatty acid profile was found in the erythrocyte membrane as well as in plasma [13, 14]; constant features were the increase in satured fatty acids and n-6 polyunsatured fatty acids (PUFA), associated with a significant decrease in n-3 PUFA and the ratio n-3/n-6 PUFA. These alterations seem to be ascribable to a dysfunction of the enzymes desaturase and elongase, that have a pivotal role in the preservation of the lipid network within biological membranes [29 , 57]. Lipid metabolism plays a central role in neoplastic cell growth and can be a target for pharmacological treatment. An increase in fatty acid synthase (FAS) expression was observed in human myeloma cell lines, and its inhibition reduced the proliferation of myeloma cells [50, 58]. As we did not assess the red cell membrane lipid composition in this study, the link between the alteration in erythrocyte deformability observed by us and an altered membrane lipid composition remains conjectural.

We did not test the microcirculatory function of MM patients, which would have been useful to determine the relevance of the impaired erythrocyte deformability. Abnormalities of microcirculation were demonstrated in MM patients with no symptoms or signs of hyperviscosity syndrome [49]. When this syndrome develops, the tissue damage can be exacerbated by a reduced erythrocyte deformability, which plays a critical role in the oxygen delivery to tissues [2 , 60].

In conclusion, in this preliminary study we found an abnormal behaviour of PV in a small group of MM patients at different stages of the disease. The abnormalities emerged when PV was measured by rotational viscometers at low shear rates. We also demonstrated an impairment of erythrocyte deformability, tested by laser diffractometry at different shear stresses. The relevance of these findings for the pathophysiology and clinical management of MM deserves further investigation.

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