Abstract
Raynaud’s phenomenon is an episodic, painful attack of the acral parts caused by local diminished blood supply. The aim of our study was to examine hemorheological parameters, cold agglutinins, cryoglobulins and their relationship in patients suffering from Raynaud’s phenomenon.
Blood was taken from 74 patients (mean age: 48 years, female/male: 56/18). Cold agglutinins and cryoglobulins were determined. Hemorheological parameters were also measured such as hematocrit, plasma and whole blood viscosity, red blood cell aggregation and deformability. Results were compared to a group of 58 healthy controls (mean age: 31.5 years, female/male: 24/34).
Cold agglutinins were positive in 70%, cryoglobulins in 43% of patients. When compared to healthy controls, increased red blood cell aggregation (64.54 ± 8.93 vs. 61.11 ± 7.05) and decreased red blood cell deformability (0.669 ± 0.002 vs. 0.681 ± 0.001) was observed in Raynaud’s patients (p < 0.05), but there were no differences in hematocrit (43.27% ± 3.85 vs. 44.10% ± 3.70), plasma (1.27 mPas ± 0.08 vs. 1.24 mPas ± 0.09) and whole blood viscosity (4.12 mPas ± 0.52 vs. 4.26 mPas ± 0.46). No differences were found between the hemorheological profile of cold agglutinin/cryoglobulin positive and negative patients. Also primary and secondary Raynaud’s patients had similar rheological profile.
Erythrocyte aggregation and deformability seems to be unfavorable in Raynaud’s patients that can play a role in the disturbance of the microcirculation.
Keywords
List of abbreviations
hematocrit
plasma viscosity
whole blood viscosity
red blood cell
aggregation index
phosphate-buffered saline
Introduction
Raynaud’s phenomenon is a group of painful symptoms of acral regions in a defined order. The affected part of the body turns white during the ischemic stage, followed by the asphyxic stage when it becomes blue due to the increased concentration of reduced hemoglobin, and finally it turns red due to reactive hyperemia. Attacks are provoked by cold exposure or emotional stress. Raynaud’s phenomenon is a clinical diagnosis based on patient’s complaints [8].
The etiology could be either unknown (primary Raynaud’s phenomenon) or known (secondary Raynaud’s phenomenon). In case of secondary Raynaud’s syndrome a wide range of underlying diseases or conditions could be present (autoimmune diseases, neurovascular compression syndromes, infections, vasospastic disorders, endocrine diseases, hematological disorders, malignant diseases, chemicals and drugs, mechanical hazards). Primary Raynaud’s disease is idiopathic, it is not associated with other disorders.
Though Maurice Raynaud reported the phenomenon in 1862, the pathophysiology remained not fully understood. The main point seems to be the imbalance between vasoconstriction and vasodilation in favor of vasoconstriction. Researchers reported three different mechanisms: neural factors, vascular and intravascular abnormalities in close connection with each other leading to vasoconstriction[4, 10].
The relationship of Raynaud’s phenomenon and hemorheological factors has been studied and conflicting results have been published. No hemorheological changes were detected in primary Raynaud’s disease by several authors [6, 13], although Ziegler et al. showed increased fibrinogen level and plasma viscosity in male patients with primary Raynaud’s [16]. Patients with secondary Raynaud’s were reported to have increased plasma viscosity [15], decreased red blood cell deformability [6], elevated fibrinogen level, and erythrocyte aggregation [9, 11]. Increased whole blood viscosity was also noticed in some cases [6], contrary to the findings of Ayres et al. [1]. Others reported no differences in hemorheological parameters between primary and secondary Raynaud’s phenomenon [13].
The aim of our retrospective study was to examine the hemorheological parameters in Raynaud’s patients that can lead us to a better understanding of the phenomenon. Hematocrit (Hct), plasma and apparent whole blood viscosity (PV, WBV), red blood cell (RBC) aggregation and deformability were studied in Raynaud’s phenomenon compared to healthy controls. Also cold agglutinins, cryoglobulins and their relationship with hemorheological parameters were evaluated in patients suffering from Raynaud’s phenomenon.
Materials and methods
Patients and healthy controls
A total of 74 patients (mean age: 48 years, female/male: 56/18) from our angiology outpatient clinic suffering from Raynaud’s phenomenon were included in our study between 2010 and 2013. The diagnosis of Raynaud’s phenomenon was based on patients’ complaints. During visits medical history was obtained, physical examination was performed, blood was taken and cold agglutinins, cryoglobulins and hemorheological parameters were determined.
The results of hemorheological measurements were compared to a group of 58 healthy, non-smoking volunteers (mean age: 31.5 years, female/male: 24/34). All were free of any chronic or acute diseases and were not taking any medication.
The study was approved by the Regional Ethics Committee and the Data Protection Service of the University of Pecs; informed consent was obtained from all patients and healthy volunteers.
Examination of cold agglutinins and cryoglobulins
Blood samples were collected into Vacutainer tubes from peripheral veins with a 21-gauge butterfly infusion set with a minimal tourniquet. Raynaud’s patients were fasting for 6 hours before the procedure.
For cold agglutinin tests (National Blood Transfusion Service, Regional Blood Transfusion Centre Pecs) patients’ blood specimens were collected in serum tubes kept warm (at 37°C) from the moment of collection until the separation of the serum from the cells. The tests were carried out in glass test tubes using 1 : 1 ratio of 3–5% PBS solution of 12 human panel RBCs (blood type O), O type cord RBC, and for autocontrol the patient’s own RBC with the patient’s serum. Furthermore when the patient’s ABO group was not O type but A, then A1, A2 adult test cells, for AB type patients A1, A2 and B adult test cells, for B type patients B type adult test cells were tested too. The test tubes were incubated at +4°C for 2 hours and at a designated temperature (15, 20, 25, 30, 35, 37°C) for 30 minutes. After incubation, the agglutination of RBCs was examined macroscopically by gentle shaking and grading (+/–/gentle/, 1+, 2+, 3+, 4+).
For cryoprotein screening (Department of Laboratory Medicine, University of Pecs), blood was collected from patients into 37°C prewarmed serum, 3.8% Na3-citrate or heparin containing blood collection tubes. The samples were allowed to clot at this temperature for 60 minutes. Serum and plasma was separated from the clot by centrifuging warm for 10 minutes at 2500×g. 1.5 ml serum or plasma was then transferred into secondary tubes, checked for lipemia and placed in 4°C refrigerator. After 7 days the samples were checked for cryoprecipitation. If refrigerated serum and citrated plasma both contained cryoprecipitate, the precipitated proteins were referred as cryoglobulins. If precipitation developed only in the refrigerated citrated plasma, the cryoprecipitate was cryofibrinogen. If cryoprecipitate was detected in heparinized plasma, we talked about cryofibronectin. Then the samples were placed for one hour in 37°C thermostat to prove that the precipitate is indeed a cryoprecipitate by demonstrating the resolubilization of precipitates with warming. The results are reported semiquantitative as: negative, slightly positive, positive and stronglypositive.
Hemorheological examinations
To measure hemorheological parameters in Raynaud’s patients and healthy controls 8 ml of blood was collected into Vacutainer tubes coated with lithium heparin. Hemorheological measurements were performed within 1 hour after blood sampling. Hematocrit was determined by a microhematocrit centrifuge (Haemofuge Heraeus Instr., Germany). Plasma viscosity and whole blood viscosity were measured with Hevimet 40 capillary viscometer (Hemorex Ltd., Budapest, Hungary) at a shear rate of 90 s–1 at 37°C. Red blood cell aggregation and deformability were determined at 37°C by LORCA (Laser-assisted Optical Rotational Cell Analyzer; R&R Mechatronics, Hoorn, Netherlands).
Statistical analysis
SPSS statistical software, version 11.0.1. was used to conduct descriptive analyses and to describe the sample. Significance level was defined as p < 0.05. Data are shown as mean±SD.
Differences in micro- and macrohemorheological parameters between Raynaud and control group, as well as between the subgroups (between cold agglutinin positive and negative, and between cryoglobulin positive and negative Raynaud patients) were evaluated by a one-way ANOVA statistical test (Tamhane post-hoc test) after using the Kolmogorov–Smirnov test to check the normality of the data distribution.
A sample size and power analysis was performed for the overall population using PASS software. The sample size of n = 71 patients is needed to detect a true difference of d = 3.5 in AI with 100% power, where type I error probability is α= 0.001.
Results
Patients characteristics
In 54 of 74 cases an underlying disease or condition was found in the background of Raynaud’s phenomenon. Most of the patients have been suffering from autoimmune diseases, occlusive vascular diseases or hematological disorders (Table 1). Other 20 patients with unknown etiology after at least two years of follow up were considered primary Raynaud’s patients.
Abnormal levels of specific proteins
In 70.5% of Raynaud’s patients cold agglutinins were found, and 43.7% of the patients were cryoglobulin positive.
Microhemorheological parameters
Red blood cell aggregation index (AI) was significantly higher in Raynaud’s patients when compared to healthy controls (Raynaud: 64.54±8.93; Control: 61.11±7.05, p < 0.05).
The deformability of RBCs at higher shear rates showed significantly lower values in the Raynaud group. Higher shear rates characterize the conditions mostly in small vessels and capillaries (Table 2).
Maximal elongation index (EI belonging to limitless shear rates) was calculated and was also significantly lower compared to control group (Raynaud: 0.669±0.002; Control: 0.681±0.001, p < 0.05).
Macrohemorheological parameters
No difference was observed in hematocrit (Raynaud: 43.27% ± 3.85; Control: 44.10% ± 3.70, p = 0.236), plasma viscosity (Raynaud: 1.27 mPas±0.08; Control: 1.24 mPas±0.09, p = 0.148) and apparent whole blood viscosity at 90 s–1 (Raynaud: 4.12 mPas±0.52; Control: 4.26 mPas±0.46, p = 0.140) between the two groups.
Raynaud’s patients
No differences were found in hemorheological parameters between cold agglutinin positive and negative, and between cryoglobulin positive and negative patients (Table 3).
Also primary and secondary Raynaud’s patients had the same rheological profile, no difference was observed (Table 4).
Discussion
The results of our study show the alteration of microrheological parameters in Raynaud’s phenomenon when compared to healthy controls. We observed increased red blood cell aggregation in Raynaud’s patients that is concordant with the results of previous studies [3, 11]. RBC aggregation is known to be influenced by the concentration of various plasma proteins as well as erythrocyte hypodeformability. Though we did not measure plasma fibrinogen levels, higher aggregation index could be explained by elevated fibrinogen levels and other pathological proteins shown by other authors [13, 16]. Red blood cell deformability was also impaired in Raynaud’s patients. Significant difference was found mostly at high and medium shear rates, which characterizes microcirculatory flow. Vasoconstriction increases shear stress, which contributes to increased platelet and leukocyte activation. Increased polymorphonuclear leukocyte activation as well as repeated periods of hypoperfusion resulting in ischemia-reperfusion injury are associated with Raynaud’s phenomenon and may contribute to an increased level of oxidative stress and thus negatively influence red blood cell deformability. Erythrocyte aggregation and deformability significantly influences microcirculatory blood flow rate [2], therefore we believe that there is a microcirculatory disorder in patients suffering from Raynaud’s phenomenon that can lead to the impairment of tissue oxygenation which can result in the worsening of complaints and the development of small ulcers and gangrene.
Cold agglutinins are usually polyclonal or monoclonal IgM molecules, but rarely they can be IgG or IgA types. After different bacterial and viral infections polyclonal antibodies can be formed in the body. The occurrence of these cold-reacting autoantibodies are very common in the population at very low titres. Monoclonal IgM molecules can be associated with chronic diseases, typically lymphoproliferative disorders. In patients with cold agglutinin disease antibodies bind to the surface of red blood cells at low temperatures and activate the complement system which can lead to hemolysis. In addition to hemolysis, other clinical manifestations like Raynaud’s phenomenon can be observed [12].
Contrary to our expectations, neither cold agglutinins nor cryoglobulins seemed to influence hemorheological parameters. According to Kroger et al. low titres of cold agglutinins or cryoglobulins have no diagnostic and prognostic relevance and are not associated with clinical symptoms. Concordantly with our results, no association was found between cold agglutinins/cryoglobulins and hemorhelological parameters in a previous study [5]. In our study population cold agglutinins occurred at low titres mostly. According to our clinical experience abnormally high level of these proteins can worsen patients’ complaints, although patient’s subjective complaints were not assessed in our study. Vaya et al. showed that both in primary and secondary Raynaud’s phenomenon a mild increase in homocysteine levels can be observed, which is related to the severity of microangiopathy [14].
Based on our examinations there were no differences between Raynaud’s patients and healthy controls in macrorheological parameters. Some authors showed increased plasma viscosity in patients with secondary Raynaud’s phenomenon [16]. Also we were unable to find any alterations in whole blood viscosity when compared to healthy controls. Viscosity measurements were performed at 37°C although at low temperatures, when patients’ complaints were usually experienced, a slight difference might have been detected.
According to our examinations no hemorheological differences were observed between primary and secondary Raynaud’s patients that is concordant with the results of others [13]. The explanation of discrepancies could be the different patient classification, different methodology, the various underlying diseases and comorbidities. It is also hard to compare primary and secondary Raynaud’s phenomenon, because there are no clear clinical delineations separating the two entities [8].
Study limitations
One limitation of this study was that hemorheological parameters were measured at 37°C because of technical reasons, though patients with Raynaud’s disease have complaints at lower temperatures. There might be a difference in macrorheological parameters when measured at low temperatures. Another limitation was the relatively small number of subjects investigated and that cold agglutinins and cryoglobulins were not quantified, which does not allow us to compare hemorheological parameters in patients with low and high titres. Also different etiology of Raynaud’s disease makes the interpretation of data more complex. Furthermore the mean age of our control group was lower than Raynaud’s patients and also there were some differences in male/female distribution.
Conclusions
Erythrocyte aggregation and deformability seems to be unfavorable in patients suffering from Raynaud’s phenomenon, which can play a role in the disturbance of microcirculation, thus the phenomenon is not only a vasospastic, but also a complex circulatory disorder.