Rouleaux Formation Research Articles

Contribution of Acute-Phase Proteins and Cardiovascular Risk Factors to Erythrocyte Aggregation in Normolipidemic and Hyperlipidemic Individuals

Numerous studies have demonstrated that elevated concentrations of acute-phase proteins affect red blood cell (RBC) aggregation. Plasma lipids and lipoproteins were also shown to be correlated with RBC aggregation in hypercholesterolemia. However, whether acute-phase proteins promote RBC hyperaggregation in hyperlipidemic patients is unknown. The main objective of the study was to identify the impact of acute-phase proteins such as fibrinogen (Fib), haptoglobin (Hp), ceruloplasmin (Cp), alpha1-acid glycoprotein (AGP), alpha1-antitrypsin (AT), immunoglobulin G (IgG), and albumin (Alb) on RBC aggregation in 35 hyperlipidemic patients. The influence of these proteins in 32 normolipidemic subjects was also determined. RBC aggregation parameters reflecting the kinetics of rouleau formation and the adhesive strength between RBCs were measured by laser reflectometry. Multivariate forward stepwise linear regression analyses were performed to study the relationship between RBC aggregation and these acute-phase proteins, total cholesterol (TC), triglycerides (TG), high (HDL-C) and low (LDL-C) density lipoprotein cholesterol, age, gender, body mass index (BMI), mean blood pressure (Mpresure), and smoking habit. The kinetics of rouleau formation was positively correlated with the linear combination of IgG and Hp (r=0.76, p <0.0001) in hyperlipidemic patients, whereas IgG, smoking, AGP and gender were significant independent predictors in healthy subjects (r=0.79, p <0.0001). The correlations obtained for the models predicting the adhesive strength between RBCs were 0.69 in patients (Alb, HDL-C, IgG, p <0.002) and 0.71 in healthy individuals (AGP, BMI, p <0.0001). This study suggests that acute-phase proteins such as IgG, Hp, AGP and Alb influence significantly and in an independent way the level of RBC aggregation. The close association between RBC aggregation and cardiovascular risk factors further strengthens its clinical importance.

Digital ischemia and gangrene due to red blood cell aggregation induced by acquired dysfibrinogenemia

Digital gangrene was observed in a patient who had angiographic findings of digital arterial occlusion. The patient's blood showed a marked red blood cell aggregation with rouleaux formation in long chains, which could not be dispersed at shear rates up to 200 sec –1. Studies of the patient's blood revealed the presence of an abnormal fibrinogen capable of aggregating normal red blood cells. This fibrinogen was found by Raman spectroscopy to have an increased α-helical content, whereas the β-sheet content was decreased. Defibrinogenation therapy with ancrod resulted in a dramatic symptomatic relief. The disappearance of the abnormal fibrinogen 6 months later and an absence of a family history indicate that this dysfibrinogenemia was acquired. (J Vasc Surg 1997;26:1061-8.)

Disruption of the murine lecithin:cholesterol acyltransferase gene causes impairment of adrenal lipid delivery and up-regulation of scavenger receptor class B type I.

Lecithin:cholesterol acyltransferase (LCAT) is the major determinant of the cholesteryl ester (CE) content of high density lipoprotein (HDL) in plasma. The selective uptake of HDL-CE is postulated to participate in delivery of tissue-derived cholesterol both to the liver and steroidogenic tissues. Recent studies comparing mice with similarly low levels of HDL, due to the absence of either of the two major HDL-associated apolipoproteins apoA-I and apoA-II, suggest that apoA-I is crucial in modulating this process, possibly through interaction with scavenger receptor class B type I (SR-BI). Because of the central role of LCAT in determining the size, lipid composition, and plasma concentration of HDL, we have created LCAT-deficient mice by gene targeting to examine the effect of LCAT deficiency on HDL structure and composition and adrenal cholesterol delivery. The HDL in the LCAT-deficient mice was reduced in its plasma concentration (92%) and CE content (96%). The HDL particles were heterogeneous in size and morphology and included numerous discoidal particles, mimicking those observed in LCAT-deficient humans. The adrenals of the male Lcat (-/-) mice were severely depleted of lipid stores, which was associated with a 2-fold up-regulation of the adrenal SR-BI mRNA. These studies demonstrate that LCAT deficiency, similar to apoA-I deficiency, is associated with a marked decrease in adrenal cholesterol delivery and supports the hypothesis that adrenal SR-BI expression is regulated by the adrenal cholesterol.

Experiments on the risk factor role of fibrinogen

Summary There is convincing evidence that fibrinogen is a major risk factor for myocardial infarction and stroke. The responsible mechanism is still uncertain; it may involve one or more of four possible fibrinogen functions: as the precursor of fibrin in clotting; as an essential co-factor of platelet aggregation; as a factor in rouleaux formation of red cells; and through increasing plasma viscosity. Plasma fibrinogen is increased in cigarette smokers and diminishes very slowly in people who have stopped smoking. Most interestingly, fibrinogen and hypertension act synergistically as risk factors. The mechanism of that is also unknown. However, a possible explanation is suggested by our discovery that different pressor agents, viz. noradrenaline, adrenaline, angiotensin, II, L-NAME and desoxycortisone plus salt are associated with increases in the uptake of fibrinogen, as well as of low density lipoprotein, by artery walls.

Analytical model for effects of shear rate on rouleau size and blood viscosity

In this paper, a theoretical model is proposed by using the reversible kinetic equation of colloid coagulation to reveal the effects of erythrocyte aggregation and shear rate on the rouleau size and the blood viscosity. With this model, shear rate γ dependences of the average size of rouleaux n e and the viscosity η in concentrated red blood cell suspension are derived analytically. In this model, we consider not only the shear rate effect but also the rouleau size effect on the aggregation and degradation mechanism. By comparing with experimental results obtained by Shiga et al., some theoretical parameters have been determined. The variation of rate of rouleau formation with shear rate derived in our model is in fairly good agreement with the experimental results, and the viscosity of the concentrated red cell suspension derived in our model showing shear thinning agrees qualitatively with the experimental results obtained by Chien and Sung.

Microscopic observation of the behavior of red blood cells with plasma proteins under strong magnetic fields

The magnetic orientations of human red blood cells (RBCs) in some media such as phosphate buffer solutions (PBS), serum and plasma were investigated to ascertain the behavior of RBCs under strong magnetic fields in the body. The effects of orientation of RBCs in suspensions were measured by changes in the electrical conductivity of suspensions. The electrical conductivity of the suspensions were increased by application of magnetic fields parallel to the electric field. This phenomenon indicates that RBCs are redirected by a magnetic field. The changes in electrical conductivity of samples of 40% RBCs suspended in PBS, serum and plasma were 19%, 9% and less than 4% respectively. The electrical conductivity of anticoagulated whole blood was not changed by application of magnetic fields up to 8 T. Through microscopic observation of RBCs suspended in plasma under 8 T magnetic field, it was revealed that the aggregation of RBCs or rouleau formation occurred in plasma and rouleaux were oriented by the strong magnetic field. The changes in the electrical conductivity of the suspensions and whole blood by the magnetic orientation are prevented by the aggregation and rouleaux formation of RBCs.

Using enzymes to degrade the red blood cell surface in order to investigate cellular factors of importance to rouleaux formation

Using enzymes to degrade the red blood cell surface in order to investigate cellular factors of importance to rouleaux formation

Increased erythrocyte aggregation in insulin-dependent diabetes mellitus and its relationship to plasma factors: A multivariate analysis

Red blood cell aggregation in vitro (kinetics and shear resistance) was studied in 13 healthy controls and 13 type I (insulin-dependent) diabetic patients free of severe degenerative complications who were matched for age, sex, and body mass index. Measurements were performed with a device that analyzes the laser light backscattered by a blood suspension. Both the velocity of rouleau formation and the cohesion of the rouleau network were significantly increased in diabetic patients. Plasma viscosity and whole-blood viscosity measured at low shear rate (0.95 s −1) were also significantly elevated in the diabetic group. Multivariate analyses of the whole population sample and the diabetic patients confirmed the influence of plasma proteins on the kinetics of aggregation. Fibrinogen levels, which were close to normal, affected mainly the shear resistance of the aggregates. Triglyceride and apolipoprotein (apo) B levels and indexes of metabolic control or protein glycation (fasting blood glucose and fructosamine) also appeared to influence markedly both the kinetics of rouleau formation and the cohesion of the rouleau networks. These rheological abnormalities occurred in diabetic patients before the appearance of any severe degenerative complications. We suggest that these rheological abnormalities are linked to plasma or erythrocyte factors, and are not due to angiopathy.

PEMPHIGUS FOLIACEUS AND NON‐HODGKIN'S LYMPHOMA

A 56‐year‐olcl Iraqi woman presented to her general practitioner with an erythematous scaly eruption on the left ear. An initial diagnosis of otitis externa was made and a 7‐day‐course of penicillin commenced. Three days later, the patient returned complaining of abdominal pain and diarrhea. Examination of the abdomen revealed a large mass. Subsequent investigation with computerized tomography (CT) scanning showed a large paraaortic mass, fine needle biopsy of which showed a diffuse non‐Hodgkins lymphoma. The majority of the cells were small follicle center cells with clear nuclei that stained positively for B‐lymphocyte antigens (L26, MB2). Bone marrow biopsy showed focal paratrabecular deposition of non‐Hodgkins lymphoma cells of a similar nature to those in the paraaortic node. The patient was started on a monthly regimen of chlorambucil 10 mg daily with prednlsolone 40 mg daily for the first 10 days and allopurinol, 200 mg daily. Fourteen days after completing the first monthly course of treatment the patient developed a generalized eruption. This was initially attributed to her allopurinol therapy, which was discontinued. The patient was given a second 10‐day course of prednisolone and chlorambucil, at the end of which the eruption had almost completely resolved. Within 9 days the eruption recurred and the patient complained of red eyes. Examination of the skin showed scattered urticated plaques with peripheral vesiculation on the trunk and annular urticated lesions without vesiculation on the legs. A scaly erythematous eruption was also noted on the left ear (Fig. 1).The hemoglobin was 10.9 g/dL, with a normochromic, normocytic picture with rouleaux formation on the blood smear. The WBC was 7.9, with lymphopenia of 1.0 (normal range 1.5–4.0 × 10−9). Lymphocyte subset analysis showed decreased numbers of cytotoxic/suppressor T cells (0.16 × 10−9; normal range 0.28–1.35 × 10−9). The helper T cell num‐bers and the platelet count were normal. The erythrocyte sedimentation rate (ESR) was 102. Urea and electrolytes, and liver function tests were normal. Total protein was 85 g/L (normal range 60–80 g/L). Serum electrophoresis showed an acute phase response with increased IgA of 6.0 g/L (normal range 0.8–4.0 g/L). Routine hematoxylin and eosin stain of a biopsy of an urticated vesicular lesion on the trunk and from the erythematous lesion on the ear showed small subcorneal blisters beginning to form that contained eosinophils together with eosinophilic spongiosis. Direct immunofluorescence of perilesional uninvolved skin showed intercellular deposition of igG and C3 typical of pemphigus. Indirect immunoflu‐orescence revealed circulating IgG intercellular antibodies to atitre of 1:160.The patient was treated with prednisolone, 80 mg daily. The previous regimen of chlorambucil, 10 days each month, was continued and daily allopurinol recommenced. This was followed by resolution of the eruption, the eruption on the ear being the last area to resolve. Ten‐day courses of chlorambucil were given monthly for a further 7 months, following which, a course of palliative radiotherapy was given to the paraaortic nodes. Although repeat CT scan after 3 months of chemotherapy showed a dramatic reduction in the paraaortic mass, this has remained unchanged 1 year later.

Enzymatic degradation of the red cell surface and its effect on Rouleaux formation

Enzymatic degradation of the red cell surface and its effect on Rouleaux formation

Deoxygenation-induced changes in sickle cell-sickle cell adhesion

The tendency for sickle cells to adhere to each other is increased in oxygenated sickle blood in parallel with cell density. The increased adherence of these cells occurred despite their reduced deformability and diminished ability to form rouleaux. Using a method developed in our laboratory, we measured the yield stress: a sensitive index of cell- cell adhesion of deoxygenated suspensions of sickle cells. Deoxygenation of whole sickle blood to 30 to 50 mm Hg caused a significant increase in yield stress of all sickle blood samples. Deoxygenation caused a significant increase in yield stress of both dense and light sickle cells. Deoxygenation-induced increases in yield stress occurred at higher oxygen tensions for dense (= 55 mm Hg) than for light sickle cells (&lt; 45 mm Hg). The increase in yield stress on deoxygenation was correlated with hemoglobin polymerization as assessed morphologically by sickling or by changes in relative viscosity. Thus, deoxygenation-induced cell sticking must involve small areas of strong membrane adhesion because the changes in yield stress occurred despite a reduction in rouleaux formation and surface area of membrane contact. Sickle trait red blood cells also exhibited increased yield stress on deoxygenation but only under hypertonic conditions where sickling occurred. Thus, deoxygenation-induced cell adhesion did not require prior membrane damage because it occurred in sickle trait cells. No change in yield stress was seen when deoxygenated sickle cells were suspended in buffer, but the addition of physiologic amounts of fibrinogen to buffer restored the deoxygenation-induced increase in cell adhesion. We speculate that the increase in sticking among sickle cells on deoxygenation results from spicule formation and may involve interaction of fibrinogen and possibly other plasma proteins with the cell membrane.

Deoxygenation-Induced Changes in Sickle Cell–Sickle Cell Adhesion

The tendency for sickle cells to adhere to each other is increased in oxygenated sickle blood in parallel with cell density. The increased adherence of these cells occurred despite their reduced deformability and diminished ability to form rouleaux. Using a method developed in our laboratory, we measured the yield stress: a sensitive index of cell-cell adhesion of deoxygenated suspensions of sickle cells. Deoxygenation of whole sickle blood to 30 to 50 mm Hg caused a significant increase in yield stress of all sickle blood samples. Deoxygenation caused a significant increase in yield stress of both dense and light sickle cells. Deoxygenation-induced increases in yield stress occurred at higher oxygen tensions for dense (> 55 mm Hg) than for light sickle cells (< 45 mm Hg). The increase in yield stress on deoxygenation was correlated with hemoglobin polymerization as assessed morphologically by sickling or by changes in relative viscosity. Thus, deoxygenation-induced cell sticking must involve small areas of strong membrane adhesion because the changes in yield stress occurred despite a reduction in rouleaux formation and surface area of membrane contact. Sickle trait red blood cells also exhibited increased yield stress on deoxygenation but only under hypertonic conditions where sickling occurred. Thus, deoxygenation-induced cell adhesion did not require prior membrane damage because it occurred in sickle trait cells. No change in yield stress was seen when deoxygenated sickle cells were suspended in buffer, but the addition of physiologic amounts of fibrinogen to buffer restored the deoxygenation-induced increase in cell adhesion. We speculate that the increase in sticking among sickle cells on deoxygenation results from spicule formation and may involve interaction of fibrinogen and possibly other plasma proteins with the cell membrane.

A Role for the WH-30 Protein in Sperm-Sperm Adhesion during Rouleaux Formation in the Guinea Pig

Mammalian spermatozoa participate in specific cell adhesion phenomena during their development and functional lifespan; this includes interaction with Sertoli cells, the zona pellucida, and the oolemma. In some species such as the guinea pig, an additional sperm-sperm adhesion occurs during epididymal maturation which results in the formation of rouleaux in which the sperm heads are stacked one upon the other and the periacrosomal plasma membranes of adjacent sperm are linked by periodic crossbridges. In this study, we have used a monoclonal antibody to investigate the role of the WH-30 protein on the sperm surface in the formation of the junctional zones between adjacent guinea pig sperm in rouleaux. WH-30 monoclonal antibodies caused a dose- and time-dependent dissociation of rouleaux and an increase in the percentage of single, acrosome-intact sperm; there were no effects on sperm motility (maintained at 80-90%) or ultrastructure during the 120-min incubations. The maximal effect of about 80% single sperm was obtained with a 1:4 dilution of the WH-30 hybridoma supernatant or 5-50 μg/ml of purified WH-30 IgG. In contrast, incubation of sperm in AH-20 IgG, myeloma cell supernatants, or purified, nonspecific mouse IgG 1 had no effect on rouleaux. Treatment of sperm with a WH-30 Fab fragment resulted in almost complete dissociation of rouleaux without any observed effect on sperm motility or acrosomal status. Surface labeling of sperm followed by immunoprecipitation and SDS-PAGE revealed that the WH-30 antibody recognizes a single polypeptide of 43-45 kDa. Using immunofluorescence, the WH-30 protein was localized over the entire surface of the sperm head (wholehead pattern), and immunogold labeling showed that WH-30 is localized in the glycocalyx on both the dorsal and ventral surfaces of the periacrosomal and postacrosomal plasma membranes. These results indicate that the WH-30 protein on the sperm surface is a cell adhesion protein which is involved in the molecular interactions that maintain guinea pig sperm in rouleaux.

Haemorheological changes in the very short term after abstention from tobacco by cigarette smokers

A study was made of the haemorheological changes that took place in the days immediately following tobacco withdrawal from cigarette smokers. In both males and females substantial and persistent reductions in blood viscosity occurred within 2 d, the fall at high shear rate corresponding to about 8% while at low shear rate it was approximately twice as much. These changes were due partly to a fall in packed cell volume (PCV), but also to reductions in total plasma protein and fibrinogen concentrations which led to reductions in plasma viscosity and rouleaux formation. These plasma protein-related changes were less pronounced in the female group. The results indicate that, even in the very short-term, abstention from cigarettes leads to substantial improvements in the haemorheological profile of heavy smokers.

5 Dyslipoproteinaemia of liver disease

Hepatic diseases differ from most other causes of secondary dyslipidaemia in that the circulating lipoproteins are not only present in abnormal amounts but they frequently also have abnormal composition, electrophoretic mobility and appearance. Pre-beta and alpha bands can be absent on electrophoresis in all types of liver disease although material in the VLDL and HDL ranges can be isolated in the ultracentrifuge. Cholestatic liver disease has been the most extensively studied and the hyperlipidaemia can be extreme with marked elevations of free cholesterol and phospholipids. This results largely from the presence of LP-X, an abnormal LDL, with a vesicular structure that appears in rouleaux formation under the electron microscope. It is virtually specific for cholestasis and familial LCAT deficiency. The LDL, however, is heterogeneous and may also contain a large triglyceride-rich particle (LP-Y) as well as more normal-looking particles, which are none the less depleted in cholesteryl esters and rich in triglycerides. Indeed, when patients with cholestasis are hypertriglyceridaemic the excess triglyceride is to be found predominantly in these two LDL fractions rather than in VLDL. HDL in cholestasis may contain disc-like particles, similar to those newly secreted by the liver and intestine, as well as more normal-looking spherical particles. In extrahepatic obstruction concentrations of HDL and its major apolipoproteins, apoAI and apoAII, are frequently reduced, although a subfraction rich in apoE is often found. In all but the latest stages of chronic intrahepatic cholestasis due to primary biliary cirrhosis, however, HDL, especially HDL2, concentrations are increased, probably due to the presence of a circulating inhibitor of HL. Many of these lipoprotein changes found in cholestasis resemble those of familial LCAT deficiency, although the hyperlipidaemia is not usually so severe in the latter condition. Indeed, in patients with cholestasis but well-preserved LCAT activity many of the characteristic lipoprotein changes, such as LP-X, LP-Y and discoidal HDL, may not be seen. In acute hepatocellular disease, such as alcoholic or viral hepatitis, it is not unusual for the patient to go through a cholestatic phase and many of the same lipoprotein changes may be seen. In cirrhosis without cholestasis the patients are not usually significantly hyperlipidaemic and in advanced cases cholesterol and apoB levels may be reduced. Although LCAT activity and the proportion of plasma cholesterol esterified may also be markedly reduced, LP-X is not usually seen, possibly because the flux of free cholesterol and phospholipid (lecithin), the LCAT substrates, is relatively low. Discoidal HDLs are often present.(ABSTRACT TRUNCATED AT 400 WORDS)

Adhesive interaction of erythrocytes in vitro in multiple myeloma

The early stage of rouleaux formation, which can be observed on a microscope slide, was assessed quantitatively for pairs of cells forming adhering doublets. Previous work on normal cells in normal plasma has been extended to blood samples from patients with multiple myeloma, a blood disorder with a large component of high molecular weight immune plasma protein. Cine film records were obtained for each of 19 pairs of cells which made adhesive contact in a cell suspension diluted by myeloma blood plasma. Analysis, restricted to the events of sliding contact between cells, revealed an approximate doubling of peak sliding velocity, a marked reduction in time to half complete sliding interaction (13 compared to 56 sec for normal cells), and a high variance when comparing sliding velocity as a function of position of overlap. We invite consideration that circumstances of low or zero fluid shear rate that can occur in the venous side of the circulation might have a transient but profound affect on local blood viscosity.

Plasma Cell Leukemia and Hyperviscosity Syndrome

A 56-year-old man had dyspnea, weight loss, hemoptysis, and a generalized bleeding diathesis. Physical examination disclosed hepatosplenomegaly, congestive heart failure, and multiple sites of bleeding. Severe anemia, thrombocytopenia, rouleaux formation, and a leukocytosis with circulating immature plasma cells were observed, along with azotemia, hyperuricemia, and marked elevation of total proteins with a monoclonal IgG kappa spike. The finding of increased serum viscosity confirmed the clinical impression of the hyperviscosity syndrome. Emergency plasma exchange produced marked improvement in the clinical manifestations of hyperviscosity syndrome. Systemic chemotherapy resulted in a partial remission of the disease, but the patient ultimately died of complications of treatment. In this review, we discuss the diagnosis and management of the hyperviscosity syndrome.

Blood Viscosity in Horses With Colic

Whole blood viscosity (WBV) was measured on six healthy horses and ten horses presented for colic surgery. A Wells-Brookfield cone-and-plate microviscometer at 6 rpm was used to determine WBV and WBV was adjusted for packed cell volume (PCV). The ten colic horses were divided into two groups: five horses that died or were euthanatized (group 1) and five horses that were discharged after surgery and medical therapy (group 2). The mean WBV for each group was compared using analysis of variance followed by Duncan multiple-range tests. Mean WBV of group 2 (5.81 +/- 1.48 centipoise [cp]) and normal horses (7.1 +/- 2.3 cp) was significantly (P less than 0.01) lower than group 1 (17.15 +/- 5.71 cp). This preliminary trial shows that horses that died or were euthanatized had a significantly higher WBV than horses surviving. Since viscosity was adjusted for PCV, the differences may represent changes in other factors such as fibrinogen, rouleaux formation, red blood cell deformability, PaO2, or intracellular calcium ion concentration. Thus, increases in WBV in horses with colic may indicate severely compromised regional perfusion that may lead to significant tissue damage. In this preliminary study, it appears that WBV may be a prognostic aid in horses with colic.

Rheological properties of erythrocytes in recombinant human erythropoietin-administered normal rat.

Recombinant human erythropoietin [rhEPO (180 iu/micrograms); 1 or 10 micrograms polypeptide equivalent/kg] was intravenously administered daily to 6-week-old normal rats for 1 week. Rheologically, (1) blood viscosity at shear rate of 40-380 s-1 increased in a manner entirely dependent upon the haematocrit, (2) no change in erythrocyte deformability was detected by high shear rheoscopy, and (3) the velocity of rouleau formation in autologous plasma (at 7.5 s-1) decreased. Haematologically, rhEPO administration induced considerable polycythaemia with reticulocytosis in a dose-dependent manner, accompanying increased cell volume and decreased intracellular haemoglobin concentration, thus the density distribution of erythrocytes shifted towards low specific gravity. Plasma viscosity and plasma protein composition were unaffected by rhEPO-administration. In erythrocyte metabolism, no drastic alteration in the level of 2.3-DPG or ATP was detected.

Application of the Fröhlich theory to the modelling of rouleau formation in human erythrocytes

The details of Frohlich's theory and some recent experiments on the rouleau formation of human erythrocytes which exhibit a strong interaction that appears to satisfy the prerequisites of the Frohlich theory, are summarized. To verify whether the Frohlich theory of long-range coherence in biological systems is applicable to the phenomenon of rouleau formation in human erythrocytes, the interactions between erythrocytes are modelled as those between two large, coupled oscillating dipoles. Relevant expressions for the resonant long-range and the van der Waals interaction are then derived. Using the available numerical data, the eigenfrequencies and the interaction energies corresponding to the experimental conditions are then derived. In the range of postulated frequencies (1011–1012 Hz) the effective interaction coefficient Ξ due to the resonant long-range forces is, indeed, found to agree with its experimental value of 3.0. However, the same value of Ξ can also be achieved through the ordinary van der Waals interactions between dipoles oscillating at lower frequencies. It is concluded that the resonant long-range interaction between erythrocytes may be responsible for the onset of rouleau formation. However, other mechanisms cannot be ruled out at this stage, especially since the Frohlich mechanism requires a number of unconfirmed preconditions.