Method for treatment of ophthalmological diseases

The invention relates to a method for the effective therapeutic treatment of ocular diseases which comprises the treatment of blood of patients suffering from ocular diseases by extracorporeal plasmapheresis techniques.

BACKGROUND OF THE INVENTION
 The present invention relates to a new method for the effective therapeutic
 treatment of ocular diseases especially maculopathy and non arteritic
 anterior ischaemic optic neuropathy (NAION).
 In the past ophthalmological diseases like age-related maculopathy (AMD)
 retinal vein occlusion, diabetic retinopathy, arterial occlusion, uveal
 effusion syndrome, NAION, Stargardt-disease, uveitis, and maculopathy of
 different origin could not be treated with a generally accepted therapy.
 For example for the treatment of AMD lasering treatments, radiation and
 operation were used. However these methods had no effect on the further
 development of the disease in many of the patients suffering therefrom.
 Out of the different ocular diseases AMD is the major disease. AMD is a
 severe progressive disease which occurs in the elderly. It is considered
 to be the most frequent cause of blindness in patients beyond an age of 65
 years. There are more than 4.5 million Americans suffering from this
 disease. Two types of AMD are known. The "dry" form develops more slowly,
 however ends up in blindness generally not later than after 12 to 14
 years.
 The second form the "wet" type progresses rapidly leading to blindness
 generally within a few up to 7 years though sometimes much shorter within
 months. The other ocular diseases which are mentioned above are not so
 common but also for these diseases no general accepted therapy exists.
 Therefore, there is a great need for a new and effective therapeutic
 treatment of the above ocular diseases. In the early '90s the inventors of
 the present invention observed that the elimination of fibrinogen and
 plasma proteins of higher molecular weight led to an increase of the
 visual acuity of patients suffering from macular disease and uveal
 effusion syndrome (Brunner, Borberg et al. Acta Medica Austriaca 1991, 18,
 supplement 1, page 63 to 65). In this document 1 patient with uveal
 effusion syndrome and 16 patients which maculopathy were treated. The
 haematocrit was reduced by erythrocyte apheresis. Fibrinogen and plasma
 proteins were eliminated by plasma exchange using a solution of 5% human
 albumin. The visual acuity of 9 of the patients with maculopathy was
 significantly increased after one therapy.
 In a further publication from 1991 (Brunner, Borberg et al., Dev.
 Ophthalmol., Karger (Public.) Basel, 1992, vol. 23, p. 275 to 284) it was
 studied whether clinical improvements could be obtained by plasma exchange
 therapy with patients suffering from intermediate uveitis using a solution
 of 5% human albumin. It was found out that both the haemorheological and
 immunomodulatory effects of this treatment could be beneficial in this
 disease. Human albumin as well as preserved serum were used as exchange
 fluids.
 However, a general concept for the effective therapeutic treatment of
 ocular diseases was not described in these documents.
 Therefore it was the object of the invention to provide a method for the
 effective therapeutic treatment of ocular diseases, especially different
 kinds of maculopathy and NAION.
 SUMMARY OF THE INVENTION
 This object was solved by a method for the effective therapeutic treatment
 of ocular diseases which comprises the treatment of blood of patients
 suffering from ocular diseases by extracorporeal plasmapheresis
 techniques. According to a preferred embodiment of the invention the
 ocular diseases which can be treated are selected from the group
 comprising age-related maculopathy, retinal vein occlusion, diabetic
 retinopathy, arterial occlusion, uveal effusion syndrome, non arteritic
 anterior ischaemic optic neuropathy (NAION), Stargardt-disease, uveitis,
 maculopathy of different origin.
 In a further preferred embodiment the plasmapheresis technique is selected
 from the following techniques: blood cell plasma separation, plasma
 differential separation, plasma differential precipitation, plasma
 differential adsorption, plasma differential filtration.
 The treatment comprises the steps of withdrawing the blood from the
 patient, treatment of the blood by the plasmapheresis techniques mentioned
 above and re-infusing the treated blood.

DETAILED DESCRIPTION OF THE INVENTION
 Hemapheresis (apheresis) is defined as the on line separation of blood into
 blood components. The process of separation refers to the blood cells or
 to the blood fluid (plasma). Haemapheresis procedures are performed using
 centrifugal or filtration techniques to modulate blood composition as for
 instance for the donation of blood components or to perform therapies. The
 modulation of the blood composition using hemapheresis procedures can
 generally be attributed to both elimination and dilution. The standard
 techniques are world wide distributed and under increasing usage.
 Rheology is defined as follows. The fluidity of blood as a non Newtonian
 fluid is characterized from Theological parameters, mainly from viscosity.
 The viscosity of the blood in the human organism is determined from
 several variables, the most important being the concentration of blood
 cells and the protein content of the plasma. Within the plasma high
 molecular weight proteins, such as low density lipoprotein cholesterol,
 alpha-2-macroglobulin, immunoglobulin M, fibrinogen, are major
 determinants. Thus, hyperviscosity syndromes are mainly either due to
 hypercellularity as it occurs for instance in leukemias or due to an
 excessively elevated concentration of plasma proteins as for instance in
 Waldenstrom's disease. The treatment of diseases characterized from
 hyperviscosity has for many years been performed using haemapheresis
 techniques by removing such an excess of cells or plasma. These therapies
 are also generally known and world wide applied.
 Ophthalmological diseases are for unknown reasons hardly or not at all
 treated with haemapheresis techniques. Some of them, such as age related
 macular degeneration are without a generally accepted therapy, though
 various attempts have without success been made to inhibit the progress of
 the disease. These failures may be due to a lack of knowledge on the
 pathogenesis of this and other ophthalmological conditions. Among several
 hypotheses one of them tries to relate the clinical deterioration with an
 insufficient and decreasing chorioretinal blood perfusion. Alternatively,
 an improvement of organ perfusion for instance due to an optimation of
 blood fluidity could well be used for the treatment of ophthalmological
 diseases associated with a disorder of the microcirculation.
 According to the above mentioned theory it was assumed from the inventors,
 that a positive clinical effect might be observed in patients suffering
 from retinal circulatory diseases and many other ophthalmological diseases
 if the haemorheology could be improved following erythrocyte apheresis in
 combination with an elimination of fibrinogen and plasma proteins of
 higher molecular weight. Preliminary observations supported this
 hypothesis indeed. (R. Brunner, H. Borberg, J. Kadar, M. Heidel, K.
 Heidner, W. Konen. Erythrocyte apheresis in combination with elimination
 of fibrinogen and plasma proteins of higher molecular weight in macular
 disease and uveal effusion syndrome. AMA 1991; 18 (suppl. 1): 63-65). As a
 correction of the haematocrit is not often necessary, the studies
 concentrated more on an optimation of plasma viscosity applying plasma
 exchange against 5% human albumin (R. Brunner, H. Borberg, J. Kadar, A.
 Hoffmann, W. Konen, M. Heidel, K. Heidner, in Intermediate Uveitis, WRF
 Boke, K. F. Manthey, R. B. Nussenblatt (eds), Dev. Ophthalmol, Karger
 (publ.), Basel 1992; 23: 275-284.
 Subsequently, several plasma differential separation techniques were
 investigated and compared with plasma exchange therapy. It could be shown,
 that plasma viscosity, standardized whole blood viscosity at an hct of
 0.45 and standardized erythrocyte aggregation at an hct of 0.30 were still
 significantly decreased one day after therapy. The values of the
 standardized whole blood viscosity showed a non-linear,
 haematocrit-dependent rise, whereas the erythrocyte aggregation revealed
 the maximum value near a haematocrit of 0.3. A reduction in all adjusted
 haematocrits was demonstrated. Also, native whole blood viscosity and
 erythrocyte aggregation were lowered within the same range while the
 haematocrit was only slightly diminished. The high molecular weight
 substances of the plasma (Lipids such as total cholesterol,
 LDL-cholesterol, HDL-cholesterol, proteins such as alpha-2-macroglobulin,
 immunoglobulin including fibrinogen) were decreased between 20-70% (R.
 Brunner, R. A. Widder, P. Walter, H. Borberg, K. Oette. Change in
 haemorheological and biochemical parameters following membrane
 differential separation. Int. J. Artif.Organs 1995: 18, 12:794-798).
 Finally, several plasma differential separation techniques were examined
 from the inventors to improve the techniques applicable for an
 extracorporeal haemorheotherapy of ophthalmological diseases.
 Techniques applicable for the use within the invention consist of blood
 cell--plasma separation, also named primary separation, plasma
 differential separation, also named secondary separation. After the
 process of separation and elimination of either cells (if necessary)
 and/or plasma proteins whole blood is returned to the patient. However,
 these techniques are not obligatory, as whole blood perfusion of filters
 or adsorbers may also be applied
 Blood Cell--Plasma Separation (Primary Separation)
 Centrifugal separation based on continuous or discontinuous flow principles
 (e.g. IBM model 2997, COBE Spectra, FRESENIUS AS 104, DIDECO Excel,
 HAEMONETICS model 50), can successfully be applied to separate blood cells
 from blood plasma. Alternatively flat sheet membrane (COBE TPE) or hollow
 fiber plasma separators (ASAHI OP-05) were and can be used to obtain cell
 free plasma. The separated plasma is discarded to be substituted with
 human albumin, a mixture or combination of human albumin with other fluids
 or other fluid alone (plasmaexchange therapy).
 For anticoagulation usually heparin, citrate or a combination of both may
 be applied. Other upcoming anticoagulation procedures are equally
 applicable. The access to the circulation can be established on a
 veno-venous basis from one antecubital vein to the other or from a central
 venous access to a peripheral vein or using an artificial access to the
 circulation applying for instance a shunt, a Shaldon catheter or any other
 catheter using for instance Seldinger's technique. Single needle
 techniques may also be used.
 Plasma Differential Separation (Secondary Separation)
 As plasma exchange therapy may be characterized from safety drawbacks as
 for instance the transfer of infectious agents such as viruses or prions,
 secondary or plasma differential separation techniques are preferred. They
 consist of plasma differential precipitation with heparin, adsorption or
 absorption of plasma proteins or plasma filtration. All technologies for
 secondary plasma separation are preferably applied on-line.
 Plasma Differential Precipitation
 Plasma differential separation is performed adding an excess of heparin to
 the separated plasma, establishing a p.sub.H of 5.12 to precipitate
 heparin binding plasma proteins, separating the precipitate from the
 plasma by hollow fiber filtration, and removing the heparin excess via
 adsorption. Any other plasma protein precipitating technique may also be
 applied.
 Plasma Differential Adsorption
 Plasma differential separation is performed permitting the separated plasma
 to pass through an adsorption column eliminating plasma proteins for
 instance using hydrophobic interaction chromatography (applying for
 instance an ASAHI TR 350 adsorption column) or affinity chromatography
 (applying for instance an LDL-apheresis column) or any other related
 adsorption technology. (As mentioned above, instead of perfusing
 adsorption columns with separated plasma, whole blood perfusion in
 biocompatible systems may also be applied).
 Plasma Differential Filtration
 Plasma differential separation is performed permitting the separated plasma
 to pass through a secondary filter eliminating plasma proteins according
 to the pore size of the membranes (for example ASAHI AC 1730, ASAHI AC
 1760, ASAHI Rheofilter 2000, ASAHI AC 1770, KURARAY Evaflux filters with
 different pore sizes, DIDECO Albusave).
 Retransfusion
 If whole blood perfusion is used, the blood cleared from the eliminated
 components is returned to the patients. If plasma differential separation
 is used, the blood components without the cells and/or plasma are returned
 to the patient.
 A typical example of plasma differential separation using filtration as an
 example is shown in FIG. 1. (Step 1: Blood is removed from a pump into the
 extracorporeal circuit, where cells are separated from the plasma in a
 plasma separator. The plasma separator may consist of either a centrifugal
 or a filtration apparatus. Step 2: The separated plasma is either
 discarded and replaced (plasma exchange), or, alternatively, permitted to
 pass through one or more secondary devices eliminating the precipitated,
 adsorbed or filtered plasma. Step 3: The purified blood is returned to the
 patient. Note that whole blood perfusion devices are correspondingly
 simpler)
 Treatments were performed processing about 50 to 200% of the patient's
 plasma volume preferably 120% on day one and about 50 to 150% preferably
 60-80% of the patient's plasma volume on day 3 or 4 with either technique
 described above. Blood samples were drawn prior to and one day after the
 second treatment to measure the efficacy parameters. Two treatments were
 considered as one cycle. The interval from one cycle to another was
 generally 4-5 weeks. A total of 5 treatment cycles was applied.
 Other treatment approaches mainly but not only used for maintenance therapy
 consisted of a single treatment or one single treatment at different
 intervals. The definition of the interval is related to the choice of the
 separation techniques, thus other approaches are equally possible.
 Patients which were treated with a method according to the invention
 reported about daily life improvements like improved reading ability, less
 problems when watching television, better phase recognition, reduction of
 distorted vision, enhanced color vision perception, and an increased
 degree of independence.
 The present invention will be further understood by reference to the
 following examples.
 EXAMPLE 1
 Treatment of patients with age related maculopathy (n=17), uveitis
 associated (n=14), myopia associated maculopathy (n=5), uveal effusion
 syndrome (n=3), central retinal vein occlusion (n=4).
 To determine the clinical efficiency of haemorheological therapy using
 plasma exchange, selective adsorption and cascade filtration patients
 suffering from maculopathy, retinal vein occlusion and uveal effusion
 syndrome were treated.
 36 patients suffering from maculopathies of different origin were treated
 age related age related (n=17, 31 eyes), uveitis associated (n=14, 26
 eyes) and myopia associated (n=5, 9 eyes). Also patients suffering from
 central retinal vein occlusion (n=4, 5 eyes) resistant to conventional
 haemodilution and uveal effusion syndrome (n=3, 4 eyes) not responding to
 steroid application were treated. The mean age of the patients was 58
 years (SD 15). The main clinical parameter was the best corrected visual
 acuity, measured by Early Treatment Diabetic Retinopathy Study charts
 (Lighthouse New York).
 For patients with maculopathy treatment consisted of two treatment sessions
 with a one day interval. The patients were hospitalized for 5 days.
 Measurements were carried out one day prior to the first treatment session
 and 24 hours after the second treatment session. Patients suffering from
 uveal effusion syndrome and retinal vein occlusion underwent a mean of 3.4
 (range 1 to 7) repetitive treatments.
 We applied the following extracorporeal treatment procedures: plasma
 exchange with 5% human albumin solution, selective adsorption using the
 trytophan-polyvinylalcohol adsorber TR-350 (Asahi Medical Tokyo) and
 membrane differential separation with the Cascadeflo AC-1760 (Asahi
 Medical Tokyo). The amount of plasma volume perfused varied between
 80-120% of the patient's plasma volume. A centrifugal blood separator (IBM
 2997, Cobe Spectra) and the OP-05 hollow fiber filter (Asahi Medical
 Tokyo) were used for primary separation. The blood flow was established
 between two antecubital veins. Anticoagulation consisted of heparin (2500
 units) injected as a bolus prior to therapy and ACD-A citrate continuously
 infused at a ratio of 1:16 during the course of treatment. Patients with
 severe systemic diseases like malignomas, cardial decompensation,
 hepatitis, HIV infection as well as severe hypotony were excluded. Plasma
 and standardized, whole blood viscosity at a haematocrit of 45% were
 measured using a cone-plate system (CS-Rheometer Carri Med Ltd., Dorking,
 UK) at 37 degrees Celsius and standardized erythrocyte aggregation at a
 haematocrit of 30% was measured applying the Mini-Aggregometer (Myrenne
 GmbH, Roetgen, Germany) (7). Measurements of the pulsatile ocular blood
 flow according to Langham were performed in 10 patients suffering from age
 related maculopathy (8). For statistical evaluation the Wilcoxon signed
 rank test was used. P&lt;0.05 was regarded as statistically significant.
 Results
 Severe side effects did not occur. Five patients showed anticoagulation
 dependent symptoms of hypocalcaemia such as tingling of the finger tips
 and the lips and received oral calcium. Three other patients experienced
 hypotonic episodes with a drop of blood pressure not below 90/60 mmHg.
 They were treated with the substitution of 250 ml 5% human albumin. No
 further medical treatment was required.
 Maculopathy
 25/36 patients (34/66 eyes) showed an improvement of visual acuity after
 therapy of at least one line. This improvement was achieved in AMD for
 11/17 patients (15/31 eyes), in uveitis for 12/14 patients (17/26 eyes),
 in myopia for 2/5 patients (3/9 eyes). A bisection of the visual angle
 (improvement of three lines or more) was achieved in 7/17 patients (age
 related, 9/31 eyes), 6/14 patients (inflammatory associated, 9/26 eyes)
 and in 0/5 patients (myopia associated). The changes in visual acuity were
 statistically significant in AMD and uveitis (FIG. 2). The
 haemorheological parameters were, significantly lowered: plasma viscosity
 to 86% (p&lt;0.001), whole blood viscosity to 87% (p&lt;0.001) and erythrocyte
 aggregation to 65% (p&lt;0.001) of their initial value. The pulsatile ocular
 blood flow was increased in 9/10 patients. The mean improvement was from
 649 (SD 188) before to 790 microliter/minute (SD 250) after treatment
 (p=0.028). Also the improvement of the pulsatile ocular blood flow as well
 as the improvement of retinal circulation time after elimination of high
 molecular weight proteins and lipids were demonstrated.
 Retinal Vein Occlusion and Uveal Effusion Syndrome
 All patients suffering from retinal vein occlusion showed an improvement of
 visual acuity of at least one line. In two patients a bisection of the
 visual angle was achieved. One patient improved his visual acuity from 0.1
 to 0.5, a second from 0.0625 to 0.5. All patients with uveal effusion
 syndrome showed a bisection of the visual angle in all eyes after multiple
 treatments.
 Discussion
 Extracorporeal haemorheological treatment led to clinical improvement in
 25/36 patients suffering from maculopathy and in all patients with retinal
 vein occlusion and uveal effusion syndrome. These changes were achieved by
 optimizing rheological parameters such as plasma viscosity, whole blood
 viscosity and erythrocyte aggregation. Serious side effects did not occur.
 Measuring the pulsatile ocular blood flow, it could be demonstrated that
 changes of blood viscosity and erythrocyte aggregation led to a
 modification of the blood supply in the ocular compartment.
 Comparing the outcome of the patients with maculopathy the main improvement
 was achieved treating age related maculopathy and inflammation associated
 maculopathy while myopia related maculopathy did not display a clinically
 relevant improvement. The pathogenesis of maculopathy is not fully
 understood. In age related and inflammation associated maculopathy as well
 as in retinal vein occlusion a disturbance of the ocular blood flow was
 found in several investigations. The treatment of choice for uveitis
 without specific pathogenesis is immunosuppression, e.g. with steroids.
 Patients in this study developed maculopathy which did not respond to
 earlier drug therapy. So far age related maculopathy remains without
 generally accepted or established therapy. Alternative therapeutic
 strategies are laser coagulation, radiation therapy and subretinal
 membrane peeling. These approaches are able to slow down the progress or
 to avoid further complications of the disease. They however fail to
 improve symptoms such as reduced visual acuity. They additionally have in
 common the destruction of retinal tissue and are only suitable for a
 minority of patients.
 Uveal effusion syndrome is caused by a decreased blood flow in the vortex
 veins on their way through the sclera out of the ocular globe. Alternative
 treatment means surgical decompression with the potential risks of surgery
 and additional therapy with steroids to which our patients did not
 respond. In retinal vein occlusion the therapy of choice is the
 rheological therapy using haemodilution. The benefical effects for the
 majority of patients were demonstrated in randomized studies. We treated
 patients who did not respond to this therapy but to extracorporeal
 treatment. This may be due to the limitations of conventional
 haemodilution in influencing plasma viscosity. Using hyper- or isovolemic
 haemodilution an evident or lasting influence on plasma viscosity could
 not be demonstrated. The effects remained limited to the time interval
 between the end of treatment and the measurements performed after therapy.
 Subsequent changes were not detectable 24 hours later. Long term effects
 have been described but the results remained within the ranges of
 shamprocedures. Extracorporeal therapy failed to improve visual acuity in
 myopia related disease. In these patients it seems that the mechanical
 component, caused by the elongation of the ocular globe, is of major
 importance. It was not possible to improve visual acuity by improving
 ocular blood supply.
 Rheological treatment is not a causative treatment but it decreases the
 patient's symptoms by improving the ocular blood supply. Based on the
 current experience it will be necessary to select those patients who may
 benefit from therapy through clinical, morphological and
 electrophysiological criteria.
 In patients with uveitis associated maculopathy and retinal vein occlusion
 an extracorporeal therapy should be carried out after conventional therapy
 such as steroid treatment or haemodilution have failed.
 In age related maculopathy and uveal effusion syndrome extracorporeal
 treatment should be taken into consideration before invasive techniques
 are applied. This excludes patients with subretinal neovascular membranes
 fulfilling the guidelines for laser coagulation of the Macular Coagulation
 Study Group. In these patients it remains to be elaborated whether
 extracorporeal treatment prior to laser'therapy is more beneficial.
 Extracorporeal treatment procedures like plasma exchange, selective
 adsorption and cascade filtration are able to improve visual acuity in
 patients suffering from maculopathy, retinal vein occlusion and uveal
 effusion syndrome by optimizing rheological parameters. They are able to
 affect the disease process without causing tissue damage as occuring after
 laser coagulation, radiation therapy or surgery.
 FIG. 2 shows the changes of visual acuity after therapy in patients
 suffering from maculopathy.
 0.1 log MAR unit is equivalent to one line on the used visual acuity
 charts. Statistically significant changes occurred in patients suffering
 from age related maculopathy (AMD) and uveitis associated maculopathy. In
 myopia no clinically relevant changes were detected.
 [also see Brunner et Borberg, Transfus, Sci., Vol. 17, No. 4, pp, 493-498,
 1996 (published 1997)]. This document is incorporated by Reference.
 EXAMPLE 2
 Improvement of visual acuity in patients suffering from diabetic
 retinopathy after membrane differential filtration.
 Six patients (4 men and 2 woman) with non proliferative diabetic
 retinopathy and clinically significant macular edema (5 patients, 9 eyes)
 according to the definition of the Early Treatment Diabetic Retinopathy
 Study (ETDRS 1991) and inactive proliferative diabetic retinopathy (1
 patient, 1 eye) underwent treatment. The mean age was 61 years (range
 56-68 years). Two patients were insulin dependent. The mean duration of
 the disease was 12 years (range 1 -24 years). One patient showed in one
 eye no signs of diabetic retinopathy, another patient had 1 eye inoculated
 after a penetrating injury. So 6 patients (10 eyes) were included into
 analysis.
 A plasmaflo OP-05 hollow fiber filter was used for primary plasma
 separation. Plasma differential filtration was performed applying the
 cascadeflo AC 1730 filter (Asahi Medical Tokyo). The blood flow was
 established by venipuncture of both forearms. Anticoagulation consisted of
 heparin (4500 units) administered as a bolus prior to treatment and ACD-A
 citrate (Acidium citrium dextrose formule A) infused during treatment at a
 ratio of 1:16. 80% of the patients plasma volume was processed during the
 treatment. A mean of 500 ml of 5% human albumin solution was administered
 to each patient.
 Whole blood viscosity was measured with a cone plate system (CS-Rheometer
 Carri Med Ltd., Dorking, UK). Plasma viscosity was determined with the
 capillary tube plasma viscosimeter (Jung 83). Eythrocyte aggregation was
 measured with the mini-aggregometer (Myrenne GmbH, Roetgen, Germany)
 (Kiesewetter 1982). The biochemical parameters, total protein, albumin,
 fibrinogen, IgG, IgM, IgA, alpha-2-macroglobulin, total cholesterol,
 triglycerides, LDL, VLDL and HDL were determined using standard procedures
 as described in detail before (Brunner 1995).
 Results
 Main parameter of the study was the best corrected visual acuity measured
 with ETDRS-charts (Lighthouse, Long Island, USA). All parameters were
 determined one day prior to and one day after therapy. Visual acuity was
 checked again on follow-up 25 days (range 4-90 days) after therapy. For
 statistical analysis the Wilcoxon signed rank test was used. P&lt;0.05 was
 regarded as statistically significant.
 No side effects occurred during or after treatment. The rheological
 parameters, plasma viscosity, whole blood viscosity and erythrocyte were
 still significantly decreased one day after therapy. The haematocrit of
 the patients was not affected by treatment. There was a significant
 reduction of total protein, fibrinogen, IgG, IgM, IgA,
 alpha-2-macroglobulin, total cholesterol, LDL and HDL. Albumin remained
 unchanged after therapy. Triglycerides and VLDL were decreased after
 therapy, but the differences were not statistically significant. The
 changes of biochemical and rheological parameters are summarized in table
 I.
 The mean visual acuity of all eyes was 0.55 log MAR (SD 0.34) before
 therapy and changed to 0.41 log MAR 8SD 0.31) after therapy. On follow-up
 mean visual acuity was still 0.41 log MAR (SD 0.32).
 Visual acuity was increased by more than one line in 8/10 eyes. Of these
 eyes three improved their visual acuity by more than two lines. One of
 these patients showed an improvement of three lines on the ETDRS-chart.
 The mean change compared to baseline was 1.4 lines (SD 0.8 lines, p=0.002)
 24 hours after therapy and was statistically significant. At follow-up the
 mean improvement compared to the values prior to therapy was still 1.4
 lines (SD 1.0 lines, p=0.002). No patient showed a deterioration of visual
 acuity after therapy. Changes in the macular region were not detectable by
 slit-lamp biomicroscopy.
 Discussion
 The results of this pilot study show that membrane differential filtration
 is a strong tool to influence blood rheology and is able to alter visual
 function in patients suffering from diabetic retinopathy.
 Diabetic retinopathy is one of the leading causes of blindness in the
 western world. Approximately 25% of diabetics have some form of
 retinopathy and 90% of all diabetics will develop retinopathy at some time
 during their lives. The leading cause of visual impairment in diabetics is
 the occurrence of macular edema (Federman 1994). There is evidence, that a
 diffuse damage of the retinal vessels affects their patency and
 permeability. Subsequently microaneurysms, hemorrhage, focal hard
 exuilates and diffuse intraretinal edema can be observed in mild
 nonproliferative diabetic retinopathy and lead to visual impairment.
 Haemorheological disturbances might play a role in the impairment of
 microcirculatory blood flow, and may contribute to the development of
 microangiopathy in diabetes mellitus. It might also contribute to
 endothelium dysfunction or injury because an increased shear stress caused
 by an increased blood viscosity can lead to endothelial injury.
 Additionally most investigators found a decreased retinal blood flow in
 diabetics.
 Membrane differential filtration eliminates high molecular weight proteins
 and lipids and is therefore able to lower rheological parameters like
 whole blood viscosity, plasma viscosity and erythrocyte aggregation. Also
 the improvement of the pulsatile ocular blood flow as well as the
 improvement of retinal circulation time after elimination of high
 molecular weight proteins and lipids were demonstrated.
 In this study it was demonstrated, that these changes led to a short term
 functional improvement in patients suffering from macular edema.
 Lowering of blood viscosity and erythrocyte aggregation may lead to a
 decreased shear stress to the endothelium and to a decreased postcapillary
 resistance both mechanisms result in a decreased pressure of fluid in the
 subretinal space. Also, improvement of endothelial function may decrease
 fluid pressure in the subretinal space. Even a single session of
 LDL-apheresis, a treatment, for which membrane differential separation
 originally was developed, is able to alter endothelial function. These
 three sequelae of membrane differential filtration may explain the short
 term effects on visual function in diabetics with diabetic retinopathy and
 clinically significant to enhance visual function in these patients and
 may therefore give more insights in the development and pathogenesis of
 visual impairment in diabetics with macular edema.
 It is important to stress that haemorheological therapy is an experimental
 approach and does not replace laser treatment which is mandatory in
 diabetic retinopathy. A current study is initiated to investigate the long
 term effects in patients undergoing repetitive treatment with membrane
 differential filtration.

Table I shows changes in biochemical and haemorheological pa-
 rameters 24 hours after membrane differential filtration.
 Before therapy After therapy Final
 Parameter Unit mean/sd mean/SD % pW
 Rheology:
 Haematocrit % 44.4 3.2 44.2 2.9 100 ns
 PV mPa s 1.37 0.13 1.19 0.11 87 *
 WBV mPa s 4.50 0.70 3.74 0.35 83 *
 EA rU 11.44 0.83 5.99 1.73 52 *
 Proteins:
 Total protein g/l 75.3 9.6 59.0 6.90 78 *
 Albumin g/l 44.17 2.48 44.67 5.82 101 ns
 Fibrinogen g/l 3.04 0.63 1.90 0.54 63 *
 IgG g/l 15.05 2.07 9.97 0.95 66 *
 IgA g/l 4.73 1.42 3.01 0.94 64 *
 IgM g/l 2.28 2.24 1.02 0.85 45 *
 A2M g/l 2.41 0.64 1.35 0.50 56 *
 Lipids:
 Triglycerides mg/dl 224.0 118.3 179.7 59.6 80 ns
 Cholesterol mg/dl 222.8 71.0 140.3 24.7 63 *
 VLDL mg/dl 29.2 21.5 24.2 8.8 83 ns
 LDL mg/dl 148.3 45.0 85.2 20.3 57 *
 HDL mg/dl 45.3 13.7 30.8 8.5 68 *
 Abbreviations: Final = final value in % of the initial value, pW* = p &lt;
 0.05 / ns = not significant according to Wilcoxon signed rank test, sd =
 standard deviation, PV = plasma viscosity, WBV = whole blood viscosity, EA
 = erythrocyte aggregation, rU = relative units, A2M =
 alpha-2-macroglobulin, n = 6 patients
 EXAMPLE 3
 Clinical improvement in patients suffering from maculopathy after plasma
 perfusion using a tryptophan polyvinylalcohol adsorber.
 Seven men and three woman suffering from maculopathy were treated.
 Maculopathy is a disorder of the central part of the retina. The
 degeneration of the retina and the adjacent pigment epithelium was
 diagnosed using indirect ophthalmoscopy and fluorescein angiography. The
 main symptom of the disease is the reduction of visual acuity which can be
 evaluated with standard visual acuity charts. In our study maculopathy
 developed after uveitis (n=6), high myopia (n=1), uveal effusion syndrome
 (n=1) and in two cases it was age related. Improvement of visual acuity
 was expected through an improvement of the microcirculation. The mean age
 of the patients was 55 (SD 11) years.
 The treatments were performed applying a plasma separator (Plasmaflo OP-05)
 and a tR-350 plasma perfusion column separator (Asahi Medical, Tokyo) and
 were carried out twice within three days with a one day interval. This
 treatment schedule was chosen empirically considering to reappearence of
 the plasma proteins as described below. The container of the column
 (length 221 mm, diameter 76 mm, weight 600 g) is made of polypropylene.
 The adsorber material consists of tryptophan bound to a polyvinylalcohol
 gel and has a volume of 350 ml. The adsorption process is mainly based on
 hydrophobic interaction chromatography. The blood flow was established
 from one forearm to the other using peripheral veins.
 The flow rates were 70-80 ml/min for whole blood and 15-20 ml/min for
 plasma. One treatment session took between two and three hours depending
 on the patients total plasma volume. Anticoagulation consisted of heparin
 (4000 units) injected as a bolus prior to the therapy and ACD-A citrate
 (Acidium citrium dextrose formule A) was continuously infused in a ratio
 of 1:16 during the course of treatment.
 Rheological and biochemical parameters were determined one day prior to the
 first treatment and one day after the second treatment. For the evaluation
 of the Theological parameters 30 ml EDTA (Ethylenediaminetetraacetic acid)
 blood was drawn. After the centrifugation and the separation of plasma,
 whole blood samples were adjusted to haematocrits of 15, 30, 45 and 60%
 using the patient's own plasma. This technique allows a person to observe
 changes of these parameters independent from the haematocrits. Then plasma
 viscosity, whole blood viscosity and erythrocyte aggregation were
 determined within three hours after venipuncture. Viscosity was measured
 using a cone-plate system (CS-Rheometer Carri Med Ltd., Dorking, UK) at 37
 degrees Celsius. Shear stress was regulated from 0 to 1,414 N/m.sup.2 over
 a period of seven minutes. The viscosity was calculated from 200 single
 measurements taken over this period. Erythrocyte aggregation was measured
 applying the Mini-Aggregometer (Myrenne GmbH, Roetgen, Germany).
 Biochemical parameters were determined with standard procedures: fibrinogen
 (turbidimetric determination along with prothrombin time), albumin, IgG,
 IgM, IgA, alpha-2-macroglobulin (nephelometric, Behring, Marburg), total
 protein (Biuret), total cholesterol (enzymatic assays,
 Boehringer-Mannheim).
 The best corrected visual acuity was measured by ETDRS (Early Treatment
 Diabetic Retinopathy Study) or Idemvisus projector charts. Plasma volume
 was determined according to the method of Dagher and coworkers.
 The aim of the study was the reduction of plasma viscosity, standardized
 whole blood viscosity at haematocrit 45% and standardized erythrocyte
 aggregation at haematocrit 30%. Statistical evaluation was carried out
 using the Wilcoxon signed rank test. Evaluation were performed on a
 personal computer with the statistical software StatView 4.01 (Abacus
 Concepts Inc.). P&lt;0.05 was regarded as statistically significant. Because
 of multiple testing a p value&lt;0.017 was mandatory for each evaluation to
 be regarded as significant. The p value of 0.017 (0.05/3) was chosen
 because three endpoints were tested and in this case a p value&lt;0.017 is
 equivalent to a p value&lt;0.05 compared to statistical evaluation of a
 single parameter.
 Results
 A mean of 89% (SD 15) of the patients' plasma volume was processed during
 the adsorption procedure.
 Two different patients experienced minor side effects shown as transient
 hypotonic episodes (RR 90/60 mmHg) and mild hypocalcemia (tingling of the
 finger tips) controlled by intravenous infusion of 250 ml 5% human albumin
 and oral calcium administration. The mean serum calcium concentration was
 not decreased in these cases, in ionized form of calcium was not measured.
 Plasma viscosity, standardized whole blood viscosity at haematocrit of 45%
 and standardized erythrocyte aggregation at haematocrit of 30% measured 24
 hours after treatment were shown to be significantly lower as shown in
 table II.

Table II shows the changes in
 biochemical and haemorheological parameters
 Before therapy After therapy Red
 Parameter Unit (mean .+-. sd) (mean .+-. SD) % pW
 Rheology:
 PV mPa s 1.49 .+-. 0.14 1.3 .+-. 0.12 87 0.005
 Sa_WBV mPa s 4.21 .+-. 0.66 3.72 .+-. 0.38 88 0.007
 Sa_EA rU 12.25 .+-. 2.35 7.93 .+-. 0.75 65 0.005
 Haematocrit % 44.7 .+-. 2.5 42.2 .+-. 4.5 94 0.06
 Proteins:
 TP g/l 78 .+-. 7.63 68.7 .+-. 7.39 88 0.03
 Albumin g/l 48.7 .+-. 10.00 46.5 .+-. 7.96 95 0.36
 IgG g/l 14.61 .+-. 4.51 9.53 .+-. 3.66 65 0.005
 IgA g/l 3.87 .+-. 2.37 2.83 .+-. 1.45 73 0.007
 IgM g/l 2.70 .+-. 2.27 1.71 .+-. 1.49 63 0.005
 Fib g/l 3.08 .+-. 0.89 1.53 .+-. 0.31 50 0.005
 A2M g/l 2.15 .+-. 0.83 1.80 .+-. 0.67 84 0.02
 Chol g/l 2.51 .+-. 0.52 2.12 .+-. 0.38 84 0.04
 Abbreviations: Red = reduction to % of the initial value, pW = p value
 according to Wilcoxon signed rank test, sd = standard deviation, PV =
 plasma viscosity, Sa_WBV = standardized whole blood viscosity at
 haematocrit 45%, Sa_EA = erythrocyte aggregation at haematocrit 30%, rU =
 relative units, TP = total protein, Fib = fibrinogen, A2M =
 alpha-2-macroglobulin,
 #Chol = total cholesterol, n = 10 (except A2M/n = 7; Chol/n = 9
 The graph of the whole blood viscosity revealed a non linear haematocrit
 dependent rise (FIG. 3), while the graph of the erythrocyte aggregation
 showed the maximum value at haematocrit 0.3 (FIG. 4). In both diagrams a
 clear reduction after therapy is demonstrated (p&lt;0.05).
 The high molecular weight proteins and lipoproteins such as
 immunoglobulins, fibrinogen, alpha-2-macroglobulin and total cholesterol
 were reduced. This resulted in a reduction of total protein while albumin
 was largely unaffected. The analysis of alpha-2-macroglobulin and total
 cholesterol values was limited to seven instead of nine patients, as some
 of the blood samples were lost.
 Leucocyte-, erythrocyte and thrombocyte counts and also potassium and
 sodium values remained unchanged, whereas the calcium level was lowered.
 An improvement of visual acuity of at least one line was achieved in six
 patients 24 hours after therapy (uveitis 4/6, age-related 1/2, uveal
 effusion 1/1, myopia 0/1) in their better eye. Three of these six revealed
 a bisection of the visual angle (improvement of three lines or more).
 These three suffered from uveitis, age-related maculopathy and uveal
 effusion syndrome.
 Discussion
 Selective adsorption using the TR-350 column turned out to be an effective
 extracorporeal treatment to reduce haemorheological determinants like
 whole blood viscosity, plasma viscosity and erythrocyte aggregation. The
 effect was shown to be present 24 hours after therapy and was accompanied
 by clinical improvement. Severe side effects did not occur. They are not
 typical for adsorption therapy in general and if they occur they appear to
 depend on the character of the different materials used.
 Haemodilution has gained widespread application as an approach of
 influencing haemorheological parameters. Randomized double blind trials
 revealed a remarkable influence on whole blood viscosity accompanied by
 clinical improvement in patients suffering from peripheral arterial
 disease and retinal vein occlusion.
 However, the impact on plasma viscosity and erythrocyte aggregation is
 still under debate. Using hyper- or isovolemic haemodilution a clear or
 lasting influence on plasma viscosity could not be demonstrated. The
 effects remained limited to the time interval between the end of treatment
 and the measurements performed after the therapy. Changes of plasma
 viscosity were no more detectable 24 hours later. Long term effects have
 been described, but the results remained within the ranges of
 sham-procedures. Similar data were found for the influence on erythrocyte
 aggregation.
 The relation of whole blood viscosity to microcirculation is under debate
 and should be discussed with caution, as it was demonstrated that the
 viscosity of whole blood tends towards the values of plasma viscosity in
 small capillary tubes. Therefore, it is possible that plasma viscosity
 plays a major role in microcirculatory blood supply. Plasma viscosity and
 erythrocyte aggregation are determined by the blood level of fibrinogen,
 high molecular weight proteins like globulins and lipoproteins. Moreover,
 proteins adhering to the capillary endothelium and therefore interacting
 with erythrocytes, may contribute to the quantity and quality of the
 capillary blood flow.
 Extracorporeal treatment is able to influence haemorheological parameters
 by erythrocyte apheresis and the elimination of high molecular weight
 proteins and lipoproteins. We were able to demonstrate that plasma
 exchange could be used as a successful treatment of ocular disorders.
 Because of the potential side effects of plasma exchange a more selective
 approach to haemorheological therapy using selective adsorption appeared
 to be attractive and provided for the capacity of the elimination of high
 molecular weight proteins and lipoproteins and could influence Theological
 parameters.
 A comparison between selective adsorption and plasma exchange, carried out
 in an earlier study, revealed distinct elimination profiles. A similar
 decrease of total protein was accompanied by a lower reduction of total
 cholesterol, alpha-2-macroglobulin and IgM when selective adsorption was
 used. However, the influence on the rheological parameters plasma
 viscosity, whole blood viscosity and erythrocyte aggregation was quite
 similar. Other investigators using another technique for the semiselective
 removal of high molecular molecules lowered the Theological parameters
 within the same range. This leads to the conclusion that the removal of
 the total amount of high molecular weight proteins and lipoproteins and
 not the selective removal of a single substrate plays the major role for
 the influence on blood rheology.
 Different elimination profiles may lead to different long term effects. A
 decrease of plasma viscosity with plasma exchange is demonstrated for at
 least six days whereas the efficacy of plasma precipitation as another
 selective approach did not last more than 24-48 hours after a single
 treatment. Our own measurements of the Theological and the biochemical
 parameters were carried out 24 hours after the second of two treatments.
 This design permitted to demonstrate significant effect on rheology.
 However, to which extent long term effects can be achieved remains to be
 determined.
 In conclusion selective adsorption using hydrophobic, interaction
 chromatography is effective influencing not only biochemical but also
 rheological parameters and leads to clinical improvement in patients
 suffering from maculopathies of different origin. According to our
 preliminary experience these changes seem to be more profound as compared
 to haemodilution where a clear or lasting influence on plasma viscosity
 could not be demonstrated as discussed before. It is assumed and currently
 under investigation that clinical effects may last longer than those
 observed for haemodilution and extracorporeal plasma differential
 precipitation. The optimal treatment schedule for lasting effects has to
 be investigated in further studies.