GB2184670A - Thermofiltration of plasma - Google Patents

Abstract

A method for more selectively removing macromolecules from a plasma solution, whereby plasma containing the macromolecules to be removed is provided and heated to a temperature near or above normal body temperature but below the boiling point of the plasma solution. The heated plasma solution is filtered while at a temperature near or above normal body temperature but below its boiling point with a membrane filter to remove selectively macromolecules from the plasma solution. An apparatus for accomplishing the foregoing is also provided. <IMAGE>

Description

SPECIFICATION Thermofiltration of plasma Background of the Invention The present invention relates to the filtration of macromolecules from fluids, and more particularly to the removal of undesirable macromolecules from plasma solutions, by the process termed thermofiltration.

The separation of undesirable solutes from blood plasma through plasma filtration is a known method of treating diseases, wherein such diseases have in common undesirable elevated levels of plasma solutes, such as toxins, excessive antibodies, and other metabolic factors. Successful treatment of such diseases involves the removal of the undesirable plasma solutes from the blood plasma by membrane filtration.

Various methods of plasma filtration, including cascade filtration, double filtration, and cryofiltration have been developed. However, these methods contain a number of undesirable characteristics which limittheir usage.

Applicants have noted a number of parameters associated with performance, including module design, membrane properties, plasma composition, and plasma and filtration temperature. Characteristics ofthe modulewhich affectflowdynamics and in turn performance include, area, fluid and film dimensions, aswell as properties of the separating membrane, including polymer type and microstructural features such as pore size, pore tortuosity, pore length, and pore number.

Variations in the plasma's composition also affects its filtration. Plasma from patient's with different disease states or with differing macromolecule contents have different filtration characteristics. Manipulation of the plasma to effect changes in pH or electrolyte composition and the addition of anticoagulants such as heparin or other macromolecule-aggregating additives such as polyethylene glycol will effect filtration performance. Generally such manipulations are carried outforthe purpose of augmenting the separation, by macromolecule aggregation or precipitation, of one or a group of solutes from the plasma.

Because of the number of parameters affecting filtration performance, temperature selection and its control has been demonstrated to be a key parameter in fluid separation. In order to augmenttheselectrive removal in a particular macromolecular range, it is extremely important to operate within the propertemperature range. In this respect, significant differences have been noted for comparable conditions offiltration (similar operating flows, modular types and plasma types) between cascade and double filtration, which operate at near ambienttemperatures, and cryofiltration, which operates at temperatures below a set physiologictemperature.

Temperature control offers many advantages overthe other parameters in thattemperature control is the easiest controlled physical parameter, and thattemperature control may be combined with the use of various complexing agents to increase the sensitivity of macromolecule removal. A specific example of this can be shown in the case of cryofiltration, where the addition of heparin aids in the formation of cryogel byforming complexes with fibronectin and fibrinogen at temperatures below 250C.

Filtration at sub-physiologic temperature is effective for the removal of plasma constituents similar in size but differing in temperature sensitivity. A number of autoimmune diseases can be treated in this fashion, as has been described in the literature. The effectiveness of the treatment is attributed to the formation and removal of cryogel, which is composed of high concentrations ofthe macromolecules associated with autoimmune disease states. Thus, the separation in cryofiltration is not based on molecular size at physiologic temperature but on molecular size at reduced temperatures.

However, operation at a reduced temperature can, in fact, reduce the selectivity of molecule separation when size differences are great since aggregation or complexformation of small molecules may also occur at reduced temperatures. Therefore, for separation based on size differences at physiologic temperatures, it may be more advantageous to avoid cryogel formation.

Accordingly, it is an object of the present invention to provide an improved means of removing undesirable macromolecules from fluids in an effective and efficient manner.

Summary of the Invention In one aspect, the present invention relates to a method of selectively removing macromolecules from a plasma solution including the steps of providing a plasma solution containing the macromolecules to be removed, heating and/or maintaining the plasma at a temperature near or above normal bodytemperature but belowthe boiling pointofthe plasma solution, and filtering the warmed plasma solution while ata temperature near or above normal body temperature but below the boiling point with a membrane filterto remove more selectively macromolecules from the plasma solution.In addition, by heating the plasma solution, various macromolecules present within the plasma solution may become inactivated or denatured, aiding in their selective removal through plasma filtration.

In another aspect, the present invention concerns a method of selectively removing macromolecules from a plasma solution including the steps of securing a blood flow from a specimen, separating the blood flow into a concentrated cellular element stream and a plasma stream containing the macromolecules to be removed, heating or maintaining the plasma stream containing macromolecules to be removed to atemperature near or above normal body temperature but below its boiling point, filtering the warmed plasma stream while ata temperature near or above normal body temperature but below its boiling point with a membrane filter to remove more selectively macromolecules from the plasma solution to form a filtered plasma stream, combining the filtered plasma stream and said cellularelementstream to form a processed plasma stream, and cooling and/or returning the processed stream plasma to the specimen.

In another aspect, the present invention concerns a method of controlling conditions of lipoprotein abnormalities in a living organism by selectively removing macromolecules from a plasma solution including the step of securing a blood flowfrom a living organism, separating the blood flow into a concentrated cellularelementstream and a plasma stream containing the macromolecules to be removed, heating or maintaining the plasma stream containing macromolecules to be removed to a temperature near or above normal bodytemperature but below its boiling point,filtering the heated plasma stream while at a tem perature near or above normal body temperature but below its boiling point with a membrane filter tore- move more selectively macromolecules from the plasma solution to form a filtered plasma stream, combining the filtered plasma stream and said cellular element stream to form a processed plasma stream, and cooling and/or returning the processed plasma stream to the living organism.

In still another aspect, the present invention concerns an apparatus for removing more selectively macromolecules from a plasma solution comprising a means of receiving and dividing a plasma containing solution containing macromolecules procured from a specimen into a concentrated cellular element stream and a plasma stream, a means of receiving, heating and/or maintaining the plasma stream to a temperature near or above normal bodytemperature but below its boiling point, a means of receiving and filtering the heated plasma stream to remove selective macromolecules, a means for receiving the filtered plasma stream from the filter means and for receiving the concentrated cellular element stream and combining the streams to form a processed stream substantially free of the macro-molecules intended to be remove and a means of receiving and/or cooling the combination stream to normal body temperature for returning said fluid to the specimen.

Brief description ofthe drawings The following is a brief description of the drawings which are presented for the purpose of illustrating the invention and notforthe purpose of limiting same.

Figure 1 shows the circuit used in in vitro filtration; Figure2 shows the extracorporeal circuit used in exvivo or clinical filtration; Figure 3shows the amount of cholesterol removed at varying temperatures for in vitro filtration; Figure 4 shows the mean sieving coefficients with various kinds of plasma (Kuraray Eval 4A module; In vitro at370C); Figure 5 shows the particle size distribution ofthe lipoprotein for the FHC plasma and the dog plasma with various cholesterol levels; and Figure 6shows the posttreatment recovery in a dog of LDL-VLDL and HDL cholesterol.

Detailed description ofthe invention Applicant has discovered the thermofiltration offers many advantages overthe conventionally known methods of plasma filtration. Thermofiltration is the removal of macromolecules from plasma by warming the plasma to a selective temperature near or above the normal physiologictemperature but not above its boiling point and filtering the warmed plasma with a membrane filter having a porosity that will remove the desired macromolecules.The critical advantages demonstrated bythermofiltration include the ability to filter a much greater volume of plasma at highertemperatures because plasma exposed to the highertem- perature has less of a tendency to form deposits of undesirable solutes on the membrane media and the more selective removal of macromolecules based upon differences in sieving coefficients at highertemperatures.

Evaluations of multiple membrane filters for plasma filtration based on filter material, pore size, and structure indictate thatthe Kuraray Eval 4A module (ethylene and vinyl alcohol copolymer, Kuraray Co., Japan) and other modules of similar properties are particularly well suited for plasma solutefractionation by thermofiltration. Other suitable filters include those which utilize filter media consisting of polysulfone, polypropylene, nylon, polyester, cellulose acetate, collagen and the like.

Applicants have demonstrated that sieving coefficients of some macromolecules are significantly higher at 370C and 420C than at 250C for the Kuraray Eval 4A module. (Table I below). Particularly noteworthy are the higher sieving of HDL cholesterol, IgG, fibringen, total protein, and albumin at 370 to 420C. In addition, as a resultofthe reduction of cryogel formation at these highertemperatures, a much greater volume of plasma can be filtered. This is because at near or above normal physiologictemperatures solute aggregation is kept to a minimum, and separation is due to the molecular size differences ofthe solutes and not the aggregate compositions.

Table l: Sieving coefficentsforvarious macromolecules.

Volume TP Alb Glb Fib IgG IgA IgM Processed (mlX 250C 0.61 0.71 0.48 < 0.06 0.36 0.21 0.15 1,000 370C 0.74 0.82 0.59 0.13 0.55 0.51 0.17 1,000#3,000 420C 0.74 0.86 0.59 0.26 0.57 0.46 0.18 1,000#3,000 Volume T Chol LDL HDL TG Processed (mi) 250C 0.07 0.03 0.71 0.13 < 1,000 370C 0.06 0.02 0.84 0.11 1,000#3,000 420C 0.04 0.03 0.97 0.13 1,000#3,000 All macromolecules are from the same plasma source. Heparin dosage: 1,000 U/L.

TP: total protein Alb: albumin; Glb: globulin; Fib: fibrinogen; TChol: total cholesterol, LDL: low density lipoprotein; HDL: high density lipoprotein; TG: triglycerides.

Moreover, thermofiltration is an effective method of removing pyroglobulins from plasma solutions. Pyroglobulins are serum globulins that precipitateorgel upon heating. Normally, pyroglobulins are not found in serum of normal individuals. Rather they are readily associated with macroglobulinemia and other lymphoproliferative or multiple myeloma disorders. Heating a serum containing pyroglobulinsto 55-56 C results in gel formation, which can be effectively removed from the serum through plasma filtration. Similarly, proteins and other immunoglobulins which effectively denature or coagulate upon heating may also be selectively removed through thermofiltration.

As a consequence of the above advantages of thermofiltration over conventionally known methods of plasma separation, thermofiltration can be used to selectively remove pathologic macromolecules from blood in on-line and off-line plasma treatments, while atthe same time allowing for the passage or return of beneficial plasma proteins. The advantage of this type of treatment can be clearly shown in the therapeutic control of cholesterol.

Cholesterol has been determined to be an important components of arterial plague formation in artherosclerosis as well as in hypercholosterolemia. Cholesterol circulates in the blood linked to large protein molecules. One form of cholesterol carrying protein, called low-density lipoprotein (LDL), is known to promote atherosclerosis. Abouttwo-thirds or more oftotal blood cholesterol istranported in LDL. Anotherform, called high-density lipoprotein (HDL), is known to be protective against the disease process. Therefore, the selective removal of LDL and maintenance of HDL is important in the treatment of atherosclerosis and the therapeutic control of hypercholesterolemia.

Recently, plasma exchange has been utilized forthe removal of plasma and its replacement with electrolyte solutions and/or plasma products in familial hypercholesterolemia patients. However, these methods are non-selective and remove proportionately low density lipoproteins (LDL) with high-density lipoproteins (HDL) and other plasma proteins which are beneficial to the patient. In addition, several methods have been studied for the selective removal of LDL, including anti-LDL-antibody Sepharose columns, and combinations of heparin precipitation and bicarbonate dialysis, however, membrane filtration offers many advantages over these methods in terms of biocompatibility and treatment cost effectiveness.

The selective removal of LDL cholesterol from plasma bythermofiltration can be demonstrated under both in vitro and ex vivo conditions. In vitro pertains to conditions of experimentation in a laboratory setting, whereas exvivo pertains to conditions of extracorporeal circulation with living organisms.

1. In Vitro Filtration Various types of plasma are used in vitro to evaluate the temperature effect on selective cholesterol removal. The in vitro filtration tests are carried out with the various types of plasma at varying temperatures according to the extracorporeal circuit demonstrated in Figure 1.

In Figure 1, one unit per ml. heparin (heparin sodium injection, Invenex Lab., OH) is added to plasma pool 10 wherein the plasma is kept at approximately 370C by heat controller 12 and magneticstirrer 14. Plasma is drawn from plasma pool 10 into line 16 and fed into plasma pump 18 of a plasma flow rate of 15ml/min.The plasma is pumped from plasma pump 18 into line 20 and then into water bath 22 which is controlled by thermo-regulater 24. Within water bath 22, the plasma passes through heat exchanger 26 and passes by pressure gauge 28 into filter 30, where the LDL cholesterol is retained and the HDL cholesterol and albumin substantially pass through. From filter 30, the filtered plasma minus LDL cholesterol flows through line 32 into filtrate collection pan 34.

The following specific examples further illustrates the practice of the present invention.

Example I Familial type II hypercholesterolemic plasma (FHC) was procured by repeated centrifugal plasma exchange. The in vitro filtration tests were carried out with the FHC plasma and the Kuraray EVAL 4A membrane filter at temperatures of 4,25,37,42 and 47"C, respectively, according to the extracorporeal circuitdemonstrated in Figure 1 and as described above. The mean sieving coefficients (sc), the plasma volume processed, andthetotal amount of LDL and HDL cholesterol were determined at the above temperatures bythefollowing calculations.

Concentration in = Concentration in Filtrate (mg/dl) sieving coefficient (sc) = Concentration in filter inlet (mg/dl) wherein a sieving coefficient of 0.9 to 1 indicates little or no separation or removal of the macromoleculefrom plasma, and a sieving coefficient of 0 to 0.1 indicates substantially total removal of macromolecules from plasma.

RemovalAmount(g) = Concentration in plasma pool (g/dl) x (1-sc) x Processed Volume (dI) Results Table II outlines the volume processed and the mean sieving coefficients for total cholesterol, HDLcholesterol, LDL cholesterol, and albumin at varying temperatures. The results indicated that greaterthan 85% ofthe total cholesterol and 90% of LDL cholesterol were removed while over70% of albumin and 60% of HDL cholesterol were passed through the filter. The sieving coefficients of HDL cholesterol and albumin increased with increasing temperature while LDL cholesterol was independent oftemperature.

Table II. Mean sieving coefficients and plasma volumes processed at varying temperatures; in vitro filtration test of Kuraray Eval 4A (1.0 M surface area) using familial hypercholesterolemic plasma.

Processed Mean Sieving Coefficients Temp Volume Total HDL LDL pcl (mI) Chol Chol Chol Alb 4 365 0.10 0.58 0.07 0.72 25 1135 0.11 0.76 0.10 0.84 37 1780 0.12 0.67 0.06 0.81 42 2150 0.16 0.72 0.08 0.91 47 2350 0.14 0.79 0.08 0.94 Figure 3 demonstrates that cholesterol removal differs at varying temperatures. At 37 to 42 C, the removed LDL cholesterol amount is largest(4.5g module), while HDL cholesterol is below 0.1 g. Removal amount per module was limited by the maximum Ptm (300 mm Hg) permitted.

Conclusion: Thermofiltration is highly effective in selectively removing large quantities of LDL cholesterol from plasma while retaining large quantities of useful HDLcholesterol and albumin under in vitro conditions.

The in vitro membrane filtration of FHC plasma with the EVAL 4Afilter permits near complete rejection of LDL cholesterol with high sieving or retainment of HDL cholesterol and albumin. The sieving coefficients of HDL cholesterol and albumin increased with increasing temperature, while the sieving coefficient of LDLcholesterol was near complete rejection at all temperatures. Thus, membrane filtration near or above physiologic temperatures, i.e., thermofiltration, improves the selectivity of LDL cholesterol removal over that of HDL cholesterol and albumin.

Moreover, thermofiltration also permits higher plasma volumes to be processed. This is a result of the reduction of cryogel formation and less removal of solutes not intended to be removed at the elevated temperatures. Also, highervolumes of plasma are processed and larger quantities of cholesterol are re- moved per unit module.

Example II In vitro module filtration testewere carried out with normal human plasma (NHP) and sclerosing cholangitis plasma (SCP) using the Eval 4A (copolymer of ethylene and vinyl alcohol; surface area 2.0 m2) at 4 ,25 , 370,420,470 and 52 C, respectively, according to the extracorporeal circuit demonstrated in Figure 1 and as described above. The NHP was procured by filtration at 370C of outdated citrated plasma using the Toray PS-05 plasma separator (polymethylmethacrylate; surface area 0.5m2; Toray Industries, Japan). The SCP was procured by membrane plasma exchange. The SCP differed from the NHP in thatthe SCP had 1.5fold higherfibrinogen and four fold higher LDL cholesterol concentrations with similar levels of albumin and antithrombin Ill when compared to NHP.

All filtration tests were carried out with a plasma flow of 30 ml/min. Changes in inlet pressure were moni tored as a function of the transmembrane pressure and reflect membrane plugging.

Samples obtained pre and postfiltration were analyzedforvarious biochemical solutes including albumin (Alb),fibrinogen (Fib), total cholesterol (T Chol), LDL cholesterol (LDLChol), HDL Cholesterol (HDL Chol), antithrombin III (AT lil) and heparin. Alb was measured wth an autoanalyzer (SMA-II, Technicon Instrument Co., Tarrytown, NY) by the bromocresol green method. Fib was measured by the Fibrosystem (BBL, Ockeys- ville, MD). T chol and triglycerides were measured with an autoanalyzer (AA II, Technicon Inst.Co.) using the cholesterol oxidase-peroxidase enzymatic method. LDL chole was calculated as: T chol - HDLchol- 1/Stri- glycerides. HDL chol was measured by the dextran-sulfate-Mg 2+ precipitation method. Antithrombin Ill and heparin were measured by the Protopath antithrombin Ill and heparin synthetic substrate assay (American Dade, Miami, FL).

Results Figures 4A and 4B show the volumes processed and the sieving coefficients for the filtration of NHP and SCP overthetemperature range of 4"to 52"C. In both plasmas highervolumeswere processed asthetem- perature was increased from 40 to 52 C. However, the volume processed did not increaseattemperatures over420C and dropped significantly at 52 C. Sievings forAlb incresed with incresing temperature from 4"to 420C and thereafter also dropped. A similar tendency can be seen in fibrinogen.Significant increases of HDL chol sieving were observed in the temperature range of 40 to 420C where no major changes in LDL chol were noted. HDL chol sieving also dropped 52"C. Total removal of LDL chol and removal ratios of HDL chol andAlb to LDLchoI were listed in Table III. Maximum removal amounts ofLDLchol and minimal removal of HDLchol and Alb versus removal were obtained at420C.

Table III: Total removal and ratios of HDL/LDL and albumin/LDLcholesterol removal using NHP and SCP patients plasma at varying temperatures.

Plasma Temp 0C 40 25 370 420 470 520 NHP LDL Chol (mg) 130 347 932 1854 1549 198 HDLchol 0.95 0.76 0.45 0.19 0.23 0.48 LDL chol Alb 31.1 18.2 10.5 5.6 6.5 14.1 LDL chol SCPLDLchoI(mg) 887 1458 2368 3130 2790 1216 HDLchol 0.016 0.021 0.008 0.001 0.007 0.05 LDL chol Alb 6.2 5.6 2.9 2.2 3.2 4.8 LDL chol NHP = normal human plasma CP = sclerosing cholangitis plasma Conclusion:These results suggest that operation at near physiologic temperature is promising for preven tion of heparin induced aggregation which occurs below physiologic temperature and filter plugging caused by these deposits when smaller pore size membranes are used to separate molecules. As shown in Figure 4A and 4B, filtration above physiologictemperature (up to 47 C) produces higher volumes processed and a higher passage of albumin and HDL chol. These results indicate that plasma filtration nearorabove370C, thermofiltration, is promising for clinical use in the separation of plasma solutes based on size differences (i.e., LDL selective separation vs. HDL sieving).

II. Ex Vivo Filtration Exvivo filtration is the continuous on-linefiltering of plasma with living organisms. The exvivafiltration tests were carried out at 370C according to the extracorporeal circuit demonstrated in Figure 2.

Referring to Figure 2, blood is drawn from the artery of a living organism into line 36 and fed into a pump38 from which it is pumped into a line 40, passing by pressure gauge 42 and into plasma separator44, where the plasma and blood cellular elements are fed into line 46, while the plasma is fed into line 48. From line 48,the plasma flows through pump 50 to line 52 where it enters water bath 54 controlled by thermo-regulator 56.

Within water bath 54, the plasma passes through heat exchanger 58 and by pressure gauge 60 into filter 62, where the LDL cholesterol, is substantially retained and the HDL cholesterol, albumin, and other low molec ularweight macromolecules substantially pass through.

From filter 62,the filtered plasma which is essentially deficient in LDL cholesterol flows through line 64and is intermixed with the blood cellular elements of line 46. The mixture then is either cooled to bodytem perature in heat exchanger 66, or passed into line 68 where is passes pressure gauge 70 and is fed into line 72 and returned to the vein of a living organism in a continuous process.

The following specific examples further illustrate the practice ofthe present invention.

Example 3 As a result of the similarity of lipoproteins and its suitable body size for on-line filtration, a hypercholesterol dog was the model chosen forexvivo filtration. The thyroidectomized/dietcanine model is well established and has been studied extensively. Using this model, three different cholesterol level ranges u p to 600mg/dl (normal, 120 mg/dl) were evaluated.

Arterio-venous (AV) fistulae were constructed in two healthy male mongrel dogs weighing from 24to 30 kg. As a control, dogs were maintained with a normal diet (lab Canine Diet 5006; Lab Chow, St. Louis, MO). As a middle cholesterol concentration model,the same dog was maintained with the same diet aftersurgical thyroidectomy. As a high cholesterol concentration model, the dog was maintained with a special dietthat consisted ofthe normal meal with 4% hydrogenated coconut oil and 0.75% cholicacid added (TD 75337, Taklad, Madison, WI) afterthyroidectomy.

Exvivo filtration tests were carried out in the dogs at different cholesterol levels under general anesthesia with nitrous oxide gas and nembutal injection (Nembutal sodium solution, Abbott Lab, IL). An AVfistula was used for blood access and 200 uniting heparin was injected as an anticoagulant. Plasma was separated in the on-line extracorporeal system using a membrane plasma separator (Mitsubishi 60TW, polyethelene, Mitsubishi Rayon Co., Japan). The separated plasma was filtered using the same method as in vitro filtration (at 37 C) and the filtered plasma was then recombined and returned to the dog (Figure 3). Blood and plasma flows were 60 and 15 ml/min, respectively. One calculated plasma volume was filtered.Eight hundred to 1000 ml of lactated Ringer's solution was infused intravenously during the extracorporeal circulation.

Samples were drawn simultaneously from the arterial and plasma lines ofthefilterinletand outletwhen one half and one volume were processed, and pre- and post-treatment. At one hourand 1,3,7,14 and 21 days posttreatment, blood samples were taken following 14to 16 hours fasting. Dogs were fed the same diet as before filtration ad libitum. Biochemical measurements included cholesterol and triglyceride (automatic ana- lysis AA II, Tech nicon Instrument Co., NY), HDL cholesterol (Dextran Sulfate MgCI2 precipitation procedure), in addition to routine serum multiple analysis (SMA-II system Technicon Instrument Co.) and hematological analysis (automated cell counter, Coulter Electronics Inc., FL).

The LDL cholesterol concentration of human plasma was calculated using the equation: total cholesterol HDL cholesterol - 1/5 triglyceride. The LDL-VLDL cholesterol concentration in dogs was calculated as follows: total cholesterol HDL cholesterol. The lipoprotein fractions were prepared for analysis by preparative ultracentrifuge (human LDL; 1.006 < d < 1.063, human HDL; 1.063 < d < 1.21, canine LDL-VLDL; d < 1.063,canine HDL; 1.087 < d < 1.21), where d = density. Lipoprotein particle sizes were measured using negative straining electron micrographs. These fractions of canine lipoproteins are not homogeneous but are comparable to the fractions obtained by precipitation methods.

Result Table IV outlines cholesterol concentrations at the various stages ofthe dog model. Total cholesterol, particularly LDL-VLDL cholesterol, was increased and the ratio of LDL-VLDL cholesterol to HDLcholesterol was increased over 10 times. Albumin showed no significant change. On-line plasma filtration was carried out at each cholesterol level. Transmembrane pressure (Ptm) of the plasma separator was stablethroughout the procedure. Sieving coefficients of albumin and total cholesterol and other macromolecules were over 95%. The Ptm of the macromolecule filter increased gradually during the perfusion. The Ptm values at one plasma volume processed ranged from 10 to 256 mmHg. Significant differences in Ptm changes were not dependent on cholesterol concentrations.

Table IV Cholesterol and albumin levels on canine hypercholesterolemic model.

CHOLESTEROL LEVEL FmgidlJ Albumin level TOTAL HDL LDL-VLDL zgidlJ l; 137+24 115+15 21 3.00#0.06(n=3) ll; 395 t 30 181 + 13 214±17 3.25#0.07(n=2) III; 600 + 14 219 + 30 382 # 382116 # 3.2510.21 (n=2) I; Normal dog with normal diet ll; Thyroidectomized dog with normal diet Ill; Thyroidectomized dog with 4% hydrogenated coconut oil and 0.75% cholic acid addition on normal diet Table V outlines mean sieving coefficients at the varying cholesterol level. LDL-VLDL cholesterol was highly rejected bythe plasma filter, whereas albumin and HDL cholesterol showed high sieving. The sc ofthe LDL-VLDL cholesterol decreased with increasing cholesterol.

Table V. Mean sieving coefficients of macromolecule filter; exvivo on-line filtration test (37 C) of Kuraray Eval4Aon differentcholesterol levels.

CHOLESTEROL TOTAL HDL LDL-VLDL ALBUMIN l; 0.6010.10 0.6310.09 0.3910.07 0.8810.07 (n=3) ll; 0.42+0.08 0.6110.11 0.32+0.01 0.9510.06(n=2) III; 0.34+0.05 0.5910.09 0.19+0.04 0.93+0.04(n=2) Mean + Standard Deviation Figure 5 shows the particle size distribution of the lipoprotein for the FHC plasma and the dog plasmas with various cholesterol levels. The size difference between the HDL and LDL of FHC plasma was greaterthan that ofthe dog plasmas.The particle diameters and deviation of LDL-VLDL in dog plasmas also increased, but not in as great degree as with the human plasma. The HDL size was not significantly different among the groups.

Figure 5 shows the post treatment recovery of HDL-VLDL and HDL cholesterols. Recovery of LDL-VLDL cholesterol was prolonged in the higher cholesterol level groups. For Groups II and lil, ittookabout2weeks to return to pre-treatment values. HDL cholesterol recovery was constant and returned to pre-values within 7 days for all groups. Figure 6 shows the changes in the LDL-VLDL cholesterol/HDL cholesterol ratios. The ratio decreased during the past filtration periods and was maintained at a lower level for 14 days. The tendencyfor a higher reduced ratio for longer periods in comparison to the pre or post treatment values was greater in Group Ill which had the highest cholesterol.

Conclusion: The data indicates that lipoprotein particle size and sieving coefficients are highly cholesterol concentration dependent. As the cholesterol concentration increases, lipoprotein particle size (particularly LDL-VLDL) increases, sieving decreases and the total cholesterol (LDL-VLDLcholesterol) removal increases.

A comparison of the sieving coefficients indicates that canine LDL-VLDL sieving is greaterthan that of human LDL. This correlation is explained by the particle size study which indicates larger deviations and overlaps between canine LDL-VLDL and HDL, making it more difficult to separate the lipoproteins in canines than in humans. These reults indicatethatthermofiltration would be quite effective in humans in regard to the selective removal of lipoproteins.

Moreover, the data indicates that there is a more prolonged recovery of the LDL-VLDL cholesterol in the more hypercholesterolemic stages, while HDL cholesterol recovery remains relatively normal. The reduction of LDL cholesterol with preservation of HDL cholesterol by thermofiltration and the prolonged recovery of LDL-VLDL cholesterol with the maintenance of lower LDUHDL ratio is highly suggestive of an effective method of treating lipoprotein abnormalities.

Example Initial clinical thermofiltration procedures were preformed on a secondary hypercholesterolemia patient.

The patient selected for the trail was a 39 year old man who had a high concentration (210-450 mg/dl) of cholesterol and a very high LDL/HDL cholesterol ratio (8-30) due to the cholestasis of sclerosing cholangitis.

The thermofiltration tests were carried out according to the on-line system exhibited in Figure 2. The blood flow was set at 100 ml/min and the plasma flow at 30 ml/min. The Toray PS-05 (Toray Industries, Tokyo, Japan), and Asahi Plasmaflo (AP0511: Asahi Medical Co..Tokyo,Japan) modules were used as the plasma separator, and the EVAL 4A2.0 m2 (Kuraray Co.. Osaka, Japan) was used as the plasma filter. The filter and a warmer plate were wrapped with an electric heating pad to maintain the temperature at 370C in the cryochamber of the Cryomax (Cryomax 360; Parker Biomedical, Irvine, CA, U.S.A.). For anticoagulation, 5000 U of heparin was injected as a bolus prior to initiation of the extracorporeal circulation.The processed plasma volume and transmembrane pressure were monitored continuously throughout the procedure. The filtration was carried out until the transmembrane pressure (Ptm) of the plasma filter reaches 500 mm Hg.

Samples were drawn simultaneously from the plasma inlet and outlet lines of the filter when the Ptm reached 150 and 300 mm Hg to calculate the sieving coefficients (sc). Solute sieving was calculated as concentration in the filtrate divided by the concentration in the plasma inlet to the plasma filter. Biochemical measurements included cholesterol and triglyceride (automatic analysis AAII, Technicon Instrument Co., Tarrytown, NY, U.S.A.), and HDL cholesterol (dextran sulfate MgCI2 precipitation procedure), in addition to routine serum multiple analysis (SMA-Il, Technicon Instrument Co.). The filter was removed from the circuit following plugging, and the plasmapheresis procedure was changed to plasma exchange, using 5% albumin solution asa substitution fluid.Plasma exchange was continued until one calculated plasma volume (2893 ml) was processed by plasma exchange alone.

In vitro filtration studies were done using the same filter, the same temperature, and plasma from the same patient as described in Example I.

Results The procedure was well tolerated bythe patient and no adverse reactions were noted. No substitution fluid was used during the thermofiltration phase. To a Ptm of 500 mm Hg. 13551275 ml (1160 and 1540) ml of plasma were filtered and 1117# 183 ml (980 and 1253 ml) were filtered to a Ptm of 300 mm Hg. The course of Ptm versus the filtered volume was comparable to in vitro studies with this patient's plasma, as was the filtered volume (1060 ml processed to 300 mm Hg of Ptm in vitro).

There was near complete rejection for LDL + VLDL cholesterol (sc = 0.02) and high passage of albumin (sc = 0.75) and HDL cholesterol sc=0.78). Fibrinogen sieving was low (0.07). These results were comparable to the in vitro filtration results. (Table VI below).

Table Vl Concentration and sieving coefficients (sc) of solute (mean t SD) Clinical (Ex Vivo)* Concentration sc Total protein (g/dl) 6.81 1.0 0.64 + 0.09 Albumin (g/dl) 3.2 1 0.2 0.75 10.06 Total Cholesterol (mg/dl) 181 1 47 0.09 10.00 HDLcholesterol (mg/dl) 1717 0.7810.09 LDLcholesterol (mg/dl) 140140 0.01 LDL-VLDL cholesterol (mg/dl) 163 141 0.02 10.01 Fibrinogen (mg/dl) 286 # 84 0.07 1 0.03 In vitro ** Concentration sc Total protein (g/di) 6.2 1 0.3 0.72 1 0.02 Albumin(g/dI) 3.6#0.1 0.82 #0.03 Total cholesterol (mg/dl) 2741 2 0.06 1 0.01 HDL cholesterol (mg/dl) 11 13 0.841 0.08 LDL-VLDL cholesterol (mg/dl) 263# 5 0.03 + 0.02 Fibrinogen (mg/dl) 298#28 0.13 + 0.03 HDL: high density lipoproteins; LDL: low density lipoproteins, VLDL: very low density lipoproteins; Ptm: transmembrane pressure.

* Mean value for fou r samples, taken at Ptm of 150 mm Hg and 300 mm Hg from each of two treatments.

** Mean value of three filtration tests at the same conditions.

Conclusion Sieving coefficients of LDL-cholesterol (0.02), HDL cholesterol (0.78) and albumin (0.75) demonstrate the selectivity ofthermofiltration. These results are comparable to in vitro filtrations tests using the plasma ofthe same patient. The advantage of this system compared to plasma exchange is the maintenance of HDL (anti- atherogenic lipoprotein) and other essential plasma solutes that would be discarded in plasma exchange.

Thermofiltration is more selective than membrane schemes without temperature control and simplerto apply, as it does not require other plasma treatment steps or the addition of potentially harmful chemical additives. Moreover, abnormal concentrations of various proteins (such as immunogiobulins) can also be effectively removed bythermofiltration.

While there have been described herein what are at present considered to be the preferred embodiments of this invention, it will be apparent to those skilled in the artthatvarious changes and modifications may be made therein without departing from the invention, and it is, therefore, intended in the appended claims to cover all such changes and modifications as fall within the true spirit and scope of the invention.

Claims (32)

1. A method of more selectively removing macromolecules from a plasma solution including the steps of: a. providing a plasma solution containing the macromolecules to be removed; b. heating said plasma to a temperature near or above normal body temperature but below the boiling point of said plasma solution, and c. filtering said heated plasma solution while at a temperature near or above normal bodytemperature but below its boiling point with a membrane filter to selectively remove macromolecules from the plasma solution.

2. The method of claim 1 wherein said heating is carried out at a temperature range from about 350to about 60 C.

3. The method of claim 2 wherein said heating is carried out ata temperature ranging from about 37" to about 520C.

4. The method of claim 2 wherein said membrane filter has a porosity less than that of the selective macromolecules to be removed from the plasma solution.

5. The method of claim 1 wherein said macromolecules are separated from said plasma solution based on differences in sieving coefficients at elevated temperatures.

6. The method of claim 1 wherein said macromolecules to be removed are selected from the group consisting of low-density lipoproteins (LDL), pyroglobulins, high molecularweight proteins and mixtures thereof.

7. The method of claim 1 wherein said plasma includes normal sclerosing cholangitis and type Ila hypercholesterolemia plasma.

8. A method of selectively removing macromolecules from a plasma solution including the steps of: a. securing a blood flow from a specimen; b. separating said blood flow into a concentrated cellular element stream and a plasma stream containing macromolecules to be removed; c. heating said plasma stream containing macromolecules to be removed to a temperature near or about normal body temperature but below its boiling point; d. filtering said heated plasma stream while at a temperature near or about normal bodytemperature but below its boiling point with a membranefilterto selectively remove macromolecules from the plasma solution to form a filtered plasma stream; and e. combining said filtered plasma stream and said cellular element to form a processed plasma stream.

9. The method of claim 8 wherein said process stream plasma is cooled before it is combined with said cellularelementstream.

10. The method of claim 8 wherein the processed stream plasma is returned to the specimen.

11. The method of claim 8 which further includes the step of pumping the blood from the patient before it is formed into a separate stream.

12. The method of claim 8 wherein the separation ofthe blood flow into a concentrated cellularelement stream and a plasma stream is effected by either a membrane filter or by a centrifuge.

13. The method of claim 8 wherein the heating of plasma stream is carried outinthetemperature range of 35 C to 60 C.

14. The method of claim 13wherein said heating is carried out at a temperature ranging from about37 C to about 52 C.

15. The method of claim 8 wherein said macromolecular membranefilter has a porosity lessthan that of the selective macromolecules to be removed from the plasma solution.

16. The method of claim 8 wherein said selective macromolecules are separated from said plasma solution based on differences in sieving coefficients at elevated temperature.

17 The method of claim 8wherein said selective macromolecules to be removed are low density lipo proteins (LDL), pyroglobulins, high molecularweight proteins,or mixturesthernof

18. The method of claim 8 wherein the process is continuous.

19. A method of controlling abnormal plasma conditions in living organism by selectively removing macromoleculesfroma plasma solution including thestep of: a. securing a blood flow from a living organism; b. separating said blood flow into a concentrated cellular element stream and a plasma stream containing macromolecules to be removed; c. heating said plasma stream containing macromolecules to be removed to a temperature near or above normal body temperature but below its boiling point; d. filtering said heated plasma stream while at a temperature near or about normal body temperature but below its boiling point with a membrane filter to remove more selectively macromolecules from the plasma solution to form a filtered plasma stream;; e. combining said filtered plasma streams and said cellular element stream to form a processed plasma stream.

20. The method of claim 19 wherein said process stream plasma is cooled before it is combined with said cellular element stream.

21. The method of claim 19 wherein the processed plasma stream to the living organism.

22. The method of claim 19which further includes the step of pumping the blood from the living organism before it is formed into a separate stream.

22. The method of claim 19 wherein the separation ofthe blood flow into a concentrated cellular element stream and a plasma stream is effected by a membrane filter.

23. The method of claim 19whereinthe heating is carried out at a temperature ranging from about35 Cto about 60 C.

24. The method of claim 23 wherein said heating is carried outatatemperature ranging from a bout 37 C to about 520.

25. The method of claim 19 wherein said macromolecular membrane filter has a porosity less than that of the selective macromolecules to be removed from the plasma solution.

26. The method of claim 19 wherein said macromolecules are separated from said plasma solution based on differences in sieving coefficients at elevated temperature.

27. The method of claim 19 wherein said selective macromolecules are low-density lipoproteins, pyroglobulins, high molecularweight proteins or mixtures thereof.

28. The method of claim 19 wherein said lipoprotein abnormalities exist in conditions of lipidemia.

29. The method of claim 19 wherein the process is continuous.

30. An apparatus for removing selective macromolecules from a plasma solution comprising: a. a means for dividing a plasma containing solution containing macromolecules into a concentrated cel lular element stream and a plasma stream; b. ameans for heating said plasma stream to a temperature above normal body temperature but below its boiling point; c. ameansforfiltering said heated plasma stream to selectively remove macromolecules; d. a means for receiving said filtered plasma stream and said concentrated cellular element stream com- bining to form a processed stream substantially free of selective macromolecules.

31. The apparatus of claim 30futher including means for cooling said combination stream to normal body temperature and for returning said fluid to the specimen.

32. The apparatus of claim 30, wherein said means for heating is adapted to warm the plasma stream to a temperature ranging from about 350Cto about 60"C.