Atherosclerosis, 86 (1991) I-8 8 1991 Elsevier Scientific Publishers ADONIS 0021915091000513
LDL-apheresis: results of longterm and vascular outcome Christiane Medizinrsche
der Universitiit, Miinchen (F. R. G.)
(Received 25 June, 1990) (Revised, received 10 September, 1990) (Accepted 14 September, 1990)
Summary LDL-apheresis (immunoabsorption, heparin precipitation (HELP), dextran sulfate cellulose binding (DSC) or filtration) is a potent therapeutic tool in familial hypercholesterolemia (FH) to eliminate LDL-cholesterol, Lp(a) or fibrinogen from the circulation and improve blood rheology. Repetitive use can deplete the cholesterol pool between 40 and 80%. As first reports showed, progression of coronary atherosclerosis can be stopped and sometimes regression can be induced. So far the domain of plasmapheresis was homozygous familial hypercholesterolemia. With several apheresis methods now available, it seems timely to define the indication of plasma therapy for heterozygous FH and the place of this potent therapeutic tool in primary and secondary prevention of atherosclerotic coronary heart disease in patients suffering from severe hypercholesterolemia resistant to diet and/or drug therapy.
Introduction Since the first experimental discontinuous plasmapheresis for homozygous familial hypercholesterolemia (FH) in a 23-year-old man with severe heart failure and far advanced atherosclerosis by de Gennes et al. [l], in 1967, there have been years of intensive work to more effectively
Correspondence to: Professor Dr. Christiane Keller, Medizinische Poliklinik der Universitlt, Pettenkoferstr. Sa, D-8000 Miinchen 2, F.R.G. Tel.: 089-51603511; Fax: 089-52 22 95.
eliminate LDL cholesterol from the circulation. The continuous elimination of LDL rich plasma and its simultaneous substitution by different protein solutions marked the breakthrough of therapeutic plasma exchange. Thompson et al.  published the first results in 1975, after 8 months of repeated treatment of two homozygous females, thus demonstrating the practicability of the method. The diversity of systems now available for effective LDL elimination justifies a consideration of the present accomplishment and of open questions for the future.
2 Methods Separation of plasma and cells Separation of blood cells and plasma is carried out either by centrifugation (cell separators of different brands) or by filtration (hollow fibre filters of different brands). Systemic heparinization of the patient is always necessary. The access to the circulation is veno-venous, rarely by arterio-venous shunt. Plasma exchange Within about 2 h 2500-3000 ml of plasma can be continuously exchanged. Total blood flow ranges from 50 to 80 ml per min. An ACD-A solution (USP; citric acid hydrate 0.8%, sodium citrate dihydrate 2.28, dextrose 2.45%) is infused during the treatment to combat platelet aggregation. The patient’s plasma is replaced by a plasma protein fraction or a human albumin solution (concentration 5%) substituted with calcium and potassium ions [3,4]. LDL-apheresis This term is used for a plasmapheresis procedure which permits a selective or specific removal of the plasma constituent LDL cholesterol. Filtration (double filtration (2 filters on-line ), cascade filtration (3 filters on-line ) and plasma filtration after termination of plasma exchange off-line with one filter of a pore size suitable to retain mainly LDL-cholesterol ) are selective procedures which make use of the large molecular weight of LDL cholesterol in comparison to plasma proteins. The pore size of the filter permits the elimination of LDL cholesterol. In addition, a small amount of the large plasma proteins is also held back by the filter. The LDL free plasma is returned to the patient during plasmapheresis or, as in case of filtration off-line, as a substituton solution instead of albumin during the next plasma exchange. Various methods adopted from affinity chromatography have been developed to specifically eliminate LDL cholesterol. A polyclonal [6,7] sheep antibody to human apolipoprotein B or only the F(ab) fragments thereof  coupled to activated sepharose are able to bind apolipoprotein B-containing lipoproteins (immunoabsorption). These affinity col-
umns can be regenerated, i.e. LDL and VLDL are washed away by an acid glycine buffer once 400 ml of plasma have accumulated in the column. For each patient two columns are employed for each treatment. They can be repeatedly used 40-50 times depending on the plasma LDL cholesterol concentration before treatment. The average blood flow during treatment is 50-60 ml/mm and 4500 ml of plasma or more are processed. The duration of the treatment is two to three hours. Membrane separation of blood cells and plasma followed by chemical precipitation of LDL with excess heparin at low pH and bicarbonate dialysis (heparin-extracorporeal-LDL-precipitation, HELP)  is carried out with disposable materials. 3000 ml of plasma are processed using a blood flow of 80-90 ml/mm. Affinity binding of LDL by dextran sulfate coupled irreversibly to cellulose beads (DSC) [lo] as disposable columns is another device to eliminate LDL efficiently. 4000 ml plasma or more are processed by DSC-apheresis with a doublecolumn system and a blood flow of 50-90 ml/mm. Patients At first mainly homozygous patients were treated by plasmapheresis, yielding longterm results .for small numbers of patients. Nine have undergone up to 9 years of continuous treatment by plasma exchange [35,42], 8 up to 7 years by immunoabsorption . With the advent of modern apheresis devices the number of patients being treated quickly increased, many of them heterozygotes for FH. The results of several multicenter studies containing up to 50 heterozygous patients are awaited at present. The HELP Study Group has completed the first two year treatment course (50 patients, secondary prevention of CHD), a study with immunoabsorption is approaching its end, while one with dextran sulfate cellulose absorption is still in progress.
Biochemical results Table 1 (lipids, lipoproteins and other plasma proteins) compiles data from the literature (54 homozygotes and 71 heterozygotes for FH, 10 with primary non-familial hypercholesterolemia,
from the literature
DUE TO EXTRACORPOREAL
and compared decrease
30 14-40 -
_ _ -
_ _ _
(HELP) 69-74 65-68
Plasma exchange Thompson  Baeyer (51 Keller 
41-55 40 53-63
Immunoabsorption Borberg  Saal  Riesen [S]
70 44-82 70
Dextran sulfate cellulose absorption (DSC) Mabuchi [lo] 64 0 Keller  60-62 15-17 Filtration Baeyer  Keller 
Heparin precipitation Eisenhauer  Keller 
to our results from more than 1500 weekly treatments
some of the patients appearing repeatedly in successive reports) and our own results obtained from over 1500 plasmaphereses since 1976 by treating 8 homozygous and 7 heterozygous patients with different methods. The acute lipid lowering effects vary within a narrow range with the different forms of plasmapheresis. Immunoabsorption and HELP result in the largest LDL reductions with a time averaged depletion of the cholesterol pool of 41-68s (immunoabsorption, [lo]) and of 37-41% (HELP, [ll]) after months of weekly treatment. At the same time other atherogenic substances, namely Lp(a) [12,13] and fibrinogen [ll] are markedly reduced. The extent of Lp(a) and fibrinogen elimination differs with the apheresis method applied (Table l), the most efficient being the HELP procedure. Small amounts of HDL are lost with apheresis in contrast to plasma exchange. In some instances an increase of HDL was observed after several months of treatment with immunoabsorption  or with
59 41 68-76
_ _ _
HELP  and related to depletion of LDL cholesterol. Accompanying plasmapheresis with lipid lowering drugs (nicotinic acid , simvastatin [ll]) enhances the acute reduction of lipids and lipoproteins. The prolongation of the interval between two treatments from 1 to 2 weeks was achieved in 2 (1 homozygote, 1 heterozygote) of 6 patients in one study [ll]. In our patients (2 homozygotes, 1 heterozygote for FH) the rebound of lipids and lipoproteins after plasma exchange accompanied by cholestyramine and/or pyridylcarbinol was not slower than without medication . Few authors have examined whether compositional changes of lipoprotein particles occur due to plasmapheresis. Jadhav et al.  reported that the elevated ratio of cholesterol to phospholipids in LDL of homo- and heterozygous patients returned to normal within 1 day after plasma exchange. Two to 3 weeks later this ratio had reversed to the former state. The decreased ratio of
4 lecithin to sphingomyelin in LDL of FH patients normalized within the first 24 h after plasma exchange for 24 h. Koizumi et al.  observed that apolipoprotein E rich HDL particles decreased during the first few days following DSC-apheresis. Franceschini et al.  measured an increase of the flotation rate of HDL, particles due to the increase of free cholesterol in HDL, plasma, and LDL. LCAT activity initially decreasing by 26% increased by 40% within 24 h after DSC-apheresis. Thanabalsingham et al.  measured a slower increase of LCAT following plasma exchange than Franceschini et al.  reported after DSCapheresis. All authors conclude that the compositional changes might induce mobilization of tissue cholesterol and regression of vascular atherosclerosis. Thompson and Myant  and Soutar et al.  measured the turnover of LDL- and VLDLapo B in normal, heterozygous and homozygous patients for FH following plasma exchange. Depending on the receptor status (negative or defective in tissue culture) they elicited differences in homozygous FH. The receptor-negative homozygote had a markedly decreased fractional catabolic rate for LDL-apo B and a slightly increased LDL-apo B production rate whereas the opposite was true for the receptor-defective homozygote. The synthesis of LDL-apo B exceeded the synthesis of VLDL-apo B by 1.5-2.0 times in homozygotes. Their VLDL-apo B synthesis rate was normal. In a heterozygote with the phenotype IIa LDL-apo B and VLDL-apo B synthesis rates were normal, but elevated in a patient with the phenotype IIb. Plasma exchange did not increase the rates of synthesis of LDL-apo B or VLDL-apo B and had no significant effect on the precursorproduct relationship between IDL-apo B and LDL-apo B in heterozygotes or homozygotes. Measuring mevalonic acid excretion during immunoabsorption apheresis, Parker et al.  concluded that synthesis of cholesterol increased transiently. Our measurements of the sterol balance during immunoabsorption LDL-apheresis and HELP-apheresis in heterozygotes and one receptor-defective homozygous patient suggested a 2-3 day increase of cholesterol synthesis following
treatment (unpublished data). In a recent study on normolipidemic volunteers it was demonstrated that the HELP procedure did not increase hepatic production of VLDL apolipoprotein B labelled with the stable isotope [1-r3C]leucin (Arends, J. et al., personal communication). Clinical results A common experience for all therapists dealing with FH patients incapacitated by angina pectoris is the rapid onset of feeling better and increasing physical fitness within a few weeks after the start of LDL-apheresis. This might be due to the reported reduction of blood and plasma viscosity , lesser aggregability of erythrocytes, increased tissue oxygen tension  and increased blood flow to cerebrum  and legs  following plasma exchange , HELP [25,28], or DSCapheresis . Tuberous xanthoma disappeared within months of treatment , the size of tendon xanthoma measured by xeroradiography decreased at a slower rate . Cardiovascular outcome Carotid atheroma documented with Duplex scan disappeared with weekly immunoabsorption treatments in one of our 2 patients . At the same time there was no evidence of progression of coronary atherosclerosis as judged by stress ECG test. In one report on immunoabsorption-apheresis the angina pectoris score (7 of 9 patients) and stress test ECG (5 out of 8) improved markedly within l-4 years of weekly treatment . Coronary atherosclerosis documented by angiography regressed in 10 of 22 stenoses and in 21 of 79 sclerotic vessel segments after a maximum of 5 years of regular immunoabsorption . Myocardial performance documented by echocardiography increased. Progression of atherosclerosis was seen in three vessels (one stenosis, 2 sclerotic segments out of 22 and 79 lesions, respectively). Following 5 years of LDL-apheresis by cascade filtration in a Japanese center  the coronary stenoses did not progress, aortic wall atherosclerotic roughness disappeared and atheroma of the renal artery were no longer visualized. In a report
Side effects (W) in relation
Plasma exchange Keller (n = 820) Immunoabsorption Borberg  (n = 465) Saal (n = 164) Heparin precipitation (HELP) HELP Study Group (n = 1000) a Keller (n = 440) Dextran sulfate cellulose absorption (DSC) Mabuchi [lo] (n = 18) Keller (n = 24) Filtration Baeyer  (n = 40) Keller (n = 60) * Personal
Nausea, vomiting, headaches
on cascade filtration with ultrafiltration of proteins regression of coronary atherosclerosis was mentioned but no details reported. An aortic valve gradient of 20 mm Hg could be corrected during 4 years of plasma exchange at two week intervals in a homozygous receptor-defective man  whereas another study reported increasing aortic gradients during monthly treatment of two homozygous women over a 4-year period . In 1985 Thompson et al.  published results of biweekly plasma exchange for 8.4 years in five homozygous patients. In comparison with their untreated homozygous siblings the treated group lived 5.5 years longer. Side effects Unwanted effects of the different methods are summarized in Table 2. Reported data are scanty. After about 500 applications of the newly developed immunoabsorption system Borberg et al.  reported 83 “minor to moderate” reactions during the treatment which did not lead to interruption of therapy. 6% of the reactions were treated by medication not specified. Among these reactions shaking chills, headache, stomach ache, hypotension and vomiting were listed. The majority of incidences were experienced by 2 out of 6
patients. Seven attacks of angina pectotis were recorded during treatment. Three severe reactions required immediate interruption of the treatment; hemolysis due to a defective hollow fiber module for plasma separation, severe headache induced by unvoluntary infusion of sodium azide used for storage of the column between two applications and hypocalcemia due to ACD-solution. One episode of prolonged bleeding from the venous puncture site was observed. Saal et al.  recently reported 12 episodes of chills and fever or cutaneous flushing in 1312 immunoabsorption procedures in 15 patients treated 8-59 months. The development of a humoral antibody response in 12 of 15 patients as a result of shedding of sheep immunoglobulin from the column was demonstrated. There was no correlation between the presence or absence of immunization and clinical reactions of the patients. Clinical reactions occurred during the first use of a new column and during subsequent therapies. They were associated with complement activation when plasma was perfused through the column. Bacterial or pyrogen contamination of the columns with toxemia in the patient and sensitization to ethylene oxide used for sterilization of the disposables could be excluded.
6 We observed 5 severe reactions to plasma exchange with human albumin solution which necessitated immediate termination of the treatment. Three patients developed urticaria and hypotension within 20 minutes of treatment, one patient cutaneous flushing, urticaria and Quincke edema on two occasions within 10 and 20 minutes of therapy. Each reaction could be promptly relieved by intravenous application of steroid and an antihistamine drug. All patients had been treated uneventfully for years when the reaction occurred. Occasional chills and fever occurring 2-3 hours after the treatment with HELP were observed in 2 patients. The origin of these reactions remains unclear. Bacterial and pyrogen contamination as well as ethylene oxide sensitization can be excluded. Pyrogen contamination occurred three times in the filtrate gained off-line and led to chills and fever in 2 patients. No measures had to be taken. No dropout rates in study populations have been reported.
Discussion The three newer methods of LDL-apheresis, immunoabsorption, HELP, and DSC-apheresis can be handled with ease, are rather specific and very effective in eliminating LDL cholesterol. Plasma exchange is too unspecific compared with the former and it depletes the patients’ plasma proteins and HDL. Protein loss to be replaced by albumin also occurs in homozygous patients treated weekly by plasmapheresis combined with selective plasma protein ultrafiltration but not in heterozygotes treated every two weeks . Severe side effects resulting from any form of extracorporeal LDL elimination and requiring interruption of the therapy, rarely occur. Mild to moderate reactions as listed in Table 2 seem to be tolerated by the patients without medication. Experimental work on macaca monkeys  and the results of the CLAS-study  demonstrated that lowering LDL to 90 mg/dl or less can induce regression of atherosclerosis. LDL-apheresis seems to be a form of treatment capable of reaching this goal. There is no evidence that the small loss of HDL
during plasma therapy is a disadvantage of apheresis. Moreover, Lp(a) and fibrinogen, the one a lipoprotein and the other a plasma protein not influenced by lipid lowering drugs and suspected to be atherogenic, decrease with apheresis, strongest with the HELP system (Table 1). This may be another positive aspect of LDL-apheresis probably inducing regression of atherosclerosis. The rapid increase of well-being and physical fitness of patients on apheresis treatment is most likely due to the profound changes of blood rheology which occur faster than changes of atherosclerosis [24281. Up to the early 1980s only homozygous FH was indicated for plasmapheresis. For homozygotes LDL-apheresis is still the only effective therapy and should be started before atherosclerosis becomes apparent. At present it seems necessary to outline the indications for the treatment of FH heterozygotes and for primary non-familial hypercholesterolemia. There is little doubt that one should treat a heterozygote of young age, with hypercholesterolemia unresponsive to drugs and with advanced coronary atherosclerosis which cannot be treated otherwise. Considering primary or secondary prevention of coronary heart disease in severely hypercholesterolemic patients, it seems necessary to define how low LDL should be and for which period of time the intensive treatment of apheresis should last. With the data available a definite answer is not yet possible. Probably it is not only the degree of LDL lowering which determines a successful treatment of patients with high LDL levels and far advanced coronary disease. With respect to the early LDL turnover data on FH patients of Thompson et al.  and recent data of James et al.  there is a lack of information on the kinetics of LDL, VLDL and apolipoproteins B turnover in patients with different forms of LDL receptor defect. Measurements with stable isotopes [23,39,40] may help to recognize the metabolic differences between normal, primary non-familial hypercholesterolemic and LDL-receptor defective patients and the complexity of the interplay of LDL and HDL in the vascular wall and the atheromatous plaque. Therefore it seems important to characterize the receptor status of any patient treated by apheresis as
7 accurately as possible in order to offer the best treatment. With better knowledge of these interferences, LDL-apheresis and the simultaneous application of lipid lowering drugs could probably be optimized. On the other hand it is desirable that non-invasive methods to document the status of the coronary arteries are able to be exactly duplicated at any time. Data from the literature suggest that atherosclerosis of the carotid artery and of the coronaries develop simultaneously and parallel. Our latest results obtained during the HELP study  do not support this view, but again it is a very small group of patients observed for two years. Nevertheless Duplex scan seems to be a useful method to closely follow patients on intensive treatment. With larger numbers of patients the question regarding the speed of atherosclerotic development and regression in different sections of the arterial tree might be answered. The profound acute and longterm change of lipids and lipoproteins by applying LDL-apheresis does not only offer better perspectives for severely ill FH patients, but is also a valuable investigative tool for better understanding the complex disturbances of lipoprotein metabolism. References 1 De Gennes, J.-L., Touraine,
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