Vox Sang 2000;79(1):40-5

Erythrocytapheresis with recombinant human erythropoietin in hereditary
hemochromatosis therapy: a new alternative.

Kohan A, Niborski R, Daruich J, Rey J, Bastos F, Amerise G, Herrera R,
Garcia M, Olivera W, Santarelli MT, Avalos JS, Findor J

Departments of Transfusion Medicine and Immunotherapy, and Gastroenterology,
Hospital de Clinicas, University of Buenos Aires School of Medicine, Buenos
Aires, Argentina. aikohan@intramed.net.ar

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Abstract
Background and objectives: The purposes of this study were to evaluate the tolerance, efficacy and safety of isovolemic erythrocytapheresis (EA) in nonanemic patients with hereditary hemochromatosis (HH), and to assess the usefulness of recombinant human erythropoietin (rHuEPO) associated with EA to reduce treatment duration.

Materials and Methods: In 10 asymptomatic patients with serum ferritin >400 microg/l, transferrin saturation >50%, and GPT elevation, EA with rHuEPO and folic acid was performed.

Results: Red cell indices, serum ferritin, and other iron metabolism parameters (serum iron, transferrin, and transferrin saturation); GPT and other laboratory data were considerably improved.

Conclusion: This method offers better results in less time than traditional phlebotomy. EA with rHuEPO is an effective therapeutic alternative for patients with HH.
====================

Introduction

Hereditary hemochromatosis (HH) is characterized by excessive iron deposits in various tissues, associated with increased intestinal absorption. This accumulation may be 10 or more times greater than usual [1]. The iron overload is toxic [2-5] and may be potentially carcinogenic [6-8]. Clinically, liver manifestations (cirrhosis and hepatocellular carcinoma) predominate and may lead to death in more than 75% of patients [6, 9-12]. Diabetes mellitus and its complications [10, 13], as well as cardiac diseases [14, 15], may cause death in 10% of cases. Less frequently, sexual dysfunction, amenorrhea [16-18] and skin hyperpigmentation may develop. Medical treatments are usually based on iron remova1 [10, 19, 20] through phlebotomy and chelating agents such as deferoxamine [21-23], often ill-tolerated, discontinued or with no appropriate follow-up. Large-volume erythrocytapheresis (EA), described by Kellner and Zoller [24], has become a useful therapeutic alternative, especially when more than 1,000 ml of red blood cells can be collected. Recombinant human erythropoietin (rHuEPO) proved to be effective in promoting hematocrit (VPRC) and hemoglobin increase in patients with inadequate endogenous production due to renal failure, or when large volumes of autologous blood must be withdrawn preoperatively [25, 26]. The purposes of this study were: to evaluate the tolerance, efficacy, and safety of isovolemic EA in nonanemic patients with HH and to assess the usefulness of rHuEPO association with EA to reduce treatment duration.

Materials and Methods

Ten nonrelated male patients (average age 48 years; range 44-54), seen at the Departments of Transfusion Medicine and Immunohematology, and Gastroenterology between October 1, 1994, and August 30, 1996, were considered. Cases selected for this study were those most likely to tolerate regular therapeutic phlebotomy, i.e., patients without circulatory overload/hypervolemia, hyponatremia, and markers of advanced hepatic disease.

Inclusion criteria were: serum iron > 168 ug/dl, transferrin saturation >65%, serum ferritin >400 ug/l, and liver biopsy consistent with hemochromatosis. Histology sections were examined with hematoxilin-eosin, Masson trichrome, and Perls stains. Iron stores were estimated by the method of Cook et al. [27]. Patients refractory to regular phlebotomy for more than 1 year were also enrolled.

Exclusion criteria were: clinical features of circulatory overload and/or hypervolemia (ascitic edematous syndrome), serum sodium > 130 mEq/l, serum potassium >5 mEq/l, BUN >40 mg/dl, serum creatinine > 1 mg/dl, creatinine clearance < 80 ml/min, alpha-fetoprotein > 15 ng/dl, alcohol consumption >40 g/day, positive serum markers of viral hepatitis (HbcAc, HbsAg, and/or anti-HCV) and autoimmunity (ANA, SMA, LKMl, and anti-mitochrondrial antibodies), anti- HIV, secondary hemochromatosis, or HH with encephalopathy. The main characteristics of this series were skin hyperpigmentation (8/10), hepatomegaly (9/10), splenomegaly (5/10), cardiac involvement (5/10), insulin-dependent diabetes mellitus (3/10), glucose overload intolerance (2/10), gonadal failure (5/10), arthralgia (4/10), asthenia (lO/lO), liver cirrhosis (10/10), and HLA-A3 (9/10).

EA was prescribed when the VPRC or hemoglobin reached 34% or 10 g/dl, respectively. The amount of red blood cells collected was based on the total volume estimated, although in no case was 30% of the circulating erythrocyte mass exceeded. Starting with the first EA, all patients received rHuEPO, 4,000 units subcutaneously, three times a week (approximately 150 U/kg/week), and folic acid, 10 mg/day per os.

Red cell indices were assessed weakly, before and after each EA; serum ferritin was measured every other week, while other iron metabolism parameters (serum iron, transferrin, and transferrin saturation), GPT, y-GT, AP, total protein, alpha 1-, alpha 2-, betha-, and y-globulin, se-um glucose and BUN, were evaluated monthly.

EA therapy was deemed complete when iron metabolism values returned to normal and remained steady for more than 30 days.

A manually operated, discontinous flow cell separator in one-way mode (Haemonetics V50 Plus) and a continous flow cell separator in two-way mode (Excel-Dideco) were used. Before each procedure, a physical examination was performed. In patients with insulin-dependent diabetes mellitus, serum glucose levels were measured before, during, and after EA.

A superficial vein of the forearm was punctured and 500-1,000 ml of 0.9 g/dl NaCl, according to the estimated erythrocyte volume to be removed, were infused. Blood was collected (50-70 ml/min) in ACD- A solution (sodium citrate, 1.64 g; citric acid, 0.6 g; dextrose, 1.84 g, in 500 ml), at a ratio of 12:1 to 14.1.

EA consisted of three phases: (1) blood collection and blood product, separation; (2) isovolemic normal saline administration to replace the removed volume, and (3) autologous plasma and buffy-coat infusion. The adjusted erythrocyte volume withdrawn was defined as the total volume multiplied by its VPRC.

Results

Laboratory findings before and after EA are compared in table 1; reticulocyte and hemoglobin data for each patient over time are illustrated in figure 1.

Adjusted red blood cell volumes collected in each EA and after treatment has been completed are shown in table 2.

In each EA, a median reduction of 11% (range 6-17) for VPRC and of 3.3 g/dl (range 1.8-5.9) for hemoglobin was noted. The median interval between EA, necessary for VPRC and hemoglobin levels to reach 34% or 10 g/dl, respectively, was 9 days (range 3-14). A monthly median of 3.1 EA (range 2.4-4.4) was obtained, and 2,098 ml (range 1,648-3,677) of red blood cells could be collected.

Biochemistry parameters of body iron reached nonnal values within 3 months. The median duration of treatment needed for serum ferritin to return to normal levels was 3.25 months (range 1.0-7.5). When these levels exceeded 2,000 ug/l, more than 6 g of iron could be withdrawn. Serum ferritin changes are shown in figure 2. Serum CPT declined in parallel with ferritin and proved to be a valuable indirect index of tissue iron depletion. A 38-51% reduction of iron deposits was documented (table 3).

Clinically, skin pigmentation was considerably attenuated. Liver and spleen enlargement was also diminished; in 1 patient (case 3), splenomegaly persisted and was associated with thrombocytopenia. This insulin-dependent diabetic patient could be later controlled with diet only, and in case 2, insulin doses could be reduced in approximately 50%. Except in case 2, with impotence for 10 years, the sexual dysfunction disappeared. Arthralgia also receded, and the general status improvement allowed patients to resume their daily activities.

Hepatic biopsies obtained 30 days after EA therapy was completed showed a net reduction of iron contents (from grade 4 to grade l or 0) and no inflammation. EA was generally well tolerated and discontinuation was never required. Only orthostatic hypotension after the fIrst EA was noted, but no treatment was needed. All patients reported fatigue after the procedure.

Discussion

HH results from an excessive intestinal absorption of iron, which accumulates in tissues and produces systernic lesions that may be irreversible, particularly when diagnosis is delayed [28-32]. These lesions appear to be related to the duration and amount of iron stored [33, 34]. To reduce iron overload, several approaches may be used. In patients with iron overload caused by multiple transfusions, the chelating agent deferoxamine is frequency employed. In HH, deferoxamine administration is limited to patients with anemia, difficult venous access, unstable cardiovascular status, or endocrine diseases, in whom phlebotomy or EA cannot be done. In addition, deferoxamine therapy to deplete iron deposits is significantly protracted and expensive; it may require several years in HH and is usually taken for life by chronically transfused patients. The drug is injected subcutaneously and is associated with adverse effects that often lead to intolerance and poor compliance [35]. Oral iron chelators may become a valuable alternative as adjuvants designed to reduce the period of time necessary to deplete iron deposits and partially revert HH manifestations; however, these agents are not yet available for clinical use [36-40].

Classically, HH was treated with regular phlebotomy. During this procedure, 180-220 ml of red b1ood ce1ls may be collected; considering that 1 ml of red blood cells contains 1 mg of iron, approximately 200 mg of iron may be removed with each blood unit. In our experience, this is not the case. In fact, HH patients usually require weekly phlebotomies, and although sustained iron elimination is obtained, in 7 days VPRC or hemoglobin levels cannot return to normal. Consequently, withdrawn volumes are considerably less than 200 ml, efficacy is often overestimated, and the period of time needed to reduce the iron overload is protracted (from 1 to 3 or more years).

Regular phlebotomy is a useful, rather simple and affordable option for HH patients [21]; however, in symptomatic cases, 100 or more procedures may be required [41]. In addition, a good venous access must be available. In many instances, frequent venipunctures are not well tolerated and lead to discontinuation of treatment [10,21]. Moreover, phlebotomy also extracts plasma, platelets, and white blood cells, and therefore, the clinical status may worsen.

In recent years, EA has been successfully used to collect massive amounts of whole blood. When performed with strict adherence to procedural standards, it has demonstrated to be effective and safe for patients with polycythemia vera, porphyria cutanea, erythropoietic porphyria, and, central retinal vein thrombosis [42-44].

In 1989, Conte et al. [45] reported a study of patients treated with EA and confirmed its value to collect red blood cells only. However, volumes were similar to those obtained with phlebotomy (200-250 ml), reduction of iron deposits was not accelerated, and frequent venipunctures were not avoided.

In 1992, Kellner and Zoller [44] described a prospective study of 8 patients with HH, who underwent repeated isovolemic large-volume EA (1,000 ml) with 28-day intervals. Around 1 g of iron could be removed in each EA. Serum ferritin level decline started after 60 days and returned to normal within 12 months, thus showing a significant difference with classic phlebotomy and chelating agents. This finding may be explained by the bone-marrow-induced mobilization of iron deposits in response to lower circulating hemoglobin levels. Induced anemia normally augments endogenous erythropoietin synthesis; an up to sixfold increase in erythropoiesis may occur [45] and hemoglobin may return to normal in 30 days. rHuEPO has proved to be highly effective in elevating hemoglobin levels when endogenous production is low (chronic renal failure) or even when it is normal, to promote erythropoiesis for preoperative collection of autologous blood [25, 26].

In our series, serum GPT values decreased in parallel with ferritin, whereas transferrin saturation and serum iron remained increased almost up to the end of EA therapy. This observation suggests the following hypothesis: it is well known that iron metabolism is based on its mobilization in three compartments: tissues (deposit), serum (ferritin) and erythropoiesis (utilization). Large red blood cell volume removal and rHuEPO-induced erythropoietic estimulation promote iron deposit utilization.

During this stage, ferritin levels decline as a result of reduced iron deposits. This iron is released from tissues as free iron, which is rapidly used to maintain serum iron and transferrin saturation levels. This event may explain the co-existence of iron deposit depletion and high levels of serum iron. Consequently, drops in serum GPT and ferritin may result from tissue iron depletion and iron injury. Therefore, serum GPT may be used as a indirect marker of iron deposit reduction.

In our study, a median red blood cell volume of 475 ml (range 400-1,400 ml) was removed in each EA; a median of 3.1 (range 2.4-4.4) EAs/month were performed, and approximately 2,000 ml of red blood cells/month could be withdrawn. EA with rHuEPO removed a volume more than three times higher than phlebotomy; moreover, the period of time needed for serum ferritin to return to normal was considerably shorter than expected according to the litera- ture data (3.5 months versus more than 12 months) [21,46, 47]. EA with 28-day intervals, as described by Kellner and Zoller [24] could collected a mean of 1,010 ml in each procedure, during a mean of 8.5 months, whereas in our series, red blood cell volume removal could be doubled and the period of time required for iron deposits to return to normal was reduced by more than 50%. In both cases, side effects were similar and not significant.

After EA therapy is completed, serum ferritin and transferrin saturation are measured monthly during the first year and at 6 month intervals thereafter. If any of these parameters increases, EA is resumed to prevent tissue injury. In patients with HH and low serum albumin levels and/or thrombocytopenia, weekly phlebotomies may aggravate the problem. In patients with HH and water and electrolyte imbalance, EA with 5% dextrose may be used.

EA may seem expensive and demands adequate equipment and a trained staff, but in our series, the period of time needed to deplete iron deposits, HH risks and working day losses were sensibly reduced. Cost-effectiveness must be evaluated in the context of all factors involved in the treatment of HH and its complications.

Conclusion

In our experience, EA with rHuEPO and folic acid is a well-tolerated and useful procedure to reduce iron deposits and thus totally or partially correct associated dysfunctions. This method offers better results in less time than traditional phlebotomy and is an effective therapeutic alternative for patients with HH

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