Discontinuous Flow Centrifugation Research Articles

ABSTRACT 604

Background and aims: Plasmapheresis (PF) is an extracorporeal blood purification technique designed for the removal of large molecular weight substances. The basic premise of the treatment is that the removal of these substances will allow for the reversal of the pathologic processes related to their presence. PF has seen an increase in usage in critically ill patients with immune dysregulation. Aims: To review the indications, number of procedures, morbidity and clinical evolution of PF in critically ill children with autoimmune diseases. Methods: Prospective study in a 8 bed PICU. A discontinuous flow centrifugation system was used (Haemonetics). Diagnosis, demographic, ICU days, outcome and complications of the technique were determined. OFI score (max score 6) was measured daily. Values are expressed in median (range). Results: 32 PF procedures on 6 patients with age 92m (48 -168), weight 34 kg(16–70), OFI score of 5(3–6) and Ferritin levels of 16803 μg/dL (1700–39000) were reviewed. The diagnosis were macrophage activation syndrome 2, hemophagocytic lymphohistiocytosis 1, anti NMDAR encephalitis 1, TPO encephalitis 1 and acute disseminated encephalomyelitis 1. All received previously metilprednisolone and immunoglobulin, 2 cyclosporine and 1 rituximab. The procedure was performed 3 days (0–5) after PICU admission. The serum ferritin and OFI score decreased after 2 PF sessions.The most frequent complication, was hypotension requiring fluid bolus in 15 procedures .5 patients survived, after a mean of 17 days (1–52) in the PICU. Conclusions: PF should be considered as rescue treatment in pediatric patients with life threatening autoimmune diseases refractory to conventional therapies.

Trend of indications for therapeutic plasma exchange at an Iranian university hospital

Background: There is neither a registry system nor center-by-center reports on therapeutic plasma exchange in Iran. We evaluated the trend of indications as well as techniques and safety of plasma exchange in a single center in Iran. Methods: All registered cases in the plasma exchange unit of Al-Zahra hospital between 1996 and 2005 accounting for 560 patients (260 men, 300 women; median age 29, range 1–95 years) who underwent a total of 3985 procedures were included. Data collection was performed using the official questionnaire of the International Apheresis Registry System. Results: The total number of procedures per year has remained stable corresponding to a median of 9 treatments per 100,000 inhabitants. Of the 560 patients, the majority had neurologic (62.8%), followed by hematologic disorders (16.9%). The three most common indications were: Guillain–Barré syndrome (27.8%), myasthenia gravis (23.0%) and thrombotic thrombocytopenic purpura (11.4%). Three hundred and seventy-nine patients (67.7%) belonged to category I of the ASFA indication classification, 28 (5%) to category II, 104 (18.5%) to category III, and five (0.9%) to category IV. Forty-four cases (7.8%) were unclassified. All plasma exchanges were performed with a discontinuous flow centrifugation method. Normal saline + albumin (5%) was the main liquid of replacement (77.9%). Conclusion: The trend of indications in our center is comparable to international data. However, the average number of procedures in our population is lower than world statistics and the method of plasma exchange has not been modified.

Continuous-flow apheresis of microfilariae in Loa loa infestations

Loiasis is endemic in the tropical rain forest of Central and some parts of West Africa and is transmitted by the insect vector Chrysops silacea and Chrysops dimidiata. Loa loa infestation causes local inflammation due to migrating adult worms in the subcutis (Calabar swelling) and in the conjunctivae (eye worm). The diagnosis is made by the demonstration of circulating blood microfilariae, the larvae of the adult worms. Since the density of the microfilariae is very low, they can only be identified by concentration methods, such as hemofiltration. During treatment with the specific antiparasitic drug (diethylcarbamazine, DEC), side effects such as allergic reactions due to the rapid disruption of circulating microfilariae may occur. Those symptoms are aggravated in patients with a high number of larvae, leading to meningo-encephalomyelitis or even death. 1 Since microfilariae accumulate in the buffy coat during leukocytapheresis, 2 it was thought that this technique might be a suitable tool to reduce the parasite load and to bypass the side effects of specific drug treatment. Muylle et al. achieved apheresis of microfilariae in two patients with Loa loa infestation by using a Haemonetics, M-300 blood processor. 3 The authors studied two patients with an excessive microfilarial count in the peripheral blood of up to 9/μL and achieved a high enrichment by discontinuous-flow centrifugation. A similar observation was reported by Saeed et al. later on. 4 We have studied two patients with loiasis and report the results of microfilarial apheresis using a continuous-flow IBM/Cobe cell separator.

Efficacy of Discontinuous Flow Centrifugation Compared with Cascade Filtration in Waldenstrom's Macroglobulinemia: A Pilot Study

In the treatment of Waldenstrom's macroglobulinemia (WM) and lipoprotein or immune complex mediated diseases, centrifugal and membrane plasma separation systems have been successfully employed over the past years. More recently, semiselective double filtration systems have been used in isolated cases. While it is hoped that cascade filtration (CF) will soon become a routine technique, a note of caution came from some investigators because of the lack of any evidence that CF does really deplete patients' plasma of known pathogenic macromolecules. The aim of this study was to test the efficacy of CF in the removal of IgM paraproteins in comparison with discontinuous flow centrifugation (DFC). Eight patients were treated by DFC and seven by CF; there were no significant differences between the two groups in terms of sex, age, clinical severity and IgM plasma concentration. After DFC or CF sessions, IgM concentration diminished by 50% and plasma viscosity by 60%, producing a comparable mean reduction in patients' clinical severity. Our in vivo results thus preliminarily answer the question whether CF is capable of removing known pathogenic macromolecules from patients' plasma, and the technique appears to warrant further investigations.

Removal by centrifugation of leukocytes and red cells from apheresis platelets

Platelets prepared by discontinuous flow centrifugation contain significant numbers of leukocytes and red cells. Platelet products often are centrifuged further to remove this cellular "contamination." A method is described by which platelet products obtained by cytapheresis were prepared using an additional slow centrifugation step (121 X g for 7.5 minutes). In a production environment, we removed an average of 97 percent of leukocytes and most of the contaminating red cells with this method, while retaining 89 percent of the platelets in the final product. This method is an improvement over methods described previously and was consistently effective in routine use.

A new method to collect granulocytes using a low dose of hydroxyethyl starch.

A new granulocyte collection method employing discontinuous flow centrifugation and low donor dose of hydroxyethyl starch (HES) is presented. Following the procedure, the increase in serum polysaccharide concentration was 100 mg/dl, about 20% of that seen after standard discontinuous flow leukapheresis. The average efficiency of granulocyte collection by this method is about 64%. The average yield of WBC from 2,600 ml of donors' whole blood is 1.64 +/- 0.54 x 10(10) (mean +/- 1 SD) and the average granulocyte yield is 1.33 +/- 0.51 x 10(10) (mean +/- 1 SD). The granulocytes were shown to migrate to the sites of infection in vivo. This approach is an improvement over standard discontinuous flow leukapheresis because of the high efficiency of granulocyte collection and because multiple procedures are possible without exposing donors to more HES than is currently given during a single procedure.

Plasma-exchange in neurological diseases.

This paper summarizes experience with plasma-exchange therapy for neurological diseases at the Saronno hospital. Most treatments were performed by discontinuous flow centrifugation, but membrane plasma separation and cascade filtration were also employed. Eighty-five patients with demyelinating diseases of the peripheral nervous system (Guillain-Barré syndrome, immune complex polyneuropathies, paraneoplastic polyneuropathies), demyelinating diseases of the central nervous system (multiple sclerosis, subacute sclerosing panencephalitis), dermatopolymyositis and myasthenia gravis have been treated so far. Particular attention is paid to the combination of plasmapheresis with lymphocytapheresis and immunosuppressive drugs. This therapeutic approach appears to bring about dramatic and sustained improvement in most patients with neurological diseases, thus altering their natural course.

Comparative morphology of granulocytes collected by three methods of leukapheresis

The morphology of granulocytes collected by continuous-flow centrifugation (CFC), discontinuous-flow centrifugation (DFC), and continuous-flow filtration (CFF) was investigated in 18 healthy donors by means of light microscopy and transmission electron microscopy. Light microscopy study of semithin sections of granulocytes collected by CFC and DFC showed minimal morphologic abnormalities, compared to granulocytes procured by CFF. Ultrastructural study of granulocytes procured by CFF showed more conspicuous qualitative and quantitative abnormalities (the most prominent being "microvilli," degranulation, and bazarre chromatin) than in granulocytes obtained by the other two methods. Controls showed that the bulk of CFF-cell abnormalities was due to the "tapping" of the filters. Factors such as the mechanical compression (plasma extractor) used in DFC method, donor pretreatment with anticoagulants and steroids, hydroxyethyl starch, and duration of leukapheresis scarcely influenced granulocyte morphology.

Evaluation of granulocyte replacement therapy.

SummaryThe authors studied the clinical and biological effectiveness of 204 leucocyte concentrates prepared by discontinuous flow centrifugation to treat 47 febrile episodes in 32 patients.Even in 21 out of 37 treated patients with granulocyte counts lower than 0.1 X 10 9/1at the start of leucocyte transfusiona good clinical outcome was noted. A hemolytic transfusion reaction due to immune anti-A is reported.Some data concerning donor care and changes in blood parameters of the donor induced by preStreatment and leukapheresis procedure are discussed.

Cryopreservation of human platelets isolated by discontinuous-flow centrifugation using the Haemonetics Model 30 Blood Processor.

Platelets were isolated from normal volunteers by discontinuous-flow centrifugation using the Haemonetics Model 30 Blood Processor. The numerical equivalent of about five single units of platelets collected at each pheresis were frozen together in a -80 C mechanical freezer with a 6% final concentration of dimethylsulfoxide (DMSO) as the cryoprotectant. Platelet freeze-thaw-wash recovery in vitro was about 80 per cent and the platelet recovery value depended upon the method used to enumerate the platelets. The 51Cr survival values in vivo were about 50 per cent less than those in fresh platelets. These values were not significantly different from those seen when platelets were isolated from single units of blood by differential serial centrifugation. Transfusion of two and one-half units of freeze-preserved platelets provided an increase in the recipient's circulating platelet count comparable with that from one unit of fresh platelets. The hemostatic effectiveness of freeze-preserved platelets isolated by discontinuous-flow centrifugation has not yet been studied.

PH, PCO2 and PO2 in "high-volume" platelet concentrates prepared by discontinuous-flow centrifugation and stored in polyvinylchloride and plyethylene containers.

High-volume (200 ml) platelet concentrates prepared by discontinuous-flow centrifugation, with counts as high as ca. 2.0 x 10(12)/l, were stored in either 600 ml-capacity polyvinylchloride bags or 800-ml capacity polyethylene bags. Both polyvinylchloride and polyethylene bags appeared adequate for 24-hour storage of platelet concentrates with counts greater than 0.5 x 10(12)/l. However, at 24 hours the platelet concentrates in polyvinylchloride bags showed a reduction in pH and an increase in PCO2 that were proportional to the platelet count. Only with storage in polyethylene bags did the pH and PCO2 of the platelet concentrates show little change for periods of as long as 120 hours.

The sterility of platelet and granulocyte concentrates collected by discontinuous flow centrifugation.

Platelet concentrates prepared by a discontinuous flow centrifugal technique (Haemonetics) were examined for evidence of bacterial contamination and subsequent growth from 3 to 11 days after collection. Of the 126 platelet concentrates examined, 4 revealed bacterial growth. However, the growth patterns indicated contamination during the microbiologic manipulations rather than contamination of the units during preparation. These studies indicate that platelet concentrates prepared using the Haemonetics Blood Processor can be safely transfused for up to three days after collection if stored at 4 to 6 C.

Local versus Regional Procurement and Distribution of Granulocytes

Granulocyte concentrates obtained by discontinuous flow centrifugation (DFC) and continuous flow centrifugation (CFC) were studied. The DFC granulocytes were obtained from a regional center and stored for 24 hours prior to transfusion. The CFC granulocytes were obtained locally and transfused within a few hours. Even at 24 hours, DFC granulocytes had significantly reduced bactericidal capacity, chemiluminescence, nitroblue tetrazolium reduction, chemotaxis and random mobility. Granulocyte kinetics utilizing DF32P and skin windows demonstrated the ability of DFC granulocytes to circulate and migrate into the tissues despite the in vitro abnormalities. Until effective storage techniques for granulocyte preservation became available, rapid transportation and processing from regional centers or local procurement of granulocytes is necessary to transfuse functional granulocytes.

Properties of neutrophils collected by discontinuous-flow centrifugation leukapheresis employing hydroxyethyl starch.

The properties of neutrophils, collected by discontinuous-flow centrifugation leukapheresis in the presence of hydroxyethyl starch and citrate were studied in an attempt to insure that cells harvested in this fashion are suitable for transfusion. Neutrophils obtained from leukocyte units prepared for transfusion by leukapheresis were found to perform similarly to those isolated from the venous blood of corresponding donors prior to the pheresis procedures when assessed by morphology, viability, hexose monophosphate shunt activity, superoxide anion generation, nitroblue tetrazolium dye reduction, chemiluminescence, adherence to nylon fibers, random mobility, chemotaxis and staphylococcal killing. The results compared favorably with values previously established for healthy control subjects. Neutrophils prepared in this manner should serve as a satisfactory blood component.

Redistribution of platelets during discontinous flow platelet pheresis.

Greater numbers of platelets are recovered during discontinuous flow centrifugation than can be simply accounted for by the decrease in total circulating platelets in the donor. There is a linear relationship between the logarithm of the circulating platelet count and the number of plateletpheresis bowls filled. The disappearance of platelets from the peripheral circulation occurs at a greater rate in splenectomized donors than in normal donors, and the rate of platelet disappearance in normal donors is less than what would be expected if there were no in vivo platelet storage pools. The data suggest the redistribution of platelets from the spleen in normal donors during the time course of the procedure.

Collection of granulocytes for transfusion. The effect of collection methods on cell enzyme release.

When granulocytes are collected by either discontinuous-flow centrifugation or filtration leukapheresis, lysozyme is released. More lysozyme is released during the filtration procedure than during the centrifugation procedure. A small amount of red cell lysis occurs during the centrifugation but not the filtration procedure. A rise in lactate dehydrogenase levels consonant with the amount of hemolysis is observed. These findings suggest that granulocytes collected by either filtration or discontinuous-flow centrifugation undergo degranulation but not lysis sufficient to cause cytoplasmic enzyme release during the donation.

Storage of platelets prepared by discontinuous flow centrifugation.

Platelet concentrates were prepared by plateletpheresis using discontinuous flow centrifugation. Platelet units were stored in PL-146 bags of 300 ml and 2,000 ml capacity, and in vitro measures helpful in predicting platelet viability were compared. Storage in bags of 300 ml capacity led to significant fall in pH, decreased recovery from osmotic stress, and deterioration of morphology in 24 hours. Storage lesions were significantly decreased by use of 2,000 ml capacity bags.

Redistribution of Platelets During Discontinous Flow Platelet Pheresis

Greater numbers of platelets are recovered during discontinuous flow centrifugation than can be simply accounted for by the decrease in total circulating platelets in the donor. There is a linear relationship between the logarithm of the circulating platelet count and the number of plateletpheresis bowls filled. The disappearance of platelets from the peripheral circulation occurs at a greater rate in splenectomized donors than in normal donors, and the rate of platelet disappearance in normal donors is less than what would be expected if there were no in vivo platelet storage pools. The data suggest the redistribution of platelets from the spleen in normal donors during the time course of the procedure.

Effect of Storage on Normal Neutrophils Collected by Discontinuous-Flow Centrifugation Leukapheresis

Storage limits for granulocytes have not been defined. The purpose of this study was to determine these limits for neutrophils collected by discontinuous-flow centrifugation and stored at 4 degrees-6 degrees C. The parameters studied were total leukocyte and absolute cell counts, viability measured by dye exclusion, morphology, percentage phagocytic neutrophils, number of Candida organisms ingested per phagocytic neutrophil, candidacidal activity by differential staining, and chemotaxis under agarose. There was a progressive loss of neutrophils on storage that was statistically significant by 48 hr. Phagocytosis was the best preserved function. Microbial killing measured by candidacidal activity was less well preserved. Chemotaxis was the most poorly maintained parameter. There was mild impairment at 24 hr and a severe functional loss at 48 hr. The data suggest the following: (1) the first functions lost on storage are the most highly integrated, i.e., chemotaxis, followed by microbial killing and then phagocytosis; and (2) assuming that these functional losses are irreversible, storage of normal neutrophils used for transfusion should be limited to approximately 24 hr because a severe defect in migration occurs between the first and second days.

Effect of storage on normal neutrophils collected by discontinuous-flow centrifugation leukapheresis

Storage limits for granulocytes have not been defined. The purpose of this study was to determine these limits for neutrophils collected by discontinuous-flow centrifugation and stored at 4 degrees-6 degrees C. The parameters studied were total leukocyte and absolute cell counts, viability measured by dye exclusion, morphology, percentage phagocytic neutrophils, number of Candida organisms ingested per phagocytic neutrophil, candidacidal activity by differential staining, and chemotaxis under agarose. There was a progressive loss of neutrophils on storage that was statistically significant by 48 hr. Phagocytosis was the best preserved function. Microbial killing measured by candidacidal activity was less well preserved. Chemotaxis was the most poorly maintained parameter. There was mild impairment at 24 hr and a severe functional loss at 48 hr. The data suggest the following: (1) the first functions lost on storage are the most highly integrated, i.e., chemotaxis, followed by microbial killing and then phagocytosis; and (2) assuming that these functional losses are irreversible, storage of normal neutrophils used for transfusion should be limited to approximately 24 hr because a severe defect in migration occurs between the first and second days.