Regional Citrate Anticoagulation Protocol For Patients With Presumed Absent Citrate Metabolism

Background: Regional citrate anticoagulation (RCA) is not recommended in patients with shock or severe liver failure. We designed a protocol with personalized pre-calculated flow settings for patients with absent citrate metabolism that abrogates risk of citrate toxicity, maintains neutral CKRT circuit calcium mass balance and normal systemic ionized calcium levels. Methods: Single center prospective cohort study of patients in five Adult Intensive Care Units triaged to the CVVHDF-RCA "Shock" protocol. Results: Of 31 patients included in the study, 30 (97%) had acute kidney injury, 16 (52%) had acute liver failure, and 5 (16%) had cirrhosis at the start of CKRT. The median (interquartile range (IQR)) lactate was 5 (3.2 to 10.7), AST 822 (122 to 2950), ALT 352 (41 to 2238), total bilirubin 2.7 (1.0 to 5.1), INR 2 (1.5 to 2.6). The median first hemofilter life censored for causes other than clotting exceeded 70 hours. The cumulative incidence of hypernatremia (Na >148 mM), metabolic alkalosis (HCO3- >30 mM) and hypophosphatemia (P< 2 mg/dL) were 1/26 (4%), 0/30 (0%), 1/30 (3%) respectively and were not clinically significant. Mild hypocalcemia occurred in the first 4 hours in 2/31 patients and corrected by hour 6 with no additional Ca-supplementation beyond the per-protocol administered Ca-infusion. The maximum systemic total Ca (tCa; mM)/ionized Ca (iCa; mM) ratio never exceeded 2.5. Conclusions: The "Shock" protocol can be used without contra-indications and is effective in maintaining circuit patency with a high, fixed ACDA infusion rate to blood flow ratio. Keeping single-pass citrate extraction on the dialyzer >0.75 minimizes the risk of citrate toxicity even in patients with absent citrate metabolism. Pre-calculated, personalized dosing of the initial Ca-infusion rate from a table based on the patient's albumin level and the filter effluent flow rate maintains neutral CKRT circuit calcium mass balance and a normal systemic iCa level.

Apheresis-Adverse Events in Man and Machine Individualities

Adverse events due to platelet pheresis are not unheard of citrate related reactions being the most common. Most of these events are mild and self limiting. The current study describes adverse events in platelet pheresis using modern apheresis systems. This prospective study included 1455 platelet pheresis procedures done from July 2016 to December 2017. Procedures were performed on Hemonetics MCS+, Trima Accel and Cobe spectra cell separators. The endpoint of each procedure was a yield of 3 × 1011 platelets (PLTs) per unit. Donor adverse reaction if any was managed, reported, and documented. The median age of donors was 31 years with male to female ratio of 13:1. The median body surface area and body mass index were 1.64 m2 and 22.4 kg/m2, respectively. The mean PLT count of donors was 199.8 × 103/uL with a mean hemoglobin value of 13.6 g/dl. ACD infusion was significantly more in the Hemonetics MCS+, (P< 0.01). Donation time was least with the Trima compared to Hemonetics MCS+ (P< 0.01) and Cobe (P< 0.001). Total whole blood volume processed was higher in Hemonetics MCS+, (P< 0.01). Paresthesia due to citrate toxicity was the most common adverse reaction (65.3%), and vascular injury was observed in only five donors. The overall incidence of adverse reaction was 3.4%. Serious adverse events were not observed. The modern generation apheresis machines are more donors friendly and cause less adverse reactions compared to the older versions. Good donor screening, optimized donor physiognomic and hematological values and skilled operators are the key factors in reaction reduction by apheresis. Faridpur Med. Coll. J. Jan 2019;14(1): 27-30

Optimization of the Spectra Optia for Leukapheresis of Very Small Subjects

Leukapheresis procedures in very small volume subjects require procedure modifications related to anticoagulant, venous access, and volume. Historically, we accomplished this procedure in 4-5 kg rhesus macaques using a modified CS3000Plus cell separator, but this instrument is no longer available. The Spectra Optia apheresis system and its mononuclear cell (MNC) collection set were evaluated to develop leukapheresis procedures for such subjects. Five rhesus macaques, weight 7-9 kg, blood volume 60 mL/kg, were studied. They pre-donated 100-120 mL of autologous blood (25-30 mL/wk x 4 wks). Aspirin 81 mg was given daily for 3 days prior to 5 of 8 procedures. AMD3100 1 mg/kg SQ 2-3 hours prior to leukapheresis was given as a mobilizing agent. Autologous blood was pooled, diluted 1:1 with saline, and washed to remove citrate and plasma. The Optia was primed first with saline, and then with a customized albumin option in which 30-35 mL of the 40-50 mL of packed RBCs were pumped into the disposable apheresis kit at 10 mL/min. The albumin option shortens procedure ramp-up time. A closed circuit was created between the return line and the packed RBC bag and the flow rate increased to 40 mL/min until 400 mL was processed. To minimize citrate toxicity and avoid volume overload, animals received heparin 50 U/kg bolus IV followed by an ACDA solution containing heparin 5U/mL at an inlet:AC ratio of 25:1. A Collection Preference (CP) setting of 20 was selected using the automatic interface management mode. In the last procedure a semi-automatic mode was utilized so that collection deeper than CP of 20 was possible. Procedures were terminated at 120 min. Animals were anesthetized and catheters placed in the saphenous and cephalic as draw and return lines. Blood counts, chemistries, and ionized calcium (iCa) levels were monitored every 30 min. The elutriation chamber, which concentrates MNCs and allows platelets to return to the donor, was filled 1-2 times per procedure resulting in a product volume of 17-34 mL. A total of eight leukapheresis procedures were performed. Inlet flow rates were typically 10.5 mL/min. In one animal, an inlet rate of 12.0 mL/min was achieved; however, at 120 minutes severe citrate toxicity developed with bradycardia and an iCa level of 0.62 mmol/L, requiring IV calcium gluconate. Excluding this case, a mean(SD) baseline iCa value of 1.17(0.2) dropped to 0.84(0.03) mmol/L at120 min. Mean hematocrit (HCT) decreased from 40.4(1.7)% to a nadir of 36.8(2.6)% at 90 min. Mean platelets declined to a greater degree, from 342(42) x103/mcL to 109(33) x103/mcL at 60 min and 121(43) x103/mcL at 120 min (p<0.001). This drop was reflected in the large number of platelets in the product, 2.49(1.09)x1010 total. Occasional platelet aggregate formation was observed in animals both receiving and not receiving aspirin. Product WBC differential was 65.5(11.1)% MNC and 34.5(10)% granulocytes. Total product MNC content was 1.95(0.54)x109 . MNC collection efficiency (CE) was 26.2(8.5)% using CP=20 and improved to 38.1% using the semi-automatic mode. Product MNC composition consisted of 5.1(2.0)x108 CD4+ , 5.6(2.1)x108 CD8+, and 3.0(2.0)x108 CD20+ lymphocytes, and 9.8(7.0)x108 CD14+ monocytes. As a measure of product contamination, the CE for granulocytes was 5.8(3.0)% and for platelets 11.2(4.4)%. In 3 procedures, two-chamber collections were obtained, the remaining 5 procedures had one-chamber collections (all within 120 min). The HCT of the product was low, 3.7(1.2)%. The product also was enriched for reticulocytes (10.57(2.44)%) and CD4+CD8+ cells(3.4(2.2)x107). Relatively low numbers of CD34+ cells were collected, 5.2(1.9)x106; however, the mobilization protocol was not optimized for CD34+ collection in that AMD3100 alone and older monkeys were used. We are currently optimizing the procedure for even smaller rhesus and combination cytokine mobilization. MNC collections through the process of leukapheresis have proven to be effective in isolating cells for transplantation and cellular therapeutics. The use of leukapheresis in a pediatric setting is difficult due to small blood volumes and a high risk of adverse events. Here we have identified a novel means to perform leukapheresis procedures safely and effectively in small volume subjects. This methodology will have numerous therapeutic applications in both human and veterinary medicine. Disclosures Parker: Terumo BCT, Inc.: Employment. Lienesch:Terumo BCT, Inc.: Employment.

A Novel Tool Using Marrow Natural Killer (NK) Cell Percentages to Predict Collection Requirements of Peripheral Blood Mononuclear Cell (MNC), Apheresis Products Used in NK Cell Adoptive Immunotherapy

Our institution is involved in two clinical trials that require a single infusion of non-expanded Natural Killer (NK) cells in recipients of haploidentical marrow transplants. NK cells are selected from non-mobilized Mononuclear cells (MNC), apheresis products collected 6 days after HPC, Marrow harvest then processed and infused the following day. The two-step processing procedure requires initial depletion of CD3+ T cells followed by enrichment of CD56+ NK cells. Results from 27 patients treated thus far show an average CD3-CD56+ NK cell recovery of 54.5 ± 8.3% with 5.46 ± 0.74 log depletion of CD3+CD56- T cells. The NK cell dose level target is now 5.0 x 106 NK cells/kg and is limited to ≤1.0 x 104 T cells/kg in these mixed pediatric and adult patients. For 21 of 26 patients (80.7%) whose adult donors underwent a standard 2-blood volume (BV) apheresis procedure, a dose level >5.0 x 106 NK cells/kg was achievable. Processing a larger BV would likely improve the percentage achieving the desired target dose but would also increase the likelihood of adverse reactions, particularly citrate toxicity. It is believed that mature lymphocytes and monocytes in marrow harvested for clinical transplant come primarily from contaminating peripheral blood. Given this, we hypothesized that NK cell content in marrow MNCs would reflect NK cell content within the apheresis product and allow us to predict when a larger BV collection is needed. Immunophenotype data from 18 donors of both marrow and MNC, Apheresis products was evaluated to determine the correlation of the percentage of NK cells within the Marrow MNC population with that within the apheresis products. The % of NK cells within the MNC population was quite variable for marrow (6.1 ±3.7%, range 2.4 to 17.7%) and apheresis products (7.3± 4.6%, range 2.4 to 26.1%) but were highly correlated for individual donors (R2=0.85, p <0.0001). Based on this data, we developed a prediction workform tool requiring 3 data points from the marrow immunophenotype: 1) % of marrow lymphocytes (defined by flow cytometry as CD45bright, CD14-), 2) the % of marrow monocytes (defined as CD45bright, CD14+), and 3) the % of CD3-CD56+ NK cells within the marrow lymphocyte gate. These data were used to calculate NK cell content in the marrow MNC fraction. That data together with the desired NK cell dose/kg, patient weight, and average post-processing NK recovery (54.5%) was used to calculate the number of MNC cells required in the apheresis product to meet target NK cell doses. The BV to be processed was then estimated using the % MNC in the peripheral blood at marrow harvest and the known MNC collection efficiency of the device used (a conservative estimate of ~35% MNC collection efficiency was used). The prediction workform indicated that additional BV needed to be processed for one of the 3 donors for whom it was used indicated. This resulted in sufficient NK cells to meet the target infusion dose. Calcium infusion was used prophylactically to prevent citrate toxicity. Similar information could be obtained using a peripheral blood sample to determine NK cell content the day of collection. However, having information in advance from the marrow harvest allows for planning on the part of the donor and the collection team when additional collection time is needed. In conclusion, when available data from a marrow harvest performed prior to MNC, Apheresis is a useful tool to estimate the BVs needed to be processed meet infusion cell target dose. Disclosures No relevant conflicts of interest to declare.

Adverse events among 2408 unrelated donors of peripheral blood stem cells: results of a prospective trial from the National Marrow Donor Program

Limited data are available describing donor adverse events (AEs) associated with filgrastim mobilized peripheral blood stem cell (PBSC) collections in unrelated volunteers. We report results in 2408 unrelated PBSC donors prospectively evaluated by the National Marrow Donor Program (NMDP) between 1999 and 2004. Female donors had higher rates of AEs, requiring central line placement more often (17% vs 4%, P < .001), experiencing more apheresis-related AEs (20% vs 7%, P < .001), more bone pain (odds ratio [OR] = 1.49), and higher rates of grades II-IV and III-IV CALGB AEs (OR = 2.22 and 2.32). Obese donors experienced more bone pain (obese vs normal, OR = 1.73) and heavy donors had higher rates of CALGB toxicities (> 95 kg vs < 70 kg, OR = 1.49). Six percent of donors experienced grade III-IV CALGB toxicities and 0.6% experienced toxicities that were considered serious and unexpected. Complete recovery is universal, however, and no late AEs attributable to donation have been identified. In conclusion, PBSC collection in unrelated donors is generally safe, but nearly all donors will experience bone pain, 1 in 4 will have significant headache, nausea, or citrate toxicity, and a small percentage will experience serious short-term adverse events. In addition, women and larger donors are at higher risk for donation-related AEs.

Complications of Pediatric Apheresis.

Objectives Even if the practice and applications of apheresis have grown over the last decade, the data in pediatrics are limited. The frequency of the complications is low in adults but there is a lack of clinical trials assessing the nature and the frequency of the complications in children. Methods We have revised the medical files of all patients (0–18 years old) who have undergone an apheresis at Ste-Justine Hospital (pediatric hospital in Montreal, Canada) between November 1994 and July 2002. The reported complications were: symptoms of hypocalcemia, hypotensions (drop of systolic BP ≥10 mmg Hg), complications of the vascular access (thrombosis and infection), reactions to blood products and severe anemia. Results A total of 1632 aphereis were performed on 186 different patients (32 distinct diagnosis). The procedures were plasma exchange (67%), peripheral blood stem cells collect (18%), red cell exchange (6,9%), cytoreduction leucapheresis (0,7%) and immunoadsorption (6,7%). Adverse reactions, although mostly minor, were reported in most of the patients. The more frequent complications were hypotensions (14% of apheresis and 48,4% of patients), hypotensions which needed bolus (4,8% of apheresis and 26,9% of patients) , symptoms of hypocalcemia (9,7% and 28,5%), allergic reactions (4,4% and 5,9%) and troubles related to the vascular access: infection (2,1% and 16,1%) and thrombosis (1,7% and 12,4%). A severe anemia (Hb < 70g/L) complicated 2,5% of apheresis (17,2% of patients). There were two deaths related to procedures. With a multivariate analysis, we have identified independent risk factors for occurence of complications: lower weight, lower hemoglobin pre-pheresis, pheresis in ICU and number of pheresis. Conclusion The incidence of complications of apheresis in our pediatric cohort is much higher than what is reported in adults. The adverse reactions can be explained by the citrate toxicity, the extravascular volume shift and the vascular access installed. We consider that apheresis in pediatrics present unique features and should be done in specialized centers.