Evaluation of the Reticulocyte Production Index in the Pediatric Population

Objectives Since hematologic values vary with age in children, we evaluated the agreement between the “traditional” reticulocyte production index (RPI) and an RPI by age (RPI/A)–adjusted normal values. Methods A retrospective, observational, and analytical study was performed on CBCs of children with anemia younger than 18 years. The agreement and clinical repercussions of the RPI values were analyzed with an RPI/A developed with theoretical values for different ages. Results A total of 5,503 tests were analyzed and no systematic error between the two indices was found; however, there were significant proportional differences at higher values that resulted in lower RPI/A in children younger than 15 days and higher RPI/A in children aged 15 days and older. No agreement was observed at any age. The proportion of arregenerative anemia diagnosed using RPI/A was higher in children younger than 15 days and lower in those 15 days and older. Conclusions RPI is not an adequate tool for evaluating the erythropoietic capacity of bone marrow in the pediatric population. The disagreement between the results can be explained by the difference in normal hematologic values between children and adults.

Reticulocytic indices in assessing erythropoiesis effectiveness in multiple myeloma.

e19510 Background: The purpose of the study was to analyze changes in reticulocytic indices and their role in assessing the effectiveness of erythropoiesis in patients with multiple myeloma. Methods: Thestudy included 10 patients with primary multiple myeloma (MM) and 16 patients with recurrent MM, stage II-III. Parameters of the peripheral blood were studied: Hb, RBC, MCV, MCH, RBC-He (red cell hemoglobin content), RET (number and percentage), IRF (immature reticulocyte fraction), high (HFR), median (MFR) and low (LFR) fluorescence reticulocytes, RET-He (reticulocyte hemoglobin content), RPI (reticulocyte production index). Results: Anemia was found in21 (80.8%) patients before treatment: grade I in 30.8%, II in 46.1%, III in 3.8%; normocytic hypochromic anemia – 6, normocytic normochromic – 15. Anemia detection rate in primary MM was 60.0%, recurrent – 93.7%. RET levels in grade I-II anemia were close to the norm, but IRF was increased due to MFR increase by 3.7 times and HFR – by 4.7 times. Grade III anemia: RET number decreased by 1.6 times due to IRF reduction by 2 times, in particular MFR and HFR fractions. As a result, RET-He was decreased to varying degrees in all cases. Patients with complete or partial remission showed no significant changes in erythrocyte parameters, but had RET level increased by 2.5 times, IRF – by 9 times due to MFR increase by 6.5 times and HFR by 30.8 times, RPI - by 2.0 times. The data showed the effectiveness of erythropoiesis during anticancer therapy. We did not observe signs of increasing anemia syndrome in patients with stabilization or patients resistant to treatment, as well as in patients with initial grade III anemia. Hb, RBC, RET and RPI were unchanged, but the ratio of fractions changed: the number of mature ones - LFR increased by 1.4 times and young ones - HFR decreased by 2.5 times, RBC-He were increased and RET-He doubled compared to the levels before treatment, which indicated the activation of processes of erythrokaryocyte hemoglobinization. Conclusions: RET, IRF, RET-He and RPI indices adequately reflect the process of recovery of erythropoiesis and its effectiveness in real time which is extremely important in tumor therapy monitoring.

Diagnostic performances of manual and automated reticulocyte parameters in anaemic cats

Objectives The objective of this study was to evaluate the diagnostic performances of manual and instrumental measurement of reticulocyte percentage (Ret%), reticulocyte number (Ret#) and reticulocyte production index (RPI) to differentiate regenerative anaemia (RA) from non-regenerative anaemia (NRA) in cats. Methods Data from 106 blood samples from anaemic cats with manual counts (n = 74; 68 NRA, six RA) or instrumental counts of reticulocytes (n = 32; 25 NRA, seven RA) collected between 1995 and 2013 were retrospectively analysed. Sensitivity, specificity and positive likelihood ratio (LR+) were calculated using either cut-offs reported in the literature or cut-offs determined from receiver operating characteristic (ROC) curves. Results All the reticulocyte parameters were significantly higher in cats with RA than in cats with NRA. All the ROC curves were significantly different ( P <0.001) from the line of no discrimination, without significant differences between the three parameters. Using the cut-offs published in literature, the Ret% (cut-off: 0.5%) was sensitive (100%) but not specific (<75%), the RPI (cut-off: 1.0) was specific (>92%) but not sensitive (<15%), and the Ret# (cut-off: 50 × 10³/µl) had a sensitivity and specificity >80% and the highest LR+ (manual count: 14; instrumental count: 6). For all the parameters, sensitivity and specificity approached 100% using the cut-offs determined by the ROC curves. These cut-offs were higher than those reported in the literature for Ret% (manual: 1.70%; instrumental: 3.06%), lower for RPI (manual: 0.39; instrumental: 0.59) and variably different, depending on the method (manual: 41 × 10³/µl; instrumental: 57 × 10³/µl), for Ret#. Using these cut-offs, the RPI had the highest LR+ (manual: 22.7; instrumental: 12.5). Conclusions and relevance This study indicated that all the reticulocyte parameters may confirm regeneration when the pretest probability is high, while when this probability is moderate, RA should be identified using the RPI providing that cut-offs <1.0 are used.

Rituximab Subcutaneous Formulation (SC-R) Is Safe, Effective and Cost-Saving in Patients Suffering from Autoimmune Hemolytic Anemia (AIHA)

Rituximab, a monoclonal antibody directed against the CD20 antigen expressed on B cells, has been shown to be effective in AIHA, both in idiopathic and secondary including those associated with autoimmune and lymphoproliferative disorders as well as in Evans syndrome. At standard dose of 375 mg/mq weekly for a median of 4 weeks , overall survival (OS), complete response (CR), desease free survival (DFS) were respectively OR 83-87%,CR 54-60% DFS 72% at one and 56% at two years. Moreover rituximab re-treatment is effective and some patients responded to re-treatment more than once. Because is available today a Rituximab solution for subcutaneous injection, we have used this formulation for the treatment of 6 patients (pts) suffering from AIHA. Methods 6 pts (M 2, F 4 ) were enrolled in this study. Median age was 58.3 yr (range, 52-82) 2 out of 6 pts were idiopathic and the remaining 4 were associated with chronic lymphoproliferative syndromes. 2 out of 6 pts had relapsed after a first-line treatment with intravenous rituximab and steroids.For 2 pts mean Hb value and Ht at presentation were 6.2 g/dL ± 1.2, and 26 mL/dL, Median reticulocyte percentage was 10%, and median reticulocyte production index was 2.9 times basal. 24 % of cases had an initial reticulocyte count less than 4%, and 40% had an initial reticulocyte production index less than 2.0 times basal. These reticulocytopenic patients were prevalent in secondary cases. Pts had altered hemolysis markers and direct antiglobulin test (DAT) was positive for both complement and IgG ( IgA 1 pts). All cases had a bone marrow examination during hospitalization Erythroid hypoplasia was seen only in CLL pts.6/6 pts had serial reticulocyte measurements, Rituximab subcutaneous formulation was administered at a fixed dose of 1400 mg weekly for 4 weeks; Before starting MabThera subcutaneous injections, all patients received beforehand, a full dose of Rituximab by intravenous infusion. Premedication consisting of an anti-pyretic and an antihistaminic was given orally in the evening before and the morning of the subcutaneous administration in order to reduce the time to stay in day hospital . All pts received prednisone 1 mg/Kg/day /for 30 days ; for these reason premedication with glucocorticoids was avoided. Results All pts completed treatment. No major infusion related side effects to subcutaneous Rituximab SC-R) were observed. Response criteria were defined as follows: Complete Response (CR): Hb >10 g/dl or Hb increase >1.5 g/dl, resolution of symptoms of anemia, transfusion independent; Partial Response (PR): Hb > 9 g/dl or Hb increase of 1-1.5 g/dl. improvement in symptoms of anemia, transfusion independent; NR (failure to meet CR/PR). 100% were eligible for response.Complete Responses were seen in 6/6 pts .At the end of treatment DAT became negative in 4/6 pts , concentration of lactic dehydrogenase , total bilirubin and indirect bilirubin began to decrease at 12 days after the first dose of rituximab, and decreased to normal range after 22 days. 3 patients required packed red cell transfusions before starting SC-R and all became transfusion-free. A moderate hemolysis still persisted only in one patient. rh-Epo has been administered in three pts initially reticulocytopenic. The reticulocyte production index rapidly increased, indicating a marrow erythropoietic response to rh-Epo Conclusion Our experience demonstrates that SC- Rituximab is an effective and safe alternative to IV formulation both in the first line and in the relapsed pts. SC-Rituximab shortens the treatment time significantly, enabling administration over approximately 5 minutes compared with 3 hours during IV infusion. The ready-to-use SC formulation significantly reduce pharmacy time and the impact on hospital resources as medicine preparation time and hospital staff time per administration are significantly reduced also in our hands. Disclosures No relevant conflicts of interest to declare.

Assessment of Bone Marrow Function in Sickle Cell Anaemia Patients Using Corrected Reticulocyte Counts

Introduction: Chronic hemolysis occurs in sickle cell anemia as a result of recurrent sickling and other abnormalities of the red blood cells including eryptosis. Exuberant reticulocytosis is anticipated to partially compensate for the resultant anemia. Sickle cell anemia patients may also have aplastic crisis, bone marrow (BM) infarction and erythropoietin deficiency which could lead to reticulocytopenia despite the anemia. High degree of reticulocytosis among asymptomatic infants with sickle cell anemia has been associated with an increased risk of death or stroke during childhood. Assessment of BM function in sickle cell anemia is important due to potential complications associated with both under-activity and hyperactivity. This study aimed at evaluating the erythropoietic function of the BM in steady state sickle cell anemia using corrected reticulocyte counts. Methods: This study was carried out at the hematology clinic in the University College Hospital, Ibadan. HbSS patients in steady state were recruited from the hematology clinic. Local ethical committee approval was obtained and all participants gave written informed consent. Patients with M. tuberculosis, Hepatitis B, HIV and P. falciparum infection were excluded. Peripheral blood samples were analyzed using Sysmex Ki-X21 for complete blood count (CBC) and standard point of care for serum electrolytes and liver function tests. The glomerular filtration rates were calculated using the Cockcroft-Gault formula. Reticulocyte counts were determined manually using fresh samples from K2 EDTA bottles and methylene blue stain. Two drops of stain were mixed with two to four volumes of anticoagulated blood and incubated at 37ºC for 15 minutes. Afterwards, the cells were re-suspended and blood films were made. Corrected reticulocyte count and reticulocyte production index were calculated. Participants were categorized according to corrected reticulocyte counts of greater than or less than 2.5%. Univariate and multivariate analyses were performed to determine variables associated with corrected reticulocyte count <2.5%. Results: 92 HbSS patients were recruited with a mean (SD) age of 19.6 (5.8) years. There was no correlation between age and eGFR (p-value: 0.227). Median (range) reticulocyte count, corrected reticulocyte count and reticulocyte production index were 5.5 (0.5 - 29.9), 3.3 (0.1 - 17.1) and 1.7 (0.2 - 8.6) respectively. 40 (43.5%) patients had corrected reticulocyte count <2.5% and 52 (56.5%) had a corrected count >2.5%. Those corrected reticulocyte count <2.5% were older (p: 0.013), taller (p: 0.041) and had higher aspartate transaminase (AST) levels (p: 0.006) than those with corrected counts >2.5% (Table 1). CBC parameters were not different when compared between both groups. Results of multivariate logistic regression analysis carried out showed that only AST was independently linked with corrected reticulocyte count <2.5% (R2: 0.172, p-value: 0.001) (Table 2). Table 1. Factors Associated with Low Reticulocyte Count Corrected count<2.5% Corrected count>2.5% p-Value Age (Mean, SD) 21.4 (6.3) 18.4 (5.0) 0.013 Gender (N, %) Male 22 (55.0) 28 (53.8) 0.912 Female 18 (45.0) 24 (46.2) Height (Mean, SD) 1.6 (0.1) 1.5 (0.1) 0.041 BMI (Mean, SD) 18.7 (3.1) 18.7 (3.0) 0.753 GFR (Mean, SD) 64.3 (37.7) 66.4 (29.3) 0.453 Bilirubin (Mean, SD) 1.7 (1.1) 1.9 (2.6) 0.674 AST (Mean, SD) 22.5 (13.5) 14.5 (6.6) 0.006 ALT (Mean, SD) 13.4 (7.7) 14.4 (11.1) 0.876 Table 2. Independent Predictors of Corrected Reticulocyte Count <2.5% or 95% CI p-Value Age 1.08 0.97 - 1.21 0.169 Height 19.8 0.11 - 366.10 0.259 AST 1.10 1.04 - 1.17 0.002 Hemoglobin 1.00 0.97 - 1.02 0.872 R2: 0.172, p: 0.001 Conclusion: Despite corrected reticulocyte count <2.5% in about half of the patients, there were similar hematological parameters and eGFR in both groups of patients. AST is a marker of hemolysis and low ALT rules out hepatic involvement. Since only 17.2% of the variability in BM response as assessed by corrected reticulocyte count could be accounted for by variables included in this study, there is a need to further evaluate the BM function of sickle cell patients to establish the causes of corrected reticulocyte count <2.5% in the setting of anemia, having ruled out erythropoietin as well as iron, folate or cobalamin deficiencies. This will aid the development of a functional algorithm for the individualized management of sickle cell disease patients with anemia. Disclosures No relevant conflicts of interest to declare.

Targeted Metabolomic Profiles Are Strongly Correlated With Metabolic Alterations In Patients With Sickle Cell/Beta Thalassemia Disease

Background The complex pathophysiology of Sickle Cell Disease (SCD) makes unlikely that a single therapeutic agent will prevent or reverse all SCD complications. Metabolomic analysis might help in the characterization of the endogenous and exogenous effects of potential new treatments. Metabolites are small molecules that are chemically transformed during metabolism and provide a functional readout of cellular state. Metabolites serve as direct signatures of biochemical activity and are therefore easier to correlate with phenotype. The metabolome is typically defined as the collection of small molecules produced by cells and offers a window for interrogating how mechanistic biochemistry relates to cellular phenotype. There are very few reports providing comprehensive measurements of metabolites present in blood and almost no reports on metabolites changes associated with SCD. In this context we aimed to detect and to quantify targeted metabolites’ abnormalities in patients with Sickle Cell/beta thalassemia disease (HbS/βThal) and their implication in pathways that might be of interest to prevent vaso-occlusion and/or to monitor the effects of new therapies on SCD. Patients and Methods Twenty adult patients with HbS/βThal were enrolled in the study, while 20 apparently healthy individuals matched for age served as controls. Targeted metabolome analyses based on aminoacids and carnitines were performed after extraction from dry blood spots (DBSs) on filter paper using High Performance Liquid Chromatography followed by tandem Mass Spectrometry (LC/MS/MS), with derivatization (AB SCIEX 5500 triple quadrupole and QTRAP® LC/MS/MS Systems, Framingham, MA, USA) with reagents provided by Chromsystems Instruments & Chemicals, Germany. The injection volume was 10 µL and the analysis lasted ca. 2 min. Results The main results of the study showed that: a) fifty metabolites were separated in patients and controls samples, b) mapping the results of analyses, patients with HbS/βThal compared to controls had 17 metabolites with significantly lower concentration, 10 metabolites with comparable concentration and 23 metabolites with significantly higher concentration, c) L-arginine and L-ornithine concentrations were significantly lower in patients HbS/βThal compared to controls, 9.3±2.6 vs 14.7±3.7 µmoles/L, (p<0.01), and 116.0±15.0 vs 211.2±19.5 µmoles/L, (p<0.001), respectively, while L-citrulline was lower in patients HbS/βThal compared to controls but no significantly 21.8±2.5 vs 25.1±2.5 µmoles/L, (p>0.06) and d) carnitine, acetylcarnitine and propionylcarnitine correlated significantly positive with reticulocyte production index (p<0.001). Conclusions Our findings showed significant alterations in whole blood metabolome of patients with HbS/βThal. Also we demonstrated the very important metabolic abnormality of Nitric Oxide biosynthesis pathway due to the low concentration of the aminoacids serving of substrates in this cycle and disturbances in carnitine and acylcarnitines homeostasis. These abnormalities in the metabolome reflected the hemolysis, inflammatory process and pulmonary hypertension observed in these patients. Disclosures: No relevant conflicts of interest to declare.

How to approach chronic anemia

We present herein an approach to diagnosing the cause of chronic anemia based on a patient's history and complete blood cell count (CBC). Four patterns that are encountered frequently in CBCs associated with chronic anemias are considered: (1) anemia with abnormal platelet and/or leukocyte counts, (2) anemia with increased reticulocyte counts, (3) life-long history of chronic anemia, and (4) anemia with inappropriately low reticulocytes. The pathophysiologic bases for some chronic anemias with low reticulocyte production are reviewed in terms of the bone marrow (BM) events that reduce normal rates of erythropoiesis. These events include: apoptosis of erythroid progenitor and precursor cells by intrinsic and extrinsic factors, development of macrocytosis when erythroblast DNA replication is impaired, and development of microcytosis due to heme-regulated eIF2α kinase inhibition of protein synthesis in iron-deficient or thalassemic erythroblasts.