NT5E gene and CD38 protein as potential prognostic biomarkers for childhood B-acute lymphoblastic leukemia.

The risk stratification of B-acute lymphoblastic leukemia (B-ALL) is based on clinical and biological factors. However, B-ALL has significant biological and clinical heterogeneity and 50% of B-ALL patients do not have defined prognostic markers. In this sense, the identification of new prognostic biomarkers is necessary. Considering different cohorts of childhood B-ALL patients, gene (DPP4/CD38/ENTPD1/NT5E) and protein (CD38/CD39/CD73) expressions of ectonucleotidases were analyzed in silico and ex vivo and the association with prognosis was established. In univariate analyses, expression of NT5E was significantly associated with worse progression-free survival (PFS) in bone marrow (BM) samples. In multivariate analyses, Kaplan-Meier analysis, and log-rank test, higher NT5E expression predicted unfavorable PFS in BM samples. Considering minimal residual disease (MRD), higher levels of cellularity were associated with the high NT5E expression at day 8 of induction therapy. In addition, we observed that white blood cells (WBC) of childhood B-ALL patients had more CD38 compared to the same cell population of healthy donors (HD). In fact, MRD > 0.1% patients had higher CD38 protein expression on WBC in comparison to HD. Noteworthy, we observed higher CD38 expression on WBC than blasts in MRD > 0.1% patients. We suggest that NT5E gene and CD38 protein expression, of the ectonucleotidases family, could provide interesting prognostic biomarkers for childhood B-ALL.

Advances in laboratory diagnosis of hereditary spherocytosis.

Among the red cell membrane disorders, hereditary spherocytosis (HS) is one of the most common causes of inherited hemolytic anemia. HS results from the deficiency or dysfunction of red blood cell membrane proteins, such as α spectrin, ß spectrin, ankyrin, anion channel protein (Band-3 protein), protein 4.1 and protein 4.2. Conventionally, HS diagnosis is established through a series of tests, which include spherocytes identification in peripheral smear, reticulocyte count, osmotic fragility, etc. Currently, different hematological analyzers provide erythrocyte indicators that estimate the presence of spherocytes and correlate that with HS, which can be useful for disease screening. The most traditional method is the osmotic fragility (OF) test, which is labor-intensive and time-consuming to perform and presents low sensitivity and specificity values. Thus, new methods have been developed for HS diagnosis, such as flow cytometry. Current guidelines recommend the use of flow cytometry as a screening test for HS diagnosis using the eosin-5'-maleimide (EMA) binding test. Thus, HS diagnosis is the result of a collaboration between clinicians and laboratories, who should take into account the family history and the exclusion of other causes of secondary spherocytosis.

Flow Cytometry in Detection of Fetal Red Blood Cells and Maternal F Cells to Identify Fetomaternal Hemorrhage.

Accurate detection and quantitation of fetomaternal hemorrhage (FMH) is critical to the obstetric management of rhesus D alloimmunization in Rh-negative pregnant women. The flow cytometry is based on the detection of fetal red blood cells using a monoclonal anti-HbF antibody, and is the method most indicated for this estimation. The objective of this study was to quantify fetal red blood cell levels of pregnant women using flow cytometry. We analyzed 101 peripheral blood samples from Rh-negative and Rh-positive women, whose mean age was 24 years (20-32 years), after vaginal delivery or cesarean section. Our study showed that 53% of pregnant women had fetal red blood cells levels <2.0 mL, 31% between 2.0-3.9 mL, 16% between 4.0-15.0 mL, and 1% >15.0 mL. Accurate quantitation of fetal red blood cells is necessary to determine the appropriate dose of anti-D (RHD) immunoglobulin to be administered to pregnant or postpartum women.

Evaluation of HLA-DR expression in monocytes and CD64 in neutrophils as A predictor of SEPSIS/sirs in the infectious-inflammatory process.

Sepsis is a highly fatal disease that affects millions of people worldwide every year. Currently, the diagnosis of sepsis is made by identifying at least two symptoms of systemic inflammatory response syndrome (SIRS), along with confirming the presence of microorganisms using a blood culture examination. Some biomarkers are already used to aid in the diagnosis, such as increased levels of C-reactive protein (CRP), leukocytes, immature granulocytes (IG), and bands. In addition, studies have shown a relationship between the expression of certain antigen receptors in the body's defense cells and its infectious state. CD64 is a receptor expressed in monocytes, and, in cases of infection, its expression is strongly observed in neutrophils. On the other hand, the class II MHC (major histocompatibility complex) marker, HLA-DR (human leukocyte antigen-DR), decreases its expression in monocytes in response to infection. This cohort study was conducted with 77 adult patients from a university hospital, divided into two groups: Non-Sepsis/SIRS and Sepsis/SIRS. The selected samples were analyzed by flow cytometry, identifying the expression of CD64 and HLA-DR according to their MFI, and calculating the sepsis index (SI) for each patient. All three parameters exhibited significant differences in expression between the two groups. When compared to the laboratory tests already in use, the utilization of HLA-DR, CD64, and the new index has shown greater sensitivity and specificity in identifying sepsis. This study contributes to knowledge about the relationship between the expression of antigens on defense cells and sepsis. The use of these biomarkers can help to improve the diagnosis and treatment of sepsis, which may contribute to the reduction of mortality related to the disease.

Interleukin-10 GCC/GCC haplotype is associated high risk of acute GVHD in patients undergoing allogeneic HSCT in the southern of Brazil.

INTRODUCTION: Hematopoietic Stem Cell Transplant (HSCT) is currently an important curative treatment for many patients with malignant and non-malignant diseases. Graft versus host disease (GVHD) represents a major complication in allogeneic HSCT recipients. Several polymorphisms in cytokine genes have recently been investigated as candidates for risk factors for acute-GVHD (aGVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). OBJECTIVE: In this study, we analyzed specific interleukin (IL)-10 haplotypes polymorphisms, in a cohort of 99 patients and their respective allo-HSCT donors for aGVHD and risk. RESULTS: An association was found between IL-10 promoter haplotype polymorphisms at positions -1082, -819 and - 592 with the occurrence of aGVHD. Patients who have the GCC/GCC haplotype are at increased risk of aGVHD (P = 0.017, HR: 5.42 (95% CI: 1.34-21.84). In the donors group and severity of aGVHD as not found statistical significancy. CONCLUSION: The results obtained show the IL-10 GCC/GCC haplotype can be an important biomarker to identify the greatest risk of aGVHD in the patient undergoing HSCT.

Flow cytometry immunophenotyping of healthy platelets and hospitalized patients with suspected platelet dysfunction: Challenges for establishing a cutoff value.

INTRODUCTION AND OBJECTIVE: Flow Cytometry (FC) is one of the techniques, which allows the identification and characterization of platelets. The detection of absent or reduced expression of the glycoproteins is the main objective of this technique. Abnormalities of glycoproteins lead to hemorrhagic syndromes. Among the main diseases, the Bernard-Soulier syndrome (BSS) and Glanzmann thrombasthenia (GT) stand out. We aimed to show a FC-based platelet assessment test for diagnostic use, which measures the expression of markers in normal patients, and evaluate these markers in patients with platelet disorders. METHODS: We examined a control group of 41 healthy adults to establish reference values and assess the variability of the relative expression of platelet markers and subsequently compared these findings to those of 30 patients with suspected platelet dysfunctions. We determined the mean fluorescent intensity (MFI) of the expressed parameters by FC using CD41, CD42a, CD42b and CD61 and SSC/FSC platelet-gated cells. RESULTS: We determined our baseline panel of markers and compared them to suspected platelet dysfunctions. Patients with suspected BSS presented increased levels of the MFI for the GPIIIa (CD61) and GPIIb (CD41). They showed significantly reduced levels of the GPIb (CD42b) and GPIX (CD42a). Patients with suspected GT showed normal expression of the GPIX (CD42a), increased expression of the GPIb (CD42b) and reduced levels of the GPIIIa (CD61). In this case, with reduced levels of only one marker, the GPIIb (CD41), values showed normal expression. CONCLUSIONS: We describe the FC assay to support the diagnosis of different platelet disorders. Our study made it possible to implement a technique that brought benefits to care.

Evaluation of RET-He values as an early indicator of iron deficiency anemia in pregnant women.

INTRODUCTION: During pregnancy, women are at an increased risk of developing iron-deficiency anemia. OBJECTIVE: The objective of this study was to assess the diagnostic performance of the reticulocyte hemoglobin equivalent (RET-He) in the early detection of iron-deficiency anemia in a group of pregnant women and to establish a reference range for this parameter in a group of control individuals. METHOD: A total of 60 patients and 130 control subjects were included in the study. Blood samples collected from the subjects were submitted to a complete blood count and a serum ferritin test and the data were analyzed by comparing the groups and ROC curves. RESULTS: The reference range found for the RET-He was between 29.75pg and 38.24pg, with a median of 35pg. The receiver operating characteristic (ROC) curve analysis for the ferritin parameter showed an area under the curve of 0.732 for the RET-He, 0.586 for hemoglobin, 0.551 for the mean corpuscular hemoglobin concentration and 0.482 for the mean corpuscular volume. CONCLUSION: Early diagnosis of iron deficiency anemia in pregnancy is essential to prevent damage to both maternal and fetal health. The RET-He presents an excellent potential as an auxiliary tool for the diagnosis of iron deficiency in pregnant women.

Comparison of Nonsequencing Techniques for Identification of NPM1 Mutations and Associated Blast Morphology in Patients With Acute Myeloid Leukemia.

CONTEXT.­: Nucleophosmin 1 (NPM1) mutations affect 20% to 30% of all acute myeloid leukemia (AML) patients; several methods are employed to analyze NPM1 mutations, each of them with its advantages and limitations. OBJECTIVE.­: To compare 3 nonsequencing protocols capable of detecting the main NPM1 mutations and to evaluate nuclear morphometric analysis (NMA) as an alternative to cuplike blast detection. DESIGN.­: We selected multiparameter flow cytometry (MFC), amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), and a quantitative PCR (qPCR) kit to identify NPM1 mutations in AML patients at diagnosis. We also evaluated the presence of cuplike blasts and assessed nuclear morphometry using NMA. RESULTS.­: MFC appears as a screening method for NPM1 mutations because of its lower specificity. ARMS-PCR demonstrated specificity similar to that of the qPCR kit, although it was more laborious. qPCR testing, conversely, is relatively fast and easy to standardize. Of these methods, qPCR was the only one capable of identifying the type of NPM1 mutation. With regard to morphology, NMA could be used as an alternative for the evaluation of cuplike blasts in AML smears. CONCLUSIONS.­: qPCR appears to be the best option to identify NPM1 mutations, with ARMS-PCR representing a cheaper alternative. MFC may be used as a screening method, in which results falling within and above the gray zone should be confirmed by molecular testing.

Evaluation of the immature myeloid information (IMI) by Sysmex XE 2100® hematology analyzer in the identification of blasts of myeloid lineage.

The immature myeloid information (IMI) provided by the Sysmex XE 2100 hematology analyzer has demonstrated that it is possible to differentiate granulocytes of immature cells in daily practice. A specific reagent lyses mature white blood cells, allowing that immature myeloid cells remain intact and consequently detectable. It is known that lymphoblasts cannot be detected in this channel. This channel does not entail additional costs, since it is provided by the traditional hematology analyzers used in blood tests and is widely useful in differentiating cell lines. This study has aimed to assess the consonance between IMI results and subtypes of acute leukemias and other hematologic malignancies in order to use it as screening test in the definition of cell lineage. A total of 141 cases of hematologic malignancies have been evaluated. Results of the IMI channel were compared using the Sudan Black cytochemical and flow cytometry. The Cohen's Kappa coefficient of agreement between IMI and flow cytometry results was 0.8%. IMI had sensibility and specificity levels of 90.7% and 90.8%, respectively; VP: 68 (91.9%); FP: 6 (8.1%); VN: 59 (89.4%) and FN: 7 (10.6%); PPV 91.9% and NPV 89.4%. The Sysmex XE 2100 analyzer showed a good analytical performance for the detection of immature myeloid cells. These results indicate that the IMI channel has sensitivity and specificity levels, consistent with previous studies. Given this situation, one may conclude that IMI was able to be used as a screening test to complement cytochemistry for identify blasts of myeloid lineage.

High plasma IL-6 levels following haploidentical allogeneic hematopoietic stem cell transplantation post-transplant cyclophosphamide as predictor of early death and worse outcome.

INTRODUCTION: Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) can be used as an alternative procedure in the absence of HLA-compatible donors. The use of high doses of cyclophosphamide after infusion improves the prognosis and eliminates the need for T cell depletion in vivo. Among the main complications of haplo-HSCT are acute graft-versus-host disease (a-GVHD) and cytokine release syndrome (CRS). This is a systemic inflammatory response that leads to the release of inflammatory proteins, including IL-6. This syndrome has several clinical features, with mild to severe symptoms. This study aimed to compare plasma IL-6 levels in patients submitted to different HSCT types and to associate them with the presence of acute graft versus host disease (a-GVHD), CRS and survival. METHODS: A total of 84 patients (22 haploidentical and 62 non-haploidentical) were evaluated at different times. The IL-6 levels in haplo and non-haplo-HSCT recipients were measured before transplantation and on days D7, D14, D28, D60, and D100. RESULTS: IL-6 levels were higher in haplo-HSCT recipients than in non-haplo-HSCT recipients, remaining elevated from D14 until D100 (P = 0.006) and a cut-off ≥11 pg/mL on D7, which is related to worse overall survival. In our study, we found no association with a-GVHD (P = 0.239), a common complication of this type of transplant, but we found a relationship between the increase in IL-6 and CRS (P = 0.021). CONCLUSION: IL6 can be used as a biomarker for patients submitted to haplo-HSCT, allowing clinical interference in patients having levels of IL-6 times larger than normality values, avoiding early death in this group of patients.

Flow cytometry as a diagnostic tool in neuroblastoma.

In recent years, there has been an expansion in the use of flow cytometry (FC) immunophenotyping in the diagnosis and monitoring of childhood solid neoplasms. Neuroblastoma (NB), in turn, is the most common extracranial solid tumor in childhood. In the present study, we sought to compare FC and anatomopathological examination (PA) / immunohistochemistry (IHC) of children diagnosed or suspected with NB. The median age was 59 months (minimum 0; maximum 325 months), of these 12 were male (57.1%, 12/21). Forty-eight samples (27 bone marrow (BM), 10 peripheral blood (PB), 8 primary tumors (PT) and 2 liver nodules (HN) and 1 rib fragment (RF)) from 21 patients were evaluated. Twenty-nine samples were from patients with clinical suspicion while 19 samples were from patients with previously confirmed diagnosis. Thirteen samples (7 BM, 5 PT and 1 HN) presented NB when analyzed in FC while 8 (3 BM and 5 PT) samples were positive for NB in the PA/IHC. They were concordant in 88.9% of the cases. No NB cells were identified in any PB. Considering the PA as the gold standard, the FC obtained a sensitivity of 100%, a specificity of 86%, a positive predictive value of 67% and a negative predictive value of 100%. This study demonstrates that FC can be used as a methodology for diagnosis and assessment of NB involvement. In addition, FC has the advantage of allowing a quick diagnosis and accurate classification of the disease, and can also assist in monitoring the treatment.

HEMOPHAGOCYTOSIS BY BLASTS IN A CHILD WITH ACUTE MONOCYTIC LEUKEMIA AFTER CHEMOTHERAPY.

OBJECTIVE: To describe the case of a child who presented hemophagocytic lymphohistiocytosis (HLH) associated with acute monocytic leukemia after chemotherapy, with hemophagocytosis caused by leukemic cells. CASE DESCRIPTION: In a university hospital in Southern Brazil, a 3-year-old female was diagnosed with acute monocytic leukemia with normal karyotype. The chemotherapy regimen was initiated, and she achieved complete remission six months later, relapsing after four months with a complex karyotype involving chromosomes 8p and 16q. The bone marrow showed vacuolated blasts with a monocytic aspect and evidence of hemophagocytosis. The child presented progressive clinical deterioration and died two months after the relapse. COMMENTS: HLH is a rare and aggressive inflammatory condition characterized by cytopenias, hepatosplenomegaly, fever, and hemophagocytosis in the bone marrow, lymph nodes, spleen, and liver. Although rare, malignancy-associated HLH (M-HLH) is fatal. The patient in this case report met five out of the eight established criteria for HLH. The evolution of the patient's karyotype, regardless of the diagnostic profile, seemed secondary to the treatment for acute monocytic leukemia. In this case, the cytogenetic instability might have influenced the abnormal behavior of leukemic cells. This is a rare case of HLH in a child with acute monocytic leukemia.

Definition of reference ranges for the platelet distribution width (PDW): a local need.

BACKGROUND: Automated hematological analyzers have contributed to more precise and faster results. They also make it possible to measure several blood cell parameters automatically. Among the parameters provided, platelet indices are probably the most ignored by clinical laboratories due to the difficulty of standardization, as well as being affected by a range of methodological problems. It has been suggested that each laboratory determines its own reference intervals with the equipment used. METHODS: Our goal was to determine the reference range of platelet distribution width (PDW) in venous blood samples from 231 patients using the Pentra 120 ABX hematology analyzer. RESULTS: The PDW median was 13.3%, with a reference range of 10.0%-17.9% for the 5th-95th percentiles, with a confidence interval of 95%. CONCLUSIONS: Among all indices, the PDW has been receiving attention due to its usefulness for distinguishing between reactive thrombocytosis and thrombocytosis associated with myeloproliferative disorder. Determination of the PDW reference range is fundamental, and the association of this parameter with the platelet number and mean platelet volume may be used for the diagnosis and differentiation of several pathologies.

Influence of minimal residual disease by multiparametric flow cytometry at day 15 of induction in risk stratification of children with B-cell acute lymphoblastic leukemia treated at a referral hospital in southern Brazil.

BACKGROUND: The minimal residual disease (MRD) is the most important prognostic factor for acute lymphoblastic leukemia (ALL) in children. This study aimed to investigate the influence of detecting the MRD by the multiparametric flow cytometry (MFC) at day 15 (D15) of the induction on the analysis of the risk group classifications of the different childhood ALL treatment protocols used in a referral hospital in southern Brazil. METHOD: We retrospectively reviewed the medical records of patients with B-cell ALL, aged 1 to 18 years, treated at a hospital from January 2013 to April 2017. MAIN RESULTS: Seventy-five patients were analyzed. Regarding the MRD by the MFC at D15, the analyses showed statistical significance when the MRD was grouped into three categories, < 0.1%, 0.1-10%, and > 10%, with the following distribution: 30.7%, 52.0%, and 17.3%, respectively. There was a significant association between D15 MRD-MFC < 0.1% and the likelihood of dying or relapsing and between D15 MRD-MFC > 10% and the likelihood of dying or relapsing. The cumulative hazard ratio for the relapse of patients with D15 MRD-MFC < 0.1%, 0.1-10%, and > 10% was 19.2%, 59.8%, and 80.1%, respectively. CONCLUSION: Our analysis suggests D15 MRD-MFC < 0.1% as a cut-off point for patients with more favorable outcomes and that the MRD at D15 in risk classifications is particularly useful for the stratification of patients with a more favorable prognosis.

Evaluation of RET-He values as an early indicator of iron deficiency anemia in pregnant women

Abstract Introduction During pregnancy, women are at an increased risk of developing iron-deficiency anemia. Objective The objective of this study was to assess the diagnostic performance of the reticulocyte hemoglobin equivalent (RET-He) in the early detection of iron-deficiency anemia in a group of pregnant women and to establish a reference range for this parameter in a group of control individuals. Method: A total of 60 patients and 130 control subjects were included in the study. Blood samples collected from the subjects were submitted to a complete blood count and a serum ferritin test and the data were analyzed by comparing the groups and ROC curves. Results The reference range found for the RET-He was between 29.75pg and 38.24pg, with a median of 35pg. The receiver operating characteristic (ROC) curve analysis for the ferritin parameter showed an area under the curve of 0.732 for the RET-He, 0.586 for hemoglobin, 0.551 for the mean corpuscular hemoglobin concentration and 0.482 for the mean corpuscular volume. Conclusion Early diagnosis of iron deficiency anemia in pregnancy is essential to prevent damage to both maternal and fetal health. The RET-He presents an excellent potential as an auxiliary tool for the diagnosis of iron deficiency in pregnant women.

Quantification of peripheral blood CD34+ cells prior to stem cell harvesting by leukapheresis: a single center experience.

BACKGROUND: Due to laboratory logistic issues, our center has traditionally scheduled peripheral blood stem cell harvests based on timing from the start of mobilization. This has proved to be useful in some cases, but also resulted in many fruitless harvests due to poor mobilization. In order to improve the efficiency of collections and compare the effectiveness of peripheral blood CD34+ cells as a predictor with data from other reports, this study analyzed the implementation of this routine. METHODS: Peripheral blood and leukapheresis samples were quantified by flow cytometry and the association between these parameters was assessed. RESULTS: Sixty-six consecutive leukapheresis samples were collected from 34 patients after the collection of peripheral blood samples for CD34+ quantification. A moderate positive correlation was observed between peripheral blood CD34+ cell count and total CD34+ cell count/kg (r = 0.596; p-value < 0.001). A multivariable regression model also confirmed this association and allowed the estimation that for every increase in five CD34+ cells/µL in the peripheral blood, a mean increase of 0.38 × 106 CD34+ cells/kg could be predicted. Demographic characteristics, baseline comorbidities and mobilization regimen did not influence final CD34+ cell count in this sample. CONCLUSIONS: As observed in other centers, quantification of peripheral blood CD34+ progenitor cells is a strong predictor of effectiveness to guide stem cell harvesting. Due to the results of this study, a modification in the peripheral blood stem cell harvesting logistics was implemented at our center in order to incorporate this routine.

Cut-off value for absolute lymphocytes as an alternative for the immunophenotypic analysis of CD3+ T cells in the monitoring of immunosuppressive therapy with thymoglobulin.

INTRODUCTION: Immunosuppression of T lymphocytes is required for preventing acute rejection after transplantation and for the treatment of chronic autoimmune and inflammatory diseases. The laboratory monitoring for this therapy is the measurement of T cells by immunophenotyping, aiming the target value of less than 20 cells per µL. OBJECTIVE: To establish a cut-off point for the total number of lymphocytes in the automated blood cell count that reflects less than twenty T cells µL by immunophenotyping. METHODS: We studied and evaluated 242 kidney transplant patients that had results of automated blood cell count and quantification of T cells by immunophenotyping technique. The patients were divided into two groups, depending on the T lymphocyte immunophenotyping rates established by lower and higher than 20 cells per µL. After, we evaluated the cut-off point for lymphocytes in the blood cell count with a specificity of 100% to exclude patients with high levels of T lymphocytes. RESULTS: We found that the cut-off point of 70 lymphocytes per µL obtained by automated blood cell count showed 100% of specificity to exclude patients with T-cell counts higher than 20 cells per µL by immunophenotyping. CONCLUSION: The results found in this study may be helpful to monitor the immunosuppressive therapy in kidney transplant patients in places where a flow cytometer is not available, or when this equipment is not present in the full routine.

Do Alpha Thalassemia, Fetal Hemoglobin, and the UGT1A1 Polymorphism have an Influence on Serum Bilirubin Levels and Cholelithiasis in Patients with Sickle Cell Disease?

BACKGROUND: Increased destruction of erythrocytes in patients with sickle cell disease results in chronic hyperbilirubinemia and leads to the formation of gallstones. OBJECTIVES: The objective of this study was to determine the combined influence of alpha thalassemia, fetal hemoglobin, and the UGT1A1 polymorphism on serum bilirubin levels and cholelithiasis in patients with sickle cell disease. METHODS: We analyzed 72 patients treated in the outpatient hematology unit of the Clinical Hospital of Porto Alegre. The alpha thalassemia trait was determined by multiplex polymerase chain reaction and the polymorphisms of UGT1A1 by capillary electrophoresis with tagged primers. RESULTS: Total and indirect bilirubin levels differed significantly between genotypes TA7/TA7 and TA6/TA6 (p < 0.05). Bilirubin levels were influenced by the UGT1A1 polymorphism but not by alpha thalassemia and fetal hemoglobin. There was no association between cholelithiasis and any of the variables studied. CONCLUSION: These preliminary findings suggest that the UGT1A1 gene can influence serum bilirubin levels in sickle cell anemia and serve as a tool to differentiate an acute hemolytic condition from a pre-existing condition of hyperbilirubinemia.

Determination of reference ranges for immature platelet and reticulocyte fractions and reticulocyte hemoglobin equivalent.

INTRODUCTION: The immature platelet and immature reticulocyte fractions represent the ratios of platelets and reticulocytes recently released into the circulation and thus with higher RNA content. They are considered early indicators of bone marrow recovery. OBJECTIVE: The aim of this study was to determine the reference ranges for the immature platelet and reticulocyte fractions of hematologically normal individuals in a university hospital. METHODS: Venous blood samples collected in ethylenediaminetetraacetic acid K3 were analyzed using a Sysmex XE-5000™ analyzer. Individuals with platelet and reticulocyte counts within the reference ranges, and a blood count within the laboratory's screening criteria were included. Individuals with clinical conditions that could affect hematological results were excluded. The immature platelet fraction, high, medium and low fluorescence reticulocyte fractions and reticulocyte hemoglobin equivalent were evaluated. The reference ranges were determined according to the recommendations of the International Federation of Clinical Chemistry. RESULTS: One hundred and thirty-two outpatients were evaluated. The mean age was 44 years (range: 13-80 years), 72 (54.5%) were women treated in a university hospital. The mean platelet count was 250.8×109/L and the mean reticulocyte count was 0.052×109/L. The following reference ranges were obtained: immature reticulocyte fraction 1.6-12.1%, the high, medium and low fluorescence reticulocyte fractions were 0.0-1.7%, 1.6-11.0% and 87.9-98.4%, respectively, the reticulocyte hemoglobin equivalent was 30.0-37.6% and immature platelet fraction was 0.8-5.6%. There was a statistically significant difference (p-value=0.006) between genders in respect to the immature platelet fraction with 0.8-4.7% for females and 0.7-6.1% for males. The immature reticulocyte fraction was directly correlated with the reticulocyte count. CONCLUSION: Determining the reference range is critical to the introduction of a new parameter. The reference ranges obtained herein corroborate those reported in previous publications and will contribute to the clinical and laboratory application of the indices.