Reticulated platelets and immature platelet fraction: Clinical applications and method limitations.

So-called "reticulated" or "immature platelets," which are newly released into the circulation, are more reactive than mature platelets, contain more RNA, and can be counted using flow cytometry after staining with thiazole orange or using some fully automated hematology analyzers, albeit with numerical disagreement. This review provides an overview of the state of the art of available technology for measuring immature or reticulated platelets (RP) with preanalytical (time stability, biological variation), analytical (methods, imprecision), and postanalytical (reference range) limitations. We also analyzed the clinical conditions in which immature/RP can be considered a diagnostic or prognostic tool (ie, differential diagnosis of thrombocytopenia, recovery after bone marrow or stem cell transplantation, risk assessment in cardiovascular diseases, response to antiplatelet drugs). They might also be of clinical utility in other settings but with lower evidence. The lack of a specific reference method and universal control material, as well as dependency of results on the measurement technique used, calls for different reference intervals and compromises comparison between clinical studies carried out using different analytical methods. To obviate lack of agreement between methods, more specific RNA dyes are necessary and the impact of the platelet size on the fluorescence signal defined. In the harmonization age, also in nomenclature field, a new definition instead of "reticulated" or "immature" platelets would be useful, and "young platelets" might be a more appropriate definition taking into account both the age and the functionality of this platelet fraction.

Effect of preanalytical and analytical variables on the clinical utility of mean platelet volume.

BACKGROUND: The study endpoint was to analyze the effect of preanalytical (time, temperature, anticoagulant) and analytical (imprecision, correlation between volume and platelet concentration) variables on mean platelet volume (MPV). A further aim was to calculate in an adult population the reference intervals using the Sysmex XE-5000 analyzer. A critical evaluation was also made of the clinical utility of these parameters. METHODS: Analyses of the above values were performed in duplicate in 170 healthy adults of both sexes: (1) within 30 min from collection, and (2) after 4 h. To evaluate stability over time, the value of the platelet parameters of 20 subjects were determined, a re-analysis being performed for a period of up to 24 h on samples maintained at room temperature and 4°C using either K2-EDTA or Na-citrate as anticoagulants. RESULTS: The stability over time of MPV closely depends on the anticoagulant used, storage temperature and time interval between venipuncture and analysis. An inverse, non-linear correlation between MPV and platelet count was also found. CONCLUSIONS: In view of their effect on MPV and other related indices, the preanalytical and analytical variables make them, little more than experimental.

Evaluation of the hypochromic erythrocyte and reticulocyte hemoglobin content provided by the Sysmex XE-5000 analyzer in diagnosis of iron deficiency erythropoiesis.

BACKGROUND: Iron deficiency represents the most frequent cause of anemia. To diagnose iron deficiency some biochemical tests such as serum ferritin and the transferring saturation percent (TSAT%) are usually used. Recently, some hematological parameters such as mean reticulocyte hemoglobin content (CHr or Ret-He) and percentage of hypochromic RBCs (Hypo% or %Hypo-He) were proposed as alternative to biochemical tests. In this study, the analytic performance and the diagnostic efficiency of these two parameters provided by Sysmex XE5000 analyzer on iron deficiency patients with or without anemia (IDA and ID, respectively) were evaluated. METHODS: One hundred and sixty-four healthy adults, 58 with IDA, 21 with iron depleted stores (ID), 23 with ß-thalassemia trait, and 24 with non iron deficiency anemia were selected. The gold standard used to define iron deficiency was the coexistence of serum ferritin below 15 µg/L (12 in women) and TSAT <16%. RESULTS: For %Hypo-He, the best cut-off value for both IDA and ID is 0.9% while for Ret-He is 30.6 pg. For both parameters the performance was better to diagnose IDA (AUC, 0.96 and 0.98) than ID (AUC, 0.93 and 0.95). The Ret-He behavior was always slightly better than that of %Hypo-He. CONCLUSIONS: The use of these two parameters is useful to detect iron deficiency conditions if the hemoglobin synthesis has already been compromised.

Extraction methods of red blood cell membrane proteins for Multidimensional Protein Identification Technology (MudPIT) analysis.

Since red blood cells (RBCs) lack nuclei and organelles, cell membrane is their main load-bearing component and, according to a dynamic interaction with the cytoskeleton compartment, plays a pivotal role in their functioning. Even if erythrocyte membranes are available in large quantities, the low abundance and the hydrophobic nature of cell membrane proteins complicate their purification and detection by conventional 2D gel-based proteomic approaches. So, in order to increase the efficiency of RBC membrane proteome identification, here we took advantage of a simple and reproducible membrane sub-fractionation method coupled to Multidimensional Protein Identification Technology (MudPIT). In addition, the adoption of a stringent RBC filtration strategy from the whole blood, permitted to remove exhaustively contaminants, such as platelets and white blood cells, and to identify a total of 275 proteins in the three RBC membrane fractions collected and analysed. Finally, by means of software for the elaboration of the great quantity of data obtained and programs for statistical analysis and protein classification, it was possible to determine the validity of the entire system workflow and to assign the proper sub-cellular localization and function for the greatest number of the identified proteins.

Diagnosis of iron deficiency in patients undergoing hemodialysis.

To diagnose iron deficiency in patients undergoing hemodialysis, the percentage of hypochromic RBCs (with cellular hemoglobin concentration <280 g/L [HYPO%]) and mean reticulocyte hemoglobin content (CHret) provided by the Siemens ADVIA 120 and 2120 analyzers (Siemens Diagnostic Solutions, Tarrytown, NY) were proposed as alternatives to biochemical tests. Sysmex, with its XE-5000 analyzer (Sysmex, Kobe, Japan), also proposed the percentage of erythrocytes with cellular hemoglobin content lower than 17 pg (%Hypo-He) and equivalent of the mean reticulocyte hemoglobin content (Ret-He) with similar clinical applications. Our aim was to verify the clinical usefulness of the biochemical and cellular parameters as predictors of iron deficiency in patients undergoing long-term hemodialysis. We studied 69 patients undergoing hemodialysis 3 times weekly. The baseline values of serum ferritin and percentage of transferrin saturation were poor predictors of iron responsiveness. Better ability was demonstrated by reticulocyte indices (area under the curve [AUC], 0.74 for CHret and 0.72 for Ret-He; best cutoff values, 31.2 and 30.6 pg, respectively) and erythrocyte parameters (AUC, 0.72 for HYPO% and 0.68 for %Hypo-He; best cutoff values, 5.8 and 2.7, respectively). The newly proposed Ret-He and %Hypo-He can provide clinicians with information equivalent to CHret and HYPO%.

Automated blood cell counts: state of the art.

The CBC count and leukocyte differential count (LDC) are among the most frequently requested clinical laboratory tests. These analyses are highly automated, and the correct interpretation of results requires extensive knowledge of the analytic performance of the instruments and the clinical significance of the results they provide. In this review, we analyze the state of the art regarding traditional and new parameters with emphasis on clinical applications and analytic quality. The problems of some traditional parameters of the CBC count, such as platelet counts, some components of the LDC such as monocyte and basophil counts, and other commonly used indices such as red cell volume distribution width and platelet indices such as mean platelet volume and platelet distribution width are considered. The new parameters, evaluated from analytic and clinical viewpoints, are the available components of the extended differential count (hematopoietic progenitor cells, immature granulocytes, and erythroblasts), the immature reticulocyte fraction, the reticulocyte indices, the fragmented RBCs, and the immature platelet fraction.

Recommendations for detection and management of unsuitable samples in clinical laboratories.

A large body of evidence attests that quality programs developed around the analytical phase of the total testing process would only produce limited improvements, since the large majority of errors encountered in clinical laboratories still prevails within extra-analytical areas of testing, especially in manually intensive preanalytical processes. Most preanalytical errors result from system flaws and insufficient audit of the operators involved in specimen collection and handling responsibilities, leading to an unacceptable number of unsuitable specimens due to misidentification, in vitro hemolysis, clotting, inappropriate volume, wrong container or contamination from infusive routes. Detection and management of unsuitable samples are necessary to overcome this variability. The present document, issued by the Italian Inter-society SIBioC-SIMeL-CISMEL (Society of Clinical Biochemistry and Clinical Molecular Biology-Italian Society of Laboratory Medicine-Italian Committee for Standardization of Hematological and Laboratory Methods) Study Group on Extra-analytical Variability, reviews the major causes of unsuitable specimens in clinical laboratories, providing consensus recommendations for detection and management.

Quality specification in haematology: the automated blood cell count.

BACKGROUND: Quality specifications for automated blood cell counts include topics that go beyond the traditional analytic stage (imprecision, inaccuracy, quality control) and extend to pre- and post-analytic phases. METHODS: In this review pre-analytic aspects concerning the choice of anticoagulants, maximum conservation times and differences between storage at room temperature or at 4 degrees C are considered. For the analytic phase, goals for imprecision and bias obtained with various approaches (ratio to biologic variation, state of the art, specific clinical situations) are evaluated. For the post-analytic phase, medical review criteria (algorithm, decision limit and delta check) and the structure of the report (general part and comments), which constitutes the formal act through which a laboratory communicates with clinicians, are considered. RESULTS: K2EDTA is considered the anticoagulant of choice for automated cell counts. Regarding storage, specimens should be analyzed as soon as possible. Storage at 4 degrees C may stabilize specimens from 24 to 72 h when complete blood count (CBC) and differential leucocyte count (DLC) is performed. For precision, analytical goals based on the state of the art are acceptable while for bias this is satisfactory only for some parameters. CONCLUSIONS: In haematology quality specifications for pre- and analytical phases are important, but the review criteria and the quality of the report play a central role in assuring a definite clinical value.

The new reticulocyte parameter (RET-Y) of the Sysmex XE 2100: its use in the diagnosis and monitoring of posttreatment sideropenic anemia.

To verify their clinical usefulness in diagnosis and early response to therapy of sideropenic anemia, we compared the behavior of the reticulocyte parameter (RET-Y), a raw measure dependent on size and content of the cell, generated by the Sysmex XE 2100, with the mean reticulocyte volume (MCVr) and mean reticulocyte hemoglobin content (CHr) from the Bayer ADVIA 120 in healthy subjects and patients with iron deficiency anemia. Correlations were high (r = 0.88 and r = 0.94, respectively). All parameters varied significantly as early as 48 hours after the start of intravenous iron therapy (mean differences of 17.4% [RET-Y], 4.5% [MCVr], and 9.5% [CHr]). Sudden decreases in those parameters at interruption of therapy indicate the reappearance of sideropenic erythropoiesis. The receiver operating characteristic curve demonstrated a high degree of efficiency in differentiating moderate or severe iron deficiency anemia from the healthy state. The best association between sensitivity and specificity was at a cutoff of channel 1,624 for RET-Y and 104.5 fL for MCVr (negative and positive predictive values, respectively, of 99.6% and 96.5% for RET-Y and 98.7% and 93.3% for MCVr). RET-Y is correlated closely with CHr and is useful for diagnosis and early monitoring after the administration of intravenous iron.

A case of adult T cell leukemia and lymphoma in an Italian woman showing different malignant clones in tumor mass and in blood.

HTLV-1 infections and their associated diseases are very rare in Italy, as well as in most parts of Europe, occurring prevalently in subjects related to endemic areas. The HTLV-1-associated leukemia/lymphoma, ATLL, is a very aggressive T-cell non-Hodgkin's lymphoma which can be difficult to recognize in non-endemic areas. Here we describe the case of an elderly Italian woman, with no apparent risk factors, affected by a rapidly fatal ATLL who presented with an abdominal lymphomatous mass and circulating leukemic cells. The simultaneous presence of different T-cell clones in the tumor mass and in the blood was demonstrated by T-cell receptor gene rearrangement analysis and HTLV-1 integration pattern studies. After surgery, all the T-cell clones were present in the blood, indicating that tumor cells had spread from the mass. Phylogenetic analysis, using the complete LTR sequence, showed that the patient's HTLV-1 isolate belongs to the cosmopolitan subtype A.

Five fully automated methods for performing immature reticulocyte fraction: comparison in diagnosis of bone marrow aplasia.

We performed a parallel evaluation of 5 automated reticulocyte counters to produce the immature reticulocyte fraction (IRF). We analyzed 225 samples from healthy control subjects, 115 from patients with various diseases, 38 with advanced aplasia, and 22 in early erythropoietic recovery after chemotherapy or bone marrow transplantation. The reference intervals were different for each instrument (ADVIA 120, 0.04-0.25; CELL DYN 4000, 0.15-0.35; GEN-S, 0.20-0.37; SE 9500 RET 0.05-0.21; VEGA RETIC: 0.06-0.23). The imprecision, obtained by 1-way analysis of variance on duplicates, was satisfactory for clinical use for all methods (coefficient of variation, 7.6%-20.5% in healthy subjects), although it was higher than the analytic goal based on biologic variability within subjects. The comparison of different methods shows that agreement is good only between SE 9500 RET CELL DYN 4000, and VEGA RETIC (r2 = 0.72-0.78). The study of diagnostic performance in distinguishing aplasia from early bone marrow recovery shows slightly different results (area under the curve from 0.70 for ADVIA 120 to 0.96 for SE 9500 RET). Even with slight differences, the fluorescence-based methods seem to be more robust than other methods for IRF measurement.