Recent advances in laboratory hematology reflected by a decade of CCLM publications.

On the occasion of the 60th anniversary of Clinical Chemistry and Laboratory Medicine (CCLM) we present a review of recent developments in the discipline of laboratory hematology as these are reflected by papers published in CCLM in the period 2012-2022. Since data on CCLM publications from 1963 to 2012 are also available, we were able to make a comparison between the two periods. This interestingly revealed that the share of laboratory hematology papers has steadily increased and reached now 16% of all papers published in CCLM. It also became evident that blood coagulation and fibrinolysis, erythrocytes, platelets and instrument and method evaluation constituted the 'hottest' topics with regard to number of publications. Some traditional, characteristic CCLM categories like reference intervals, standardization and harmonization, were more stable and probably will remain so in the future. With the advent of important newer topics, like new coagulation assays and drugs and cell population data generated by hematology analyzers, laboratory hematology is anticipated to remain a significant discipline in CCLM publications.

Basophil counting in hematology analyzers: time to discontinue?

Basophils (basophilic granulocytes) are the least abundant cells in blood. Nowadays, basophils are included in the complete blood count performed by hematology analyzers and therefore reported in practically all patients in whom hematologic investigations are requested. However, hematology analyzers are not reliable enough to report clinically useful results. This is due to a combination of very high analytical imprecision and poor specificity, because the chemical and physical methods used for basophil counting in hematology analyzers are ill-defined and thus basophils are not well recognized by the analyzers. As a result, false basophil counts are quite common. In view of increasing analytical performance demands, hematology laboratories should stop reporting basophil counts produced by hematology analyzers. Suggestions for alternative pathways are presented for those situations where basophils are of clinical relevance.

Role of RDW in mathematical formulas aiding the differential diagnosis of microcytic anemia.

Many mathematical formulas containing simple red blood cell parameters have been proposed for differentiating between iron deficiency and thalassemia in patients with microcytic anemia. Approximately half of these formulas do include red cell distribution width (RDW), along with other red cell parameters. In the present study we investigated the role of RDW, expressed in relative or in absolute units in relation with the formulas' discriminant performance. We used a database containing over 2200 subjects with microcytic anemia, for whom a final diagnosis (iron-deficiency anemia, thalassemia, both or other) was available. Performance of the discriminant formulas was assessed by Receiver Operator Curve analysis. Substitution of relative by absolute RDW resulted in statistically significant performance increase (area under the ROC curve) in 16 out of 23 formulas, predominantly due to increased specificity. Relevant performance deterioration was seen in only three formulas that had low initial performance already with the original relative RDW. For optimal differential diagnostic performance, an RDW-based formula for distinguishing thalassemia from iron-deficiency anemia in microcytic anemia should contain 'absolute' instead of relative RDW.

Multicenter performance evaluation of the Abbott Alinity hq hematology analyzer.

Background Alinity hq (Abbott) is a new high-throughput hematology analyzer that exclusively employs optical principles for detecting and enumerating blood cells. It reports 29 parameters, including a six-part white blood cell (WBC) differential. The aim of this multicenter study was to evaluate the analytical and clinical performance of the Alinity hq. Methods Complete blood count (CBC) results and morphological flagging were compared to that of CELL-DYN Sapphire (Abbott) and 2 × 200-cell manual differential results, on 1473 whole-blood samples from a well-defined patient population from three different clinical laboratories in the Netherlands. In addition, within-run and within-laboratory precision, linearity, limit of quantitation, carryover and sample stability were assessed. External quality assessment samples were also evaluated. Results Data analysis demonstrated strong concordance of Alinity hq results with those of CELL-DYN Sapphire for all CBC parameters, except for basophil granulocytes. Alinity hq WBC differential showed high level of agreement with manual differential results and exhibited a better agreement with manual basophil results than CELL-DYN Sapphire. The sensitivity of the Alinity hq Blast flag was 57.6%, equal to the 57.6% sensitivity of the CELL-DYN Sapphire's Blast Alert. When considering samples with ≥5% blasts, the sensitivity of the Alinity hq Blast flag was 70.0%. Analytical performance of Alinity hq was shown to be consistent with state-of-the-art (SOTA) performance characteristics. Conclusions Alinity hq CBC measurands demonstrated good overall agreement with results obtained with CELL-DYN Sapphire, as well as manual WBC differential. The analytical and clinical performance characteristics of Alinity hq make it well suited for clinical laboratories.

Assessment of iron-restricted erythropoiesis in chronic renal disease: evaluation of Abbott CELL-DYN Sapphire mean reticulocyte hemoglobin content (MCHr).

We studied the performance of mean reticulocyte hemoglobin content (MCHr), measured on Abbott CELL-DYN Sapphire analyzer for the detection of functional iron deficiency. Patients with anemia and with renal disease were prospectively selected from the outpatient population of our hospital, 512 subjects were studied. Diagnoses and other medical data were retrieved from the hospital information system. MCHr was measured using a CELL-DYN Sapphire hematology analyzer (Abbott Diagnostics). Standard laboratory and statistical tests were used. Receiver operating characteristic (ROC) analysis was used to establish the diagnostic performance of MCHr for detecting iron-restricted erythropoiesis, with transferrin saturation <20% as the gold standard. Patients in the iron deficiency anemia group all had a state of iron depletion and iron-restricted erythropoiesis (median MCHr 25.4 pg). Patients with anemia of chronic disease showed also low MCHr, median 27.6 pg, but not statistically different from the iron deficient group (p = .0585). Renal patients with iron restriction (n = 66) had significantly lower MCHr (p < .0001) than those receiving adequate iron supply (n = 100): median MCHr were 27.9 and 32.5 pg, respectively. ROC analysis gave sensitivity 84.4% and specificity 80.1% with area under curve 0.863 (95% CI 0.823-0.902) at an MCHr cut-off 30.0 pg. MCHr on CELL-DYN Sapphire has equivalent clinical performance for detecting absolute or functional iron deficiency in patients with chronic kidney disease as previously published for another type of analyzer.

Comparative evaluation of platelet counts in two hematology analyzers and potential effects on prophylactic platelet transfusion decisions.

BACKGROUND: Decisions on prophylactic platelet (PLT) transfusions are generally based on the recipient's PLT count, but few clinicians are aware of precision and accuracy of the PLT counting methods used by the clinical laboratory. Each PLT counting technology has its specific inaccuracy, especially in thrombocytopenic samples and therefore may impact decisions on PLT transfusions. STUDY DESIGN AND METHODS: Five routine PLT counting methods available in two hematology analyzers (Sysmex XN-2000 and Abbott CELL-DYN Sapphire) were investigated (impedance and optical on both analyzers and fluorescent on XN-2000), using the CD61 immunologic PLT method as a reference. The impact of counting inaccuracy on imaginary transfusion decisions was examined at various common PLT thresholds. RESULTS: In total 341 samples were analyzed, 178 of which had PLT counts of less than 35 × 109 /L. Despite excellent overall correlation with the reference method (r > 0.99), thrombocytopenic samples showed only modest correlation for impedance and XN-2000 optical methods. Sapphire optical and XN-2000 fluorescent methods correlated very well with the reference, albeit with bias in the very low range. We noticed potential risk of undertransfusion (ranging from 2% to 90%), depending on the threshold used. The risk of overtransfusion was small (<10%). CONCLUSIONS: The XN-2000 fluorescent PLT counting method showed excellent correlation with the CD61 reference count, closely followed by the CELL-DYN Sapphire optical method. XN-2000 impedance and optical and Sapphire impedance methods are not accurate enough for basing transfusion decisions on. Only XN-2000 fluorescent, Sapphire optical, and CD61 methods are sufficiently accurate for making appropriate clinical decisions in patients with severe thrombocytopenia.

Critical appraisal of discriminant formulas for distinguishing thalassemia from iron deficiency in patients with microcytic anemia.

BACKGROUND: Many discriminant formulas have been reported for distinguishing thalassemia trait from iron deficiency in patients with microcytic anemia. Independent verification of several discriminant formulas is deficient or even lacking. Therefore, we have retrospectively investigated discriminant formulas in a large, well-characterized patient population. METHODS: The investigational population consisted of 2664 patients with microcytic anemia: 1259 had iron deficiency, 1196 'pure' thalassemia trait (877 ß- and 319 α-thalassemia), 150 had thalassemia trait with concomitant iron deficiency or anemia of chronic disease, and 36 had other diseases. We investigated 25 discriminant formulas that only use hematologic parameters available on all analyzers; formulas with more advanced parameters were disregarded. The diagnostic performance was investigated using ROC analysis. RESULTS: The three best performing formulas were the Jayabose (RDW index), Janel (11T), and Green and King formulas. The differences between them were not statistically significant (p>0.333), but each of them had significantly higher area under the ROC curve than any other formula. The Jayabose and Green and King formulas had the highest sensitivities: 0.917 both. The highest specificity, 0.925, was found for the Janel formula, which is a composite score of 11 other formulas. All investigated formulas performed significantly better in distinguishing ß- than α-thalassemia from iron deficiency. CONCLUSIONS: In our patient population, the Jayabose RDW index, the Green and King formula and the Janel 11T score are superior to all other formulas examined for distinguishing between thalassemia trait and iron deficiency anemia. We confirmed that all formulas perform much better in ß- than in α-thalassemia carriers and also that they incorrectly classify approximately 30% of thalassemia carriers with concomitant other anemia as not having thalassemia. The diagnostic performance of even the best formulas is not high enough for making a final thalassemia diagnosis, but in countries with limited resources, they can be helpful in identifying those patients who need further examinations for genetic anemia.

Discriminant indices for distinguishing thalassemia and iron deficiency in patients with microcytic anemia: a meta-analysis.

BACKGROUND: More than 40 mathematical indices have been proposed in the hematological literature for discriminating between iron deficiency anemia and thalassemia trait in subjects with microcytic red blood cells (RBCs). None of these discriminant indices is 100% sensitive and specific and also the ranking of the discriminant indices is not consistent. Therefore, we decided to conduct the first meta-analysis of the most frequently used discriminant indices. METHODS: An extensive literature search yielded 99 articles dealing with 12 indices that were investigated five or more times. For each discriminant index we calculated the diagnostic odds ratio (DOR) and summary ROC analysis was done for comparing the performance of the indices. RESULTS: The ratio of microcytic to hypochromic RBCs (M/H ratio) showed the best performance, DOR=100.8. This was significantly higher than that of all other indices investigated. The RBC index scored second (DOR=47.0), closely followed by the Sirdah index (DOR=46.7) and the Ehsani index (DOR=44.7). Subsequently, there was a group of four indices with intermediate and three with lower DOR. The lowest performance (DOR=6.8) was found for the RDW (Bessman index). Overall, the indices performed better for adults than for children. CONCLUSIONS: The M/H ratio outperformed all other discriminant indices for discriminating between iron deficiency anemia and thalassemia trait. Although its sensitivity and specificity are not high enough for making a definitive diagnosis, it is certainly of value for identifying those subjects with microcytic RBC in whom diagnostic tests for confirming thalassemia are indicated.

Effect of age and gender on reference intervals of red blood cell distribution width (RDW) and mean red cell volume (MCV).

BACKGROUND: Red blood cell distribution width (RDW) was recently shown to be age-dependent when using Sysmex XE-2100 hematology analyzers. As measuring RDW is subject to technology, we have investigated if this relation also exists when using a different hematology analyzer, Abbott CELL-DYN Sapphire. In addition, as RDW is generally expressed relative to mean red blood cell volume (MCV), we have explored how MCV influences the age-dependency of RDW. METHODS: We measured RDW and MCV in a large cohort and calculated RDW-SD (the "absolute" RDW), which does not contain MCV. For establishing reference intervals we used Bhattacharya statistics. RESULTS: In our study cohort of 8089 individuals we found a strong association between RDW and age, but not with gender. Also MCV showed an age-related increase over the entire age range. The conventional RDW increased by 6% from the youngest to oldest age class, whereas RDW-SD increased by nearly 15%. This difference was caused by a mean age-related increase in MCV of 6.6%. Age-dependent reference intervals were established for RDW, RDW-SD and MCV. CONCLUSIONS: The age-dependency of RDW seems to be a universal biological feature rather than related with a single type of hematology analyzer. As not only RDW, but also MCV increases with age, we propose that future studies on the prognostic significance of RDW take its age-dependency into account and focus on RDW-SD as a potential marker of adverse events in many clinical conditions.

Iron depletion in blood donors - Have extended erythrocyte and reticulocyte parameters diagnostic utility?

BACKGROUND: Blood donation is associated with iron depletion, but donor iron status is not usually investigated, as such tests are cumbersome and costly. It would therefore be desirable to have simple, fast and inexpensive tests that give information on a donor's risk of developing iron depletion. In a pilot study we investigated whether novel erythrocyte and reticulocyte parameters can serve this goal. METHODS: In regular blood donors extended red cell parameters were measured using the Abbott CELL-DYN Sapphire hematology analyzer and conventional biochemical tests of iron status. Donors were compared with a regionally matched group of non-donating controls. RESULTS: In the controls, the reference ranges of extended RBC parameters were well comparable to published data. Donors had significantly more microcytic RBC than controls (median 0.9 vs 0.6%), lower serum ferritin concentration (median 43 vs 91 mg/L) and higher soluble transferrin receptor/ferritin index (median 1.60 vs 1.27). Overall 18-28% of the donors were iron depleted. Moreover, 3.3% of donors had iron-restricted erythropoiesis. Microcytic RBC and reticulocyte mean cell hemoglobin content predicted iron depletion with 70% and 64% sensitivities and specificities of 72% and 78%, respectively. When combined these two parameters increased the sensitivity to 82%. CONCLUSIONS: Our results in Swedish blood donors confirm a high prevalence of iron depletion, despite iron supplementation used by about half of the donors. Microcytic RBC and MCHr appeared to be helpful in identifying iron-depleted donors, who might benefit from iron supplementation. We recommend larger prospective investigations in order to confirm and extend the findings of this pilot study.

Reticulated platelets: analytical aspects and clinical utility.

Reticulated platelets are immature platelets circulating in blood; they reflect the activity of megakaryopoiesis in the bone marrow. Therefore, they can be used as a non-invasive test in patients with thrombocytopenia in various clinical conditions. The preferred method of analysis is by flow cytometry. However, there is an evident lack of analytical standardization, making it difficult to compare results obtained in different laboratories. Currently, two types of hematology analyzers are on the market offering fully automated measurement of reticulated or immature platelets: the high end analyzers manufactured by Sysmex (XE- and XN-series) and Abbott (CELL-DYN Sapphire). Although the methods are essentially different and cannot be used interchangeably, both have been proven to have clinical utility. Reticulated or immature platelet assays are useful for the differential diagnosis of thrombocytopenia and for monitoring bone marrow recovery after chemotherapy or stem cell transplantation. These assays may aid clinicians in platelet transfusion decisions when recovery from thrombocytopenia is imminent. In addition, preliminary findings indicate that there is a rationale for reticulated or immature platelets for risk stratification in acute coronary syndromes and for monitoring the effect of treatment with antiplatelet drugs in patients with coronary artery diseases. The aim of this paper is to present the present technology available for measuring reticulated platelets as well as an overview of the current status of clinical application. This overview also indicates that more research is needed before reticulated or immature platelet assays can be applied in other clinical conditions than thrombocytopenia and after transplantation.

Immunological screening for tumor cells in serous body fluids has added value with the CELL-DYN Sapphire.

BACKGROUND: Conventional cytological examination has limited sensitivity for detecting tumor cells in serous body cavity effusions and therefore, adjuvant techniques are necessary for a reliable diagnosis. Flow cytometry has proven benefit in these circumstances. The aim of our study was to explore the feasibility of CELL-DYN Sapphire, an advanced hematology analyzer with flow cytometric capabilities, for detecting tumor cells in serous body fluids, using CD326 monoclonal antibodies, which are directed against the epithelial marker EpCAM. METHODS: One hundred and five serous fluids (39 peritoneal and 66 pleural effusions) were analyzed by the CELL-DYN Sapphire using monoclonal antibody combinations CD3/CD19 and CD45/CD326. Of all samples a cytospin preparation was made and microscopically examined; the pathology findings served as a reference. RESULTS: Using a threshold of 1% CD326+ cells, CELL-DYN Sapphire identified nine out of 12 cases with tumor cells in the serous effusions (sensitivity 75%), whereas routine cytology found eight cases (sensitivity 67%). The combination of immunophenotyping and cytology identified all 12 cases with tumor cells in the effusion fluid (sensitivity 100%). The specificities were 92% and 100%, respectively. CONCLUSIONS: We demonstrated that it is feasible to run an immunophenotypic assay on CELL-DYN Sapphire for detecting tumor cells in serous body fluids. In addition, this study confirmed that a combination of conventional cytology and flow cytometry had a very high diagnostic yield in cases of carcinomatous effusions.

Laboratory hematology in the history of Clinical Chemistry and Laboratory Medicine.

BACKGROUND: For the occasion of the 50th anniversary of the journal Clinical Chemistry and Laboratory Medicine (CCLM), an historic overview of papers that the journal has published in the field of laboratory hematology (LH) is presented. METHODS: All past volumes of CCLM were screened for papers on LH and these were categorized. Bibliographic data of these papers were also analyzed. RESULTS: CCLM published in total 387 LH papers. The absolute number of LH papers published annually showed a significant increase over the years since 1985. Also the share of LH papers demonstrated a steady increase (overall mean 5%, but mean 8% over the past 4 years). The most frequent category was coagulation and fibrinolysis (23.5%). Authors from Germany contributed the most LH papers to the journal (22.7%), followed by the Netherlands and Italy (16.3 and 13.2%, respectively). Recent citation data indicated that other publications cited LH review papers much more frequently than other types of papers. CONCLUSIONS: The history of the journal reflects the emergence and development of laboratory hematology as a separate discipline of laboratory medicine.

Analysis of serous body fluids using the CELL-DYN Sapphire hematology analyzer.

BACKGROUND: Correct cell enumeration and differential analysis of body fluids are important in the diagnosis and management of several diseases. Currently, microscopic analysis is still considered the "gold standard". The aim of the present study was to evaluate the analytical performance of the CELL-DYN Sapphire hematology analyzer for automated differentiation of cells in serous fluids and to explore whether manual analysis of the raw data files could improve the differential count compared with reference microscopy. METHODS: A total of 105 serous fluids (39 peritoneal and 66 pleural effusions) were analyzed by the CELL-DYN Sapphire using standard whole-blood algorithm. Additionally, we performed optimized manual gating of the Sapphire raw data file using standard flow cytometry software. RESULTS: The standard Sapphire algorithm showed substantial deviations from the reference microscopic differentiation: polymorphonuclear cell counts were too high because they contained some monocytic cells. However, when optimized manual gating strategy is used, a good correlation and negligible bias were found. CONCLUSIONS: We have demonstrated that with a modified algorithm, CELL-DYN Sapphire will provide reliable identification and enumeration of blood cells in peritoneal and pleural fluids.

Reference intervals of extended erythrocyte and reticulocyte parameters.

BACKGROUND: Optical analysis of erythrocytes can provide information on the hemoglobin concentration and content of reticulocytes and mature erythrocytes. Such parameters have proven clinical utility in anemia diagnosis and therapy monitoring. For interpretation, reliable reference ranges are needed. The aim of this study was to establish reference intervals for extended erythrocyte and reticulocyte parameters as measured with the Abbott CELL-DYN Sapphire hematology analyzer. Secondary aims were to study sample stability and to investigate gender- and age dependency of the reference ranges. METHODS: Extended RBC parameters were measured in routine samples of a primary health care laboratory. The study cohort included 8161 samples of unique individuals, which were analyzed using Bhattacharya statistics. As a comparison, reference intervals were calculated in a subset of individuals without iron depletion. RESULTS: The majority of erythrocyte and reticulocyte parameters were normally distributed, allowing calculation of reference intervals. Only for hypo- and hyperchromic erythrocytes non-parametric statistics had to be used. The reference range for mean cellular hemoglobin content of reticulocytes (MCHr) was 28.5-34.5 pg (1.77-2.14 fmol) in the entire study group and 26.0-35.1 pg (1.60-2.17 fmol) in the non iron-depleted subgroup. No differences between sexes were found. Most parameters showed significant age effects in children and adolescents. CONCLUSIONS: Reference intervals have been established for extended RBC and reticulocyte parameters for the CELL-DYN Sapphire. Gender effects could not be demonstrated and age effects were of limited size, except for individuals younger than 18 years. Extended RBC parameters are stable for at least 6 h after blood collection.