Evaluation of the Primus Ultra2 HPLC system for HbA2 measurement and β-thalassemia screening

Uvod: Primus Ultra2 je analizator na kojem se metodom tekućinske kromatografije visoke djelotvornosti (engl. high-performance liquid chromatography, HPLC) pomoću ionske izmjene obavlja kvantitativna i kvalitativna analiza frakcija hemoglobina: u 4 minute moguće je odrediti točnu koncentraciju HbF, HbA2, HA0 i detektirati druge vrste hemoglobina. Cilj istraživanja je ocjena analitičkog učinka analizatora Primus Ultra2 u rutinskoj procjeni sadržaja HbA2, najvažnijeg testa za identifikaciju nosioca β-talasemije. Materijali i metode: Provedena su istraživanja nepreciznosti, linearnosti i netočnosti prema CLSI standardima (engl. Clinical Laboratory Standards Institute, CLSI) i smjernicama proizvođača. Pouzdanost mjerenja koncentracije HbA2 u svrhu određivanja β-talasemije ispitana je na 300 uzoraka bolesnika s prethodno postavljenom dijagnozom β-talasemije. Rezultati: Nepreciznost. Koeficijent varijacije (engl. coeficient of variation, CV) unutar serije kod normalne koncentracije HbA2 (2,2%) iznosio je 1,9%, a kod povišene koncentracije (5,5%) 1,3%. CV iz serije u seriju iznosili su 2,4% (normalna koncentracija) i 1,7% (povišena koncentracija). CV iz dana u dan bili su 3,0% (normalna koncentracija) i 2,7% (povišena koncentracija). Ukupni koeficijenti varijacije bili su 4,4% (normalna koncentracija) i 3,5% (povišena koncentracija). Linearnost. Y = 1,089x + 0,01; R2= 0,992 (raspon 2,4-5,0%). Izmjerene su vrijednosti dobro korelirale s očekivanima; prosječno iskorištenje bio je 99%. Netočnost. Kod kontrolnog uzorka normalne koncentracije greška je iznosila 1,6% ± 0,26%, a kod kontrolnog uzorka povišene koncentracije 5,2% ± 0,19%. Referentni raspon bio je 0,9-3,0% (srednja vrijednost 1,9%, ± 2,5 SD). Svi ispitani nosioci β-talasemije imali su povećanu koncentraciju HbA2 u krvi [4,7% (3,3-6,9%)]. Zaključak: Analizator Primus Ultra2 omogućuje brzo i pouzdano razdvajanje različitih frakcija hemoglobina. Mjerenje koncentracije HbA2 točno je i ponovljivo, što je neophodno zbog male razlike između normalnih i patoloških vrijednosti. Postoji jasna razlika u koncentracijama HbA2 između zdravih ispitanika i nosioca β-talasemije. Granična se vrijednost za zdravu populaciju može se postaviti na 3,3% HbA2. Određivanje koncentracije HbA2 na analizatoru Primus Ultra2 je pogodna metoda za brzo probiranje unutar populacije na nosioce β-talasemije.Background: Primus Ultra2 is a HPLC ion exchange analyzer for qualitative and quantitative hemoglobin fraction analysis: in 4 minutes it quantifies HbF, HbA2, HA0 and flags abnormal hemoglobins. The aim of the study was to evaluate analytical performance of Primus Ultra2 analyzer for routine estimation of HbA2, a critical test to identify β-thalassemia carriers. Methods: The imimprecision, linearity and inaccuracy studies were performed according to CLSI and manufacturer\u27s guidelines. The reliability of HbA2 measurements for detection of β-thalassemia was studied by analysis of 300 samples from patients with previous diagnosis of the disease. Results: Imprecision. Within run coefficient of variation (CV) was 1.9% for normal HbA2 level (2.2%) and 1.3% for elevated level (5.5%). Between run CV was 2.4% for normal level and 1.7% for elevated level. Between-day CV was 3.0% for normal level and 2.7% for elevated level. Total CV was 4.4% for normal level and 3.5% for elevated level. Linearity. Y = 1.089x + 0.01; R2 = 0.992 (range 2.4-5.0%). The measured values correlated with the expected ones; the mean proportion recovered was 99%. Inaccuracy. The mean was 1.6% (SD 0.26%) for normal control and 5.2% (SD 0.19%) for high control. Reference range was 0.9-3.0% (mean 1.9%±2.5 SD). All β-thalassemia carriers had elevated HbA2 blood concentrations (mean 4.7%, range 3.3-6.9%). Conclusions: Primus Ultra2 provides a rapid and reliable separation of different hemoglobins. HbA2 measurement is accurate and reproducible, which is needed due to the slight difference between normal and pathologic values. There is good differentiation of HbA2 values between normal subjects and β-thalassemia carriers. The cut off limit can be set at 3.3% HbA2. This is a suitable method for rapid population screening for β-thalassemia carriers

Erythrocyte and Reticulocyte Indices on the LH 750 as Potential Markers of Functional Iron Deficiency

Reticulocyte hemoglobin content (CHr) and percentage of hypochromic cells (%Hypo) are restricted to the Siemens analysers. The aims of the study were to investigate the correlation of Red cells size factor (RSf) and low Hemoglobin density (LHD%), reported by Beckman-Coulter analysers, with CHr and %Hypo in the assessment of iron status in the presence of inflammation. 381 samples were run on both LH 750 (Beckman-Coulter) and Advia 2120 (Siemens) analysers. Correlation between parameters were calculated and the diagnostic performance of the new parameters was assessed. Results. Correlation between RSf and CHr, r = 0.85. ROC curve analysis for RSf in the diagnosis of iron restricted erythropoiesis defined as CHr < 28 pg: AUC 0.983; Cutoff 91.1%; Sensitivity 98.8%; Specificity 89.6% Correlation between LHD% and %Hypo, r = 0.869. ROC curve analysis for LHD% in the diagnosis of iron deficiency defined by %Hypo >5%: AUC 0.954; Cut off 6.0%; Sensitivity 96.6%; Specificity 83.2% Conclusions. RSf and LHD% could be reliable parameters for the study of iron metabolism status

Discriminant Value of %Microcytic Cells/%Hypochromic Cells Ratio in the Differential Diagnosis of Microcytic Anemia

The Mindray 6800 Plus analyzer reports red cells (RBC) extended parameters, which represent the subsets of erythrocytes. We aimed to evaluate the reliability of RBC extended parameters in the differential diagnosis of microcytic anemia. The learning set comprised samples from 250 patients with microcytic anemia mean cell volume &lt;80 fL. MH ratio (%microcytic cells/%hypochromic cells) and other discriminant functions were calculated. Optimal cut offs were established using receiver operator curves. This value was used in the validation set of 135 patients 50 carriers and 85 with mild iron deficiency anemia (IDA). Area under the curve 0.945 (95% confidence interval 0.890 to 0.977), cut off &gt;10 rendered the best Youden index (0.798), sensitivity 93.2%, specificity 86.2%. In the validation set using MH ratio &gt;10, 45 in 50 patients were correctly classified as carriers. All of 40 beta carriers were correctly classified, while the 5 false negatives resulted to be alpha carriers. In the IDA group 5 patients had MH ratio &gt;10 and thus considered carriers, but all of them had Hyper &lt;3%. The combination of MH ratio &gt;10 and %Hyper &lt;3% correctly classified 100% of IDA patients. An algorithm derived from RBC extended parameters provided by the Mindray 6800 Plus analyzer could be a useful tool in the differential diagnosis of microcytic anemia

Multivariable Discriminant Analysis for the Differential Diagnosis of Microcytic Anemia

Introduction. Iron deficiency anemia and thalassemia are the most common causes of microcytic anemia. Powerful statistical computer programming enables sensitive discriminant analyses to aid in the diagnosis. We aimed at investigating the performance of the multiple discriminant analysis (MDA) to the differential diagnosis of microcytic anemia. Methods. The training group was composed of 200 β-thalassemia carriers, 65 α-thalassemia carriers, 170 iron deficiency anemia (IDA), and 45 mixed cases of thalassemia and acute phase response or iron deficiency. A set of potential predictor parameters that could detect differences among groups were selected: Red Blood Cells (RBC), hemoglobin (Hb), mean cell volume (MCV), mean cell hemoglobin (MCH), and RBC distribution width (RDW). The functions obtained with MDA analysis were applied to a set of 628 consecutive patients with microcytic anemia. Results. For classifying patients into two groups (genetic anemia and acquired anemia), only one function was needed; 87.9% β-thalassemia carriers, and 83.3% α-thalassemia carriers, and 72.1% in the mixed group were correctly classified. Conclusion. Linear discriminant functions based on hemogram data can aid in differentiating between IDA and thalassemia, so samples can be efficiently selected for further analysis to confirm the presence of genetic anemia

Potential utility of the new Sysmex XE 5000 red blood cell extended parameters in the study of disorders of iron metabolism

Background: New erythrocyte parameters are reported by the Sysmex XE 5000 analyzer. This instrument measures the hemoglobin (Hb) content of individual red cells, calculates the percentage of hypochromic red cells (%Hypo He) and the percentage of hyperchromic red cells (%Hyper He) and quantifies the proportion of marginally sized erythrocytes (%Micro R and %Macro R). The goals of the study were to establish the reference range for erythrocyte extended parameters, their value in different types of anemia and to investigate their reliability in the study of disorders of iron metabolism. Materials: Three hundred and ninety samples were analyzed. The Kolmogorov-Smirnoff test, independent samples t-test and Pearson correlation were calculated; receiver operating characteristic (ROC) curve analysis was used to determine their diagnostic performance. Results: The values of the four parameters studied were normally distributed and statistically different (p&#60;0.0001) in the different groups of patients; the only exception was %Hypo He in cases of iron deficiency and thalassemia (p=0.3758). Results of ROC curve analysis for %Hypo He in the diagnosis of restricted erythropoiesis reticulocyte Hb content (reticulocyte hemoglobin content, Ret He &#60;29 pg) were: area under the curve 0.963; cut-off 1.8%; sensitivity 98.3%; specificity 91.1%. Conclusions: The new parameters appear to be sensitive for detecting small changes in the number of red cells with inadequate hemoglobinization and volume. Clin Chem Lab Med 2009;47:1411–6.Peer Reviewe

Biomarkers of Hypochromia: The Contemporary Assessment of Iron Status and Erythropoiesis

Iron status is the result of the balance between the rate of erythropoiesis and the amount of the iron stores. Direct consequence of an imbalance between the erythroid marrow iron requirements and the actual supply is a reduction of red cell hemoglobin content, which causes hypochromic mature red cells and reticulocytes. The diagnosis of iron deficiency is particularly challenging in patients with acute or chronic inflammatory conditions because most of the biochemical markers for iron metabolism (serum ferritin and transferrin ) are affected by acute phase reaction. For these reasons, interest has been generated in the use of erythrocyte and reticulocyte parameters, available on the modern hematology analyzers. Reported during blood analysis routinely performed on the instrument, these parameters can assist in early detection of clinical conditions (iron deficiency, absolute, or functional; ineffective erythropoiesis, including iron restricted or thalassemia), without additional cost. Technological progress has meant that in recent years modern analyzers report new parameters that provide further information from the traditional count. Nevertheless these new parameters are exclusive of each manufacturer, and they are patented. This is an update of these new laboratory test biomarkers of hypochromia reported by different manufactures, their meaning, and clinical utility on daily practice

Red cell cytogram in CELL-DYN® Sapphire: a ready-to-use function for recognizing thalassemia trait

Single-cell optical analysis of red blood cells provides information on the cellular hemoglobin concentration and volume of red cells. We evaluated the reliability of the typical profiles of the cytogram hemoglobin concentration/ volume (Mie Map), produced by the CELL-DYN® Sapphire analyzer (Abbott Diagnostics, Santa Clara, CA, USA) in the discrimination of iron deficiency anemia (IDA) and thalassemia trait. A total of 380 patients with microcytic anemia were studied: 220 with IDA, 101 β-thalassemia trait, 30 β-thalassemia trait with concomitant iron deficiency, 29 α-thalassemia trait. Three professionals reviewed the Mie maps, with no information regarding the disease of the patient. The observers made a presumptive diagnosis (genetic or acquired anemia) and the percentages of correct classifications were recorded. IDA showed broad shaped shift of the cytogram while carriers presented narrow clustering in the lower microcytic area: 100 % IDA were correctly classified and 96-82% of carriers were recognized. Visual inspection of the Mie map reveals different profiles in IDA and thalassemia trait; those patterns are in concordance with the numerical data Mie map helps in the evaluation of large amounts of data. 红细胞单细胞光学分析提供了关于细胞血红蛋白浓度及红细胞体积的信息。 我们评价了典型的细胞图血红蛋白浓度/体积分布（Mie Map）在缺铁性贫血（IDA）和地中海贫血特征的识别方面的可靠性，分布曲线由CELL-DYN® Sapphire分析仪（Abbott Diagnostics, Santa Clara, CA, USA）生成。 一共对380例小细胞性贫血进行了研究：220例患有IDA，101例β有地中海贫血特征，30例β有地中海贫血特征合并缺铁性，29 α例地中海贫血特征。 由三名专业人员在没有任何患者病情信息的情况下进行Mie map读图。 读图者作出初步诊断（遗传性或获得性贫血），记录正确分类的百分比。 IDA表现出细胞图较宽发散形状的偏移，而基因携带者在更低的小细胞区域呈现较窄的聚集：100%的IDA被正确分类，96-82%的基因携带者得到确认。 Mie map的目测检查揭示了IDA和地中海贫血特征方面不同的分布；这些模式与数值数据相一致。Mie map有助于大量数据的评估。</p