Ann Hematol DOI 10.1007/s00277-014-2127-8
ORIGINAL ARTICLE
Automated reticulocyte parameters for hereditary spherocytosis screening Elena Lazarova & Olivier Pradier & Frédéric Cotton & Béatrice Gulbis
Received: 2 April 2014 / Accepted: 1 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The laboratory diagnosis of hereditary spherocytosis (HS) is based on several screening and confirmatory tests; our algorithm includes clinical features, red blood cell morphology analysis and cryohaemolysis test, and, in case of positive screening, sodium dodecyl sulphate polyacrylamide gel electrophoresis as a diagnostic test. Using the UniCel DxH800 (Beckman Coulter) haematology analyser, we investigated automated reticulocyte parameters as HS screening tool, i.e. mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF) and mean sphered cell volume (MSCV). A total of 410 samples were screened. Gel electrophoresis was applied to 159 samples that were positive for the screening tests. A total of 48 patients were diagnosed as HS, and seven were diagnosed as acquired autoimmune haemolytic anaemia (AIHA). Some other 31 anaemic conditions were also studied. From the receiver operating characteristic (ROC) curve analysis, both delta (mean cell volume (MCV)-MSCV) and MRV presented an area under the curve (AUC) of 0.98. At the diagnostic cut-off of 100 % sensitivity, MRV showed the best specificity of 88 % and a positive likelihood ratio of 8.7. The parameters IRF, MRV and MSCV discriminated HS not only from controls and other tested pathologies but also from AIHA contrary to the cryohaemolysis test. In conclusion, automated reticulocyte parameters might be helpful for haemolytic anaemia Electronic supplementary material The online version of this article (doi:10.1007/s00277-014-2127-8) contains supplementary material, which is available to authorized users. E. Lazarova (*) : F. Cotton : B. Gulbis Department of Clinical Chemistry, Erasme Hospital, Université Libre de Bruxelles, Route de Lennik, 808, 1070 Brussels, Belgium e-mail: [email protected] E. Lazarova : O. Pradier Laboratory of Haematology, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium
diagnostic orientation even for general laboratories. In combination with cryohaemolysis, they ensure an effective and time-saving screening for HS for more specialised laboratories. Keywords Hereditary spherocytosis . Cryohaemolysis . Reticulocyte parameters . Mean reticulocyte volume . Mean sphered cell volume
Introduction Hereditary haemolytic anaemia diagnosis is a complex and laborious procedure that takes into account the clinical features and laboratory findings. This group of pathologies is very heterogeneous according to disease severity, age of onset and diagnostic tools applied. Several screening and confirmatory tests are largely used in order to rule out haemoglobinopathies, intra-erythrocyte enzyme deficiencies and acquired haemolytic anaemia. When these pathologies have been excluded, the group of hereditary red blood cell (RBC) membrane pathologies comes into attention in the diagnostic process. In this group, hereditary spherocytosis (HS) is the most frequent RBC membrane pathology [1–3]. Guidelines on HS diagnostic testing have been recently updated [3], and key recommendations are as follows: HS diagnosis can be made on a simple base of family history, typical clinical features and simple laboratory investigations like the presence of spherocytes, raised mean corpuscular haemoglobin concentration (MCHC) and increased reticulocyte number. The principle of the traditional laboratory screening tests for HS lies on the reduced area-to-volume ratio, as found in spherocytes. Abnormal RBC indices like MCHC >36 g/dL, due to a relative dehydration, have been proposed, but their sensitivity is low [4]. Generally, fully expanded cells, like microspherocytes, have increased
Ann Hematol
osmotic fragility. Based on that characteristic, many tests have been developed with also variable sensitivity [5, 6]. The oldest one is the osmotic fragility test [7]. Being cumbersome and requiring a high volume of blood, it was abandoned and has been replaced by alternative tests, i.e. the glycerol lysis test [8], the acidified glycerol lysis test [9] and the Pink test [10]; their usefulness as screening tests for HS has been recently evaluated [6]. Two other screening tests, cryohaemolysis [11] and eosin 5-maleimide (EMA)-binding test [12], have been also introduced. Being related to red cell membrane protein defects, they were suggested to be more specific to HS and have been recommended as screening tests if the diagnosis of HS is equivocal [3, 13]. Unfortunately, with reported sensitivities of 53 to 95 %, none of those tests performed alone could recognise all cases of HS. To increase the negative and positive predictive values of the tests, it has been suggested to associate two screening tests, for example the EMA-binding test and the acidified glycerol lysis test [6]. Evidence is accumulating that the spheroid shape, result of membrane surface area loss, is a multi-factorial phenomenon. On the one hand, there is a progressive release of microvesicles once the erythrocyte is in the circulation and further loss of membrane because of reduced deformability as it passes through the spleen, i.e. splenic conditioning, reviewed by Perrotta et al. [14]. On the other hand, the decreased surface area is also a reticulocyte feature [15] as it occurs already during erythropoiesis due to various mechanisms: abnormal protein assembly on the erythroblast membrane [16] and protein missorting during erythroblast enucleation or remodelling processes [17]. Moreover, it has been shown that reduced membrane surface area-to-volume ratio and increased haemoglobin concentration are specifically present at the reticulocyte stage in HS but not in normal conditions or in autoimmune haemolytic anaemia [15]. Consequently, it was hypothesised that automated reticulocyte parameters could be of great interest for hereditary spherocytosis screening especially if able to demonstrate the presence of small dehydrated reticulocytes coexistent with spherocytes [15]. Last generation haematological equipment offer new parameters derived from RBC and reticulocyte analysis such as hyperchromic RBC percentage [18], mean reticulocyte volume (MRV or MCVr), immature reticulocyte fraction (IRF), reticulocyte haemoglobin equivalent or content (Ret-He or CHr) and reticulocyte distribution width (RDWR), reviewed recently by Piva et al. [19]. There are only few published data concerning the utility of those reticulocyte parameters in the screening for HS. One study showed that IRF, in combination with reticulocyte count, might be useful in HS diagnosis, as HS is characterised by a high reticulocyte count without an equally elevated IRF [20]. In addition to MRV and IRF, Beckman Coulter instruments provide an artificial volume measurement during the reticulocyte count, i.e. the mean sphered corpuscular volume (MSCV). The whole RBC
population is analysed under the hypo-osmotic conditions of the specific ghosting solution that is used to slightly swell the RBC and to leak out the haemoglobin before the reticulum definition of the reticulocytes. A preliminary study based on parameters of the Coulter GEN•S haematology analyser concluded that in case of observation of MSCV smaller than mean cell volume (MCV), the diagnosis of HS could be suggested with a sensitivity of 100 % [21]. Later, MSCV was evaluated on the Beckman Coulter LH 750 haematology analyser: when the delta (MCV-MSCV) value was greater than 9.6 fL, HS could be suspected and differential diagnosis with AIHA by the anti-globulin test was proposed [22]. Our laboratory acquired new routine UniCel DxH800 haematological analysers (Beckman Coulter), and we were interested in investigating the new automated reticulocyte parameters as a screening tool for HS. The objectives of the present study were (a) to establish reference values for our population of the “research only” automated reticulocyte parameters, (b) to analyse the diagnostic performances of these parameters for HS compared to the cryohaemolysis test as a well-defined screening test, (c) to estimate the efficiency of those new reticulocyte parameters, i.e. MSCV, MRV and IRF, to differentiate HS from other conditions that affect erythropoiesis, and (d) if relevant, to introduce those parameters in a new screening algorithm for HS.
Methods and materials Patients In order to provide HS diagnosis on a routine basis, our working algorithm was as follows: in case of negative results of the screening tests, i.e. RBC morphology and cryohaemolysis, and no family history of HS, the patient is considered as negative for HS. If one of the screening tests is positive, as well as in the case of extended family studies, the confirmatory test, i.e. sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), is performed. For the period from June 2010 till May 2013, a total of 668 samples were received in our laboratory for RBC membrane pathology screening and diagnosis. Among them, 452 samples (68 %) were tested with the “research only” RBC and automated reticulocyte parameters on UniCel DxH800 haematology analysers within 24 h of blood sampling. Fortytwo samples were excluded from the analysis: 21 because of incorrect sampling for the confirmatory SDS-PAGE test, 4 from patients with a sickle cell syndrome and 17 from 13 patients because of the presence of doubles or triples. Finally, a total of 410 samples with available results of the new haematological parameters and cryohaemolysis test, with or without the confirmatory diagnostic test, according to our algorithm, were included in this study. Additionally, a control
Ann Hematol
group of 82 samples of the routine work flow with normal haematological and biochemical parameters was constituted, and the cryohaemolysis test and the new automated parameters on UniCel DxH800 haematology analysers were performed. Population for establishing adult reference values and stability of MRV, MSCV, RDW, RDWR, delta (MCV-MSCV), IRF and Ret/IRF Our local haematology reference values of the adult population (>18 years of age), in agreement with published data [23], are as follows: RBC count (×1012/L) 3.8–4.8 for females and 4.5–5.5 for males, haemoglobin (g/dL) 12.0–15.5 for females and 13–17 for males, reticulocyte count (103/μL) 20–110, MCV (fL) 82–100 and MCH (pg) 27–33. We applied these criteria for non-anaemic sample selection on a 2-month database extracted from our UniCel DxH800 instruments. A total of 2,400 (7 %) samples from 32,614 patient samples were analysed during the routine workflow on reticulocyte mode, so data for reticulocyte count, MRV, IRF, RDWR and MSCV were available. A total of 522 non-anaemic samples from females and 375 non-anaemic samples from males were selected in order to calculate the reference values of MRV, MSCV, red cell distribution width (RDW), RDWR, delta (MCV-MSCV), IRF and ratio of reticulocyte count/IRF (Ret/IRF), in agreement with the local and published reference values for the non-anaemic population. The stability of the automated parameters was analysed for different conservation temperatures, 4 and 20 °C by a Student paired t test. Assays Cryohaemolysis test The cryohaemolysis test was performed following the method of Streichman et al. [11] with slight modifications. Briefly, 50 μL of washed red cells was dispensed into 2 mL of hypertonic preheated (37 °C) sucrose solution. Following 10 min of incubation at 37 °C, each tube was transferred to an ice-cold (0–4 °C) water bath for another 10 min of incubation. The tubes were then centrifuged, and the absorbance of the supernatant was measured at 540 nm [DO TEST]. In parallel, 25 μL of washed red cells was completely lysed in 2 mL of distilled water and served as 100 % lysis value [DO 100 % lysate]. The percentage value of cryohaemolysis was obtained by the formula: % cryohaemolysis=[DO TEST]/ [DO 100 % lysate]×50. All measurements were done in duplicate, and a control sample was run in each experiment. The ranges of the cryohaemolysis test, established in our laboratory and in agreement with published data from other groups, were as follows: a result 15 % as positive [24].
RBC membrane protein analysis Erythrocyte ghosts for membrane protein quantification were prepared from heparinised blood samples by hypotonic lysis followed by washing according to Dodge et al. [25]. The pH and the concentration of lysing and washing buffers were locally adapted in order to remove the maximum amount of haemoglobin from the ghosts. SDS-PAGE was performed using a continuous buffer system (Fairbanks system) in a 4– 12 % acrylamide linear concentration gradient gel according to Fairbanks et al. [26]. The amounts of the major membrane proteins were expressed as ratios of α-spectrin to β-spectrin, spectrins to band 3, ankyrin to band 3, ankyrin to protein 4.1, protein 4.2 to band 3 and protein 4.1 to protein 4.2. Pre-stained SDS-PAGE molecular weight standards (BioRad Laboratory, Nazareth, Belgium), as well as two to four control samples, were run in each gel. Study sample ratios were compared to control sample ratios in order to identify the exact protein deficiency. DxH800 automated reticulocyte analysis The UniCel DxH800 (Beckman Coulter) instruments were regularly and constantly controlled by internal and external quality control schemes. The analytical variability of the instruments during the study period was less than 1 % for all parameters considered. We measured and reported data of MRV, MSCV, RDW, RDWR, IRF, delta (MCV-MSCV) and Ret/IRF. During the reticulocyte analysis, the Beckman Coulter UniCel DxH800 instrument uses staining with new methylene blue, followed by slight swelling with an acidic, hypotonic ghosting solution that clears the haemoglobin while preserving the stained RNA within the reticulocytes [27]. The sphered volume of the whole RBC population, MSCV, is measured by the unique Beckman Coulter VCS technology (volume, conductivity and light scatter). The resulting sphered cells are also classified as either mature red cells or reticulocytes. The following reticulocyte parameters are measured: RDWR, MRV and the count of reticulocytes with the greatest RNA quantity. The IRF is thus calculated as the ratio of the reticulocytes with the highest RNA content and the total number of reticulocytes. Statistical analysis Statistical analysis was performed using Analyse-it Software, Ltd. (Leeds, UK) and GraphPad Prism software (San Diego, USA). Reference values were defined as 2.5–97.5 percentile intervals [28]. A Student t test was used to evaluate statistical significance for the parameters with normal distribution. A Mann-Whitney t test or one-way ANOVA Kruskal-Wallis test was used for parameters with a non-Gaussian distribution.
Ann Hematol
One-way analysis of variance (Dunnett’s multiple comparison test) was used to compare values among different groups.
Results Reference values and stability of MRV, MSCV, RDW, RDWR, delta (MCV-MSCV), IRF and Ret/IRF After applying the local and published reference values for the adult non-anaemic population (see “Methods and materials”), we selected 522 samples from females and 375 samples from males over a 2-month period of routine work flow. Our adult reference values of MRV, MSCV, RDW, RDWR, delta (MCVMSCV), IRF and Ret/IRF are presented on Table 1. The stability of all automated parameters for 24 h at 4 °C was warranted by statistically non-significant Student t test; the same was observed at 20 °C for Ret/IRF and MRV. On the opposite, MCV, MSCV and delta (MCV-MSCV) presented statistically significant Student t test in the 24-h stability study at 20 °C with respective mean differences of 3.13, 7.14 and 10.3. Patients The distribution of the cohort of patients studied (a total of 410) was as follows: 216 females (53 %) and 194 males (47 %), mean age 30 (range 0.01–89). A total of 251 samples were considered negative on the basis of both negative screening tests, another 159 samples were analysed by SDS-PAGE and different pathologies were diagnosed or excluded (Fig. 1). The results of a total of 26 samples (16 % of all SDS-PAGE electrophoresis) were not interpretable, i.e. 19 samples presenting typical SDS-PAGE profile of reactive reticulocytosis (increased ankyrin and/or spectrin) and 7 samples showing degraded electrophoretic profile. Following our working algorithm, 319 patients were found negative for HS: 251 had negative screening tests and 68 showed a non-pathological SDS-PAGE electrophoretic profile. Haematological parameters of the studied cohort Table 2 shows data obtained for the classical automated haematological parameters for the following groups: (a) control group consisting of 82 negative controls and 131 negative samples presenting negative screening tests and haematology results within the reference values; (b) cryohaemolysis negative samples, consisting of 82 negative controls and some other 292 samples: 251 with negative screening tests plus 41 SDS-PAGE negative samples with cryohaemolysis test result less than 10 %; (c) a total of 48 HS cases confirmed by SDSPAGE; (d) 7 cases of autoimmune haemolytic anaemia; (e) 2
Table 1 Reference interval (2.5–97.5 percentile distribution) of RBC and reticulocyte parameters Parameter
Women
Men
RDW (%) MRV (fL) IRF Ret/IRF (%)
12.3 to 16.9 97.6 to 122.1 0.25 to 0.52 0.71 to 2.34
12.5 to 16.4 98.6 to 123.2 0.24 to 0.5 0.8 to 2.69
RDWR (%) MSCV (fL) Delta (MCV-MSCV)
22.2 to 30 74.3 to 95.7 −0.02 to 16.9
21.6 to 30.4 72.9 to 94.9 −0.2 to 16.8
cases of hereditary elliptocytosis (HE); and (f) 2 cases of hereditary pyropoikilocytosis (HPP). Additionally, patients with confirmed hereditary spherocytosis were grouped according to the haemoglobin level in the moderate/severe anaemia group (Hb 11.5 g/dL for adult females and Hb >13.5 for adult males). Haematological and biochemical data and defective proteins for HS patients are presented in Table 3. When analysing the different groups, a rise in the reticulocyte count, oppositely proportional to the haemoglobin level, was observed and a statistically significant difference was found between the mild anaemia group and compensated forms of the HS group. However, RDW was the only haematological parameter that showed a strong statistically significant difference (p0.7
0.69 0.6–0.76 >13.3 >22
0.91 0.87–0.94 >26.4 (28.5) 10.4 fL,
that gives 100 % sensitivity in our study, is close to the one (>9.6 fL), published by Broséus et al. [22]. We also report for the first time the performances of another parameter for HS screening that is directly linked to the surface area loss at the reticulocyte stage, i.e. MRV. For MRV, at the cut-off
ORIGINAL ARTICLE
Automated reticulocyte parameters for hereditary spherocytosis screening Elena Lazarova & Olivier Pradier & Frédéric Cotton & Béatrice Gulbis
Received: 2 April 2014 / Accepted: 1 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The laboratory diagnosis of hereditary spherocytosis (HS) is based on several screening and confirmatory tests; our algorithm includes clinical features, red blood cell morphology analysis and cryohaemolysis test, and, in case of positive screening, sodium dodecyl sulphate polyacrylamide gel electrophoresis as a diagnostic test. Using the UniCel DxH800 (Beckman Coulter) haematology analyser, we investigated automated reticulocyte parameters as HS screening tool, i.e. mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF) and mean sphered cell volume (MSCV). A total of 410 samples were screened. Gel electrophoresis was applied to 159 samples that were positive for the screening tests. A total of 48 patients were diagnosed as HS, and seven were diagnosed as acquired autoimmune haemolytic anaemia (AIHA). Some other 31 anaemic conditions were also studied. From the receiver operating characteristic (ROC) curve analysis, both delta (mean cell volume (MCV)-MSCV) and MRV presented an area under the curve (AUC) of 0.98. At the diagnostic cut-off of 100 % sensitivity, MRV showed the best specificity of 88 % and a positive likelihood ratio of 8.7. The parameters IRF, MRV and MSCV discriminated HS not only from controls and other tested pathologies but also from AIHA contrary to the cryohaemolysis test. In conclusion, automated reticulocyte parameters might be helpful for haemolytic anaemia Electronic supplementary material The online version of this article (doi:10.1007/s00277-014-2127-8) contains supplementary material, which is available to authorized users. E. Lazarova (*) : F. Cotton : B. Gulbis Department of Clinical Chemistry, Erasme Hospital, Université Libre de Bruxelles, Route de Lennik, 808, 1070 Brussels, Belgium e-mail: [email protected] E. Lazarova : O. Pradier Laboratory of Haematology, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium
diagnostic orientation even for general laboratories. In combination with cryohaemolysis, they ensure an effective and time-saving screening for HS for more specialised laboratories. Keywords Hereditary spherocytosis . Cryohaemolysis . Reticulocyte parameters . Mean reticulocyte volume . Mean sphered cell volume
Introduction Hereditary haemolytic anaemia diagnosis is a complex and laborious procedure that takes into account the clinical features and laboratory findings. This group of pathologies is very heterogeneous according to disease severity, age of onset and diagnostic tools applied. Several screening and confirmatory tests are largely used in order to rule out haemoglobinopathies, intra-erythrocyte enzyme deficiencies and acquired haemolytic anaemia. When these pathologies have been excluded, the group of hereditary red blood cell (RBC) membrane pathologies comes into attention in the diagnostic process. In this group, hereditary spherocytosis (HS) is the most frequent RBC membrane pathology [1–3]. Guidelines on HS diagnostic testing have been recently updated [3], and key recommendations are as follows: HS diagnosis can be made on a simple base of family history, typical clinical features and simple laboratory investigations like the presence of spherocytes, raised mean corpuscular haemoglobin concentration (MCHC) and increased reticulocyte number. The principle of the traditional laboratory screening tests for HS lies on the reduced area-to-volume ratio, as found in spherocytes. Abnormal RBC indices like MCHC >36 g/dL, due to a relative dehydration, have been proposed, but their sensitivity is low [4]. Generally, fully expanded cells, like microspherocytes, have increased
Ann Hematol
osmotic fragility. Based on that characteristic, many tests have been developed with also variable sensitivity [5, 6]. The oldest one is the osmotic fragility test [7]. Being cumbersome and requiring a high volume of blood, it was abandoned and has been replaced by alternative tests, i.e. the glycerol lysis test [8], the acidified glycerol lysis test [9] and the Pink test [10]; their usefulness as screening tests for HS has been recently evaluated [6]. Two other screening tests, cryohaemolysis [11] and eosin 5-maleimide (EMA)-binding test [12], have been also introduced. Being related to red cell membrane protein defects, they were suggested to be more specific to HS and have been recommended as screening tests if the diagnosis of HS is equivocal [3, 13]. Unfortunately, with reported sensitivities of 53 to 95 %, none of those tests performed alone could recognise all cases of HS. To increase the negative and positive predictive values of the tests, it has been suggested to associate two screening tests, for example the EMA-binding test and the acidified glycerol lysis test [6]. Evidence is accumulating that the spheroid shape, result of membrane surface area loss, is a multi-factorial phenomenon. On the one hand, there is a progressive release of microvesicles once the erythrocyte is in the circulation and further loss of membrane because of reduced deformability as it passes through the spleen, i.e. splenic conditioning, reviewed by Perrotta et al. [14]. On the other hand, the decreased surface area is also a reticulocyte feature [15] as it occurs already during erythropoiesis due to various mechanisms: abnormal protein assembly on the erythroblast membrane [16] and protein missorting during erythroblast enucleation or remodelling processes [17]. Moreover, it has been shown that reduced membrane surface area-to-volume ratio and increased haemoglobin concentration are specifically present at the reticulocyte stage in HS but not in normal conditions or in autoimmune haemolytic anaemia [15]. Consequently, it was hypothesised that automated reticulocyte parameters could be of great interest for hereditary spherocytosis screening especially if able to demonstrate the presence of small dehydrated reticulocytes coexistent with spherocytes [15]. Last generation haematological equipment offer new parameters derived from RBC and reticulocyte analysis such as hyperchromic RBC percentage [18], mean reticulocyte volume (MRV or MCVr), immature reticulocyte fraction (IRF), reticulocyte haemoglobin equivalent or content (Ret-He or CHr) and reticulocyte distribution width (RDWR), reviewed recently by Piva et al. [19]. There are only few published data concerning the utility of those reticulocyte parameters in the screening for HS. One study showed that IRF, in combination with reticulocyte count, might be useful in HS diagnosis, as HS is characterised by a high reticulocyte count without an equally elevated IRF [20]. In addition to MRV and IRF, Beckman Coulter instruments provide an artificial volume measurement during the reticulocyte count, i.e. the mean sphered corpuscular volume (MSCV). The whole RBC
population is analysed under the hypo-osmotic conditions of the specific ghosting solution that is used to slightly swell the RBC and to leak out the haemoglobin before the reticulum definition of the reticulocytes. A preliminary study based on parameters of the Coulter GEN•S haematology analyser concluded that in case of observation of MSCV smaller than mean cell volume (MCV), the diagnosis of HS could be suggested with a sensitivity of 100 % [21]. Later, MSCV was evaluated on the Beckman Coulter LH 750 haematology analyser: when the delta (MCV-MSCV) value was greater than 9.6 fL, HS could be suspected and differential diagnosis with AIHA by the anti-globulin test was proposed [22]. Our laboratory acquired new routine UniCel DxH800 haematological analysers (Beckman Coulter), and we were interested in investigating the new automated reticulocyte parameters as a screening tool for HS. The objectives of the present study were (a) to establish reference values for our population of the “research only” automated reticulocyte parameters, (b) to analyse the diagnostic performances of these parameters for HS compared to the cryohaemolysis test as a well-defined screening test, (c) to estimate the efficiency of those new reticulocyte parameters, i.e. MSCV, MRV and IRF, to differentiate HS from other conditions that affect erythropoiesis, and (d) if relevant, to introduce those parameters in a new screening algorithm for HS.
Methods and materials Patients In order to provide HS diagnosis on a routine basis, our working algorithm was as follows: in case of negative results of the screening tests, i.e. RBC morphology and cryohaemolysis, and no family history of HS, the patient is considered as negative for HS. If one of the screening tests is positive, as well as in the case of extended family studies, the confirmatory test, i.e. sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), is performed. For the period from June 2010 till May 2013, a total of 668 samples were received in our laboratory for RBC membrane pathology screening and diagnosis. Among them, 452 samples (68 %) were tested with the “research only” RBC and automated reticulocyte parameters on UniCel DxH800 haematology analysers within 24 h of blood sampling. Fortytwo samples were excluded from the analysis: 21 because of incorrect sampling for the confirmatory SDS-PAGE test, 4 from patients with a sickle cell syndrome and 17 from 13 patients because of the presence of doubles or triples. Finally, a total of 410 samples with available results of the new haematological parameters and cryohaemolysis test, with or without the confirmatory diagnostic test, according to our algorithm, were included in this study. Additionally, a control
Ann Hematol
group of 82 samples of the routine work flow with normal haematological and biochemical parameters was constituted, and the cryohaemolysis test and the new automated parameters on UniCel DxH800 haematology analysers were performed. Population for establishing adult reference values and stability of MRV, MSCV, RDW, RDWR, delta (MCV-MSCV), IRF and Ret/IRF Our local haematology reference values of the adult population (>18 years of age), in agreement with published data [23], are as follows: RBC count (×1012/L) 3.8–4.8 for females and 4.5–5.5 for males, haemoglobin (g/dL) 12.0–15.5 for females and 13–17 for males, reticulocyte count (103/μL) 20–110, MCV (fL) 82–100 and MCH (pg) 27–33. We applied these criteria for non-anaemic sample selection on a 2-month database extracted from our UniCel DxH800 instruments. A total of 2,400 (7 %) samples from 32,614 patient samples were analysed during the routine workflow on reticulocyte mode, so data for reticulocyte count, MRV, IRF, RDWR and MSCV were available. A total of 522 non-anaemic samples from females and 375 non-anaemic samples from males were selected in order to calculate the reference values of MRV, MSCV, red cell distribution width (RDW), RDWR, delta (MCV-MSCV), IRF and ratio of reticulocyte count/IRF (Ret/IRF), in agreement with the local and published reference values for the non-anaemic population. The stability of the automated parameters was analysed for different conservation temperatures, 4 and 20 °C by a Student paired t test. Assays Cryohaemolysis test The cryohaemolysis test was performed following the method of Streichman et al. [11] with slight modifications. Briefly, 50 μL of washed red cells was dispensed into 2 mL of hypertonic preheated (37 °C) sucrose solution. Following 10 min of incubation at 37 °C, each tube was transferred to an ice-cold (0–4 °C) water bath for another 10 min of incubation. The tubes were then centrifuged, and the absorbance of the supernatant was measured at 540 nm [DO TEST]. In parallel, 25 μL of washed red cells was completely lysed in 2 mL of distilled water and served as 100 % lysis value [DO 100 % lysate]. The percentage value of cryohaemolysis was obtained by the formula: % cryohaemolysis=[DO TEST]/ [DO 100 % lysate]×50. All measurements were done in duplicate, and a control sample was run in each experiment. The ranges of the cryohaemolysis test, established in our laboratory and in agreement with published data from other groups, were as follows: a result 15 % as positive [24].
RBC membrane protein analysis Erythrocyte ghosts for membrane protein quantification were prepared from heparinised blood samples by hypotonic lysis followed by washing according to Dodge et al. [25]. The pH and the concentration of lysing and washing buffers were locally adapted in order to remove the maximum amount of haemoglobin from the ghosts. SDS-PAGE was performed using a continuous buffer system (Fairbanks system) in a 4– 12 % acrylamide linear concentration gradient gel according to Fairbanks et al. [26]. The amounts of the major membrane proteins were expressed as ratios of α-spectrin to β-spectrin, spectrins to band 3, ankyrin to band 3, ankyrin to protein 4.1, protein 4.2 to band 3 and protein 4.1 to protein 4.2. Pre-stained SDS-PAGE molecular weight standards (BioRad Laboratory, Nazareth, Belgium), as well as two to four control samples, were run in each gel. Study sample ratios were compared to control sample ratios in order to identify the exact protein deficiency. DxH800 automated reticulocyte analysis The UniCel DxH800 (Beckman Coulter) instruments were regularly and constantly controlled by internal and external quality control schemes. The analytical variability of the instruments during the study period was less than 1 % for all parameters considered. We measured and reported data of MRV, MSCV, RDW, RDWR, IRF, delta (MCV-MSCV) and Ret/IRF. During the reticulocyte analysis, the Beckman Coulter UniCel DxH800 instrument uses staining with new methylene blue, followed by slight swelling with an acidic, hypotonic ghosting solution that clears the haemoglobin while preserving the stained RNA within the reticulocytes [27]. The sphered volume of the whole RBC population, MSCV, is measured by the unique Beckman Coulter VCS technology (volume, conductivity and light scatter). The resulting sphered cells are also classified as either mature red cells or reticulocytes. The following reticulocyte parameters are measured: RDWR, MRV and the count of reticulocytes with the greatest RNA quantity. The IRF is thus calculated as the ratio of the reticulocytes with the highest RNA content and the total number of reticulocytes. Statistical analysis Statistical analysis was performed using Analyse-it Software, Ltd. (Leeds, UK) and GraphPad Prism software (San Diego, USA). Reference values were defined as 2.5–97.5 percentile intervals [28]. A Student t test was used to evaluate statistical significance for the parameters with normal distribution. A Mann-Whitney t test or one-way ANOVA Kruskal-Wallis test was used for parameters with a non-Gaussian distribution.
Ann Hematol
One-way analysis of variance (Dunnett’s multiple comparison test) was used to compare values among different groups.
Results Reference values and stability of MRV, MSCV, RDW, RDWR, delta (MCV-MSCV), IRF and Ret/IRF After applying the local and published reference values for the adult non-anaemic population (see “Methods and materials”), we selected 522 samples from females and 375 samples from males over a 2-month period of routine work flow. Our adult reference values of MRV, MSCV, RDW, RDWR, delta (MCVMSCV), IRF and Ret/IRF are presented on Table 1. The stability of all automated parameters for 24 h at 4 °C was warranted by statistically non-significant Student t test; the same was observed at 20 °C for Ret/IRF and MRV. On the opposite, MCV, MSCV and delta (MCV-MSCV) presented statistically significant Student t test in the 24-h stability study at 20 °C with respective mean differences of 3.13, 7.14 and 10.3. Patients The distribution of the cohort of patients studied (a total of 410) was as follows: 216 females (53 %) and 194 males (47 %), mean age 30 (range 0.01–89). A total of 251 samples were considered negative on the basis of both negative screening tests, another 159 samples were analysed by SDS-PAGE and different pathologies were diagnosed or excluded (Fig. 1). The results of a total of 26 samples (16 % of all SDS-PAGE electrophoresis) were not interpretable, i.e. 19 samples presenting typical SDS-PAGE profile of reactive reticulocytosis (increased ankyrin and/or spectrin) and 7 samples showing degraded electrophoretic profile. Following our working algorithm, 319 patients were found negative for HS: 251 had negative screening tests and 68 showed a non-pathological SDS-PAGE electrophoretic profile. Haematological parameters of the studied cohort Table 2 shows data obtained for the classical automated haematological parameters for the following groups: (a) control group consisting of 82 negative controls and 131 negative samples presenting negative screening tests and haematology results within the reference values; (b) cryohaemolysis negative samples, consisting of 82 negative controls and some other 292 samples: 251 with negative screening tests plus 41 SDS-PAGE negative samples with cryohaemolysis test result less than 10 %; (c) a total of 48 HS cases confirmed by SDSPAGE; (d) 7 cases of autoimmune haemolytic anaemia; (e) 2
Table 1 Reference interval (2.5–97.5 percentile distribution) of RBC and reticulocyte parameters Parameter
Women
Men
RDW (%) MRV (fL) IRF Ret/IRF (%)
12.3 to 16.9 97.6 to 122.1 0.25 to 0.52 0.71 to 2.34
12.5 to 16.4 98.6 to 123.2 0.24 to 0.5 0.8 to 2.69
RDWR (%) MSCV (fL) Delta (MCV-MSCV)
22.2 to 30 74.3 to 95.7 −0.02 to 16.9
21.6 to 30.4 72.9 to 94.9 −0.2 to 16.8
cases of hereditary elliptocytosis (HE); and (f) 2 cases of hereditary pyropoikilocytosis (HPP). Additionally, patients with confirmed hereditary spherocytosis were grouped according to the haemoglobin level in the moderate/severe anaemia group (Hb 11.5 g/dL for adult females and Hb >13.5 for adult males). Haematological and biochemical data and defective proteins for HS patients are presented in Table 3. When analysing the different groups, a rise in the reticulocyte count, oppositely proportional to the haemoglobin level, was observed and a statistically significant difference was found between the mild anaemia group and compensated forms of the HS group. However, RDW was the only haematological parameter that showed a strong statistically significant difference (p0.7
0.69 0.6–0.76 >13.3 >22
0.91 0.87–0.94 >26.4 (28.5) 10.4 fL,
that gives 100 % sensitivity in our study, is close to the one (>9.6 fL), published by Broséus et al. [22]. We also report for the first time the performances of another parameter for HS screening that is directly linked to the surface area loss at the reticulocyte stage, i.e. MRV. For MRV, at the cut-off
