analysers in samples showing cold agglutination ... The term auto immune haemolytic anaemia (AIHA) ... acterised by an immune reaction against red blood.
Original article
Short preheating at 41°C leads to a red blood cells count comparable to that in RET channel of Sysmex analysers in samples showing cold agglutination
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Docemus Onlus “Theoretical and Practical Training School for Improving Specialty Medicine”, Torrevecchia Teatina, Italy 2 U.O. Patologia Clinica A.O.R.N. “G.Rummo”, Benevento, Italy 3 UOC Medicina di Laboratorio Azienda USL Toscana Nord Ovest, Pontedera, Italy 4 Centro Regionale Controllo di Qualità AOU Careggi, Firenze, Italy 5 Laboratorio Analisi IFCA (Istituto Fiorentino di Cura ed Assistenza, Firenze, Italy 6 UOC Medicina di Laboratorio Azienda USL Toscana Nord Ovest, Livorno, Italy 7 UOC Medicina di Laboratorio Azienda USL Toscana Nord Ovest, Versilia, Italy 8 UOC Laboratorio Analisi I - Policlinico Vittorio Emanuele, zienda Universitaria Ospedaliera, Catania, Italy Correspondence to Dr Antonio La Gioia, Docemus Onlus “Theoretical and Practical Training School for Improving Specialty Medicine”, Torrevecchia Teatina, Italy ; ant. lagioia@gmail.com Received 8 December 2017 Revised 13 February 2018 Accepted 14 February 2018
Abstract Aims The presence of cold agglutinin in blood samples can cause a spontaneous agglutination of red blood cells (RBCs) when low temperature occurs. This phenomenon causes a spurious lowering of RBC count on the automated haematological analysers that are detected by incongruous values (≥370 g/L) of the mean cellular haemoglobinconcentration (MCHC). A preheating at 37°C can remove the RBC agglutination generally resulting in a reliable count. It has been reported that the same result can be reached by using the optical reticulocyte (RET) channel of Sysmex analysers where the RBC count is not influenced by the presence of cold agglutinin. This study aims to evaluate these data in a larger population, with regard to environmental conditions on Sysmex analysers. We have also evaluated the influence of different thermal pretreatments on the RBC count. Methods This study was performed on 96 remnants of peripheral blood samples (48 with MCHC in normal range and 48 with MCHC>370 g/L) which have been analysed in different preanalytical conditions on the Sysmex analysers. Results A preheating of samples at 41°C for 1 min leads to a reversibility of the cold agglutination comparable to the one observed in the RET channel and yields better results compared with 37°C for 2 hours. Conclusions None of described procedures assure the complete cold agglutination reversibility in every case. Consequently, since the haematological analysers not yet provide reliable parameters to confirm the complete resolution of agglutination, further verification of RBC count accuracy needs to be performed.
Aims
To cite: La Gioia A, Fumi M, Fiorini F, et al. J Clin Pathol Epub ahead of print: [please include Day Month Year]. doi:10.1136/ jclinpath-2017-204954
The term auto immune haemolytic anaemia (AIHA) defines a group of immunological disorders characterised by an immune reaction against red blood cell (RBC) self-antigens. Based on the optimal temperature to which the autoantibodies react with the self-antigens, they are defined as warm or cold antibodies. The presence of these antibodies can be associated with clinical forms of warm AIHA or cold AIHA that includes cold agglutinin disease (CAD) and the paroxysmal cold haemoglobinuria. Mixed types of AIHA (warm/cold) are also described.1 Different antigen/antibody couples are involved in different AIHA types: Rh/anti-Rh IgG in the warm
AUTHOR PROOF
Antonio La Gioia,1 Maurizio Fumi,2 Fabiana Fiorini,3 Paola Pezzati,4 Fiamma Balboni,5 Maria Bombara,6 Alessandra Marini,7 Ylenia Pancione,2 Leonardo Solarino,8 Elisa Marchese,8 Silvia Sale,2 Vincenzo Rocco,2 Marcello Fiorini6
type; I-antigen/anti-I IgM in the CAD; both couples in the mixed type. The avidity and ability to initiating the classical complement pathway (higher in the CAD) as well as the titre of cold agglutinin (CA) into the blood determine the different degree of haemolysis.2 CAD has been classified into a primary form which is not associated with other diseases and into a secondary form, associated with solid organ or haematological malignancies (chronic lymphocytic leukaemia; non-Hodgkin’s lymphoma and others) as well as with autoimmune and infectious diseases (by Epstein-Barr virus, Mycoplasma or, less frequently, Legionella pneumophila, Chlamydia and others).1–3 Unlike the pseudo-thrombocytopenia, RBC agglutination is not only an ‘in vitro’ phenomenon: depending on the titre and on the thermal amplitude of CA, it can occur even ‘in vivo’ where there can be the initial phase of haemolytic anaemia. Because of this, the agglutination in vitro occurs as a result of the lowering of temperature inside the tubes and in the external environment, regardless of the type of anticoagulant. This phenomenon has been reported since the 1970s,4–8 when it was noticed that the presence of agglutination causes a false decrease of RBC and a variable increase of mean cell volume (MCV), when automatic cell counters were used. Furthermore, Wintrobe indices mean haemoglobin content (MCH) and mean cellular haemoglobin concentration (MCHC) appear to be misleadingly increased. The same occurrence has been reported on the widely used modern haematological analysers.9–14 In each of these analysers, the influence of cold agglutination on RBC count as well as on MCV, MCH and MCHC can be different due to operative situations in the RBC counting channels such as the upper threshold for the size discrimination and/or the temperature of reagents on board.15 The correct RBC count as well as the correction of MCV, MCH and MCHC can be obtained by (i) preheating at 37°C of the tube before collection and maintaining temperature control during cell count in positive history patients and (ii) preheating at 37°C/2 hours if RBC count showed an unexpected agglutination in negative history patients. In both cases, the restoration of the RBC derived indices within their respective reference limits can be interpreted as a preventive inhibition or as the disappearance of
La Gioia A, et al. J Clin Pathol 2018;:1–6. doi:10.1136/jclinpath-2017-204954
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Original article
AUTHOR PROOF
the cold agglutination. The replacement of plasma with warm isotonic solution has been proposed,16 but this bothersome and time-consuming procedure may introduce variables which are not easy to control. Recently, it has been reported that in the dedicated channel for reticulocytes (RET) counting of the Sysmex XE and XN series (Sysmex, Kobe, Japan), the separation of agglutinated cells occurs spontaneously, that is, without prewarming, yielding a correct RBC count.14 17–20 We have evaluated these promising data in a larger population, with regard to the environmental conditions in the RET channel of these analysers in samples presenting CA.
Methods
Sysmex XE and XN analysers use the same methodologies and reagents for RBC and for RET count, as follows: 1. RBCs are counted with an impedance method (RBC-I) in which the hydrodynamic focusing avoids or minimises the possibility of coincidence. Haematocrit (HTC) is measured as cumulative height of electrical pulse produced by each cell passing through two electrodes, and MCV is calculated as [HTC/RBC].20 MCH and MCHC are calculated according to Wintrobe formulas.21 An additional parameter available in the ‘service’ page is the RBC-most frequent volume (R-MFV), which represents the median value of RBC volumes. 2. RET are counted as a fraction of total RBC in a dedicated optical channel (O-channel) in which cell counting is performed by flow cytometry. Fluorocell-RET and RET-Search-II, which are the main reagents for RET count, respectively, in the XN and XE analysers, contain polymethine dye 0.03%; methanol 7.9% and ethylene glycol 92%. In this channel, the sample is heated at 41°C for 30 s during the transfer to the reading chamber. In addition to RET count, in the service page, optical RBC count (RBC-O) and delta RBC (DLT-RBC) are provided. DLT-RBC is the ratio between RBC-O and RBC-I [RBC-O/RBC-I] counted at the same time in both I-channel and O-channel. RET value is provided as percentage of RBC-O, while absolute RET count is calculated from RBC-I count. This study was performed on 96 anonymised leftovers of peripheral blood (PB) samples collected for complete blood count (CBC) in K2EDTA tubes (Vacutest Kima, Arzergrande, Italy). After processing on the Sysmex analysers XN-9000 or XE-2100, the samples were selected according to the following criteria: 1. Group A: 48 samples (22 from XE and 26 from XN) presenting MCHC values in the normal range (320–360 g/L). An aliquot of 2.0 mL of each sample was used to perform two successive countings: a. at room temperature (RT) in both impedance and optical channel b. after 2 hours at 37°C in a water bath, in the I-channel. 2. Group B: 48 samples with MCHC >370 g/L were studied as described in group A. All samples were collected from March to June 2016 and from January to February 2017 in the participating laboratories that used either XE (three labs; 22 samples) or XN analysers (two labs; 26 samples). In total, 37 patients had negative history for CA and 11 had positive history. Also, 36 out of 48 samples had low haemoglobin levels (median 99 g/L; range 52–115). Furthermore, to evaluate the environmental influences in the RET channel on reversibility of cold agglutination, additional counting was performed on Sysmex XN analyser as follows: 2
c. Twenty-one samples (1.0 mL aliquot) were read simultaneously in both I-channel and O-channel, after preheating in a water bath at 45°C for about 1 min, until reaching an internal temperature of 41°C (41°C/1 min) (subgroup B1). d. Twenty samples (1.0 mL aliquot) were read simultaneously in both I-channel and O-channel after addition of 5 µL of Fluorocell RET 1 min before counting (subgroup B2). Finally, to study the influence of temperature on cellular morphology, a blood smear was prepared by the automatic slide maker for each subgroup B1 sample, before preheating and immediately after counting. The slides were evaluated microscopically by a skilled operator. After the last counting, all samples have been centrifuged to evaluate the presence of haemolysis or plasma turbidity which resulted as absent in all selected samples. Statistical analysis was performed by MedCalc software V.11.4.2.0 (MedCalc, Ghent, Belgium). To assess reliability and agreement of measurements between groups being studied, we have used the interclass correlation coefficient (ICC), Passing & Bablok regression. To test the significance of the difference between parameters according to different categories, we performed the Mann-Whitney test because the data did not fit a normal distribution (D'Agostino Pearson test P
