Red Blood Cells. Mean cellular Hemoglobin Concentration: Wintrobe MM. A simple and accurate hematocrit. J Lab Clin Med 1929;15:287 9.

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1 CORRECTION WITHOUT PRE-WARMING OF COLD AGGLUTINATION IN THE RET OPTICAL CHANNEL OF SYSMEX XE-2100/XN 9000 AND MINDRAY BC 6800 HEMATOLOGICAL ANALYZERS. PRELIMINARY DATA A. La Gioia 1, F. Fiorini 1, M. Lorubbio 3, M. Bombara 2, F. Balboni 8, A. Marini 4, P. Pezzati 5, M. Quercioli 5, M. Fiorini 2, L. Francioni 1, E. Marchese 6, F. Maglione 7 1 Patologia Clinica Ospedale Lotti, Pontedera 2 U.O.C. Medicina di Laboratorio Ospedale di Livorno 3 Laboratorio Generale Azienda Ospedaliero Universitaria Careggi Firenze 4 Patologia Clinica Ospedale Versilia 5 Centro Regionale Controllo di Qualità AOU-Careggi - Firenze 6 Laboratorio Analisi I V. Emanuele AUO Policlinico-V. Emanuele - Catania 7 U.O.C. Patologia Clinica Venosa-Melfi. ASP - Potenza 8 Laboratorio Analisi Istituto Fiorentino di Cura e Assistenza Firenze 1

2 Background 1. Autoimmune hemolytic anemia is characterized by shortened red blood cell survival and a positive direct Coombs test. The responsible autoantibodies may be either warm reactive or cold reactive. 2. The autoantibodies may be idiopathic (primary) or related to an underlying condition such as infection, malignancy, or immune disease (secondary) 3. Diagnosis is made in the laboratory by the findings of anemia, reticulocytosis, a positive Coombs test, and specific serologic tests. 4. The presence of cold reactive agglutinin can be discovered even when clinical picture in not evident by a laboratory artifact 2

3 Wintrobe MM. A simple and accurate hematocrit. J Lab Clin Med 1929;15: Wintrobe MM. Anemia Classification and Treatment on the Basis of Differences in the Average Volume and Hemoglobin Content of the Red Corpuscles. Arch Intern Med (Chic). 1934;54(2): Wintrobe s Indices: Mean Cellular Volume (MCV) = Packed Cell Volume (PCV>>> HTC) / RBC Mean Cellular Hemoglobin (MCH) = HBG/ RBC Red Blood Cells Mean cellular Hemoglobin Concentration: 3

4 * * *RBC and MCHC after 2hrs in 37 degrees incubator were 2.24x10 6 and 34.2 g/dl, respectively & & & RBC and MCHC after 2hrs in 37 degrees incubator were 3.64x10 6 and 34.6 g/dl, respectively 4

5 Ferrero-Vacher C, Senlis JÉ, Loustaunau D, Aquaronne D, Jeandel PY, Sudaka I. Erythrocytic parameters Sysmex in a case of severe haemolysis. Ann Biol Clin (Paris) Nov- Dec;73(6): Research design. 1. Samples: from April to now, 37 discarded remnants of peripheral blood (PB) samples collected for clinical purposes showing a MCHC higher than 370 g/l (range g/L; median 540g/L) were used. 2. Hemathological Analyzers: Sysmex XE-2100 (Livorno, Versilia and Careggi Hospitals); Sysmex XN 9000 (Pontedera Hospital and Florence IFCA); Mindray BC-6800 (Livorno, Venosa and Catania Hospitals) 3. Analysis protocol: a. in all samples a second CBC was performed using RET channel only (to collect in the same time both impedentiometric and optical data). b. Later, samples were placed in a 37 degrees incubator for 2hrs. c. A new count was performed in RET channel. d. MCHC in optical count (MCHC-O) was calculated by using HGB-O value (derived mainly from RBC-O count and RBC-He) and HCT obtained from RBC-O and R-MFV (RBC- Most Frequent Value) as surrogate of MCV in Sysmex analyzers. For Mindray analyzers instead of the R-MFV the MCV obtained in the 37 degrees samples were used. 4. Statistical analysis was performed using the sw MetComp ver

6 RBC-I 37 C vs RBC-O Results The RBC-O counting resulted in a resolution of the agglutination in 35 out of 38 samples. The comparison from RBC-O and warmed RBC-I counting in 30 samples analyzed with Sysmex technology showed a Passing Bablok regression y = -0, ,1009 x (95% CI of slope 0,9837-1,3796 and intercept -1,3788 to 0,1562 ). The Passing Bablok regression was resulted acceptable even by adding 8 Mindray samples to Sysmex samples: y = -0, ,0516 x (95% CI of slope 0,9701-1,2275 and intercept - 0,7906 to 0,1800 ) 6

7 RBC-I 37 C vs RBC-O 7

8 RBC-I 37 C vs RBC-O + 8

9 MCHC-I 37 C vs MCHC-O 9

10 MCHC-I 37 C vs MCHC-O + 10

11 Discussion and conclusions. The different environmental conditions of the RBC counting in the optical respect to impedentiometric channel in both Sysmex and Mindray analyzers are the factors able to made reversible the agglutination in cold agglutinins containing samples. As the time spent by the samples in the count line is very low, the influence of temperature does not matter. So the chemical composition of reactants should be studied. Despite a probable difference between Mindray and Sysmex analyzers the effect on the reversibility of the cold agglutination is very similar. 11

12 Conclusions A recent survey of the Diagnostic Hematology Study Group of SIBioC showed that in many Italian Clinical Laboratory cold agglutination in blood samples is referred like as an analytical artifact just as the pseudo thrombocytopenia. So the clinical relevance of the cold agglutinin presence is not transferred to clinicians. For exemple. 1. Parametri eritrocitari non valutabili per marcata agglutinazione eritrocitaria: si consiglia ripetizione dell'emocromo a caldo; 2. Conteggio degli eritrociti ed ematocrito sottostimati per forte agglutinazione Eritrocitaria 3. Causa la marcata agglutinazione eritrocitaria il campione è stato riportato alla temperatura di 37 C e riprocessato: presenza di agglutinine a frigore. Si consiglia di prelevare e di avvolgere la provetta dell emocromo immediatamente nel cotone caldo inviando velocemente al settore Ematologia 4. Presenza di agglutinazione eritrocitaria reversibile dopo termostatazione a 37 C 5. Altri. For these reasons, the evidence that the RBC-O counting cancels all pre-analytical and analytical problems due to the presence of cold agglutinins, could be an opportunity to focused ours attention on the clinical significance as well as on the clinical use of the cold agglutination. 12

13 La Gioia A, Balboni F. Buoro S. I commenti interpretativi nel referto ematologico di laboratorio. Biochimica Clinica 2016; 40 (3) COMMENTO SUL REFERTO E' stata osservata agglutinazione eritrocitaria reversibile dopo incubazione a 37 C, verosimilmente dovuta alla presenza di agglutinine fredde. Si consiglia approfondimento clinico-diagnostico. NOTA INFORMATIVA PER IL MEDICO CURANTE La presenza di agglutinine fredde è spesso un rilievo casuale di laboratorio associato a difficoltà di conteggio degli eritrociti ed ad un aumento fittizio della concentrazione media di emoglobina nei GR (MCHC). Sebbene non costantemente, può essere associata ad anemia emolitica cronica moderata o lieve spesso evidenziabile solo per modesti aumenti di reticolociti, bilirubina ed LDH, per la riduzione anche notevole di aptoglobina e per la positività del test di Coombs indiretto. Tali dati complessivamente orientano per una delle diverse forme di Anemia Emolitica Autoimmune (AIHA), rappresentate essenzialmente dalla malattia primitiva da agglutinine fredde (e dalla stessa forma da agglutinine calde); dalla sindrome da agglutinine fredde, spesso secondaria ad infezioni, malattie autoimmuni o neoplasie. 13

14 Grazie per l attenzione 14