Selected CD molecules and the phagocytosis of microvesicles released from erythrocytes ex vivo.

BACKGROUND AND OBJECTIVES: The accumulation of microvesicles in erythrocyte concentrates during storage or irradiation may be responsible for clinical symptoms such as inflammation, coagulation and immunization. Our aim was to determine whether any of the cluster of differentiation (CD) molecules responsible for important functions are present on microvesicles, and if their expression level is dependent on the storage period of erythrocyte concentrates. MATERIAL AND METHODS: Erythrocyte microvesicles were isolated from 'fresh' (2nd day) and 'old' (42nd day) stored erythrocyte concentrates. Qualitative cytometric analysis of 0·5 µm, erythrocyte-derived, PS-exposing vesicles was performed using the annexin V-FITC, anti-CD235a-PE antibody and calibrated beads. The microvesicles were also visualized under a confocal microscope. The expression of the molecules CD235a, CD44, CD47, CD55, CD59 and of phosphatidylserine (PS) was compared using flow cytometry. Measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. RESULTS: The analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0·5 µm in the 'fresh' and 'old' samples. At day 2, the microvesicles had elevated expression levels of CD47, reduced expression levels of PS, CD55 and CD59. The phagocytosis index was higher for the microvesicles isolated from the 42-day-old erythrocyte concentrates. CONCLUSION: This research may bring us closer to understanding the factors responsible for erythrocyte ageing and to evaluate the quality of stored red blood concentrates intended for transfusion.

3D visualization and quantitative analysis of human erythrocyte phagocytosis.

Since the erythrophagocytosis of opsonized erythrocytes is investigated mainly by calculating the phagocytic index using subjective light microscopy evaluation, we present methods for the quantitative and qualitative analysis of human cell erythrophagocytosis. Erythrocytes from two storage periods were used. Using Imaris software, we were able to create a three-dimensional model of erythrophagocytosis. The use of microscopy instead of cytometry revealed a significantly higher number of monocytes and erythrocytes that appeared active in phagocytosis. Spatial reconstruction allowed for detailed analysis of the process by precisely locating erythrocytes in phagocytes. Additionally, a technique of sequential image registration using Nis Elements software allowed for observation of the course of phagocytosis over a range of time intervals. This in vitro research may be helpful for understanding the cellular interactions between monocytes and erythrocytes. The cytometric method-being relatively rapid, sensitive, and specific-can serve as an alternative technique to microscopy in the quantitative analysis of erythrophagocytosis. This allows us to avoid counting the erythrocytes nonspecifically attached to monocytes and gives objective results.

Feto-maternal haemorrhage assessment in a woman with a large population of red blood cells containing fetal haemoglobin.

BACKGROUND: FMH quantification is necessary to calculate an individual dose of prophylactic anti-RhD immunoglobulin and to diagnose fetal anaemia causes. We encountered a healthy woman with a numerous RBCs containing fetal haemoglobin (HbF). AIMS: To investigate the cause of this sign and the correct evaluation of fetal RBCs in maternal circulation. MATERIALS AND METHODS: Patients samples and artificial mixtures were tested by microscopic Kleihaur-Betke (KB) and flow cytometric (FC) tests with anti-HbF + anti-CA (carbonic anhydrase), and with anti-D. The patient's blood count with reticulocyte parameters, and concentration of bilirubin, haptoglobin, iron, transferrin, ferritin, hepcidin, sTR, HbF, HbA2 were measured. Genes coding the beta- and gamma-globin were sequenced. RESULTS: It was impossible to distinguish the population of fetal and maternal HbF positive cells using KBT and FC with anti-HbF. Application of anti-CA and anti-D allowed to separate them. Maternal blood haematological and biochemical parameters were normal but HbF was 3.3% of total Hb concentration (normal < 1%). There were no mutations in the beta- and gamma-globin genes, but Xmn I polymorphism at -158 position in gamma-globin gene was detected in the homozygous state. CONCLUSION: A very large population of HbF positive cells sometimes can be detect in a healthy woman. Implementation of the various procedures for FMH assessment is necessary in the such case, otherwise, the detection of fetal erythrocytes may not be possible or can give false results.

[Foeto-maternal haemorrhage--detection and quantification]. / Plodowo-matczyny przeciek krwi--wykrywanie i ocena ilosciowa.

Quantification of foetal RBCs in maternal blood samples should be essential to establish the dose of prophylactic anti-RhD immunoglobulin. In practice, the volume of foetomaternal haemorrhage (FMH) is rarely calculated and routine anti-RhD doses vary in the world from 100 microg to 300 microg. In Poland the postpartum dose of IgG anti-D is 150 microg, and there is no antepartum prophylaxis. The aim of this review paper is to present that detection and quantification of FMH are important and introduction of some tests for it evaluation is necessary in Poland. Taking into consideration many reports and our preliminary experiences we show principles of some methods and techniques, their advantages and limits. As immunohaematologists we indicate very important multidisciplinary problem which concerns transfusiologists, gynaecologists and haematologists.

[Calculation of feto-maternal haemorrhage volume using various morphological parameters and various formulas]. / Obliczanie objetosci krwawienia plodowo-matczynego z zastosowaniem róznych parametrów morfologicznych i róznych wzorów.

UNLABELLED: Quantification of FMH is essential for determination of an accurate dose of anti-RhD Ig in antepartum and postpartum immunoprophylaxis of HDFN. Doses of anti-RhD are established with reference to the volume of RhD positive foetal red blood cells in the circulation of RhD negative voluntaries. Results obtained from flow cytometry, microscopic Kleihauer-Betke test or serological test indicate percentage of these cells. THE AIM OF THE STUDY: To compare results of FMH volume calculation using various formulas. MATERIAL AND METHODS: EDTA blood samples from 51 mothers and 51 cord blood samples from their newborns were analyzed. Five formulas were based on following parameters: percentage of foetal red blood cells in the blood sample from mother, mother's body weight and Hct, maternal and foetal MCV. RESULTS: The range of maternal Hct was 25.7-46.2% (mean 34.3%), ratio of foetal MCV to maternal MCV was 1.09-1.41 (mean 1.19), body weight of mothers 56-99 kg (mean 73.5 kg) and calculated volume of their blood 3600-7425 mi (mean 5100 ml). For 0.4% FMH we predicted 2, 3 or 4 doses of anti-RhD Ig 500 IU (100 microg) and 1, 2 or 3 doses of anti-RhD Ig 750 IU (150 microg) depending on the woman. CONCLUSIONS: The formula with individual maternal and foetal morphological parameters is much more accurate for calculation of the volume of FMH and doses of anti-RhD Ig than often used formula with average values of the parameters.

[Potentially pathogenic changes in red blood cells during their storage]. / Potencjalnie patogenne zmiany w krwinkach czerwonych podczas ich przechowywania.

Blood fractionation and storage improve the availability of blood products, e.g., packed red blood cells (RBCs) for transfusion. But the following question appears: how long-stored RBCs are safe and effective? In many clinical retrospective studies authors indicated risk of transfusion of stored RBCs, especially in critically ill patients eg. with acute myocardial infarction syndrome. They observed higher frequency of mortality, morbidity, infections, renal and lung failure, inflammation and thrombosis when RBC units were long-stored and non-leucoreduced than when they were fresh and leucoreduced. Responsible mechanisms are unknown but some factors are suspected eg.: RBCs do not deliver oxygen, because they have low concentration of ATP and 2.3 DPG and their shape changes. Also cytokines, enzymes and ions (K, Ca) from white blood cells (WBCs) can influence RBCs and transfused patients. Changes involve some membrane molecules associated with adhesion, oxygen transport, complement regulation during the storage of RBCs. It is interesting how the long-period storage and presence of WBCs can influence the membrane surface of RBCs and then their biological functions. Proposed study can improve our knowledge about changes in RBCs during their storage and then may be safety of blood transfusion.