Erythrocyte Morphology in Neonatal Rhesus Factor and ABO Isoimmunization

Цель: исследование методом атомно�силовой микроскопии наноструктуры эритроцитов у новорожденных с изоимму� низацией по АВО�системе и резус�фактору. Материал и методы. В исследование включено 27 новорожденных, из них 13 детей с резус�сенсибилизацией и 14 — изоиммунизацией по АВО�системе. Течение беременности, осложнив� шейся резус�сенсибилизацией, характеризовалось появлением в крови титра RhD�антител и иммуноглобулинов (Ig) подкласса G: IgG1, IgG2, IgG3. Увеличение продукции IgG происходило к 34 неделям беременности, когда опреде� лились все подклассы в различных титрах. У новорожденных имело место несовпадение с матерью или по резус�фак� тору или по АВО�системе. В паре О(I)Rh�отрицательная мать — А(II) Rh�положительный ребенок проводилась диф� ференциальная диагностика изоиммунизации. Исследовались общий анализ, биохимические показатели крови, проводилось определение титра RhD�антител и иммуноглобулинов класса G, а у 11 новорожденных проводилось ис� следование эритроцитов в поле атомно�силового микроскопа (АСМ). Объектом исследования явились: остаточная пуповинная кровь (ОПК), центральная венозная кровь во время лечения новорожденных. Результаты. Комплекс� ная терапия изоиммунизации новорожденного по резус�фактору или АВО�системе прекращает каскад иммунологи� ческих реакций, приводит к прекращению гемолиза эритроцитов, снижению билирубина, влияет на морфоло�гичес� кий состав и макроструктуру мембран эритроцитов. Последствия перинатального воздействия на мембрану эритроцитов сохраняются в течение определенного времени и выходят за рамки раннего неонатального периода. Ключевые слова: наноструктура мембран, эритроциты, резус�конфликт, АВО�конфликт, новорожденные Objective: to conduct an atomic force microscopy (AFM) study of the red blood cell nanostructure in neonatal infants with ABO and rhesus (Rh) isoimmunization. Subjects and methods. The investigation included 27 neonates, including 13 infants with Rh sensitization and 14 with ABO isoimmunization. The course of pregnancy complicated by Rh sensi� tization was characterized by the emergence of the blood titers of Rh D antibodies and immunoglobulin G (IgG) sub� classes: IgG1, IgG2, and IgG3. IgG production increased at 34 weeks' gestation when all the subclasses were detectable in different titers. There was either Rh or ABO incompatibility between the newborns and their mothers.


Introduction
Despite the successes in the study of pathogenesis and clinic of hemolytic disease of a fetus and newborn the studies of immunological interaction of the fetus and the mother remains actual that challenge prevention, diagnos tics and treatment strategies [1,2].
One effective method to prevent the isoimmunisa tion is to inject Rh immuneglobulin to unsensitized Rh negative women.In case of rhesus (Rh) sensitization the administration of anti -RhD by Rh negative mothers prevents their primary sensitization by the Rh -positive RBCs of the fetus due to the elimination of the antigen from mother's blood.In developed countries they succeed ed to reduce the number of women with Rh sensitization to 0,2 0,1% due to the introduction of Rh immunoglobulin into clinical practice In Russia the number of women with Rh sensitisa tion remains as high as 1.2%, the hemolytic disease occurs in 0.6% of newborns [1][2][3].
The Rh antigen D is the most immunogenic of all the RBC antigens.It is responsible for 80% cases of hemolytic disease of fetus and newborn.The antigens are associated with membrane proteins that are expressed on RBC sur face.They are encoded by two linked loci -RhD and RhСсЕе, 92% of which are homologous.Due to the high immunogenicity the RhD antigen is of most important for the development of the disease.
In RhD negative individuals the product of RhD locus is completely absent, and the RhСсЕе locus encodes a molecule expressing RhС/s and RhЕ/E epitopes.Rhesus incompatibility occurs, when the mother is sensi tized to the RBC antigens of fetus RBC and the produc tion of anti RBC IgG antibodies is becoming possible .The antibodies are capable of crossing through the placenta and interact with RBCs of the fetus that causes the damage of the latter.The probability of appearance of antibodies in mother's body depends on various factors: the phenotype of the fetus, immunogenicity of RBC antigen, volume of transplacental blood flow, mmunological ability of the mother to produce antibodies.
The IgG, which binds to RBC antigen forming a complex recognized by the Fc receptors on effector cells of the reticulo endothelial system (RES), is of primary importance.The RES cells destroy RBCs of fetus by phagocytosis.This process depends on the type of IgG sub classes.All four IgG subclass are actively transferred to the fetus and increase the level of maternal antibodies in the fetus blood.However, IgG1 and IgG3 are much easier interact with Fc cell receptors than IgG2 and IgG4.Therefore, only the level of IgG1 and IgG3 have the diag nostic value [2][3][4][5][6][7].
The isoimmunization of newborn occurs due to other blood group antigens, for example Kell, but it results in low frequency (9%) and decreased antigenicity [5,6,8].
Цель работы -исследование методом атомной силовой микроскопии наноструктуры мембран эритро цитов у новорожденных с изоиммунизацией по АВО системе и резус фактору.RBCs.The can be found on other cells and in carbohydrate components of glycoproteins.Most people have antibodies to allogeneic antigens without prior sensitization by for eign RBCs.This kind of sensitization is possible in case of contact with identical epitopes.Antibodies to the antigens of ABO system belong to IgM, which do not cross the pla cental barrier, therefore no intrauterine sensitization occurs [6,9].

Материал и методы
Currently, there are non invasive methods to treat pregnant women with isoimmunization.They include desensitizing therapy by other antigens, therapy by lym phocytes and other methods to treat hypoxia and reduce permeability of placenta.However, they are not patho genetically justified and clinically effective [10,11].
Hemolytic disease of the newborns is characterized by hyperbilirubinemia, which occurs due to alterations of RBC structure and hemolysis.High bilirubin level causes bilirubin induced brain dysfunction -kernicterus and toxic encephalopathy, which causeirreversible damage of CNS and disability [2,3,12,13] The investigation of ery throcyte membranes allows to evaluate morpho functional patterns of the cells [14].
The objective of the study: to reveal the RBC mem brane nanostructure of neonates following ABO/Rh isoimmunization.

Materials and Methods
The study included 26 mothers and 27 infants (one case of bichorionic biamniotic twins), including 13 children with Rh sensitization and 14 with ABO isoimmunization The mean age of the mothers was 30.3±5.7 G.The pregnancy complicated by rhe sus sensitization was characterized by the appearance RhD anti bodies and IgG: IgG1, IgG2, IgG3 in blood (table.1).As a rule, RhD antibodies appeared first and then appeared IgG.The titer of RhD antibodies was determined in blood of 10 (38.4%) mothers.In 5 (19,2%) cases the antibodies appeared first on week 16 of the pregnancy.The earliest appearance of IgG was observed on the 25 week of gestation.IgG1 was determined in a titre of 1:100 and IgG3 in a titer of 1:10.In 3 (11,5%) cases there was a combination of antibody titers and all Ig classes G1, G2 and G3 in various titers.The increase of IgG production occurred by the 34 th week of gestation, when IgG was determined in different titers.
The general parameters of the newborns are presented in table 2. Most of the newborns were in term, 18% of newborns were born at gestational age of 35-36 weeks.The first and the fifth minute Apgar scale score were 7.8±0,5 and 8.2±0.7,respectively; there were no signs of acute intrapartum hypoxia.All the newborns had either Rh or ABO incompatibility with their mothers.In a pair of O(I)Rh negative mother and A(II) Rh positive newborn isoim munization was diagnosed.The study of RBCs of 11 newborns was performed by the atomic force microscopy (AFM).
Paying attention to the clinical history of mothers, Rh and ABO incompatibility of the mothers and the infants, the results of clinical and laboratory analysis, all the newborns were transferred to the intensive care unit for treatment.
The residual umbilical cord blood (RUCB) of 14 full term newborns with favorable course of pregnancy and urgent delivery was used to obtain normal RBCs for the AFM aided observation.The mean gestational age of the newborns was 39.4±0.5 weeks, their mean body weight at birth was 3131.7±588.8g, the first minute Apgar score was 8±0.4 points.
The study of newborns RBCs was performed in the field of AFM.The objects of the study were residual umbilical blood and central venous blood of newborns.The picture of ery throcytes was received using the atomic force microscope «NTE GRA prima» (NT MDT, Russia) in semi contact mode.Cantilevers NSG01 A were used as probes.The numbers of scan dots were 512 and 1024, the area of the scan was 100 sq.microns (10x10 microns).Detailed methodology for the analysis of nanos tructures described in previously published studies [14,15].D. The width of the RBC by volume -RDW distribution, which includes two parameters: the percentage deviation of RBC volume from the mean one in the population -RSW CV (%) = (SD*100%)/MCV, SD is the standard deviation of RBC volume from the mean one; the relative width of RSC distribution by vol ume -RDW SD.
The study was conducted using the analyzer «Hemolux 19 тм » (China) on 1, 3, and 7 days after the birth.
To determine the Rh -factor zoliclon anti D IgM monoclonal reagent was used. 6.
The determination of RhD antibodies titer in the moth er's blood during pregnancy was performed using the test RBCs ID Diacell of immunoglobulin G and a set of DAT IgG1/G3. 7.
The statistical processing of the data was carried out using the standard program of Origin 6.1 (Microsoft Office, USA).The significance of differences was estimated by the factor analysis of variance (One way ANOVA).The difference was con sidered to be significant at P<0.05.

Results and Discussion
The AB0 and rhesus isoimmunization of neonates has overall clinical and laboratory symptoms: icteric skin caused by hyperbilirubinemia with unconjugated bilirubin, hemolytic anemia and reticulocytosis of varying severity.
The skin of all the newborns was subicteric, the level of total bilirubin was elevated in residual umbilical cord blood (75.1±15 μmol/l), the peripheral blood was charac
On the first day of treatment the levels of total bilirubin in blood and their rate increases per hour were determined.Later, when assessing the effectiveness of treatment, only the assessment of a total bilirubin was per formed because the assessment of its increase per hour was not informative.The dynamics of bilirubin is presented in Fig. 1.The graph shows that the increase of bilirubin level per hour was minimal as a result of the therapy.Later, the changes of bilirubin corresponded to physiological values.
The main goal of the therapy was the cessation of RBC hemolysis and decrease of bilirubin level in blood.The methods of treatment were performed in accordance with the level of bilirubin at birth: phototherapy, intra venous infusion of immunoglobulins or operation of blood transfusion (Fig. 4).All the neonates included in our study received a continuous phototherapy.In 17 cases (63%) phototherapy was combined with the intravenous admin istration of immunoglobulin in order to block Fc receptors of the reticulo endothelial system cells and to stop the RBC hemolysis [2,3].The dose of intravenous immunoglobulins was 1000 mg/kg, the first infusion was performed 3.9±2.3hours after birth.The immunoglobulins were injected repeatedly 16.5±3 hours after birth in 22.2% of cases due to the high risk of severe illness (high baseline rates of bilirubin level , complicated pregnancy due to Rh sensitization).
2 (7,4%) infants were characterized by icteric skin and laboratory signs of jaundice -anemic form of rhesus incompatibility; bilirubin level in their blood was 110 μmol/l, RBCs -2,48•10 12 /l.It was an absolute indication for blood transfusion.The operation was carried out in standard mode.
Analysing the RBC nanostructure, the individual quantitative data (heights and the periods of the orders) were obtained.The height of the first order (h 1 ) was 1.24±0.3nm, the height of the second order (h 2 ) was 0.8±0.3nm, the height of the third order (h 3 ) was 0.17±0.04nm, which were considered to be the initial parameters in the study f [14].
RBC polymorphism (Fig. 2, a) and plots RBC mem brane destruction (Fig. 2, b) were observed in case of rhesus immunization accompanied by early appearance of antibody titers and IgG.The RBCs were found to be polymorphous regardless of the etiological factor of isoimmunization.
Planocytes,, stomatocytes, echinocytes and cell of transitional shape were the major forms of RBCs in the early period of the disease.Planocytes were visualized in 72.7% of the newborns.The mean planocytosis reached 54.6±28% of cells.Stomatocytes were observed in all the newborns, but their number varied from 18% to 100%.Absolute stomatocytes was revealed in a premature infant, born from a mother with early titre of RhD antibodies and IgG3 in the titer of 1:100, IgG1 in titer of 1:10 in the 34 th week of gestation.Echinocytes were typical for 45.5% of all the newborns with isoimmunization.The RBC morpholo gy in neonates with isoimmunization at birth was variable and depended on many factors.Isoimmunization was asso ciated with the appearance of defects on the surface of RBC membrane (Fig. 3).
Течение заболевания характеризовалось нормали зацией уровня билирубина, достоверным уменьшением The following correlation links were found: • a strong negative correlation between bilirubin level in blood and the and the height h 1 (r= 0,8; P=0.007); • a strong positive correction between the number of reticulocytes and the height parameter h 3 (r=0,8; P=0.01).
Thefore, the RBC nanostructure is affected by bilirubin level and reticulocytosis, which reflects the severity of the dis ease.The maximum increase in height h 1 occured on the 2 nd day of life.The height parameters h 2 and h 3 did not differ sig nificantly from the ones of the comparison group (P>0.05),i.e. spectrin matrix and the structural state of RBC proteins were stable (Fig. 4).Thus, isoimmunization of newborns is characterized by intrauterine poikilocytosis of RBCs and the violation of RBC membrane macrostructure at the level of h 1 .
RBCs and relative width of RBC distribution by volume by 5-7 th day of life (P<0.05).The state of RBC nanos tructures changed, a decrease of the height h 1 was observed, but its value was still significantly higher if com pared to the control group and similar data before the treatment (P<0.05).The inner cytoplasmic surface was resistant to the toxic effects of bilirubin and other factors, since the height parameters h 2 and h 3 were equail versus those from healthy newborns.There were minor changes of height parameters h 2 and h 3 , but no significant differences were detected (P>0.05) (Fig. 4).
Combination therapy for newborn Rh factor or ABO system isoimmunization stopped the cascade of immunological reactions and RBC hemolysis, increased the bilirubin level but does not affect the morphological composition and macrostructure of RBC membranes.The consequences of perinatal exposure of RBC membranes was remaining for a certain time and went beyond the early neonatal period.

Conclusion
1. AFM revealed that infants with isoimmunization are characterized by cell shape polymorphism of the cell membrane and destruction of RBCs regardless of the etio logical factor.
2. In the early stages of isoimmunization develop ment of the main forms of RBCs include planocytes, stom atocytes, echinocytes and transitional cell shape.
3. When analysing the RBC membrane nanostruc ture the parameter «height of the first order» was found to undergo the greatest changes.
Note (примечание):w w w .r e a n i m a t o l o g y .c o m

Table 4 . The methods of isoimmunisation treatment of the newborns (n=27).
1 1 ; 2 30 w w w .r e a n i m a t o l o g y .c o m Critical Illness in Newborns