COMPARISON BETWEEN FLOW CYTOMETERIC METHOD AND OTHER METHODS FORRETICULOCYTE COUNT IN DIAGNOSIS OF PHENYLHYDRAZINE-INDUCED HEMOLYTIC ANEMIA

Reticulocyte count is the salient evidence of the effectiveness of bone marrow to produce red blood cells. Currently, the reticulocyte counting is a challenge for clinical laboratoriesmainly for the ordinary ones, which still use the manual method.This study was designed to evaluate the performance of flow cytometer for reticulocytes counting comparing to traditional and optimized manual methods which helpful in diagnosis of phenylhydazine-induced anemia.For that 45 male white Albino rats were divided into 5 groups, control group,phenylhydrazine group (PHZ) which injected by phz(20 mg/kg b.w, I/P),quercetin+phz group (quercetin, 50 mg/kgb.w per os), silymarin+phz group (silymarin, 100 mg/kgb.w per os) and quercetin group. Whole blood samples of these groups were collected at day 3, 5 and 10 after 1st injection of phz which used for reticulocyte counts by flow cytometeric method and other manual methods in addition to measurement ofCBC and osmotic fragility. Analysis of the results showed that phenylhydrazine injection induced hemolytic anemia with significant reticulocytosis and using of flow cytometer in reticulocyte count more precise, easy and fast than traditional and optimized manual methods. Furthermore, degree of hemolysis was significantly increases in phz group comparing to other groups. Therefore, we concluded that flow cytometric method for reticulocyte counts was simple, fast and highly reliable comparable to traditional and optimized manual methods. Also optimized manual showed that more perfect than traditional manual method and nearly to accuracy of flow cytometeric method.

Reticulocyte count is the salient evidence of the effectiveness of bone marrow to produce red blood cells. Currently, the reticulocyte counting is a challenge for clinical laboratoriesmainly for the ordinary ones, which still use the manual method.This study was designed to evaluate the performance of flow cytometer for reticulocytes counting comparing to traditional and optimized manual methods which helpful in diagnosis of phenylhydazine-induced anemia.For that 45 male white Albino rats were divided into 5 groups, control group,phenylhydrazine group (PHZ) which injected by phz(20 mg/kg b.w, I/P),quercetin+phz group (quercetin, 50 mg/kgb.w per os), silymarin+phz group (silymarin, 100 mg/kgb.w per os) and quercetin group. Whole blood samples of these groups were collected at day 3, 5 and 10 after 1 st injection of phz which used for reticulocyte counts by flow cytometeric method and other manual methods in addition to measurement ofCBC and osmotic fragility. Analysis of the results showed that phenylhydrazine injection induced hemolytic anemia with significant reticulocytosis and using of flow cytometer in reticulocyte count more precise, easy and fast than traditional and optimized manual methods. Furthermore, degree of hemolysis was significantly increases in phz group comparing to other groups. Therefore, we concluded that flow cytometric method for reticulocyte counts was simple, fast and highly reliable comparable to traditional and optimized manual methods. Also optimized manual showed that more perfect than traditional manual method and nearly to accuracy of flow cytometeric method.

Blood sample collection:
Blood samples were collected by mean of capillary tube from retroorbital venous plexus from 3 rats from each group at theday 3, 5 and 10 after the first injection of PHZ.

Blood sample examination: Reticulocyte count:
The reticulocyte count with traditional manual method was performed according to Rowan, (1991).Moreover,Reticulocyte count was quantified by an optimized manual method described byMoradabad et al, (2019). For Flow Cytometeric method,reticulocyte count was quantified as the described by Uppal et al., (2020) using flow cytometry (FAC-SCalibur; BD Biosciences, Franklin Lakes, NJ).

CBC:
Complete blood count of all collected blood samples were evaluated by using Automatic cell counter (Hematological analyzer, MINDRAY 20S) which depend on both electrical and optical techniques according to coulter principle (1953).

Osmotic fragility:
The erythrocyte osmotic fragility (EOF) of rats from each group (n = 3) was determined as an index of hemolysis according to the method described by

Statistical analysis:
Statistical analysis was performed using the statistical software package SPSS for Windows (Version 16.0; SPSS Inc., Chicago, Ill.). Student's t-test was used to determine significant differences between two experimental groups. The significance of differences between more than two groups was evaluated by one-way analysis of variance (ANOVA). If one-way ANOVA indicated a significant difference, then differences between individual groups was estimated using Fishe's least significant difference (LSD) test.Results are expressed as the mean ± standard error of mean (SEM). A P-value of less than 0.05 was considered significant.A two-way ANOVA was done with repeated measurements or replications (more than one observation for each combination of the nominal variables) on one factor, i.e. two independent variables (factors) were regulated to just one of the treatment factors. The two independent variables in a two-way ANOVA were called factors. The idea is that there were two variables, factors, which affect the dependent variable. Each factor will have two or more levels within it, and the degrees of freedom for each factor is one less than the number of levels.

Reticulocytes changes:
The recorded data demonstrated in table (1) and figures(1&2) showed significant increase in reticulocytes values in phenylhydrazine injected rats comparing to control rats at 1 st , 2 nd and 3 rd chick points by 3 different methods. While,quercetin administrated rats and injected with phenylhydrazine showed a significant decrease in reticulocytes values when compared to phenylhydrazine. Moreover,silymarine administrated rats and injected with phenylhydrazine showed no significant changes in reticulocytes values when compared to phenylhydrazine injected rats at 1 st chick point while, there was significant decrease at 2 nd and 3 rd chick points. Furthermore, Administration of quercetin aloneto rats showed no significant changes in reticulocytes values comparing to control rats.According to differential between three different methods of reticulocytes count, flow cytometreric method showed significant increase in reticulocytes values comparing to optimized and traditional manual methods respectively in most groups at different chick points. Optimized manual method of reticulocytes count showed significant increase comparing to traditional manual method while, showed significant decrease comparing to flow cytometeric method.

Erythrogram changes:
The recorded data demonstrated in figures (3)(4)(5)(6)(7)(8) showed significant decrease in RBCs count, HCT andHb in phenylhydrazine injected rats with significant increase in MCV, MCH and MCHC at 1 st , 2 nd and 3 rd chick points (3, 5 and 10 days after injection of phenylhydrazine) when compared to control rats. Moreover, our results showed that quercetin treatment significantly reversed phenylhydrazine-induced alteration ofRBCs, Hb, Hct, MCV, MCH, and MCHC values. Silymarin administrated rats and injected with phenylhydrazine showed significant increase in RBCs, Hb and HCT values comparing to phenylhydrazine injected rats at 1 st , 2 nd and 3 rd chick points except HCT value at 2 nd chick point showed no significant changes. Furthermore, administration of quercetin to normal rats showed no significant changes in RBCs, Hb, MCH and MCHC values comparing to control rats at 1 st and 2 nd chick points.While for both HCT and MCV values, there were significant increases at 1 st chick point. Moreover, there were no significant changes in HCT and MCV values at 2 nd chick point. At 3 rd chick point, there was significant increase in RBCs, Hb and HCT while, there was significant decrease in MCV, MCH and MCHC values.

Leukogram changes:
The recorded data demonstrated in figures (9-13) showed leukocytosiswithneutrophilia, lymphocytosis,monocytosis and esinophilia in phenylhydrazine injected ratscomparing to control rats at 1 st , 2 nd and 3 rd chick points (3, 5 and 10 days after injection of PHZ) except at 1 st and 3 rd chick points, there no significant changes in eosinophils. While,quercetin treatment significantly reversed phenylhydrazine-induced alteration of WBCs, neutrophils, lymphocytesand monocytes values at 1 st , 2 nd and 3 rd chick points except at 3 rd chick point, there no significant changes in esinophils. Rats administrated silymarin and injected with phenylhydrazine showed no significant changes in WBCs at 1 st and 3 rd chick points when compared to PHZ group while there was significant decrease at 2 nd chick point. Neutrophils values showed no significant changes at 1 st chick point while there was significant decrease at 2 nd and 3 rd chick points. Moreover, lymphocytes values showed significant decrease at 1 st , 2 nd and 3 rd chick points while there were no significant changes in esinophils. Monocytes values showed significant decrease at 1 st and 2 nd chick points while there were no significant changes at 3 rd chick point.Furthermore,administration of quercetin to normal rats showed no significant changes in WBCs at 1 st chick point when compared to control rats while showed significant increase at 2 nd and 3 rd chick points. Neutrophils, monocytes and esinophils values showed no significant changes at 1 st , 2 nd and 3rd chick points. Moreover, lymphocytes values showed significant increase at 1 st , 2 nd and 3 rd chick points.

Osmotic fragility changes:
The recorded data demonstrated in figures (14-16) showed significant increase in degree of hemolysis of RBCs in phz injected rats comparing to control rats at 1 st and 2 nd chick points while, there no significance at 3 rd chick point. While, quercetin administrated rats and injected with phenylhydrazine showed significant decrease in degree of hemolysis of RBCs comparing to phz injected rats at 1 st and 2 nd chick points, while there were no significant changes at 3 rd chick point.Also, silymarin administrated rats and injected with phenylhydrazine showed significant decrease in degree of hemolysis of RBCs comparing to phz injected rats at 1 st and 2 nd chick points, while there were no significant changes at 3 rd chick point. Furthermore administration of quercetin to normal rats showed no significant changes in degree of hemolysis of RBCs comparing to control rats at 1 st , 2 nd and 3 rd chick points.

Discussion:-
Reticulocytes are immature red blood cells carrying ribosomes that spend the final stages of their maturation in peripheral blood. Reticulocyte counting is a direct indicator test for the assessment, classification and responsiveness to anemia therapy (Koepke, 1999). There are different methods for enumerating reticulocytes. Usually, reticulocyte counts are performed manually in clinical laboratories since 1940 (Piva et al., 2010).Also, there is an optimized manual method in which there is modification in manually staining method to achieve better counting (Moradabad et al., 2019).Furthermore, automated methods, in which flow cytometry technology is mainly used (Karina et al., 2014). Regarding to reticulocytes changes,Phenylhydrazine injected rats showed significant increase in reticulocytes value comparing to control rats at 1 st , 2 nd and 3 rd chick points by 3 different methods. These results were agree with Criswell et al., (2000) who found that PHZ group showed a dramatic increase in reticulocyte. karfka, (1930) has shown that a marked hemolysis is followed by a marked reticulocytosis. At a time interval corresponding to red blood cell life span, this yield of reticulocytes reaches senescence and is simultaneously destroyed, producing a "spontaneous anemia." On this interpretation hemolysis will in time produce an extremely high proportional reticulocytosis. The reticulocyte count was the first parameter to be significantly elevated and the last to return to the normal reference range. This shows the sensitivity of this parameter when assessing bone marrow responsiveness (Kjeldsberg, 1993).Quercetin administrated rats and injected with phenylhydrazine showed 942 significant decrease in reticulocytes values when compared to phenylhydrazine injected rats at 1 st , 2 nd and 3 rd chick points. Furthermore, silymarine administrated rats and injected with phenylhydrazine showed no significant changes in reticulocytes values when compared to phenylhydrazine injected rats at 1 st chick point while, there was significant decrease at 2 nd and 3 rd chick points. The significant decrease of reticulocytes count in quercetin and silymarin groups was owed to antioxidant effect of them. Administration of quercetin in normal rats showed no significant changes in reticulocytes values at 1 st , 2 nd and 3 rd chick points.According to differential between three different methods of reticulocytes count, flow cytometreric method showed significant increase in reticulocytes values comparing to optimized and traditional manual method respectively in most groups at different chick points. Optimized manual method of reticulocytes count showed significant increase comparing to traditional manual method while, showed significant decrease comparing to flow cytometeric method. These results agree with Moradabad et al, (2019)who found that flow cytometeric method for reticulocyte count more effective than optimized and traditional manual methods. Also, optimized method was more effective than traditional method.Many studies have compared different methods of reticulocyte counting , Pappas et al.,(1992), Nobes and Carter, (1992) and Scherer et al., (2015)found that flow cytometric method could be counted 30,000 or more cells, howevertraditional manual method could be counted 1,000 cells, revealing the higher statistical accuracy of the flow cytometry method. As far as the optimized method is concerned, interference with dye deposits has been minimized due to additional bleaching steps so the accuracy of this method has remained high and comparable to that of flow cytometric method (Moradabad et al,  2019). Regarding to erythrogram changes, phenylhydrazine injected rats (PHZ group) showed macrocytic anemia at 1 st , 2 nd and 3 rd chick points (3, 5 and 10 days after injection of phenylhydrazine) when compared to control rats. These results are in accordance with these reported by Crieswell et al, (2000) who found macrocytic anemia as RBCs count, HCT, Hb content were decrease with significant increase in MCV, MCH and MCHC. Hemolytic anemia is a well-known effect that occurs as consequence to phenylhydrazine administration which causes significant disturbances in hematological parameters (Giffin and Allen 1993). This anemia owed to phenylhydrazine induces oxidative stress, free radical production, lipid peroxidation, oxidative degradation of the cell membrane, and red blood cell lyses (Ferrali et al., 1997). In addition to oxidative stress within erythrocytes resulting oxidation of oxyhemoglobin leading forming methemoglobin which is subsequently converted into irreversible hemichromes that lead to the precipitation of hemoglobin in the form of Heinz bodies which could responsible for hemolysis (Shukla et  al., 2012).Crieswell et al, (2000) showed that 90 percent of the PHZ-treated animals had MCHC values that were substantially higher than normal limit. Most possibly, this was a false elevation due to the presence of free hemoglobin in the plasma. Also increases in MCV paralleled the amount of circulating reticulocytes. This is predicted because reticulocytes are younger and thus larger than mature erythrocytes. Our results showed that quercetin treatment significantly reversed phenylhydrazine-induced alteration ofRBCs, Hb  (Valko et al., 2006). Luckily, peroxidation can be prevented by antioxidant such asquercetin, which can interact with peroxidation by reacting with the radicals produced (Wang et al, 2016).Silymarin administrated rats and injected with phenylhydrazine showed significant increase in RBCs, Hb and HCT values comparing to phenylhydrazine injected rats at 1 st , 2 nd and 3 rd chick points except HCT value at 2 nd chick point showed no significant changes. These results agree with El-Tantawy and Temraz (2009)who found that silymarin treatment with phenylhydrazine significantly improved RBCs, Hb and HCT. Silymarin modulates the imbalance between cell survival and apoptosis by altering with the expressions of the cell cycle regulators and proteins involved in apoptosis (Comelli et al., 2007), protects cells from ROS damages by increasing endogenous antioxidant enzymes such as glutathione peroxidase (GPx) and superoxide dismutase (SOD). In addition, it also inhibits the activation of the nuclear factor-κ-gene binding NF-B (Khezri et al., 2016). to combat cell infiltration by foreign agents or infections; thus the, increased proliferation may indicate an immunological response due to acute infections, cell damage or inflammation (Atawodi et al., 2011)which might have been caused by the introduction of toxic substance such asphenylhydrazine.This leukocyte response is mainly due to an increase in circulating mononuclear cells, primarily lymphocytes. The neutrophil count is also elevated, but to a much lower degree, and there is a small but negligible rise in the number of circulating neutrophils. The increase in WBC reflected the increase in reticulocytes, indicating a potential relationship (Crieswell et al, 1998).Our results showed that quercetin treatment significantly reversed phenylhydrazine-induced alteration of WBCs, neutrophils, lymphocytesand monocytes and esinophils values at 1 st , 2 nd and 3 rd chick points except at 3 rd chick point there no significant changes in esinophils values. These results agree with Bahar et al, (2017) who found that quercetin administration significantly improved WBCs, neutrophils, lymphocytesand monocytes values. Ruma et al., (2013) noted that quercetin has a good anti-inflammatory ability. Alsoquercetin can suppress the development of lipopolysaccharides (LPS)-induced cytokines in different cells. For example, quercetin may inhibit the production of LPS-induced tumor necrosis factor in macrophages. The anti-inflammatory activity of quercetin is correlated with its antioxidant and free radical scavenging properties (Comalada et al., 2005). Reactive oxygen species are not only present in the oxidation phase, but are also involved in inflammatory reactions through activation of transfer factors such as the nuclear factor-κ-gene binding (NF-κB) (MacNee, 2001).In addition, NF-κBcould induce the development of TNF-α cytokines (Xu et al., 2007). Therefore, reducing reactive oxygen species could prevent oxidation and inhibit inflammation at the same time. Furthermore, Nair et al. (2006) interpreted that quercetin could inhibit the gene expression of TNF-α cytokines by modifying NF-κBin peripheral blood mononuclear cells.Rats administrated silymarin and injected with phenylhydrazine showed significant decrease in WBCs at 2 nd chick point comparing to phz group while there were no significant changes in WBCs at 1 st and 3 rd chick points. Neutrophils values showed significant decrease at 2 nd and 3 rd chick points, while no significant changes at 1 st chick point. Moreover, lymphocytes values showed significant decrease at 1 st , 2 nd and 3 rd chick points.Monocytes values showed significant decrease at 1 st and 2 nd chick points while there were no significant changes at 3 rd chick point. These results were partially in accordance with these reported byYakubuet al., (2020) as they found that administration of silymarin decreased lymphocytes and neutrophils values with decreasing WBCs values. Which contributed to antiinflammatory effects of silymarin (Colturato et al., 2012). Furthermore,administration of quercetin to normal rats showed no significant changes in WBCs at 1 st chick point when compared to control rats while showed significant increase at 2 nd and 3 rd chick points. Neutrophils, monocytes and esinophils values showed no significant changes at 1 st , 2 nd and 3rd chick points. Moreover, lymphocytes values showed significant increase at 1 st , 2 nd and 3 rd chick points. These results partially agree with Selvakumar et al, (2012) who reported that administration of quercetin showed no significant alterations in WBCs and differential leukocytic count except eosinophil counts were decreased. Increase in lymphocytes values contibutred to flavonoids that generally have immunomodulatory effects on the immune system. It has been observed that quercetin can regulate leukocyte biology with a stimulus-specific action and affects the balance between Th1 and Th2 (Chirumbolo, 2010). Concerning to osmotic fragility changes, phz injected rats showed significant increase in degree of hemolysis of RBCs comparing to control rats at 1 st and 2 nd chick points while, there no significance at 3 rd chick point. These results were in accordance with Gbolahan et al., (2020) who reported that phz induced oxidative stress in erythrocytes that considered as an important mechanism of hemolysis. As the membrane integrity is disrupted due to increased production of reactive oxygen species.Quercetin administrated rats and injected with phenylhydrazine showed significant decrease in degree of hemolysis of RBCs comparing to phz injected rats at 1 st and 2 nd chick points, while there were no significant changes at 3 rd chick point. These results agree with Asgari et al., (2005) who found thatthe highest concentration ofpure quercetin inhibited hemolysis of RBCs by 35.5%in vitro as, quercetin decreased hemolysis in a dosedependent manner. On the other hand, Razi et al., (2016) found that concurrent administration of quercetin do not make any protective effects on erythrocyte osmotic fragility.Also, silymarin administrated rats and injected with phenylhydrazine showed significant decrease in degree of hemolysis of RBCs comparing to phz injected rats at 1 st and 2 nd chick points, while there were no significant changes at 3 rd chick point. These results agree with Valenzuela et al, (1985) who they found silymarin act on the surface of the membrane or may be included in the matrix, avoiding the initiation and propagation of unsaturated fatty acid peroxidation. The increased resistance to osmotic shock of erythrocytes from silymarin pretreated rats is further evidence of the membrane stabilizing properties of flavonoid.Administration of quercetin to normal rats showed no significant changes in degree of hemolysis of RBCs comparing to control rats at 1 st , 2 nd and 3 rd chick points. These results were in accordance withDonaldson and Erlwanger (2019)who found that administration of quercetin to normal rats didn'thave effect on degree of hemolysis of RBCs.

Conclusion:-
From the previous results, we can conclude that reticulocyte count is an important in diagnosis of hemolytic anemia. Using of flow cytometer for reticulocyte counting was simple, fast and highly reliable comparable to traditional and optimized manual methods.