Immature Erythrocytes Research Articles

Changes in non-specific biomarkers in the Mediterranean barbel ( Barbus meridionalis) exposed to sewage effluents in a Mediterranean stream (Catalonia, NE Spain)

The effluents from sewage treatment plants strongly influence the water quality and flow of Mediterranean streams. These effluents play a crucial role in maintaining the aquatic communities of these ecosystems, particularly in the absence of natural flow resulting from climate constraints or intensive water use. To detect the ecological effects of these effluents, we used non-lethal biomarkers in Barbus meridionalis and traditional assessment protocols in the Ripoll River (NE of Spain). Our results demonstrate the utility of haematological parameters. In comparison with fish at reference sites, we detected a decrease in haematocrit and haemoglobin, neutrophilia, lymphopenia, monocytosis, a rise in the nucleo-cytoplasmatic ratio of erythrocytes and an increase in the frequency of abnormal, immature and senescent erythrocytes. Many haematological parameters correlated significantly with the environmental parameters measured. In addition to these haematological alterations, histopathological examination also revealed damage in fish livers but no impact was detected by the regional index of biotic integrity using fish as bioindicators. To the best of our knowledge, this study is the first to report this battery of haematological parameters as biomarkers on a freshwater fish in a Mediterranean stream. In a region in which more than 50% of native fish species are classified as endangered or vulnerable, there is an urgent need to find biomarkers that do not harm the animals.

Cardiac remodelling, blood chemistry, haematology and oxygen consumption of Atlantic cod, Gadus morhua L., induced by experimental haemolytic anaemia with phenylhydrazine

Anaemia is a common pathology associated with many infectious and non-infectious diseases. The effects of haemolytic anaemia induced by i.p. injection of phenylhydrazine (PHZ) were studied in Atlantic cod. Phenylhydrazine injection (0.3mgkg(-1)) in a DMSO and saline vehicle induced a reproducible and stable anaemia reducing haematocrit, (Hct) by 62% over 3weeks. Controls consisted of fish injected with saline and DMSO/saline vehicle with minimal effects on Hct or whole blood haemoglobin (Hb). Although anaemia resulted in reduced blood lactate and glucose in PHZ injected fish, there were no effects of anaemia on blood, sodium, chloride or potassium. Similarly, there were no changes in the relative proportions of leucocytes in the blood although an increase in the number of immature erythrocytes was observed in the anaemic fish. Anaemic fish showed a 29 and 22% increase in cardiac somatic index (CSI) relative to saline and vehicle controls, respectively, although there were no significant differences in the linear dimensions of the ventricle. Changes in cardiac somatic and ventricular somatic index correlated positively and significantly with Hct but not with whole blood Hb concentration. Anaemic fish had significantly reduced resting routine oxygen consumption compared with vehicle controls but were not able to increase oxygen consumption following a bout of exhaustive exercise. Plasma lactate concentrations increased significantly after exercise to a greater extent in anaemic fish compared with vehicle control fish. Phenylhydrazine is a useful model for studying haemolytic anaemia in Atlantic cod with minimal effects on blood biochemistry and haematology and clearly reduces the aerobic capacity in Atlantic cod.

Description of peripheral blood cells and differential blood analysis of captive female and male leopard grouper Mycteroperca rosacea as an approach for diagnosing diseases

The peripheral blood cells and differential blood profile of captive female and male leopard grouper Mycteroperca rosacea are described for aquacultural purposes. Basophilic, polychromatic, and orthochromic erythroblasts were observed as immature erythrocytes that develop into mature erythrocytes. Young erythrocytes were not evident, and bi-lobed erythrocytes were extremely scarce. Types of leukocytes included lymphocytes; three types of granulocytes (basophiles, eosinophiles, and neutrophiles); monocytes; and a specialized amoeboid-like macrophage in the blood, which has not been previously described in fish-blood literature. Thrombocytes were commonly observed. There was significantly higher erythropoiesis in males. Granulocytes and lymphocytes of females were significantly higher than males, whereas monocytes and thrombocytes were not.

Haematological health assessment in a passerine with extremely high proportion of basophils in peripheral blood

Haematological methods are widely utilised among avian ecologists as a means for individual health assessment. However, the technical simplicity of some of the tests may easily lead to oversimplification of the evaluation. Here, we show in the Scarlet Rosefinch (Carpodacus erythrinus) that haematological parameters other than the widely used heterophil/lymphocyte (H/L) ratio may be important to investigate. We give the full description of seven basic haematological traits (leukocyte differential count, immature erythrocyte count, haematocrit, mean cell volume, total red and white blood cell count and blood parasite occurrence). Most remarkably, the examination of 178 adults and 155 nestlings has revealed that this species has an extraordinarily high proportion of basophils among the peripheral blood leukocytes (on average about 42 and 56%, respectively). Although the high basophil count is a general trait even in healthy individuals of this species, the proportion of these cells is condition-dependent and is further increased by Haemoproteus infection. Our results also suggest that the immature erythrocyte count in the peripheral blood is a good predictor of the nestlings’ growth rate. We conclude that the Rosefinch haematology differs strikingly from other avian species with known values of basic haematological parameters. We therefore emphasise the importance of a general haematological examination, based on material obtained by an appropriate method (e.g. for smear preparation, we recommend using differential staining and avoiding prior methanol fixation).

Placenta growth factor in sickle cell disease: association with hemolysis and inflammation

AbstractPlacenta growth factor (PlGF) is released by immature erythrocytes and is elevated in sickle cell disease (SCD). Previous data generated in vitro suggest that PlGF may play a role in the pathophysiology of SCD-associated pulmonary hypertension (PHT) by inducing the release of the vasoconstrictor, endothelin-1. In this cross-sectional study of 74 patients with SCD, we confirm that PlGF is significantly elevated in SCD compared with healthy control subjects. We found significantly higher levels of PlGF in SCD patients with PHT but observed no association of PlGF with the frequency of acute pain episodes or history of acute chest syndrome. The observed correlation between PlGF and various measures of red cell destruction suggests that hemolysis, and the resultant erythropoietic response, results in the up-regulation of PlGF. Although relatively specific, PlGF, as well as N-terminal pro-brain natriuretic peptide and soluble vascular cell adhesion molecule, has low predictive accuracy for the presence of PHT. Prospective studies are required to conclusively define the contribution of PlGF to the pathogenesis of PHT and other hemolytic complications in SCD.

Reticulocyte profile in top-level alpine skiers during four consecutive competitive seasons

The role of reticulocytes (Ret) in sports medicine became clear when the count of immature erythrocytes was introduced in protocols used for anti-doping purposes. Because specific research regarding seasonal variations in Ret is lacking, we assessed Ret (and [Hb]) in top-level male and female skiers during four consecutive competitive seasons. A difference (P < 0.05) between males and females was found for [Hb] and Ret values: [Hb] was lower and Ret was higher in females. The difference was maintained across all four competitive seasons. Marked within-subject differences in [Hb], Ret and immature reticulocyte fraction values were noted; the within-subject variability was greater than the between-subject variability in both genders. For instance, a difference for Ret was consistently shown between first and second blood drawings, i.e. between basal value, before the start of training and competition, and the value at middle of season, when training workload was at highest level. Unlike Ret%, the analysis of variance showed significant changes in [Hb] values across competitive seasons for both genders. Comparison between consecutive seasons (e.g., 2005-2006 vs. 2006-2007) showed significant differences for both parameters. The behaviour of [Hb] and Ret during the various seasons was parallel in females, whereas a discrepancy existed in males. In general, inter-individual variability is quite high, thus, Ret and [Hb] modifications should be referred only to the single athlete. We confirm the validity of the use of Ret counts for anti-doping purposes.

Histopathological Changes of Erythrocyte and Quantity of immature Erythrocytes in Tegal Duckling (Anas Javanica) Induced by Lead (Pb)

The aim of this research is to find out the histopathological changes of erythrocyte and the quantity of immature erythrocyte of tegal duckling treated with lead (Pb). Twenty five young female ducks with the age of 7 days and 60.55.07 g body weight alloted into 4 treatment groups and 1 control group (Ko) with 5 replication each. The treatment group fed with K1 (5.7), K2 (11.4), K3 (17.1), and K4 (22.8) mg Pb/kg body weight for 10 days, respectively. Young ducks showed clinical symptoms such as the color of feces were brown to black, green color of diarrhea, anemic and decreasing body weight. Histopathologically, erythrocyte showed that immature and abnormal in shape (like teardrops, dumb bell, and bottles). The result of variant analysis followed by linier regression shows that the higher the dosage of Pb in diet the higher the quantity of immature erythrocyte andabnormal erythrocyte shape found (P0.01).

Modulation of doxorubicin-induced genotoxicity by squalene in Balb/c mice

The present study aims to evaluate the protective effect of squalene against the genotoxicity of the chemotherapeutic agent doxorubicin (Dox) using two genotoxicity assays, the micronucleus assay and the comet assay. Different groups of mice were fed squalene at the doses of 1 and 4 mmol g(-1) body weight (100 or 400 μl as squalene oil) either at 4 h before or 1 h after Dox (20 mg kg(-1)) treatment. 24 h after the Dox treatment, bone marrow erythrocytes were evaluated for the incidence of micronuclei, and the induced DNA strand breaks were examined in heart tissue by the alkaline comet assay. As expected, Dox significantly induced micronuclei in polychromatic (immature) erythrocytes, as well as in total erythrocytes. The frequency of Dox-induced micronucleated erythrocytes was significantly reduced in the mice treated with squalene both before and after Dox administration. Squalene itself obviously did not induce any micronuclei in bone marrow erythrocytes. The comet assay also demonstrated a significant increase in DNA damage, especially DNA single strand breaks in the Dox-treated group of mice as compared to the control. The Dox-induced DNA damage was also effectively reduced by squalene when it was administered either before or after the Dox treatment. Squalene did not induce any significant DNA damage by itself. Compared to the pre-treatment of squalene, post treatment gave rise to more effective prevention against Dox-induced DNA damage. The data suggest that the complimentary use of squalene with Dox will be beneficial to reduce the adverse effect of Dox in cancer chemotherapy, such as the increased incidence of undesirable mutagenic side effects.

Changes of gas exchange parameters and of functional-biochemical properties of erythrocytes in dynamics of experimental anemia in rats

In experiments on Wistar male rats, on a specially constructed computer installation, O2 consumption by the animals in comparison with changes of hematological, biochemical, and rheological blood properties is studied after anemization--acute bloodletting (12-15% of the total blood mass). An increase of the O2 consumption by the organism and tissues by 18-28% has been revealed for the first 7 days after the bloodletting, in spite of a pronounced decrease of hematocrit, and of the amount of erythrocytes and hemoglobin in peripheral blood by 20-25% of the initial level. There was a 5-10-fold increase of the content of immature erythrocyte forms--reticulocytes and a progressive rise of cell acidic resistance, which is characteristic of young erythrocyte forms. An increase of O2 consumption at a decrease of the blood oxygen capacity (a low hemoglobin level) seems to be due to the more efficient transport and yield of O2 to tissues. At the 3rd 7th day after the bloodletting, activity of Na,K-ATPase has been shown to increase by 60% and 20%, respectively. Analysis of the erythrocyte rheological properties has shown that the maximal firmness of aggregates (Uq) and the aggregation rate (1/T) decrease progressively beginning from 3 days after the bloodletting; index of deformability (I(max)) turned out to be elevated by 7-11%, probably due to an increase of the cell membrane elasticity. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are directed to optimization of blood circulation in large vessels and the capillary network.

The rat bone marrow micronucleus test--study design and statistical power

Although the rodent bone marrow micronucleus test has been in routine use for over 20 years, little work has been published to support its experimental design and all this has used the mouse rather than the rat. When it was decided to change the strain of rat routinely used in this laboratory to the Han Wistar, a preliminary study was performed to investigate the possible factors influencing experimental variability and to use statistical tools to examine possible study designs. Subsequently, a historical database comprising of vehicle controls accumulated from 65 studies was used to establish test acceptance criteria and a strategy for analysing equivocal results. The following conclusions were made: (i) no statistically significant differences were observed in experimental variability within or between control animals; although not statistically significant, the majority of experimental variability seen was found to be between separate counts on the same slide, with minimal differences found between duplicate slides from the same rat or between individual rats; (ii) power analyses showed that, if an equivocal result is obtained after scoring 2000 immature erythrocytes (IE), it is appropriate to re-code the slides and score an additional 4000 IE, i.e. analysing a total of 6000 IE; no meaningful increase in statistical power is gained by scoring >6000 IE; this is consistent with the variability observed between separate counts on the same slide; (iii) there was no significant difference between the control micronucleated immature erythrocyte (MIE) values at 24 and 48 h after dosing or between males and females; therefore, if an unusually low control value at either time point results in apparent small increases in MIE in a treated group, it is valid to pool control values from both time points for clarification and (iv) similar statistical power can be achieved by scoring 2000 IE from seven rats or 4000 IE from five rats, respectively. However, this is based only on control animals and does not consider possible differences in responses between animals to treatment with a potential genotoxin. In order to minimize the possible influence of responders and non-responders, the preferred study design in this laboratory is to score 2000 IE from groups of seven rats. Study data obtained over time confirmed observations made in the control study. Also from an ethical viewpoint, clarifying equivocal responses by combining control data from the 24- and 48-h time points and/or increasing the number of IE scored per animal has minimized the numbers of repeat studies necessary to determine the genotoxic status of a novel compound. However, before any laboratory can use these procedures, experimental data must be generated to demonstrate their validity.

Environmental genotoxicity and cytotoxicity in the offshore zones of the Baltic and the North Seas

Micronuclei (MN), nuclear buds (NB) and fragmented-apoptotic cells (FA) were analyzed in mature peripheral blood and immature cephalic kidney erythrocytes of flounder ( Platichthys flesus), dab ( Limanda limanda) and cod ( Gadus morhua) from 12 offshore sites in the Baltic Sea (479 specimens) and 11 sites in the North Sea (291 specimens), which were collected during three research vessel cruises in December 2002, 2003 and in September 2004. The highest levels of environmental genotoxicity (frequencies of MN up to 0.5‰, NB – up to 0.75‰) and cytotoxicity (FA – up to 0.53‰) were observed in flatfishes from areas close to oil and gas platforms in the North Sea and in zones related to the extensive shipping and potentially influenced by contamination from large European Rivers (Elbe, Vistula, Oder). In dab from the offshore zones of the North Sea, the levels of nuclear abnormalities were higher as compared to those in dab from the Baltic Sea. Responses in immature kidney erythrocytes were higher than in mature erythrocytes from peripheral blood. MN frequency lower than 0.05‰ (the Baltic Sea) and lower than 0.1‰ (the North Sea) could be suspected as a reference level in the peripheral blood erythrocytes of flatfish.

Experimental transmission of Sparicotyle chrysophrii (Monogenea: Polyopisthocotylea) to gilthead seabream (Sparus aurata) and histopathology of the infection

The polyopisthocotylean Sparicotyle chrysophrii (Van Beneden et Hesse, 1863) was experimentally transmitted to gilthead seabream (Sparus aurata L.) by exposure to eggs (EGT) and by cohabitation with naturally parasitized fish (CT). In EGT trials, the infection was successfully transmitted by introducing containers with monogenean eggs in the fish tanks, with the highest infection level (85.7% prevalence, 3.3 mean intensity) achieved at 6 weeks post exposure (p.e.) to the infection dose of 650 eggs per tank. In CT trials, the progression of the infection was faster and reached higher levels than in EGT. When using small fish juveniles (30 g) (CT-2), infection reached 100% prevalence (mean intensity 8 monogeneans/fish) at 5 weeks p.e., but no eggs could be found in the fish even 10 weeks p.e. By contrast, when larger juveniles (150 g) were used (CT-1), infection levels were lower, but mature adults with eggs were detected starting from 8 weeks p.e. The effect of the parasite on the condition factor, haematocrit, haemoglobin concentration (Hb), red blood cell counts, mean corpuscular haemoglobin concentration (MCHC), mean corpuscular haemoglobin content (MCH) and mean cellular volume (MCV) of infected fish was studied in CT trials. The infection produced hypochromic anaemia, since Hb concentration significantly decreased at 5 and 10 weeks p.e. in CT-2 and at 8 weeks p.e. in CT-1. MCHC was significantly lower in parasitized than in control fish at 5 and 8 weeks p.e. in CT-2 and CT-1, respectively. Also in CT-1, MCH was lower and circulating immature erythrocytes, granulocytes and plasma cells were higher in infected fish than in control ones at 8 weeks p.e. The histopathological effects of the monogenean on the gills of naturally infected fish consisted of lamellar shortening, clubbing and synechiae. The proliferation of the epithelial tissue produced fusion of secondary lamellae, and abundant chloride cells were observed.

Association of Placenta Growth Factor with Hemolysis and Inflammation in Sickle Cell Disease (SCD)

BACKGROUND: Placenta Growth Factor (PlGF) is a member of the vascular endothelial growth factor family of cytokines. Although expressed at high levels by the placenta during pregnancy, PlGF is also released by immature erythrocytes and is elevated in SCD. PlGF appears to play a role in inflammation and may be associated with the frequency of pain episodes in SCD. More recently, PlGF has been reported to increase the expression of endothelin-1 and endothelin-B receptor in human pulmonary microvascular endothelial cells and monocytes, and therefore may play a role in the development of SCD-associated pulmonary hypertension (PHT).OBJECTIVE: We have previously reported that SCD patients with PHT exhibit higher levels of inflammatory markers compared to patients without PHT. The purpose of this study was to evaluate the association of PlGF with PHT (and other SCD-related complications), and to explore the link between PlGF and laboratory markers of hemolysis and inflammation in SCD patients.METHODS: We performed a cross-sectional study of patients followed at the Sickle Cell Clinic at UNC-Chapel Hill. Doppler echocardiography was used to determine the pulmonary artery systolic pressure, and PHT was subsequently defined using an age-, sex-and BMI-adjusted reference range. Nitric oxide metabolites (NOx), PlGF and soluble VCAM (all quantified by ELISA), as well as laboratory markers of hemolysis were obtained at the time of patient evaluation. Student's t-tests (a = 0.05) were used to compare mean values of PlGF in patients with SCD-related complications. Furthermore, Spearman's correlation coefficient was used to assess for correlations between PlGF and laboratory variables.RESULTS: Plasma samples were available in 71 of the 74 subjects evaluated. Levels of PlGF were significantly higher in patients with PHT (12.3 ng/ml vs. 8.9 ng/ml, p = 0.005), and patients with a history of stroke (12.8 ng/ml vs 9.3 ng/ml p = 0.027) compared to those patients without these complications. There was however no association of PlGF with the frequency of acute pain episodes, acute chest syndrome, leg ulcers, priapism or retinopathy. We observed significant positive correlations between lactate dehydrogenase (r2 = 0.13, p = 0.002), reticulocyte counts (r2 = 0.08, p = 0.02), soluble VCAM (r2 = 0.09, p = 0.009), NT-proBNP (r2 = 0.13, p = 0.003), and absolute monocyte count (r2 = 0.08, p = 0.02) with PlGF. There were significant negative correlations between PlGF and hemoglobin (r2 = 0.19, p < 0.0001), and borderline significant negative correlations between PlGF and NOx (r2 = 0.05, p = 0.08).CONCLUSIONS: Our results indicate that PlGF is strongly associated with PHT and stroke, and correlates with soluble VCAM in SCD patients. This suggests that inflammation may play a role in the pathogenesis of SCD-associated vasculopathy. The correlation of PlGF with laboratory measures of hemolysis and NOx suggests that hemolysis, possibly with associated scavenging of nitric oxide, contributes to the inflammatory state in SCD.

Clonal diversity within infections and the virulence of a malaria parasite,Plasmodium mexicanum

Both verbal and mathematical models of parasite virulence predict that genetic diversity of microparasite infections will influence the level of costs suffered by the host. We tested this idea by manipulating the number of co-existing clones of Plasmodium mexicanum in its natural vertebrate host, the fence lizard Sceloporus occidentalis. We established replicate infections of P. mexicanum made up of 1, 2, 3, or >3 clones (scored using 3 microsatellite loci) to observe the influence of clone number on several measures of parasite virulence. Clonal diversity did not affect body growth or production of immature erythrocytes. Blood haemoglobin concentration was highest for the most genetically complex infections (equal to that of non-infected lizards), and blood glucose levels and rate of blood clotting was highest for the most diverse infections (with greater glucose and more rapid clotting than non-infected animals). Neither specific clones nor parasitaemia were associated with virulence. In this first experiment that manipulated the clonal diversity of a natural Plasmodium-host system, the cost of infection with 1 or 2 clones of P. mexicanum was similar to that previously reported for infected lizards, but the most complex infections had either no cost or could be beneficial for the host.

Ectoparasites affect hemoglobin and percentages of immature erythrocytes but not hematocrit in nestling Eastern Bluebirds

Abstract Populations of the hematophagous mite, Dermanyssus prognephilus, within Eastern Bluebird (Sialia sialis) nests were manipulated using a pyrethrin-based insecticide to examine effects on nestling growth, selected hematological parameters, and fledging success. There was no difference in body mass, fledging success, estimated white blood cell counts, or hematocrits of infested versus non-infested nestlings. However, infested nestlings had a 25.0% reduction in hemoglobin and a 20.5% increase in immature erythrocytes. These findings suggest a physiological response to parasite feeding that may affect post-fledging survival.

Circulating siderophagocytes and erythrophagocytes in a corn snake (Elaphe guttata) after coelomic surgery

Leukocytes containing nonheme iron and phagocytosed fragments of erythrocytes were found in blood smears from a corn snake (Elaphe guttata) collected 20 and 79 days after coelomic surgery (ovariosalpingectomy). Numerous immature and mitotic erythrocytes also were seen in the sample taken 20 days postsurgically. Siderophagocytes and erythrophagocytes had not been observed before surgery and were not found in multiple subsequent blood samples collected 112-602 days after surgery. Other than these hematologic abnormalities, laboratory findings were unremarkable and the snake recovered uneventfully. Based on examination of sequential blood smears, the circulating siderophagocytes were interpreted as recirculating macrophages involved in the removal of blood from the coelomic cavity after mild postsurgical hemorrhage.

Reticulocyte Enrichment of Zinc Protoporphyrin/Heme Discriminates Impaired Iron Supply During Early Development

In infants and children, elevated whole blood zinc protoporphyrin/heme (ZnPP/H) measures iron-deficient (ID) erythropoiesis. Because immature erythrocytes are less dense than mature erythrocytes, we hypothesized that the sensitivity of ZnPP/H is improved if measured in the least dense cells. Blood was collected from control suckling, mildly and severely ID suckling rats. Cord blood was collected after uncomplicated pregnancies (control), diabetic pregnancies (severe ID) and after pregnancies at-risk for iron deficiency (mild ID). ZnPP/H was measured before and after a two-step density centrifugation to obtain the lightest 6.25% of erythrocyte (top fraction). The difference between whole blood and top fraction was defined as DeltaZnPP/H. In rats, although the whole or top ZnPP/H differed by postnatal age, DeltaZnPP/H was greatest after the interval with least body iron accrual. In either rats or humans with mild ID, whole blood ZnPP/H was similar to, but DeltaZnPP/H was greater than controls. In rats and newborn humans, DeltaZnPP/H is more sensitive than whole blood ZnPP/H in identifying conditions associated with impaired erythrocyte iron delivery and may become a useful tool in measuring erythrocyte iron incorporation in early development.

Cutaneous extramedullary hematopoiesis in a patient with idiopathic myelofibrosis

Idiopathic myelofibrosis (IM) is a chronic myeloproliferative disorder and some cases of IM have extramedullary hematopoiesis. Extramedullary hematopoiesis is commonly seen in the liver, spleen and lymph nodes, but cutaneous extramedullary hematopoiesis (CEH) is very rare in cases of IM. We report a case of CEH in a 65-year-old Japanese woman with IM. This patient had many hard brownish nodules on her chest, abdomen and scalp. Histopathological examination of the nodule on her chest showed the existence of various stages of immature erythrocytes, leukocytes and megakaryocytes indicating that these hard nodules showed extramedullary hematopoiesis in the dermis. The proliferation of these immature cells in the dermis plays an important role in the pathogenesis of CEH. CEH is a very rare manifestation of IM and progresses slowly.

Pulmonary Extramedullary Haematopoiesis After Allogeneic Stem-Cell Transplantation

Practitioners performing stem-cell transplantation (SCT) sometimes experience temporary hypoxia during stem-cell infusion, which is recognized subjectively as chest discomfort and resolves completely after the infusion. Some physicians believe that this hypoxia is caused by a temporary embolism because of the presence of infused stem cells in the pulmonary artery. To the best of our knowledge, however, it remains unproven that stem cells aggregate in the small vessels of the pulmonary artery during stem-cell infusion. This infusion-related pulmonary event could be prevented by other interventions, such as filtration of the infusion stem cells to release cell clusters. A 52-year-old male aplastic anemia patient underwent allogeneic SCT from a sex-mismatched related donor. The stem-cell source was bone marrow from an human leukocyte antigen full-matched related donor with a conventional cyclophosphamide/total body irradiation regimen. Nine years after SCT, he suffered from late-onset noninfectious pulmonary complications constituting severe restrictive lung disease with pneumothorax. He underwent video-assisted thoracoscopic surgery for lung biopsy. Before video-assisted thoracoscopic surgery, his pulmonary function test showed a severe restrictive pattern, as following %VC was 45.8%, FEV1.0% was 100%, and FEV1.0 was 1.72 L. Chest x-ray photograph and chest computed tomography showed a broad low-attenuation area in the whole lung reflecting emphysema, and a reticulo-nodular shadow predominantly along the pleura. Computed tomography imaging specified bronchoectasia mainly in the bilateral upper lungs. Pathological findings of his right lung incidentally showed intravascular hematopoiesis containing immature granulocytes, erythrocytes, and megakaryocytes in his pulmonary artery (Fig. 1a). Evidence of the existence of stem cells was gained by partial staining by CD34 immunostaining in a few cells of the extramedullary hematopoiesis (EMH) site. The hematopoietic cells had XX signals detected by fluorescent in situ hybridization (Fig. 1b). There was no obvious hematological abnormality in his peripheral blood, and the proportion of XX and XY cells in the peripheral blood analyzed by fluorescent in situ hybridization revealed a 99.6% donor (XX) pattern with 0.0% XY signals. Bone marrow findings were normal, and no peripheral lymphadenopathy or hepatosplenomegaly was detected.FIGURE 1.: Pathological findings of lung biopsy (a) (×100) and sexual chromosome fluorescent in situ hybridization (FISH) in the same tissue (b) (×400). Extramedullary hematopoiesis (EMH) in the pulmonary artery was detected by XX chromosomal signals.EMH is a rare condition defined as the appearance of hematopoietic elements outside of the bone marrow. EMH is generally assumed to be a compensatory hyperplastic phenomenon when associated with anemia or space-occupying bone marrow disease (1). Though the presence of EMH in the lung is much less common (2), it is occasionally associated with traumatic events, such as a complicating bone fracture or cardiac surgery, leading to bone marrow embolism (3). In the present case, the patient’s aplastic anemia was in remission and the pathogenesis described above was not found. Peripherally transfused stem cells circulate to the lung arteries, where they may survive and continue hematopoiesis for several years after SCT. Spontaneous hypoxia during peripheral blood progenitor cell (PBPC) infusion may be recognizable if a massive cluster of stem cells occludes pulmonary circulation for a time and most of the cells recirculate while some stay to seed. Sale and Storb (4) reported the death of a dog due to extensive ossification of the lungs after dog leukocyte antigen-matched allograft transplantation. They explained that EMH needs not only hematopoietic cells but also an in vivo microenviroment including osteoblasts, fat cells, and fibroblasts. Mesenchymal cell growth could have resulted in the respiratory failure of the dog. This experimental phenomenon might provide the explanation for late-onset noninfectious pulmonary complications in the present case. And the early report concerning to the experimental model for chasing the transfused stem cells that initial sojourn of transplanted cells in the lungs last for 1 or 2 days (5). This might be associated to the early adverse events of respiratory system after transplantation. In conclusion, an influence of stem cells for lungs should be researched farther. To the best of our knowledge, this is the first documented case in the literature of isolated EMH in the lungs after SCT. Osamu Imataki Hiroaki Ohnishi Division of Haematology Department of Internal Medicine Faculty of Medicine Kagawa University Kagawa, Japan Yoshio Kushida Division of Pathology Kagawa University Hospital Kagawa, Japan Akira Kitanaka Department of Laboratory Medicine Faculty of Medicine Kagawa University Kagawa, Japan Yoshitsugu Kubota Transfusion Medicine Faculty of Medicine Kagawa University Kagawa, Japan

Nucleic acid distribution pattern in avian erythrocytes and mammalian lymphocytes: Comparative studies by fluorescence microscopy and digital imaging analytical techniques

Nucleated erythrocytes of healthy domestic chicken and ducks, and lymphocytes of healthy Sprague Dawley rats were evaluated for nucleic acid distribution pattern, employing light and fluorescence microscopy procedures, as well as digital imaging analytical methods. The results demonstrate a unique organization of nuclear DNA of mature chicken and duck erythrocytes, as well as immature duck erythrocytes, as delineated spherical nuclear bodies that mostly corresponded with euchromatin zones of the cells in routine Wright-stain blood smears. The nuclear DNA of the rat lymphocytes, on the other hand, was observed as a more diffuse green fluorescing nuclear areas, with punctate variably-sized diffuse areas of RNA red fluorescence. RNA red color fluorescence was also evident in the narrow cytoplasm of the lymphocytes, especially in large lymphocytes, in comparison with the cytoplasm of the mature avian erythrocytes that completely lacked any nucleic acid fluorescence. Nuclear RNA fluorescence was lacking in the mature chicken erythrocytes, compared with those of the mature and immature duck erythrocytes as well as lymphocytes of both avian and rats blood. The significance of these findings lies in the establishment of normal benchmarks for the nuclear and cytoplasmic nucleic acid pattern in eukaryotic cells. These normal benchmarks become valuable in rapid diagnostic situations associated with pathologies, such as the presence of viral nuclear and cytoplasmic inclusion bodies that can alter the nucleic acid pattern of the host cells, and in conditions of cellular abnormal protein aggregations. Variability of cellular nucleic acid pattern can also aid in prognostic assessments of neoplastic conditions.