Validation of the mindray BC6000 hematology analyzer for erythroblast counting in peripheral blood / Validação do analisador hematológico Mindray BC6000 para a contagem de eritroblastos em sangue periférico

ABSTRACT Introduction Differential counting of erythroblasts in blood samples by hematology analyzers still has limitations. Technological advances in blood count equipment have proposed the fully automated counting of these cells, however, not without its validation. Objective Validate the automated count of erythroblasts in peripheral blood in the Mindray BC6000 hematology analyzer and verify the existence of correlation of the maturation stages of erythroblasts with the equipment's graphics. Material and Methods Prospective study with peripheral blood samples from the Clinical Pathology Laboratory Dr Paulo C. Azevedo, regardless of age and gender, to validate the erythroblast count in the Mindray BC6000 hematology analyzer compared to the manual method (gold standard), in the period of June 2019 to December 2020. Results Seventeen peripheral blood samples were analyzed from newborns (09/17 - 52.3%) and from patients older than 2 years (08/17 - 47.7%) who had more than 18% of erythroblasts after morphological analysis of the sample. Statistical analysis of erythroblast counts by the two methodologies showed that the Mindray BC6000 hematological counter has good reproducibility, precision and linearity. There was no correlation between the maturation stages of erythroblasts and the equipment graphics. Conclusion The proposed validation showed that the Mindray BC6000 hematological counter has good analytical performance for counting erythroblasts in peripheral blood. However, there is no correlation between the maturation stages of erythroblasts with the graphics generated by the equipment.

RESUMO Introdução A contagem diferencial de eritroblastos em amostras de sangue por analisadores de hematologia ainda apresenta limitações. Os avanços tecnológicos nos equipamentos de hemograma têm proposto a contagem totalmente automatizada dessas células, porém, não sem sua validação. Objetivo Validar a contagem automatizada de eritroblastos no sangue periférico no analisador hematológico Mindray BC6000 e verificar a existência de correlação dos estágios de maturação dos eritroblastos com os gráficos do equipamento. Material e Métodos Estudo prospectivo com amostras de sangue periférico do Laboratório de Patologia Clínica Dr. Paulo C. Azevedo, independente de idade e sexo, para validação da contagem de eritroblastos no analisador hematológico Mindray BC6000 em comparação ao método manual (padrão ouro), no período de junho de 2019 a dezembro de 2020. Resultados Foram analisadas 17 amostras de sangue periférico de recém-nascidos (17/09 - 52,3%) e de pacientes maiores de 2 anos (17/08 - 47,7%) que apresentavam mais de 18% de eritroblastos após análise morfológica da amostra. A análise estatística das contagens de eritroblastos pelas duas metodologias mostrou que o contador hematológico Mindray BC6000 tem boa reprodutibilidade, precisão e linearidade. Não houve correlação entre os estágios de maturação dos eritroblastos e os gráficos do equipamento. Conclusão A validação proposta mostrou que o contador hematológico Mindray BC6000 apresenta bom desempenho analítico para contagem de eritroblastos em sangue periférico. Porém, não há correlação entre os estágios de maturação dos eritroblastos com os gráficos gerados pelo equipamento.

Estimating biological reference intervals for cell blood counts in a dog population / Estimación de los intervalos biológicos de referencia para conteos celulares sanguíneos en una población de perros / Estimando intervalos de referência biológica para contagens de células sanguíneas em uma população de cães

Abstract Background: The results from automated equipment and peripheral blood smears allow correlating clinical data with cellular blood counts (CBC), generating information on pathologies of hematological and non-hematological origin in dogs. Objective: To describe qualitative and quantitative magnitudes of CBC in healthy donor dogs of a blood- bank in Medellín (Colombia). Methods: A descriptive-prospective study was carried out from 146 records of CBC results from a blood-bank. The samples were processed by automated equipment, and trained personnel performed the observation of peripheral blood smears to collect qualitative information. Variables such as age, sex, breed, quantitative results, and observations of the peripheral blood smears were considered. For the definition of biological intervals (BIs), the mean and two standard deviations were used for the data with a normal distribution. Otherwise, the 2.5th and 97.5th percentiles were used. Results: The size, granularity, and cytoplasmic vacuoles in monocytes and polymorphonuclear eosinophils, as well as the presence of Barr chromatin and occasional circulating erythroblasts, were remarkable findings to the peripheral blood smear. With the standardization of data obtained from dogs of large and giant breeds, the present work provides BIs for some of the CBC data in the studied population. Conclusion: The findings of the present study allow approximations to the definition of alterations in blood cells and their counts, which can guide the veterinarian towards an early diagnosis in dogs.

Resumen Antecedentes: Los datos obtenidos de equipos automatizados y extendidos de sangre periférica permiten correlacionar la clínica y los resultados del hemograma, generando información de interés sobre patologías de origen hematológico y no hematológico en perros. Objetivo: Describir las magnitudes cualitativas y cuantitativas del hemograma en perros sanos, donantes de un banco de sangre en Medellín (Colombia). Métodos: Se realizó un estudio descriptivo-prospectivo a partir de 146 registros de resultados de hemograma remitidos por un banco de sangre. Las muestras fueron procesadas por un equipo automatizado y personal entrenado realizó la observación del extendido de sangre periférica para la colección de información de corte cualitativo. Variables como edad, sexo, raza, resultados cuantitativos de las muestras y observaciones al extendido de sangre periférica fueron consideradas. Para la definición de intervalos biológicos (IBs) se utilizaron la media y dos desviaciones estándar para los datos con distribución normal. De lo contrario, se utilizaron los percentiles 2,5 y 97,5. Resultados: El tamaño, granularidad y vacuolas citoplasmáticas en monocitos y polimorfonucleares eosinófilos, así como la presencia de Cromatina de Barr y eritroblastos circulantes ocasionales fueron hallazgos llamativos al extendido de sangre periférica. Con la normalización de los datos para caninos de razas grandes y gigantes, el presente trabajo aporta IBs para algunos de los datos del hemograma en la población estudiada. Conclusión: Los hallazgos del presente estudio permiten aproximaciones a la definición de alteraciones en las células hemáticas y sus conteos, que pueden orientar al médico veterinario hacia un diagnóstico temprano en perros.

Resumo Antecedentes: As informações obtidas de equipamentos automatizados e esfregaços de sangue periférico permitem correlacionar os resultados clínicos e de hemogramas, gerando informações interessantes sobre patologias de origem hematológica e não hematológica em cães. Objetivo: Descrever as magnitudes qualitativa e quantitativa do hemograma em cães saudáveis, doadores de um banco de sangue em Medellín (Colômbia). Métodos: Foi realizado um estudo descritivo-prospectivo a partir de 146 registros de resultados de hemograma encaminhados por um banco de sangue. As amostras foram processadas por uma equipe automatizada e pessoal treinado observou o esfregaço de sangue periférico para a coleta de informações qualitativas. Foram consideradas variáveis como idade, sexo, raça, resultados quantitativos das amostras e observações do esfregaço de sangue periférico. Para a definição de intervalos biológicos (IBs), foram utilizados a média e dois desvios-padrão para os dados com distribuição normal. Caso contrário, foram utilizados os percentis 2,5 e 97,5. Resultados: O tamanho, a granularidade e os vacúolos citoplasmáticos dos monócitos e das células polimorfonucleares dos eosinófilos, bem como a presença da cromatina de Barr e ocasionais eritroblastos circulantes foram achados marcantes na disseminação do sangue periférico. Com a normalização dos dados para cães de raças grandes e gigantes, o presente trabalho fornece IBs para alguns dos dados de hemograma na população estudada. Conclusão: Os achados do presente estudo permitem aproximações à definição de alterações nas células sanguíneas e suas contagens, o que pode orientar o médico veterinário responsável pelo diagnóstico precoce em cães.

Establishment of a predictive model for outcomes in patients with severe acute pancreatitis by nucleated red blood cells combined with Ranson score and APACHE Ⅱ score / 中华检验医学杂志

Objective@#To explore a predictive model for outcomes of severe acute pancreatitis (SAP) patients.@*Methods@#A retrospective study was conducted of 102 SAP patients from January 1,2016 to April 30,2018 from Taizhou Hospital in this study. The participants were divided into survival group and death group according to the outcome of 90 days after admission,88 cases were in survival group including 57 males and 31 females, aged 55.5 (40.3-69.8) years; 14 cases were in death group including 5 males and 9 females, aged 63.0 (50.8-80.8) years. Clinical data and laboratory indicators were compared between the two groups. Statistical analyses were performed to compare categorical variables. Chi-square automatic interaction detector (CHAID) was used to construct the prediction model of SAP patients′ outcomes. The study cohort consisted of SAP patients from August 1st 2018 to July 1st 2019 were collected to validate the prediction model.@*Results@#(1)Statistical analyses were performed by chi square test and Mann-Whitney U test. There were statistically significant differences in the proportion of cardiovascular and cerebrovascular diseases, mechanical ventilation and septic shock, and also the Charlson complication index (CCI), the Ranson score and APACHEⅡscore(χ2=5.554, P=0.018; χ2=5.585,P=0.018;P=0.008;Z=-3.007,P=0.003;Z=-2.982, P=0.003; Z=-3.257, P=0.001), death group were higher than survival group. (2) The MPV, CRP, MCHC, pH, pCO2 and positive rates of NRBC were statistically different between survival group and death group(Z=-2.466,P=0.014;Z=-2.689,P=0.007;Z=-2.238,P=0.025;Z=-1.977,P=0.048;Z=-2.239, P=0.025;P=0.000).The NRBC-positive rate in the death group was higher than that in the survival group, while the other indexes were lower in the survival group.(3)The decision tree CHAID method obtains the prediction scheme: when it meets the Ranson score≤3, the SAP patients were judged to be alive; when it meets the Ranson score>3 and the NRBC in peripheral blood was negative, the SAP patients were judged to be alive; when it meets the Ranson score>3, the NRBC was positive and the APACHEⅡscore≤21, the SAP patients were judged to be alive; when it meets the Ranson score>3, NRBC was positive and APACHE Ⅱ>21, the SAP patients were adverse prognosis. (4) 50 SAP patients were collected in the validation group, with 43 actual survivors and 7 deaths. The accuracy rate of predicting the outcomes of SAP patients in validation group with the scheme was 94.0% (47/50).@*Conclusion@#The NRBC combined with Ranson score system and APACHE II score system can predict the outcomes of SAP patients.

Changes of peripheral and bone marrow erythrocytesin plateau pikasand rats after hypoxia exposure / 中国组织工程研究

BACKGROUND: Changes of red blood cell in peripheral blood and bone marrow erythropoietic system in plateau pikas are of great significance for hypoxic adaptation. OBJECTIVE: To explore the hypoxic adaptation of erythropoietic system in plateau pikas by comparing the morphological changes of peripheral blood and bone marrow between plateau pikas and rats exposed to hypoxia. METHODS: There were 12 healthy wild plateau pikas and 12 clean Sprague-Dawley rats and were randomly divided into experimental and control groups, 6 in each group. The experimental group animals were fed in a simulated 5 000 m altitude hypobaric hypoxia chamber for 28 consecutive days, and the control group animals were fed in the laboratory at 2 260 m altitude. RESULTS AND CONCLUSION: (1) The diameter of the erythrocytes was smaller and red blood cell count was higher in plateau pikas than those in the rats of control group. After 28 days of hypoxia, red blood cell count, hemoglobin and hematocrit were increased in both experimental groups (P < 0.001), but the increased rate of plateau pikas were less than that of the rats. The mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration showed no significant changes in plateau pikas. (2) Results of bone marrow smear showed that the proportion of polychromatic and orthochromatic erythroblasts had no significant changes in plateau pikas after hypoxia, but increased significantly in the rats (P < 0.05). (3) Hematoxylin-eosin staining results of the sternum indicated that the immature erythroblasts islands did not change significantly in plateau pikas, but increased significantly in rats. (4) So the erythroid changes in peripheral blood and bone marrow of plateau pikas before and after hypoxia are significantly lower than those of the Sprague-Dawley rats, and they may be related to the hypoxia adaptation mechanism.

Reticulocyte parameters and their application / 中国基层医药

Reticulocyte is the immature stage of red blood cell,which is an important indicator to reflect the hematopoietic function of bone marrow and to determine the efficacy of anemia and related diseases.In recent years, full-automatic blood cell analyzer provides reticulocyte count with advanced detection means, and more related parameters.In this paper,the correlation detection of reticulocyte parameters and its clinical application were summa-rized.

Genetically Engineered In Vitro Erythropoiesis

BACKGROUND: Engineered blood has the greatest potential to combat a predicted future shortfall in the US blood supply for transfusion treatments. Engineered blood produced from hematopoietic stem cell (HSC) derived red blood cells in a laboratory is possible, but critical barriers exist to the production of clinically relevant quantities of red blood cells required to create a unit of blood. Erythroblasts have a finite expansion capacity and there are many negative regulatory mechanisms that inhibit in vitro erythropoiesis. In order to overcome these barriers and enable mass production, the expansion capacity of erythroblasts in culture will need to be exponentially improved over the current state of art. This work focused on the hypothesis that genetic engineering of HSC derived erythroblasts can overcome these obstacles. OBJECTIVES: The objective of this research effort was to improve in vitro erythropoiesis efficiency from human adult stem cell derived erythroblasts utilizing genetic engineering. The ultimate goal is to enable the mass production of engineered blood. METHODS: HSCs were isolated from blood samples and cultured in a liquid media containing growth factors. Cells were transfected using a Piggybac plasmid transposon. RESULTS: Cells transfected with SPI-1 continued to proliferate in a liquid culture media. Fluorescence-activated cell sorting (FACS) analysis on culture day 45 revealed a single population of CD71+CD117+ proerythroblast cells. The results of this study suggest that genetically modified erythroblasts could be immortalized in vitro by way of a system modeling murine erythroleukemia. CONCLUSION: Genetic modification can increase erythroblast expansion capacity and potentially enable mass production of red blood cells.

Exogenous c-Myc Blocks Differentiation and Improves Expansion of Human Erythroblasts In vitro

BACKGROUND: Engineered blood has the greatest potential to combat a predicted future shortfall in the blood supply for transfusion treatment. The production of red blood cells from hematopoietic stem cells in the laboratory is possible but the mass production of red blood cells to the level present in a blood transfusion unit is currently not possible. The proliferation capacity of the immature red blood cell will need to be increased to enable mass production. This work focused on the hypothesis that exogenous c-Myc can delay the differentiation process of highly proliferative immature erythroblasts, and increase the proliferation capacity of erythroblast cell cultures. OBJECTIVES: The objective of this research effort was to improve in vitro erythropoiesis from stem cells without gene transfection with the eventual goal of producing blood for transfusion treatment in a manner that could be easily translated into clinical medicine. METHODS: The hematopoietic stem cell containing mononuclear cell fraction of venous blood samples was cultured in a liquid media containing erythroblasts growth factors with and without exogenous c-Myc combined with a cell-penetrating peptide. The cells were maintained in the liquid culture media for 23 days. Viable cells were counted and analyzed with flow cytometry. RESULTS: Our results show a 4 fold increase in expansion of the erythroblasts grown in the c-Myc containing growth media compared to the control. Eighty percent of these cells retained the CD117 surface receptor, indicating immature cells. CONCLUSION: Exogenous c-Myc blocks the differentiation and improves in vitro expansion of human erythroblasts.

Diagnóstico presuntivo de histoplasmosis en un frotis sanguíneo / Presumptive diagnosis of histoplasmosis in a blood smear / Diagnóstico presuntivo de histoplasmose em um esfregaço de sangue

Se comunica el diagnóstico de un caso de histoplasmosis asociada al SIDA a partir de la microscopía de un extendido hemático realizado en oportunidad del procesamiento de una muestra enviada al laboratorio para un estudio hematológico. El extendido, fijado con metanol y teñido con solución de Giemsa al 10% reveló, con objetivo de 100X, estructuras levaduriformes de 2-4 µ de diámetro dentro de las células leucocitarias sanguíneas, con la típica tinción en casquete y la presencia de un halo claro periférico, que caracterizan microscópicamente a Histoplasma capsulatum. Luego, para confirmar el hallazgo micológico, se procedió a teñir el mismo preparado con la técnica de Grocott, la cual puso de manifiesto levaduras de color pardo, dentro de los leucocitos sanguíneos. El paciente, deteriorado clínica e inmunológicamente (<50 linfocitos T CD4+/µL), falleció al día siguiente de efectuado el diagnóstico, a pesar de las medidas terapéuticas tomadas. Este hallazgo pone de manifiesto la necesidad de contar, en un Centro de Referencia de Enfermedades Infecciosas, con operadores entrenados para reconocer estructuras microbianas de importancia diagnóstica en extendidos hemáticos, aún en el Laboratorio General. El diagnóstico inmediato de ésta y otras infecciones graves, como el paludismo, a partir de estas muestras permite instalar un tratamiento etiológico y mejorar las posibilidades de éxito terapéutico.

A case of AIDS-associated histoplasmosis diagnosed by microscopy from a blood smear performed during a hematologic study was reported. The smear, fixed with methanol and stained with 10% Giemsa solution, revealed with a 100X objective, 2-4 µ-diameter yeast - like structures within the leuMicrobiología contakocytes, showing a typical staining in cap and a peripheral clear halo, characteristic of Histoplasma capsulatum. To confirm the mycological finding, the same smear was stained with the Grocott technique, showing brownish yeasts in the leucocytes. The patient, clinically deteriorated and with advanced immunological disorder (<50 T CD4+ lymphocytes/µL), died the next day after the diagnosis was made, in spite of the established treatments. This finding highlights the need to have operators trained in the recognition of microbiological structures of diagnostic importance in hematological smears in a Reference Center of Infectious Diseases, and even in the General Laboratory. The immediate diagnosis of this and other serious infections, as Malaria, enables the etiologic treatment and increases the possibilities of therapeutic success.

Comunica-se o diagnóstico de um caso de histoplasmose associada à AIDS, de uma microscopia de extensão sanguínea realizada por ocasião do processamento de uma amostra enviada ao laboratório para um estudo hematológico. A extensão, fixada com metanol e corada com solução de Giemsa a 10%, revelou com objetivo de 100X, estruturas leveduriformes de 2-4 µ diâmetro dentro de células leucocitárias do sangue, com a típica coloração em tampão e a presença de um halo claro periférico, os quais caracterizam microscopicamente a Histoplasma capsulatum. Em seguida, para confirmar o achado micológico, foi corada a mesma preparação com a técnica de Grocott, que mostrou leveduras de cor parda, dentro dos leucócitos sanguíneos. O paciente clínica e imunologicamente deteriorado (<50 células T CD4+/ µL) morreu um dia após o diagnóstico, apesar das medidas terapêuticas adotadas. Este achado evidencia a necessidade de contar, em um Centro de Referência para Doenças Infecciosas, com operadores treinados para reconhecer estruturas microbianas de importância diagnóstica em extensões sanguíneas, mesmo no Laboratório Geral. O diagnóstico imediato desta e de outras infecções graves como a Malária, a partir de uma extensão sanguínea torna possível instalar um tratamento etiológico e melhorar as chances de êxito terapêutico.

Radiological protection effect of vanillin derivative VND3207 against radiation-induced cytogenetic damage in mouse bone marrow cells / 中华放射医学与防护杂志

Objective To study the protection of vanillin derivative VND3207 on the cytogenetic damage of mouse bone marrow cell induced by ionizing radiation.Methods BALB/c mice were randomly divided into five groups:normal control group,2 Gy dose irradiation group,and three groups of 2 Gy irradiaiton with VND3207 protection at doses of 10,50 and 100 mg/kg,respectively.VND3207 was given by intragastric administration once a day for five days.Two hours after the last drug administration,the mice were irradiated with 2 Gy γ-rays.The changes of polychromatophilic erythroblasts micronuclei (MN),chromosome aberration (CA) and mitosis index (MI) of mouse bone marrow cells were observed at 24 and 48 h after irradiation.Results Under the protection of VND3207 at the dosages 10,50,100 mg/kg,the yields of poly-chromatophilic erythroblasts MN and CA of bone marrow cells were significantly decreased(t = 2.36-4.26,P ＜ 0.05),and the marrow cells MI remained much higher level compared with the irradiated mice without drug protection (t = 2.58,2.01,P ＜ 0.05).The radiological protection effect was drug dose-dependent,and the administration of VND3207 at the dosage of 100 mg/kg resulted in reduction by 50% and 65% in the yields of MN and CA,respectively.Conclusions VND3207 had a good protection effect of on γ-ray induced cytogentic damage of mouse bone marrow cells.

Enrichment of circulating fetal nucleated red blood cell for non-invasive prenatal diagnosis with a new polyclonal antibody specific to fetal hemoglobin / 中华检验医学杂志

Objective To investigate the feasibility of a new polyclonal antibody specific to fetal hemoglobin (HbF) and its application in enrichment of circulating fetal nucleated red blood cell(NRBC) for non-invasive prenatal diagnosis. Methods A polyclonal antibody against a synthetic peptide comprising residues 69-78 of the γ-chain of HbF was prepared and conjugated to carrier protein KLH as the immunogen according to the specific antigenic determinant. The peptide-KLH solution was mixed with freund's complete or incomplete adjuvant and immunized goat to prepare specific polyclonal antibody against the γ-chain of fetal hemoglobin. After purification with protein G, maternal blood was obtained from 32 pregnant women at 22 to 39 weeks of gestation. NRBCs were separated and then stained with antibody against the γ chain of HbF. All the positive cells were collected by micromanipulator under microscopic observation, and whole genome was amplified by improved primer extension preamplification (PEP). Multiplex polymerase chain reaction amplification at nine different polymorphic short tandem repeat (STR) loci was also used to determine origin of the positive cells isolated from maternal blood. Results NRBCs stained with antibody against the γ chain of HbF were found in all of the blood from the 32 cases. Attached positive cells with anti-HbF staining have unique morphological characteristics, low nucleus-to-cytoplasm ratio, brown cytoplasm and blue dense nucleus after hematoxylin counterstain under microscopic observation, which can distinguished NRBCs with other cells. A total of 183 NRBCs were found in all of 32 pregnant women at a range of 0.6～1.8 cell/ml venous blood. The accurate rate was 90.6% by the STR genotype identification. Conclusion The antibodies specific to fetal γ-chain of fetal hemoglobin with synthetic peptide technology may have wide clinical utility in identification of fetal NRBCs from maternal circulation for non-invasive prenatal genetic diagnosis.

The evaluation of the immune status and the detection of the fetal cells in peripheral blood of patients with pregnancy induced hypertension syndrome / 中华检验医学杂志

Objective To investigate various types and quantities of fetal cells getting into peripheral blood of pregnant women and immune status related to pregnancy induced hypertension syndrome (PIH) and analyze the etiology of PIH.Methods (1) Three markers of fetal nucleated red blood cells (fNRBCs) in normal pregnant women(n=43)and PIH patients (n=39)were measured by flow cytometry: CD71,HbF/iAg and HbF/CA.(2)We detected the levels of sub-groups of lymphocytes and some plasma cytokines.such as TNF-α and IL-6.The blood samples were from normal pregnant subjects and patients of PIH.Results In the peripheral blood of patients of PIH and normal pregnant subjects,(1) The quantities of fetal cells using three different methods in PIH [6.56(11.37)%、0.09(0.16)%、0.06(0.11)%]were significantly different from normal pregnant subjects[1.58(3.35)%、0.04(0.08)%、0.02(0.06)%],Z= -5.31,-2.97,-4.13 respectively,P＜0.01.(2)Except CD8,the levels of CD3 (76.4±8.5)%,CD4(42.6±6.4)%,CD4/CD8(1.5±0.4)%,CD19(10.5±3.9)%,CD16/CD56 (12.2±7.7)%,TNF-α (1.4 ±0.6)μg/L and IL-6(89.6±12.9)μg/L in PIH were significantly different from normal pregnant subjects[CD3(70.4±8.3)%,CD4(35.3±6.9)%,CD4/CD8(1.2±0.4)%,CD19(8.2±2.8)%,CD16/CD56(20.5±8.9)%,TNF-α(0.5±0.2)μg/L and IL-6(22.0±5.7)μg/L,respectively,P＜0.001].There were no significant differences observed in the level of CD8(P＞0.05).Conclusion The increment of fNRBCs getting into peripheral blood of pregnant women and associated immune status were implicated in the development of PIH.

Number of Fetal Nucleated Red Blood Cell in Maternal Blood and Placenta in Pregnancies with Intrauterine Growth Retardation / 中华围产医学杂志

Objective To examine the number of fetal nucleated red blood cell (NRBC) in maternal blood and placenta tissue in fetal growth restriction(FGR) pregnancies. Methods 20 women of 28-36 weeks' gestation at age of 21~30(including 9 FGR pregnancies)were chosen. Fetal cells were isolated from maternal blood with discontinuous density gradient centrifugation. The isolated cells were made smear and counted under the microscope; After delivery, the placenta tissue were made into sections and also counted under the microscope; To determine the origin of the NRBC , the single NRBC was analysed by primer extension preamplification (PEP) and polymerase chain reaction (PCR). Results The number of NRBC in 9 FGR pregnancy women's peripheral blood ranged from 12/7 ml~40/7 ml,(average 22.6/7 ml). The number of NRBC in the control pregnancies ranged from 0/7 ml~10/7 ml, (average 5.4/7 ml). Significant difference was shown between the two groups; The number of NRBC in 9 FGR pregnancy women's placenta tissue was significantly higher than the median value in the control pregnancies (2.8/20HP compared with 0.6/20HP, P