Idiopathic aplastic anemia: Diagnosis and classification

Aplastic anemia (AA) is a disease characterized by pancytopenia and hypoplastic bone marrow caused by the decrease of hematopoietic stem cells. The pathogenesis of AA is complex and involves an abnormal hematopoietic microenvironment, hematopoietic stem cell/progenitor cell deficiencies and immunity disorders. Survival in severe aplastic anemia (SAA) has markedly improved in the past 4 decades because of advances in hematopoietic stem cell transplantation, immunosuppressive and biologic drugs, and supportive care. Herein, we will update the main issues concern AA according to our literature review.     

当归多糖干预线粒体凋亡改善骨髓衰竭的机制

文题释义： 再生障碍性贫血：①血象：呈全血细胞减少,少数病例早期可仅1系或2系细胞减少。贫血属正常细胞型,亦可呈轻度大红细胞。红细胞轻度大小不一,但无明显畸形及多染现象,一般无幼红细胞出现。网织红细胞绝对值和比例显著减少,中性粒细胞减少低于0.5×10⁹ L^-1,血小板低于20×10⁹ L^-1。②骨髓象：骨髓涂片大体观察油滴增多,骨髓小粒镜检空虚,非造血细胞和脂肪细胞增多,一般在50%以上。 线粒体凋亡：当线粒体膜电位下降,线粒体膜通透性增加,线粒体内促凋亡因子(例如Cyt C、AIF、SMAC/DIABLO、HTRA2/OMI、ENDOG)释放到胞质中。Cyt C与Apaf-1相互作用,在ATP和dATP的协助下形成凋亡复合体,凋亡复合体通过招募并激活Pro-Caspase9,形成Caspase9全酶。Caspase9全酶进一步激活效应Caspase3和Caspase7,启动Caspase级联反应,切割细胞中如α-tubulin、Actin、PARPA、Lamin等超过100种的底物,最终导致细胞凋亡。 背景：再生障碍性贫血的重要发病机制之一是造血干细胞衰竭,如何逆转骨髓衰竭是目前的热点问题。 目的：探讨当归多糖对骨髓衰竭小鼠线粒体功能异常的调控作用。 方法：将72只ICR小鼠随机分为对照组、模型组和治疗组,每组24只,后两组通过⁶⁰Co γ射线照射、腹腔注射环磷酰胺和灌胃氯霉素制备再生障碍性贫血模型,然后分别给予生理盐水或200 mg/kg当归多糖喂养2周,对照组不造模,正常喂养。造模后第1,7,14天进行外周血细胞、骨髓单个核细胞计数,并用透射电子显微镜观察骨髓的线粒体超微结构。采用免疫磁珠与流式结合的分选技术从骨髓单个核细胞中分离并纯化Lin^-Sca-1⁺c-Kit⁺(LSK)细胞,即造血干细胞,检测LSK细胞线粒体膜电位、活性氧水平以及Bcl-2、Bax、cleaved caspase-9、cleaved caspase-3、p38蛋白表达水平。 结果与结论：与对照组相比,模型组和治疗组血常规明显异常,骨髓单个核细胞计数明显下降,LSK细胞中线粒体数量减少,线粒体膜电位以及Bcl-2/Bax比值降低,活性氧水平以及cleaved caspase-9、cleaved caspase-3、p38蛋白表达水平升高(P < 0.05)。与模型组相比,治疗组上述异常得到明显改善(P < 0.05)。结果提示,当归多糖可能通过上调造血干细胞的线粒体膜电位、增加线粒体膜稳定性,逆转其异常凋亡水平,改善造血功能。 ORCID: 0000-0002-8771-5898(崔兴) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

氯甲基苯甲酰氨标记骨髓间充质干细胞在免疫介导骨髓衰竭小鼠模型体内的归巢

背景：再生障碍性贫血是T淋巴细胞免疫亢进破坏造血干祖细胞的一种异常免疫反应性疾病。骨髓间充质干细胞具有支持造血功能同时具有免疫调控作用。 目的：观察骨髓间充质干细胞移植骨髓衰竭模型小鼠体内的归巢情况。 方法：将BALB/c小鼠随机分成正常对照组、骨髓衰竭模型组和骨髓间充质干细胞移植组,于第6天将已标记氯甲基苯甲酰氨的BALB/c小鼠骨髓间充质干细胞经尾静脉注射途径移植给骨髓衰竭模型小鼠,采用流式细胞术和组织学荧光显微镜观察标记细胞在不同组织的动态分布。造模第14 天,观察各组小鼠的平均生存时间、外周血血象和骨髓形态学特征。 结果与结论：骨髓间充质干细胞经尾静脉输注后24 h在受体小鼠骨髓中可发现氯甲基苯甲酰氨阳性标记细胞,72 h时阳性标记细胞数量增多(P < 0.05)。骨髓间充质干细胞移植组小鼠濒死或处死前的白细胞、血红蛋白、血小板、骨髓单个核细胞数与模型组小鼠濒死时相比显著升高(P < 0.01)。骨髓衰竭模型组比骨髓间充质干细胞移植组平均生存时间短(P < 0.05)。结果证实,骨髓间充质干细胞输入骨髓衰竭模型体内能归巢至骨髓,促进造血恢复,减轻骨髓衰竭程度,显著延长生存时间。       

真性红细胞增多症患者骨髓单个核细胞植入再生障碍性贫血小鼠

背景：真性红细胞增多症患者骨髓的高增殖低凋亡特性使其造血干细胞植入NOD/SCID小鼠后成功分化出粒红系细胞,但其能否植入并改善再生障碍性贫血小鼠造血功能,目前国内外尚未有报道。 目的：探讨JAK2阳性真性红细胞增多症患者骨髓单个核细胞植入后对再生障碍性贫血小鼠造血重建的影响。 方法：应用注射用重组人γ-干扰素联合白消安的方法建立再生障碍性贫血小鼠模型,随机分为实验组(n=10)和对照组(n=10),给药结束后第5天实验组经尾静脉输注真性红细胞增多症患者骨髓单个核细胞悬液,对照组同法输注等容积生理盐水。输注后第14天检测小鼠外周血常规、骨髓细胞形态、骨髓组织病理变化以及小鼠外周血和骨髓内CD45+细胞的百分含量。 结果与结论：输注后第14天,实验组小鼠血细胞计数三系减少,骨髓涂片可见散在淋巴细胞和早期造血细胞,骨髓组织活检可见骨髓增生减低,少量粒系细胞及幼红细胞,未见巨核细胞,与对照组比较,造血功能无明显改善。对照组小鼠外周血及骨髓均未检测到CD45+细胞,实验组小鼠外周血及骨髓均可检测到人源化的CD45+细胞,且骨髓中CD45+细胞比外周血高,提示JAK2V617F阳性真性红细胞增多症患者骨髓单个核细胞能成功植入再生障碍性贫血小鼠体内,但血常规、骨髓涂片及活检结果显示未能明显改善骨髓造血功能。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

A study of bone marrow failure syndrome in children

Background: Bone marrow failure syndrome (BMFS), or aplastic anemia, includes peripheral blood single cytopenias, as well as pancytopenia due to inability of the marrow to effectively produce blood cells. Aim: To study the clinico-hematological profile and etiological factors of bone marrow failure syndrome in children. Setting and Design: This prospective study was carried out in the Department of Pediatrics of a university teaching hospital over 36 months. Materials and Methods: Children with pancytopenia (Hb 9 /L, platelet count < 100 x 10 9 /L) and bone marrow cellularity < 25% were included in the study. History of exposure to drugs, socioeconomic status, ethnicity and occupation of father were noted. Bone marrow aspiration; trephine biopsy; Ham test; viral studies for hepatitis A, B and C; and cytogenetic investigations were carried out. Statistical Analysis: Relative risk was estimated by odds ratio (OR) with 95% confidence interval (CI) in matched cases and controls. Results: Of the 53 children studied, 6 (11.3%) were diagnosed as Fanconi anemia. Two cases had features of myelodysplastic syndrome. Forty-five children were labeled as acquired aplastic anemia, of whom one had evidence of hepatitis B infection and two patients (5.8%) had paroxysmal nocturnal hemoglobinuria. Aplastic anemia was more common in children from family with lower socioeconomic status; in Muslims; and where the father's occupation was weaving, dyeing and painting. However, the number was small to make statistically significant conclusions. No correlation could be established with exposure to drugs. Conclusion: Fanconi anemia was responsible for approximately one-tenth of the cases of bone marrow failure syndrome. Majority of the patients had acquired aplastic anemia. Hepatitis B infection was an uncommon cause of acquired aplastic anemia.     

Severe aplastic anemia induced by ticlopidine: report of two cases.

Aplastic anemia is a rare side-effect associated with ticlopidine therapy. We report two cases of severe aplastic anemia developed after the use of ticlopidine. A 51-year-old woman took ticlopidine at 500 mg/day for 49 days to prevent a secondary stroke. She developed fever and dizziness within 49 days of initiating ticlopidine therapy. A 70-year-old woman was started on ticlopidine after coronary stent insertion. Fifty days after starting ticlopidine, she developed fever and dizziness. Both patients showed pancytopenia and were diagnosed as aplastic anemia which were confirmed by bone marrow examination. Both patients were hospitalized and received antibiotics, blood products and hematopoietic growth factors. Four and seven weeks after the withdrawal of ticlopidine, the hematologic parameters of each patient improved. A complete blood count should be monitored during ticlopidine therapy to check for cytopenia.     

Allogeneic stem cell transplantation for aplastic anemia.

Aplastic anemia encompasses a heterogeneous group of diseases with distinct pathophysiologies and a common clinical endpoint of marrow failure. Patients with severe aplastic anemia can be treated with immunosuppressive therapy (IST) or hematopoietic stem cell transplantation (HSCT). Over the last 30 years, advances in both treatment modalities have significantly improved the prognosis for this disease; yet this evolution complicates the central therapeutic question in aplastic anemia: which patients should receive IST and which ones should receive HSCT as front-line therapy? In this review, we describe the major improvements that have occurred in transplantation for aplastic anemia in the last 3 decades. We then outline a framework for deciding which patients should be considered for upfront transplantation.     

p53 expression in myeloid cells of myelodysplastic syndromes. Association with evolution of overt leukemia.

To assess p53 expression in the hematopoietic cells of the bone marrow in premalignant as well as malignant conditions, we examined immunohistochemically bone marrow biopsies from patients with myelodysplastic syndromes (MDS, n = 51), acute myeloid leukemia (n = 42) and as a nonneoplastic condition, aplastic anemia (n = 20) and samples from individuals who had no hematological disorder (control, n = 12). Nuclear accumulation of p53 protein was found in seven of 51 patients with MDS (14%) and two of 42 acute myeloid leukemia patients (5%), whereas patients with aplastic anemia and control subjects were uniformly negative for p53 protein. In the bone marrow of patient with MDS, p53-positive cells constituted about 5 to 30% of the total bone marrow cells. Two-color immunohistochemical analysis revealed that the p53-positive cells were also positive for the myeloid cell marker. Half of the MDS cases that evolved to overt leukemia (seven of 14) exhibited positive p53 reaction in the bone marrow at the time of initial diagnosis. This frequency (50%) was significantly higher than that in de novo acute myeloid leukemia cases. All of the seven MDS cases that exhibited p53 expression at the time of initial diagnosis developed overt leukemia later, and p53 expression was maintained throughout the progression of MDS. The results suggest that p53 mutations that occur in the myeloid cells in MDS may confer a growth advantage to these cells resulting in the progression to overt leukemia. Thus, immunohistochemical examination for p53 is very useful for predicting the evolution to overt leukemia from MDS.     

Spontaneous remision of aplastic anemia occurring during a pregnancy

Aplastic anemia is a rare disease caused by destruction of pluripotent stem cells in bone marrow. The occurrence of aplastic anemia during pregnancy is rare and can be fatal for both mother and child. The association is not well explained and there is no consensus on optimal management. We report the case of 30 years-old women treated for aplastic anemia during pregnancy, the evolution is favorable.     

再生障碍性贫血的研究

近二十年,再生障碍性贫血(AA)的研究取得重大突破.中国AA年发病率约0.73/10万,病毒感染等危险因素与AA发病明显相关.AA已由"骨髓衰竭综合征"逐步被认知为自身免疫性T细胞功能异常相关的骨髓衰竭症.诊断AA关键依据骨髓造血功能低下、T细胞功能亢进及除外检查.治疗AA以强化免疫抑制和促造血为主且疗程必须充分.AA是可以治愈的.     

Aplastic anemia and monosomy 7-associated dysmegakaryocytopoiesis

Aplastic anemia (AA) is marrow failure due to an inadequate number of hematopoietic cells in the marrow. Prior reports have described a more aggressive clinical course in aplastic anemia with monosomy 7. We report 3 pediatric cases of AA with normal cytogenetics followed by acquisition of monosomy 7. Bone marrow biopsies were initially diagnostic of AA but later showed monosomy 7 and an increased number of megakaryocytes with small hypolobated nuclei. Immunohistochemical stains for CD61 highlighted the marked dysmegakaryocytopoiesis. The marrow blast percentage was increased in only 1 patient with 4.6% blasts. The 3 patients underwent bone marrow transplantation, and each has remained disease free for 7 to 18 months after transplantation. Pediatric patients with AA and normal cytogenetics may develop monosomy 7 with a myelodysplastic syndrome, unclassified. Patients with AA and monosomy 7 should be evaluated for dysmegakaryocytopoiesis.     

Trisomy 1q in a patient with severe aplastic anemia

Aplastic anemia is a rare, serious disease characterized by hypocellular bone marrow and pancytopenia in the peripheral blood. Most cases are acquired, idiopathic, and without gross cytogenetic abnormalities. A few chromosome abnormalities have recurred among a small subset of patients, most commonly trisomy 8 and monosomy 7. Some of these chromosome abnormalities have prognostic and therapeutic significance, although for most the clinical relevance is not known. We present the case of a 40-year-old man with idiopathic severe aplastic anemia in bone marrow cells with trisomy of the whole long arm of chromosome 1 due to an unbalanced translocation between chromosomes 1 and 15 at breakpoints of q10 and 15q10. This clonal abnormality (which, to our knowledge, has not been previously reported in a patient with aplastic anemia) suggests that genes on 1q may be involved in marrow aplasia.     

Stimulation of myelopoiesis in patients with aplastic anemia by recombinant human granulocyte-macrophage colony-stimulating factor

Aplastic anemia is a syndrome in which pancytopenia occurs in the presence of hypocellularity of the bone marrow. To assess the biologic activities of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) in aplastic anemia, we gave GM-CSF (60 to 500 micrograms per square meter of body-surface area) to 10 patients with moderate or severe disease, by continuous intravenous infusion daily for two weeks, and repeated the treatment after a two-week rest period. The treatment increased the white-cell count (1.6- to 10-fold) in all patients, primarily because of an increase in the numbers of neutrophils (1.5 to 20-fold), eosinophils (12- to greater than 70-fold), and monocytes (2- to 32-fold). Rates of hydrogen peroxide production in purified granulocyte fractions increased during GM-CSF treatment. Increases in bone marrow cellularity, myeloid precursor cells, and myeloid:erythroid cell ratios accompanied the white-cell response. Despite the in vivo response of the white-cells, the concentration of colony-forming cells remained the same. Measurable concentrations of interleukin-2 (2 to 15 units per milliliter) were found in the serum of 8 patients, and high levels of erythropoietin (81 to 1200 IU per liter) were found in 10 patients. The predominant side effects were constitutional symptoms. These results indicate that recombinant human GM-CSF is effective in stimulating myelopoiesis in patients with severe aplastic anemia and may benefit some patients in whom the disorder is refractory to standard forms of therapy.     

Vitamin C inhibits p53-induced replicative senescence through suppression of ROS production and p38 MAPK activity

We previously reported that tumor cells expressing p53 increase intracellular levels of reactive oxygen species (ROS). In this study, we described an inhibitory effect of vitamin C on replicative senescence. Vitamin C was found to inhibit p53-induced senescence in human bladder cancer EJ cells. The senescence-like phenotype (SLP) induced by p53, which showed a morphological change and an irreversible cell cycle arrest, was not observed in vitamin C-treated EJ cells. In addition, vitamin C did not significantly affect normal cell proliferation. We investigated the molecular mechanisms of the inhibitory effect of vitamin C on the development of replicative senescence in EJ cells. We found that vitamin C inhibited this p53-induced ROS generation. Moreover, p38 kinase which was activated during p53-induced senescence was not observed in vitamin C-treated EJ cells. SB203580, a chemical inhibitor of p38 kinase, was found to consistently inhibit p53-induced senescence. Therefore, it is suggested that vitamin C inhibits p53-induced senescence by preventing ROS generation, which in turn leads to the activation of p38 MAPKinase. These results reveal the inhibitory mechanism of vitamin C on cellular senescence.     

再生障碍性贫血患者骨髓单个核细胞端粒长度及P53、P21的表达

目的:研究再生障碍性贫血(AA)患者骨髓单个核细胞(BMMNC)的端粒长度以及P53和P21表达水平,探讨它们与AA发病的关系。方法:采用实时荧光定量聚合酶链反应(RT-qPCR)检测60例AA患者[其中非重型再障(NSAA)38例,重型再障(SAA)22例]和25例对照者BMMNC中端粒长度以及P53和P21的mRNA表达情况;采用Western blot法检测P53和P21蛋白表达情况;并进行相关性比较。骨髓活检术检测骨髓造血细胞成分分布情况;流式细胞术检测CD34~+细胞占有核细胞百分比情况。结果:AA患者端粒长度较对照组明显缩短骨髓造血细胞成分及CD34~+细胞占有核细胞百分比较对照组明显减少(P0.05);NSAA、SAA患者与对照组相比端粒长度均明显缩短,骨髓造血细胞成分及CD34~+细胞占有核细胞百分比均明显减少(P0.05);SAA与NSAA比较,端粒长度明显缩短,骨髓造血细胞成分及CD34~+细胞占有核细胞百分比明显减少(P0.05)。AA患者P53和P21的mRNA及蛋白表达水平均较对照组明显升高(P0.05);NSAA和SAA患者P53和P21的mRNA及蛋白表达水平与对照组相比均明显升高(P0.05);SAA患者P53和P21的mRNA及蛋白表达水平较NSAA明显升高(P0.05)。端粒长度与P53表达水平或P21表达水平之间没有相关性(P0.05);P53表达水平与P21表达水平之间呈正相关(P0.05)。结论:端粒长度的改变以及P53和P21可能参与了再障的发病经过,推测可能是通过抑制造血干细胞的增殖和分化,从而引发造血细胞凋亡。     

乙酰苯肼、X射线和环磷酰胺联合复制小鼠再生障碍性贫血模型

目的以乙酰苯肼、X射线、环磷酰胺联合应用的方法建立小鼠再生障碍性贫血动物模型。方法BALB/c小鼠,采用皮下注射乙酰苯肼100mg/kg,次日X射线2.0Gy照射后,于试验第5天环磷酰胺80 mg/kg腹腔注射,第15天重复以上步骤但不给予射线处理。观察试验小鼠的血象、骨髓象、造血细胞内的线粒体、造血干细胞集落的形成及肝脏、脾脏的病理变化。结果与正常对照组相比,再障模型组小鼠外周血象三系细胞均有显著降低:促红细胞生成素(EPO)含量升高;骨髓有核细胞数显著降低,造血干细胞集落的形成减低,肝脾出现再障的病理改变。结论本方法建立的再生障碍性贫血小鼠模型,简便,成功率高,外周血和骨髓的病理变化持续、稳定,符合再生障碍性贫血的临床表现。     

Hematologic side effects of drugs

Bone marrow and peripheral blood cells may be adversely affected by drugs. Although the risk from most drugs is very small, many cases are reported because of the millions of doses of drugs taken each year by the population. Neutropenia, thrombocytopenia, hemolytic anemia, aplastic anemia, and macrocytic anemia are the commonest effects, in that order. Aplastic anemia is rare, but very serious when it does occur. Adverse effects may be produced by a direct toxic action of the drug or its metabolites on the bone marrow or, less often, on circulating cells. Antineoplastic drugs and chloramphenicol are examples. Most drugs produce their adverse effects through an immunological mechanism. The drug may act as a hapten or may affect the immune system leading to the production of antidrug antibodies and sometimes autoantibodies. Hemolytic anemia may result. Penicillins may behave in this manner. Some drugs act on erythrocytes with enzyme defects, e.g. glucose-6-phosphate dehydrogenase (G-6-PD) abnormalities, to produce hemolysis. In many cases, the mechanism underlying the adverse effect is unknown. The paper lists the drugs reported to have caused some hematological adverse effect and describes the mechanisms in those cases where they are known.     

Hematopoietic progenitor cells in the blood and bone marrow in various hematologic disorders

Hematopoietic progenitor cells are present in the blood and the bone marrow. Changes in the numbers of hematopoietic progenitor cells reflect alteration of pluripotent stem cells. We discuss such changes in common hematologic diseases including aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH) and thalassemia. In aplastic anemia, the numbers of burst forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) are much decreased; the decrease still exists after recovery from therapy. In PNH, the numbers of progenitor cells are low, even in the presence of marrow hypercellularity. In thalassemia, the numbers of progenitor cells are much increased; more pronounced in splenectomized patients.     

p53 protein overexpression in bone marrow biopsies of patients with Shwachman-Diamond syndrome has a prevalence similar to that of patients with refractory anemia

CONTEXT: Shwachman-Diamond syndrome (SDS) is a rare inherited disorder characterized by pancreatic insufficiency, neutropenia, and in some patients, metaphyseal dysostosis. Patients with SDS are at a high risk for development of bone marrow failure, myelodysplastic syndrome, and acute leukemia. The p53 gene plays a major role in cell-cycle regulation, particularly in the presence of a genetic alteration in DNA, a critical step for the initiation of leukemogenesis. p53 gene up-regulation and p53 protein overexpression may occur as a cellular reaction to significant DNA damage. Shwachman-Diamond syndrome and refractory anemia patients have close similarities in the prevalence of acute leukemia and in cell-cycle changes in bone marrow cells. This similarity was further investigated for p53 protein overexpression using archived tissue from patients with hematologic diseases having various leukemic propensities, including SDS and refractory anemia. METHODS: Immunohistochemical staining for p53 protein overexpression was performed on bone marrow biopsies from 9 patients with SDS. These specimens were compared with biopsies from 71 patients with acquired hematologic disorders with variable risk levels for leukemia, including acquired aplastic anemia (n = 14), refractory anemia (n = 46), and various acquired cytopenias (n = 11), as well as 37 control subjects. RESULTS: p53 protein overexpression was identified only in patients with SDS and in patients with refractory anemia; these groups exhibited comparable prevalences of 78% and 72%, respectively. None of the patients with acquired aplastic anemia, acquired cytopenias, or in the control group showed overexpression of p53 protein. CONCLUSION: The prevalence of p53 protein overexpression in SDS is significantly different from that in acquired aplastic anemia and acquired cytopenias, but it is similar to the prevalence in refractory anemia. We speculate that p53 protein overexpression in this bone marrow failure syndrome may represent an early indicator of significant DNA genetic alteration, which is a crucial step in the process of leukemogenesis.     

Blockade of alpha6-integrin reveals diversity in homing patterns among human, baboon, and murine cells

Our understanding of the mechanisms by which intravenously transplanted hematopoietic stem/progenitor cells (HSPCs) home to and engraft the bone marrow (BM) remains incomplete, but participation of adhesion molecules has been documented. We here demonstrate that blockade of the alpha6-integrin enhanced BM homing of human and nonhuman primate BM-derived HSPCs by >60% in the xenogeneic transplant model and led to significantly improved engraftment. The effect was limited to BM-derived HSPCs, as granulocyte-colony-stimulating factor mobilized peripheral blood or cord blood HSPCs express little or no alpha6 integrin. By contrast, despite high alpha6 integrin expression, no effect of alpha6 blockade on murine BM-HSPCs homing/engraftment was observed.