小鼠骨髓成熟与未成熟树突状细胞的生物免疫学功能比较研究

目的比较成熟和未成熟状态小鼠骨髓树突状细胞（Dc）的生物免疫学功能的差异。方法粒细胞一巨噬细胞刺激因子（GM—CSF）和白细胞介素（IL）-4联合诱导培养小鼠骨髓未成熟Dc，经LPS刺激制备成熟Dc。分别收集培养第6天的两组Dc并用小鼠CD11c免疫磁珠纯化。电镜下观察成熟和未成熟DC的细胞形态。经流式细胞术检测两组细胞表面分子（MHC—Ⅱ、CD86和CD40）的表达水平。另将收集的DC分别与异基因小鼠脾脏T细胞共培养72h，四唑蓝（MTF）比色法检测T细胞增殖程度。取细胞上清液，液相芯片法检测Th1／Th2细胞因子表达水平。结果电镜下可见两组Dc在形态上存在很大差异。同成熟DC相比，未成熟DC细胞表面较光滑，细胞内存在大量的吞噬小泡。流式细胞术分析显示未成熟DC细胞表面3类分子（MHC—Ⅱ、CD86和CD40）的表达水平均明显低于成熟DC组。混合淋巴细胞反应（MLR）显示．未成熟DC刺激T细胞增殖显著低于相应反应比例的成熟DC，差异有统计学意义（P〈0．05）。细胞因子表达谱表明未成熟DC组分泌Th1细胞因子（IL-2和IFN-γ）较成熟DC组显著下降（P〈0．01）．Th2细胞因子相对占主导。结论未成熟DC倾向于诱导T细胞免疫耐受，有效抑制DC的成熟并维持DC的未成熟状态将有望用于治疗类风湿关节炎等由Th1细胞介导的自身免疫性疾病。     

人脾脏树突状细胞大量培养的新方法

目的：建立人脾脏单核细胞体外培养生成大量树突状细胞的新方法。方法：人脾脏细胞悬液培养2h获得贴壁的单核细胞,加入重组人粒细胞－巨噬细胞集落刺激因子（rhGM-CSF)100μg/L或rhGM-CSF100μg/L 重组人白细胞介素－4（rhIL-4)500kU/L,体外培养7天,收集悬浮细胞,以流式细胞仪分析细胞表型及抗原内蚕能力,体外混合淋巴细胞反应检测细胞抗原呈递功能。结果：以细胞因子培养7天生成的悬浮细胞,20％－80％表达树突状细胞（DC）特异性标志CD1a,表达高水平的HLA－DR、B7－1、B7－2分子,具有较强的抗原内吞能力,体外可以强烈刺激同种T细胞增生,其形态,表型与人外周血单核细胞获得的DC相似。结论：从人脾脏获得的贴型的单核细胞,体外以GM-CSF rhIL-4培养,可以获得极其大量的DC（＞10^9细胞／个脾脏）为进一步研究和应用打下了基础。     

K562细胞可溶性抗原负载树突状细胞体外诱导CTL作用的研究

目的探讨经K562细胞裂解物冲击致敏的外周血单个核细胞衍生的树突状细胞（DC）的生物特性及体外诱导抗原特异性CTL应答的能力。方法采集健康人抗凝外周血分离单个核细胞，贴壁细胞用含rhGM—CSF、rhIL-4、TNF—α的RPM1640＋10％FBS培养基体外诱导培养产生DC，5天收获细胞并将细胞分组：A组：未负载抗原DC；B组：加入K562细胞裂解液脉冲DC。7天后用流式细胞仪检测成熟DC免疫表型，并将非贴壁细胞（淋巴细胞）作为效应细胞与各组DE共育，以产生细胞毒性T淋巴细胞（CTL）。12天用LDH释放试验测定对K562细胞的杀伤活性。并用ELISA方法测定细胞上清液中IL-12的含量。结果（1）经细胞因子联合体外诱导的各组DC较培养前在数量，形态及免疫表型上差异有统计学意义，CD86、CD83、CD40、CD1a表达增加，其中经K562细胞裂解液冲击的DC的CD83CD86表达率明显升高。（2）效应细胞与K562细胞混合培养时，负载K562细胞裂解液的DC刺激后的T细胞比单独DC刺激后的T细胞对K562细胞的杀伤作用更明显。（3）负载K562细胞裂解液的DC细胞培养上清液中产生IL-12含量较未负载抗原的DC明显增加。结论用GM—CSF、IL-4以及TNF—α诱导培养健康人外周血单个核细胞可以得到成熟的DC，且经K562细胞裂解液致敏可以进一步促进DC的成熟并体外诱导特异性杀伤靶细胞的CTL。     

体外培养的树突状细胞与细胞因子诱导的杀伤细胞相互作用的研究

目的 观察同源的树突状细胞（DC）与细胞因子诱导的杀伤（CIK）细胞于体外同一培养体系共同培养时的相互影响,为临床联合应用DC和CIK细胞进行肿瘤生物治疗提供依据.方法 用无血清培养基进行DC和CIK细胞的体外培养制作,共同培养1 w后,检测CIK细胞免疫表型、杀瘤活性的变化以及DC分泌IL-12的变化.结果 DC与CIK细胞的共培养会增加CIK细胞的CD3CD56双阳性细胞的比例和非特异性增强对K562细胞的杀伤活性;同时增强DC分泌IL-12的能力.结论 体外共培养DC与CIK细胞可相互增强抗肿瘤免疫活性.     

树突状细胞增强细胞因子诱导的杀伤细胞抗神经胶质瘤细胞作用及机制研究

目的探讨细胞因子诱导的杀伤细胞（CIK）与树突状细胞（DC）共培养后DC-CIK混合细胞抗神经胶质瘤细胞的免疫作用。方法分离健康人外周血单个核细胞,分别于体外诱导DC和CIK,然后共培养成DC-CIK细胞。实验分DC组、DC-CIK组、DC-T组和CIK组。Elisa试剂盒检测各组培养上清中IL-12和IFN．1的含量,流式细胞仪检测细胞表型,CCK一8法体外检测对神经胶质瘤细胞的杀伤活性。结果DC-CIK组培养上清中IL-12和IFN-1的含量分别为（110．24±2．22）mg／L和INF·Y／（913．46±20．64）mg／L,明显高于其它三组（P〈0．05）。DC—CIK组cDi细胞（61．34-1．31）％、CD3／CD56细胞（29．4±1．03）％也明显增加（P〈0．05）。对神经胶质瘤细胞的杀伤活性,DC—CIK组为（54．67±2．62）％,与DC组（19．44±1．07）％、DC—T组（21．27±1．85）％和CIK组（36．52±2．06）％比较,差异有统计学意义（P〈0．05）。结论DC—CIK细胞能诱导明显的神经胶质瘤细胞杀伤活性,为颅内肿瘤的免疫治疗提供了依据。     

HepG2细胞抗原对脐血CD34^＋造血干细胞诱导分化的树突细胞免疫功能的影响

背景：树突细胞（DC）是体内功能最强的抗原呈递细胞,可激活初始型T细胞,生成辅助性T细胞和杀伤性T细胞。DC具有特异性呈递肿瘤抗原的能力,在肿瘤免疫中发挥重要作用。目的：探讨HepG2细胞抗原对脐血CD34^＋造血干细胞诱导分化的DC免疫功能的影响。方法：分离培养脐血CD34^＋造血干细胞后,加入细胞因子组合诱导生成DC并将其分成HepG2细胞抗原负载组和对照组．以流式细胞仪测定DC生成率和免疫表型,以酶联免疫吸附测定（ELISA）检测干扰素-γ（IFN-γ）含量,以MTT法检测细胞毒性T淋巴细胞（CTL细胞）对HepG2细胞的杀伤作用。结果：DC生成率为60．2％±9．4％。与对照组相比,HepG2细胞抗原负载组DC免疫表型CD1a^＋／CD40^＋、CD83^＋／CD86^＋、CD14^＋／HLA-DR^＋比例显著增高（57．6％±5．4％对33．2％±6．0％、32．5％±3．9％对26．0％±2．8％、38．1％±2．6％对29．1％±2．1％,P〈0．01）;IFN-γ含量呈时间依赖性增高;CTL细胞对HepG2细胞的杀伤作用显著增强（43.3％±11.3％对13．9％±4．6％,P〈0．01）。结论：应用HepG2细胞抗原孵育脐血CD34^＋造血干细胞可诱导分化成熟DC,DC可促进异基因淋巴细胞活化分泌IFN-γ,并产生特异性CTL细胞,杀伤肝癌HepG2细胞。     

树突状细胞调节和细胞因子诱导的杀伤细胞的抗肿瘤机制及其在结直肠癌治疗中的应用前景

利用细胞因子诱导树突状细胞（dendritic cell，DC）和细胞因子诱导的杀伤细胞（cytokine induced killer，CIK细胞），以肿瘤抗原冲击DC，将经过抗原冲击的DC和CIK细胞按一定比例混合进行共培养，得到一种新的免疫效应细胞群，即DC调节和细胞因子诱导的杀伤细胞（DCIK细胞）。DCIK细胞具有识别和特异性杀伤恶性细胞的作用，打破了放化疗“敌我不分”的状况。DCIK细胞不但存活率高，增殖能力强．而且对肿瘤细胞具有特异性杀伤能力，为肿瘤免疫治疗开创了一条新的途径。     

树突状细胞体外分离扩增技术研究进展

树突状细胞（DC）是目前所知的体内功能最强的专职抗原呈递细胞,是机体免疫反应的始动者,被称为机体防御病原微生物侵袭的重要“哨兵”,在移植排斥、抗肿瘤、抗病毒感染和自身免疫疾病中发挥着重要作用。获得大量高纯度且表型、功能典型的DC是上述基础和临床研究的前提。从直接分离提取到添加细胞因子诱导扩增DC研究获得很大进展,成为当今免疫学及肿瘤治疗学等相关学科的研究热点。本文对DC体外分离、纯化及扩增方法作一简要综述。     

脐血DC与CIK共培养对白血病K562细胞杀伤活性的影响

目的观察细胞因子诱导的杀伤细胞（CIK）与同源树突状细胞（DC）共培养后DC—CIK细胞的增殖活性、表型的变化及其对白血病K562细胞杀伤活性的影响。方法采集健康产妇分娩正常足月胎儿脐血50ml,密度梯度离心法分离出脐血单个核细胞培养。收集非贴壁细胞用于诱导培养CIK,贴壁细胞诱导分化出成熟DC;将成熟DC和CIK按1：5的比例混合培养3d,用MTT法检测Dc—CIK共培养细胞对白血病K562细胞杀伤活性。结果DC与CIK共培养后,DC—CIK细胞群的增殖活性和杀伤活性明显高于单纯CIK。结论DC—CIK共培养可明显提高CIK增殖活性和细胞毒作用。     

小鼠髓源性半成熟树突状细胞致免疫耐受的实验研究

三组体外培养的小鼠骨髓细胞,分别采用低剂量粒细胞巨细胞集落刺激因子（GM-CSF）诱导、高剂量GM—CSF诱导及脂多糖（LPS）刺激、低剂量GM—CSF诱导及LPS刺激,使其分化为树突状细胞（DC）。流式细胞术检测DC表面CD11c、CD25、CD40、CD80和CD86、MHC-Ⅱ,ELISA法检测细胞培养上清液中IL-10、IL-12,初次和再次混合淋巴细胞实验检测DC刺激T细胞增殖能力,术前回输DC行同种异位心脏移植并观察术后移植心存活时间。结果显示,低剂量GM—CSF能诱导骨髓细胞分化为CD11c^＋、CD25^-、CD40^low、CD80^low和CD36^low、MHC-Ⅱ^low未成熟表型DC,经LPS刺激后分化为CD11c^＋、CD25^-、CD40^mid、CD80^low和CD86^low、MHC-Ⅱ^mid。半成熟表型DC;半成熟表型DC培养上清液中IL-10／IL-12明显升高,该DC可明显抑制同种异型T细胞增殖反应,较未成熟DC更能延长移植心存活时间。认为小鼠骨髓细胞经低剂量GM—CSF体外诱导可分化为未成熟DC,经LPS刺激可转化为半成熟DC,拥有更强的致耐受性。     

Erythrocyte-endothelial cell adherence in sickle cell disorders

Detachment of individual sickle erythrocytes from cultured endothelial cell monolayers has been evaluated by a fluid-shearing technique in an effort to quantitate adherence at shear forces that would be anticipated in the in vivo circulation. Nonirreversibly sickled cells (non-ISC) were more adherent at normal oxygen tensions than control cells. More than 1% non-ISC remained attached to the monolayer at forces greater than physiologic shear stresses in capillary and venous circulations, and many of the most avidly attached cells, once separated, immediately reattached to adjacent endothelial cells. These data suggest that hemoglobin S-containing erythrocytes may have a higher frequency of adherence in vivo in regions of low shear stress where prolonged erythrocyte-endothelial cell contact could occur. Some of these cells detached by shear force would subsequently reattach in in vivo conditions. Plasma-enhanced attachment frequency and plasma from blood in a case of sickle crisis caused further increase. These observations further support the concept that sickle erythrocyte- endothelial cell interaction may be a significant factor in initiation of vascular occlusive events in sickle cell disease.     

Liver cell adenoma and liver cell adenomatosis

During the last three decades liver cell adenoma and liver cell adenomatosis have emerged as new clinical entities in hepato-logical practice due to the widespread use of oral contraceptives and increased imaging of the liver. On review of published series there is evidence that 10% of liver cell adenomas progress to hepatocellular carcinoma, diagnosis is best made by open or laparoscopic excision biopsy, and the preferred treatment modality is resection of the liver cell adenoma to prevent bleeding and malignant transformation. In liver cell adenomatosis, the association with oral contraceptive use is not as high as in solitary liver cell adenomas. The risk of malignant transformation is not increased compared with solitary liver cell adenomas. Treatment consists of close monitoring and imaging, resection of superficially located, large (>4 cm) or growing liver cell adenomas. Liver transplantation is the last resort in case of substantive concern about malignant transformation or for large, painful adenomas in liver cell adenomatosis after treatment attempts by liver resection.     

Red cell alloimmunization in sickle cell disease

Alloimmunization to red cell antigens contributes to morbidity in transfused patients. It has been recommended that blood for sickle cell patients need not be matched for antigens other than ABO and Rh(D), as there is no greater incidence of antibody production than in other multitransfused patient populations. Post transfusion alloimmunization was studied in a group of 34 sickle cell disease patients attending a U.K. haemoglobinopathy clinic. Red cell antibodies were formed in 17.6% of the transfused patients and Rhesus and Kell antibodies accounted for 66% of this total. In order to reduce alloimmunization, a policy of performing extended red cell phenotyping on the patients, and providing blood matched for Kell, and in certain circumstances the Rhesus antigens other than Rh(D), is recommended.     

Misdiagnosis of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (RCC) represents the most common histological subtype of malignant kidney tumors. Based on symptoms alone, clear cell RCC is indistinguishable from other histological classes of RCC unless the tumor is present in the context of an RCC syndrome. Histopathological examination is, therefore, important to accurately identify clear cell RCC. Clear cell RCCs have characteristic morphological criteria; these tumors can be easily identified upon typical presentation, but diagnosis can be challenging when tumor cell pattern is unusual or when availability of tissue samples is limited. In this Review, the clinical, radiological and pathological characteristics of clear cell RCCs are described, as well as the potential tumors that can be confused with clear cell RCC and need to be considered in the differential diagnoses. Finally, the importance of an accurate diagnosis is highlighted in the context of the increasing use of preoperative tissue sampling and the prevalence of clear cell tumors associated with hereditary syndromes, which could have different therapeutic and prognostic implications for patients and their families.     

Red cell exchange in sickle cell disease

Red cell exchange transfusion is frequently of use in the management of patients with sickle cell disease either electively or therapeutically. Modern cell separators allow this procedure to be performed rapidly, effectively and safely. These machines have a number of advantages over manual exchange procedures. The patient remains isovolaemic, there is little loss of plasma or platelets, the procedure is relatively short and in elective circumstances can be performed on an outpatient basis. In this series 66 exchanges were performed on 21 patients with an overall increase in HbA of 70%. The COBE Spectra gave a mean increase in HbA of 77%, with the majority of patients achieving an HbA of > 90% post exchange. Automated redcell exchange was well tolerated by most patients, and adverse effects were limited to symptoms of hypocalcaemia which were easily treated, and to transfusion reactions. Cell separators can therefore be recommended for exchange transfusion in patients with sickle cell disease, who require an increase in HbA levels either prophylactically or therapeutically. They are safe, effective, easy and quick to use.     

Actin filament debranching regulates cell polarity during cell migration and asymmetric cell division

The formation of the branched actin networks is essential for cell polarity, but it remains unclear how the debranching activity of actin filaments contributes to this process. Here, we showed that an evolutionarily conserved coronin family protein, the Caenorhabditis elegans POD-1, debranched the Arp2/3-nucleated actin filaments in vitro. By fluorescence live imaging analysis of the endogenous POD-1 protein, we found that POD-1 colocalized with Arp2/3 at the leading edge of the migrating C. elegans neuroblasts. Conditional mutations of POD-1 in neuroblasts caused aberrant actin assembly, disrupted cell polarity, and impaired cell migration. In C. elegans one-cell−stage embryos, POD-1 and Arp2/3, moved together during cell polarity establishment, and inhibition of POD-1 blocked Arp2/3 motility and affected the polarized cortical flow, leading to symmetric segregation of cell fate determinants. Together, these results indicate that F-actin debranching organizes actin network and cell polarity in migrating neuroblasts and asymmetrically dividing embryos.

Cell polarity is a fundamental feature of virtually all eukaryotic cells and plays crucial roles in a wide range of cellular processes, including cell motility, asymmetric cell division, and cell signaling (1). The establishment of cell polarity involves the asymmetric assembly of distinct cellular components to perform specialized functions. The actin-related protein (Arp) 2/3 complex-dependent branched actin networks and the pushing force they produce provide the principal means for cells to remodel the plasma membrane during cellular polarization (2). For example, in the leading edge of a migrating cell, the local Arp2/3-nucleated actin polymerization powers asymmetric projections of the plasma membrane (3). During asymmetric cell division of the Caenorhabditis elegans zygote, an actomyosin flow is central to the transport of the polarity PAR proteins into defined subcellular domains (4).Actin filaments'' continuous assembly must be balanced by actin depolymerization to ensure a constant supply of actin monomers for new growth. The Arp2/3 complex potency in actin nucleation empowers this complex as an essential regulator to organize the actin cytoskeleton. While Arp2/3 by itself is biochemically inactive, interactions with nucleation-promoting factors (NPFs) such as the Wiskott Aldrich syndrome protein (WASP)/WASP family verproline-homologous (WASP/WAVE) family proteins shift the Arp2/3 complex from its open, inactive conformation to a closed, active conformation (5, 6). The conformationally activated Arp2/3 complex then binds to the side of preexisting actin filaments to nucleate a branch from the mother filament (7–12). Conversely, nucleation by Arp2/3 can be inhibited by several binding partners, including glia maturation factor (GMF), Gadkin, Arpin, and Coronin, whose activities replenish available pools of actin monomers and Arp2/3 complexes for sustained actin assembly (13–18).The coronin family proteins are conserved actin regulators (19). The phylogenetic analysis grouped coronin genes into three types (19, 20). The best-characterized coronin is the Type I coronin (e.g., Coronin 1B) that binds to actin filaments through the β-propeller structure and to the Arp2/3 complex via its N terminus. These interactions block the docking of Arp2/3 onto actin filaments or facilitate debranching the existing actin network (20). Coronin 1B simultaneously interacts with the Slingshot phosphatase to dephosphorylate and activate ADF/Cofilin proteins that sever actin filaments, thereby promoting the actin network disassembly (13). Despite significant progress on Type I coronin, the activity and function of other coronins remain unclear. In particular, Type III coronins, known as POD-1 in C. elegans and Drosophila or Coronin7 in Dictyostelium and humans, contain two tandem coronin repeats, making them distinct from other coronins (19–21). POD-1 was biochemically isolated from C. elegans oocytes (22), and its mutations disrupted the polarity and architecture in early C. elegans embryos and impaired midlife touch sensitivity of the nematode (21, 23). However, it remains unclear how the Type III coronin functions. The Drosophila homolog of POD-1 is required for correct axon guidance, and the purified Dpod-1 cross-links the actin and microtubule cytoskeletons (24), whereas the mammalian Coronin7 was implicated in the Golgi morphology and function (25, 26), demonstrating the functional divergence of this family of coronin. Here, we show that the C. elegans POD-1 debranches Arp2/3-nucleated actin filaments in vitro and that POD-1 regulates cell polarity by remodeling the actin cytoskeleton during cell migration and asymmetric cell division.     

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

Background

Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.

Methods

Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs.

Results

The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability.

Conclusions

Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.     

Mechanism of red blood cell aging: Relationship of cell density and cell age

The human red cell has a life span of 120 days. The mechanism that determines cell removal from the circulation with such precision remains unknown. Most studies of red cell aging have been based on analysis of cells of progressively increasing age separated by density. The relationship between red cell age and density has been recently challenged, and the hypothesis has been put forward that cell death is not the result of a progressive deterioration of essential cell constituents. This theory was based on preliminary observations in transient erythroblastopenia of childhood, which could not later be confirmed. When the relationship between cell aging and increasing density is critically reviewed, it appears to be based on firm experimental evidence, confirmed by in vivo demonstration of decreasing survival of cells of increasing age. Analysis of studies using buoyant density gradients reveals that this technique can easily distinguish the single exponential slope of decline for those cell components that change progressively throughout the red cell life span from the biphasic decline of those that decrease drastically at the reticulocyte-mature red cell transition. The view that the aging of the red cell and its removal from the circulation result from a progressive series of events during the 120 days of its life span appears to be the most consistent with the available data. Density separation, validated by much experimental evidence, remains a most useful technique for the study of the mechanism of aging of the red cell. © 1993 Wiley-Liss, Inc.     

Red cell rheology in stomatocyte-echinocyte transformation: roles of cell geometry and cell shape

The influence of the shape of the red blood cell during stomatocyte- echinocyte transformation on its deformability was studied by microsieving through pores with diameters of 2.6, 4.5, and 6.9 micron. A stomatocytic transformation was produced by chlorpromazine (0.02, 0.1, and 0.5 mmol/L) and an echinocytic transformation by sodium salicylate (7.5, 30, and 120 mmol/L). For spherostomatocytes, an increase in filtration resistance through 2.6 and 4.5 micron pores was observed, whereas for spheroechinocytes, a decrease in filtration resistance through 2.6 micron pores was found. Larger pores (6.9 micron) were not sensitive to those shape changes. The changes in deformability can be explained by the fact that the surface area of (sphero)-stomatocytes decreased, whereas that of (sphero)-echinocytes increased; the cell volume remained essentially constant. Echinocytes produced by 24-hour adenosine triphosphate depletion differed from drug- induced echinocytes: they had an increased cell volume at constant surface area and consequently an increased filtration resistance through 2.6- and 4.5-micron filter pores. Shape changes with spicule formation are therefore not a homogeneous entity, and cell geometric factors (eg, surface area and volume) must be assessed with care. The viscosity of red cell suspensions at a hematocrit level of 45% was higher for drug-induced echinocytes than discocytes or stomatocytes at all shear rates tested. We conclude that the normal discocyte represents an optimum shape for the flow in vivo since a stomatocytic transformation could impair the passage through the microcirculation (decrease in cell filterability) and an echinocytic transformation could impair the flow in larger vessels (increase in blood viscosity).     

Automated red cell exchange in sickle cell disease

We used multiple optical trapping to study the mechanism of red cell (dis)aggregation. Two sets of optical 'tweezers' were used to bring two red blood cells together to form a two-cell aggregate and then to pull them apart, to study the interaction between the cells.
We found that cross-bridging occurred in normal reversible aggregation as we observed binding and the occurrence of small tethers between opposite cell membranes. Furthermore, the cells could only be parted by sliding them side by side with a maximum velocity in the order of μm/s indicating accumulation of the cross-bridges.