静注人免疫球蛋白中IgG含量检测方法的探讨

为了更好地进行生产质量控制,快速、准确的测定静注人免疫球蛋白中的IgG含量。实验中对2001~2008年的静注人免疫球蛋白中的IgG含量检测结果进行了抽样统计分析。分析结果表明:样品40倍稀释后测得的吸光值与标准曲线第三点的吸光值无显著性差异;样品40倍稀释后测得的IgG含量与样品进行20、30、40倍三个稀释度测得的IgG含量的平均值无显著性差异。从而证明,样品40倍稀释后测得的IgG含量可以代表该批样品的IgG含量,无需进行20、30倍的稀释。检测方法会更省时、省力、省材料。     

静脉输注丙种球蛋白与新生儿溶血病患儿坏死性小肠结肠炎发生的关系

目的探讨静脉输注丙种球蛋白(IVIG)与新生儿溶血病(HDN)患儿坏死性小肠结肠炎(NEC)发生的关系。方法收集重庆医科大学附属儿童医院2013年1月1日至2014年1月1日确诊为HDN患儿的临床资料,采用倾向评分配比法建立回顾性队列研究,根据是否使用IVIG分为IVIG组和非IVIG组,比较两组患儿NEC发生情况。进一步以配比后病例建立病例对照研究,探讨溶血病患儿发生NEC的危险因素。结果共1 217例HDN患儿纳入研究,其中40例合并NEC,309例进行IVIG。进行1∶2的倾向评分配比后,共有199对匹配成功,其中NEC组25例,非NEC组572例。结果显示,倾向评分配比后,IVIG治疗并未增加溶血病患儿NEC的发生率,4.5%(9/199)vs 4.0%(16/398),χ2=0.083,P=0.773。单因素分析显示,早产、低出生体重、多胎、胎膜早破、产前糖皮质激素、静脉营养、先天性心脏病、最高胆红素值与溶血病患儿发生NEC有关。二元Logistic回归显示,先天性心脏病是溶血病患儿发生NEC的独立危险因素(OR:4.021,95%CI:1.286~12.578)。结论 IVIG治疗不增加溶血病患儿发生NEC的概率,合并先天性心脏病的HDN患儿更易发生NEC。     

Hypotension with intravenous immunoglobulin therapy: importance of pH and dimer formation

Therapy with intravenous immunoglobulin preparations has been used effectively in a wide range of conditions. Although generally well tolerated, intravenous immunoglobulin preparations may be associated with transient hypotension in some patients. This study examined the role of different immunoglobulin G fractions in the development of intravenous immunoglobulin-induced hypotension in an anaesthetized rat model and assessed the effects of a new liquid immunoglobulin prepared at a low pH on both the formation of immunoglobulin G dimers and the development of hypotension. The effects of this new preparation in an experimental autoimmune encephalomyelitis model were also evaluated.Results from the haemodynamic studies indicated that immunoglobulin G dimers in polyclonal immunoglobulin G are responsible for the hypotensive events associated with some immunoglobulin preparations. They also showed that adjustment to an acidic pH results in the rapid dissociation of immunoglobulin G dimers and prevents the development of hypotension. Additional experiments demonstrated that only immunoglobulin G dimers with a functional Fc fragment can bind to Fcgamma receptors on macrophages to induce the release of blood pressure-lowering mediators. Moreover, essentially monomeric Fc fragments can block the blood pressure-lowering effects of immunoglobulin G dimers.Preparation of a new liquid intravenous immunoglobulin with the pH adjusted to 4.3 prevents the formation of immunoglobulin G dimers even over long-term storage and does not significantly affect blood pressure in a rat model. This preparation is as effective as other intravenous immunoglobulin preparations in ameliorating symptoms of experimental autoimmune encephalomyelitis. These results, like those from previous studies, indicate that preparation of intravenous immunoglobulin at a low pH substantially reduces immunoglobulin G dimerization; this effect significantly decreases the potential for intravenous immunoglobulin to induce hypotension without reducing its clinically relevant biological activity.     

Capacity of the manufacturing process of Flebogamma DIF, a new human high purity intravenous immunoglobulin, to remove a TSE model-agent

The variant Creutfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy (TSE) associated with the ingestion of cattle derived products affected with bovine spongiform encephalopathy. vCJD emerged in the UK, where most of the cases occurred (170 of 217 cases worldwide). Manufacturers of biological products must investigate the ability of their production processes to remove TSE agents. Two manufacturing steps (polyethylene glycol-PEG precipitation and nanofiltration down to 20 nm) of Flebogamma^® DIF, were evaluated by western blot and bioassay to measure the prion protein (PrP^Sc) and infectivity clearance capacity, respectively. A laboratory scale model representative of the industrial process and a (experimentally) spiked TSE model-agent (hamster scrapie strain 263 K) were employed. Both steps showed a significant capacity to clear the TSE model-agent used since no PrP^Sc signal or infectivity was detected in the resulting product of each step. PEG precipitation and nanofiltration provided reduction factors of ≥6.19 log₁₀ID₅₀ and ≥5.45 log₁₀ID₅₀ respectively. Both steps showed consistency between western blot and bioassay results. These results demonstrate the ability of the Flebogamma^® DIF manufacturing process to clear TSE agents beyond the limit of detection of the assays, by several orders of magnitude.     

Long-term follow up of patients with common variable immunodeficiency treated with intravenous immunoglobulin: Reevaluation of intravenous immunoglobulin replacement therapy

Five patients with common variable immunodeficiency treated in our hospital between December 1979 and December 1990 were given six kinds of intravenous immunoglobulin preparations (pepsin treated, S-sulfonated, polyethylene glycol treated, pH4 treated, alkylated, and pH4.25 formulation preparation) for replacement therapy. Duration of the therapy ranged from 7.6 to 11 years. Incidences of fever and acute infections were variable among patients, but no significant differences were seen in the incidences among periods given each preparation. Three cases revealed abnormal pulmonary functions in tests. Adverse reactions were rarely seen in our study periods, and no severe reactions were observed. No significant differences were seen in incidences of adverse reactions. Postinfusion levels of serum complement slightly decreased from preinfusion levels. However, the decrease in complement was not related to any adverse reaction. No long-term complications such as transmission of hepatitis have been observed. Our data suggest that no obvious differences exist between the efficacy and safety of each IVIG preparation. Differences of efficacy of IVIG replacement therapy may be due to the variable pathophysiology of each patient.Abbreviations CVID common variable immunodeficiency - IVIG intravenous immunoglobulin     

Identification of target antigens of self‐reactive IgG in intravenous immunoglobulin preparations

Intravenous immunoglobulin (IVIg) contains a wide range of self‐reactive immunoglobulins (Ig) G. Acidic pH is known to increase the reactivity of purified IgG with self‐antigens. We describe here the target antigens of IgG autoantibodies in IVIg and analyze the influence of acidic pH on IgG reactivities. We used 2‐DE and immunoblotting with protein extracts of human umbilical vein endothelial cells (HUVEC) and HEp‐2 cells. Two IVIg preparations obtained by ethanol fractionation [one with an acidic pH step (acidic‐IVIg) and one with β‐propiolactone (propiolactone‐IVIg)] and a pool of sera from 12 healthy individuals were tested. Serum IgG of 3 healthy individuals and IgG purified from the same sera with elution at pH 2.8 were also tested individually. Finally, propiolactone‐IVIg was acidified at pH 2.8. IgG obtained with a step at low pH recognized many more target spots than IgG obtained without acidic pH. Our findings demonstrate that an acidic pH step artificially enlarges the repertoire of self‐reactive IgG. Thus, protein spots recognized by IgG in propiolactone‐IVIg represent the core set of self‐antigens targeted by IVIg. Overall, 96 proteins were identified by MS. Fourteen were recognized in both extracts including glycolysis proteins such as α‐enolase, RNA processing and cytoskeletal proteins such as lamin‐A/C.     

低pH孵放法灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证低pH孵放法对不同厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG的pH值为3.8～4.4,在23～25℃环境中,孵放21天可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥5.50～6.62和≥5.38～6.62logTCID50/0.1ml。因此,低pH孵放法是一种安全、有效且简便实用的灭活脂包膜病毒的方法。     

Monitoring of the serum proteome in Kawasaki disease patients before and after immunoglobulin therapy

Kawasaki disease (KD) is a systemic vasculitis that mainly affects children younger than 5 years. The causal pathogen is unknown, therefore specific diagnostic biomarkers and therapy are unavailable. High-dose intravenous immunoglobulin (IVIG) is considered as the most effective therapy to reduce the prevalence of coronary artery lesion (CAL) in KD; however, it has side effects. This study aimed to (1) determine whether IVIG therapy is effective at the molecular level; (2) provide the first serum proteomic profile of KD under IVIG therapy; and (3) screen for monitoring biomarker candidates. We extracted serum proteins from samples of healthy individuals and from KD patients before and after IVIG therapy, and employed two-dimensional electrophoresis and MALDI-TOF/TOF mass spectrometry to identify differentially expressed proteins. The identifications were validated by Western blotting. We identified 29 differentially expressed proteins in KD patients and found that IVIG therapy restored most of these proteins to near-normal levels. Tracing the protein levels of single patients before and after IVIG therapy showed that the proteins, especially Transthyretin (TTR), are potential markers for therapeutic monitoring. Functional analyses of these proteins by PANTHER and String suggested that the key influence of KD lay in the immune system, which was targeted by IVIG.     

微生态制剂防治婴幼儿肺炎继发腹泻的卫生经济学评价

目的 评价预防性应用微生态制剂(培菲康)防治婴幼儿肺炎继发腹泻的疗效及成本效益.方法 2008年1月到2010年9月住院治疗的婴幼儿肺炎966例为研究对象,对患儿的临床特征、微生态制剂(培菲康)预防性应用情况、是否出现继发腹泻及住院治疗费用的关系进行分析.结果 干预组594例,对照组372例,两组住院治疗期间继发腹泻的发病率分别为18.69％、39.78％,两组比较,x2 =51.889,P ＜0.01,RR=0.470;干预组99例发生继发腹泻后继续原有微生态制剂治疗,对照组134例因继发腹泻才开始应用同类微生态制剂治疗,两组腹泻治疗总有效率分别86.87％、74.63％,差异无统计学意义(x2=5.299,P=O.021).干预组、对照组每例(次)患儿住院总费用分别为(2775.0±1724.2)元和(3130.7±1899.8)元,预防性应用微生态制剂(培菲康)次均节省直接医疗费用支出355.6元,差异有统计学意义(t =2.998,P=0.003);预防性应用微生态制剂(培菲康)的效益成本比为13.02.结论 预防性应用微生态制剂(培菲康)不仅能降低肺炎继发腹泻的发病率,缩短住院病程,提高腹泻治疗总有效率,同时,明显降低患儿的次均医疗费用,效益成本比高.     

Cytokine profiles in pemphigus vulgaris patients treated with intravenous immunoglobulins as compared to conventional immunosuppressive therapy

Pemphigus vulgaris (PV) is a potentially fatal blistering disease of the skin and mucous membranes, characterized by the presence of autoantibodies against adhesion molecules (desmoglein, Dsg3) present on the surface of keratinocytes, which lead to the loss of cellular adhesion or acantholysis. The mainstay of treatment is conventional immunosuppressive therapy (CIST), i.e. high dose, long-term systemic corticosteroids with or without immunosuppressive drugs. Intravenous immunoglobulin (IVIg) has been used in patients refractory to CIST, and its use has resulted in long-term clinical remission. Since cytokines play an important role in the immunopathogenesis of PV, it would be useful to compare how both IVIg and CIST therapies affect cytokine levels in the serum of PV patients. Thus, the goal of this study was to conduct a comparative analysis of levels of various cytokines, during an 18 month consecutive period, after the initiation of CIST or IVIg treatment in PV patients, with similar extent and severity of disease in the two study groups, with 11 patients in each group. The cytokines measured were IL-1β, IL-6, IL-8, IFN-γ, IL-4 and IL-10. The levels of most of these cytokines were higher in the sera of untreated patients in both groups, compared to normal controls. The cumulative data collected over an 18 month period of treatment demonstrates that there is a gradual reduction in the levels of these cytokines, until they are at levels observed in normal individuals. The conclusions from this limited number of patients, prospectively studied, would suggest that both CIST and IVIg therapies are similar in their ability to influence a panel of cytokines in patients with pemphigus vulgaris.     

Post‐marketing observational study on 5% intravenous immunoglobulin therapy in patients with secondary immunodeficiency and recurrent serious bacterial infections

Secondary hypogammaglobulinemia is one of the factors responsible for the increased susceptibility to infection in patients with chronic lymphocytic leukemia (CLL). This study assessed the therapeutic results, concomitant medication and tolerance of administering 5% intravenous immunoglobulin, secondary immunodeficiency and recurrent serious bacterial infections. A single center, post‐marketing, observational clinical study was performed on 10 patients with a variety of hematological malignancies (CLL, follicular non‐Hodgkin lymphoma, IgM‐secreting immunocytoma, IgA plasmacytoma and myelodysplastic syndrome/non‐Hodgkin lymphoma) who had been infused with IVIG from June 1994 to May 2009. The clinical benefit of IVIG was assessed by comparing the incidence of bacterial infections before and after starting this therapy. Plasma immunoglobulin concentrations and relevant hematological variables were recorded. For safety assessment, adverse events were monitored. The standard IVIG dosage was approximately 0.35 g/kg body weight every 3–4 weeks. Most patients had normal IgG trough values of >600 mg/dL during the IVIG treatment period. The rate of bacterial infections was reduced from 2.4 per patient in the 3 months before IVIG to 0.7 (0–1.5) per patient per year during IVIG treatment. All patients received concomitant medication, mainly anticancer and anti‐anemia therapy (100%). No serious adverse events related to IVIG were observed. The frequency of at least one minor adverse reaction was 1.44% (8/556 infusions). In conclusion, the investigated IVIG preparation was well tolerated and clinically beneficial in reducing the long term rate of serious bacterial infections in patients receiving concomitant treatment for malignant diseases.     

肺炎患者与健康人口咽部菌群分析

目的观察肺炎患者口咽部菌群分布情况,探讨肺炎患者与健康人群口咽部菌群分布差异。方法采集细菌性肺炎稳定期患者(29例)和健康人群(31例)咽拭子进行细菌培养,并对其咽部菌落进行计数和菌种鉴定。结果肺炎患者咽拭子培养中主要致病菌和条件致病菌检出种类和检出率较健康者明显增加,肺炎组与健康组相比需氧菌与厌氧菌的菌群密度具有上升趋势,使用t检验计算结果差异有统计学意义(P0.05)。在可培养的肺炎组人群口咽菌群中肺炎链球菌检出率最高,其次为微球菌、副溶血链球菌、甲型链球菌;在健康体检人群口咽部菌群中甲型链球菌检出率最高,其次为奇异变形杆菌、微杆菌属、轻型链球菌、颗粒链球菌。结论与健康对照组相比,肺炎患者口咽部菌群分布紊乱,且肺炎组患者呼吸道菌群数量明显增多。     

降低静脉注射用人免疫球蛋白抗补体活性的试验观察

采用CH50试验法测定静脉注射用人免疫球蛋白(IVIG)抗补体活性(ACA),在中性pH条件下,比较了不同的Na^ 含量及不同种类的糖对ACA测定结果的影响。结果表明,NaCl含量由0．2％上升至1．0％时,ACA呈逐渐下降趋势;用5％葡萄糖作稳定剂时ACA最低。IVIG在37℃条件下放置一月后,ACA有明显下降趋势。在半成品配制过程中,pH及各种成份的加入顺序对ACA也有一定影响。     

社区获得性肺炎非典型病原体分布及其流行特征

目的 了解社区获得性肺炎(CAP)非典型病原体感染的分布情况及其流行特征.方法 收集确诊为社区获得性肺炎患者278例,间接免疫荧光法(IFA)检测人血清中呼吸道9种主要的非典型性病原体的IgM抗体.结果 病原体检测阳性者150例,总阳性率54.O％.单一病原体感染中,肺炎支原体(MP) 125例(45.0％)、呼吸道合胞病毒( RSV) 27例(9.7％)、腺病毒22例(7.9％)、副流感病毒1、2和3型19例(6.8％)、乙型流感病毒16例(5.8％)、嗜肺军团菌血清1型13例(4.7％)、肺炎衣原体2例(0.7％)和甲型流感病毒1例(0.4％).混合感染共63例(22.7％),其中61例(21.9％)为MP与其他病原体的混合感染,病毒感染以RSV最常见,共27例(9.7％).CAP患者患有基础疾病共139例(50％),其余为无基础疾病者.基础疾病中以循环疾病和呼吸疾病最常见,各占总CAP患者的15,1％和13.0％.所有受检者MP阳性率最高,达45％,其中未成年组3～18岁中MP阳性率高达60.2％,而成人组18 ～50岁中MP阳性率高达81.8％.CAP春季病原体阳性检出率为46.9％,冬季病原体阳性检出率为63.8％(x2=7.752,P＜0.05).结论 非典型性病原体(特别是MP)感染在CAP患者中比例较大,其流行与分布跟病原体种类、基础疾病、年龄、季节等有一定的关系.     

采用多种抗原测定静注人免疫球蛋白(pH4)中抗体Fc段生物学活性的初探

目的初步探讨采用多种抗原测定静注人免疫球蛋白(IVIG)(pH4)中抗体的Fc段生物学活性,了解IVIG中抗体的Fc段生物学活性。方法采用补体活化的经典途径中免疫复合物激活补体的方法,将不同浓度的麻疹病毒、风疹病毒、乙肝表面抗原(HBsAg)、破伤风类毒素、脑膜炎球菌P64k外膜蛋白和白喉类毒素6种抗原分别致敏人O型血红细胞形成红细胞-抗原结合物;然后,6种致敏红细胞分别与IVIG孵育,与特异性抗体形成红细胞-抗原-抗体复合物;最后,此复合物与补体反应,在541 nm波长处读取吸光值,并绘制溶血反应动力学曲线,分别计算IVIG中针对上述6种抗原IgG的Fc段生物学活性。采用此方法,用6种抗原致敏的红细胞测定IVIG的Fc段生物学活性10次,验证此方法的重复性。结果麻疹病毒、风疹病毒、HBsAg、破伤风类毒素和脑膜炎球菌P64k外膜蛋白致敏的红细胞分别与供试品和补体反应后,测定的溶血反应动力学曲线较平缓,而白喉类毒素致敏的红细胞与供试品和补体反应后,测定的溶血反应动力学曲线下降明显,呈典型的"S"型曲线。计算结果显示,IVIG中针对此六种抗原的抗体Fc段生物学活性相对于参考品均大于80%。Fc段生物学检测方法重复性较好。结论采用多种抗原分别致敏红细胞,可以用来检测IVIG中多种抗体的Fc段生物学活性,为深入了解IVIG制品中的多种抗体的Fc段生物学活性奠定了基础。     

Detection of disease-specific augmentation of abnormal immunoglobulin G in sera of patients with rheumatoid arthritis

Galactose-free immunoglobulin G (IgG), which is known to be higher in the sera of patients with rheumatoid arthritis, was prepared from IgG of healthy volunteers using enzymes. Its reactivity to lectins was analyzed. The galactose-free IgG showed no reactivity to Ricinus communis agglutinin 120 but displayed greater reactivity to concanavalin A and Lens culinaris lectin than did intact human IgG. Then, IgG in serum samples was bound to protein A immobilized on a nitrocellulose membrane, and its reactivity to biotinylated concanavalin A was measured with streptavidin-conjugated horseradish peroxidase. When the reactivity to concanavalin A of IgG in sera from healthy individuals and patients with rheumatoid arthritis (RA), osteoarthritis, systemic lupus erythematosus, or hepatic disease was compared, higher levels were shown in patients with RA, notably in 60% of the seronegative patients and 80% of the early phase patients. Therefore, it was suggested that augmentation of the abnormal IgG in sera was highly specific to patients with RA and that this novel serum test could be very useful for an accurate diagnosis of this disease.     

社区获得性肺炎痰菌群特征与嗜酸性粒细胞水平的相关性

目的 探究社区获得性肺炎（CAP）患者痰液菌群特征及其与嗜酸性粒细胞水平的相关性。方法 对2018年9月—2018年11月间于我院就诊的63例患者（CAP组）和63例同时期、同年龄段体检者（对照组）的痰液菌群进行16S rDNA检测,比较两组患者痰液菌群特征、痰液和血液嗜酸性粒细胞百分比（Eos%）水平差异,应用Pearson模型和多因素线性回归模型探究患者痰液菌群与嗜酸性粒细胞水平的相关性。结果 CAP组患者的外周血和痰液的Eos%水平明显低于对照组（t=9.486,6.436,Ps0.05）,CAP组的Shannon指数明显低于对照组,Simpson指数显著高于对照组,差异有统计学意义（Ps4log10）,而对照组中优势菌为链球菌属、厌氧菌、奈瑟菌属和韦荣球菌属等。Pearson相关分析显示,肺炎链球菌、金黄色葡萄球菌、流感嗜血杆菌、肺炎克雷伯菌、铜绿假单胞菌、大肠杆菌和嗜肺军团菌等病原菌相对丰度与外周血和痰液的Eos%水平均呈负相关（Ps<0.05）。多因素线性回归分析显示,肺炎链球菌水平与痰液和血液Eos%水平呈明显的独立负相关（β= -0.633,-0.525,P=0.000）。结论 CAP患者痰液菌群特征与健康人群存在明显差异,其中肺炎链球菌水平与痰嗜酸性粒细胞百分比呈明显的负相关。     

Predicting treatment failure in patients with community acquired pneumonia: a case-control study

Introduction

Treatment failure in community-acquired-pneumonia (CAP) patients is associated with a high mortality rate, and therefore are a matter of great concern in clinical management. Those patients have increased mortality and are a target population for randomized clinical trials.

Methods

A case–control study was performed in patients with CAP (non-failure cases vs. failure cases, discriminating by late and early failure). CRP, PCT, interleukin 1, 6, 8 and 10 and TNF were determined at days 1 and 3 of hospitalization.

Results

A total of 253 patients were included in this study where 83 patients presented treatment failure. Of these, 40 (48.2%) had early failure. A discriminative effect was found for a higher CURB-65 score among late failure patients (p = 0.004). A significant increase on day 1 of hospitalization in CRP (p < 0.001), PCT (p = 0.004), IL-6 (p < 0.001) and IL-8 (p = 0.02), and a decrease in IL-1 (p = 0.06) in patients with failure was observed compared with patients without failure. On day 3, only the increase in CRP (p < 0.001), PCT (p = 0.007) and IL-6 (p < 0.001) remained significant. Independent predictors for early failure were higher IL-6 levels on day 1 (OR = 1.78, IC = 1.2-2.6) and pleural effusion (OR = 2.25, IC = 1.0-5.3), and for late failure, higher PCT levels on day 3 (OR = 1.60, IC = 1.0-2.5), CURB-65 score ≥ 3 (OR = 1.43, IC = 1.0-2.0), and multilobar involvement (OR = 4.50, IC = 2.1-9.9).

Conclusions

There was a good correlation of IL-6 levels and CAP failure and IL-6 & PCT with late CAP failure. Pleural effusion and multilobar involvement were simple clinical predictors of early and late failure, respectively.

Trial registration

IRB Register: http://2009/5451.     

Comprehensive characterization of immunoglobulin gene rearrangements in patients with chronic lymphocytic leukaemia

Previous studies have suggested a geographical pattern of immunoglobulin rearrangement in chronic lymphocytic leukaemia (CLL), which could be as a result of a genetic background or an environmental antigen. However, the characteristics of Ig rearrangements in the population from the South of France have not yet been established. Here, we studied CLL B‐cell repertoire and mutational pattern in a Southern French cohort of patients using an in‐house protocol for whole sequencing of the rearranged immunoglobulin heavy‐chain genes. Described biased usage of variable, diversity and joining genes between the mutated and unmutated groups was found in our population. However, variable gene frequencies are more in accordance with those observed in the Mediterranean patients. We found that the third complementary‐determining region (CDR) length was higher in unmutated sequences, because of bias in the diversity and joining genes usage and not due to the N diversity. Mutations found in CLL followed the features of canonical somatic hypermutation mechanism: preference of targeting for activation‐induced cytidine deaminase and polymerase motifs, base change bias for transitions and more replacement mutations occurring in CDRs than in framework regions. Surprisingly, localization of activation‐induced cytidine deaminase motifs onto the variable gene showed a preference for framework regions. The study of the characteristics at the age of diagnosis showed no difference in clinical outcome, but suggested a tendency of increased replacement and transition‐over‐transversion mutations and a longer third CDR length in older patients.