several studies reported histophatological observations during influenza a / h1n1 2009 pneumonia : the major finding was diffuse alveolar damage ( dad ) [ 14 ] . type ii pneumocytes were considered the main target of influenza a / h1n1 infection . a limited number of observations described in vivo samples , such as bronchoalveolar lavage ( bal ) [ 68 ] . therefore , it may play a role in further understanding the pathogenesis of new viral strains . in this case series we present the results of bal performed in three patients with severe influenza a / h1n1 2009 pneumonia complicated by acute respiratory distress syndrome ( ards ) . between august and december 2009 we performed a bronchoscopy - guided bal of three patients admitted to the san gerardo hospital , monza , italy , for influenza a / h1n1 virus pneumonia with subsequent development of ards . all three patients required intensive care unit ( icu ) admission and received mechanical ventilation ( mv ) and extracorporeal respiratory support ( ecmo ) . influenza a / h1n1 diagnosis was confirmed by a / h1n1 virus rna detection on nasal swab samples using influenza a / h1n1 2009 rrt - pcr ( real - time reverse - transcriptase - polymerase - chain - reaction ) assay ( genexpert ) . bal differential cell count on cytocentrifugate was performed in the light microscopy ( lm ) by counting about 300 cells in random fields at 400x magnification . demographics , comorbidities , severity on admission , microbiological isolations , and outcome for each patient are described in table 1 . antiviral therapy with oseltamivir ( 150  mg twice daily ) was started at hospital admission simultaneously with rrt - pcr assay and was continued until rrt - pcr assay on nasal swab turned negative . large , atypical cells with plasmocytoid appearance were observed in all three specimens with similar percentages ( 8 , 9 , and 6% , resp . ) . at lm , they appeared as large , plasmoblastic / plasmocytoid - like cells with eccentric nuclei and paranuclear vacuoles , high ratio nucleus / cytoplasm , and intensely basophilic cytoplasm . immunocytochemical analysis on cytocentrifugate showed that these cells were negative for cd20 ( b lymphocyte marker ) and cd138 ( plasma cell marker ) . ultrastructural examinations at transmission em allowed us to observe multilamellar osmiophilic bodies in the cytoplasm of plasmoblastic / plasmocytoid - like cells . em showed in these cells small round cytoplasmic inclusions with irregular surface and a diameter around 60  nm ( figure 2 ) . they also showed cytoplasmic vesicles with a diameter around 100  nm and an irregular electron - dense core ( figure 3 ) . the main characteristic of the bal of our patients with influenza a / h1n1 pneumonia associated with ards was the presence of large cells with a plasmoblastic / plasmocytoid - like appearance , identified at em as atypical type ii pneumocytes . the first hypothesis regards the reparative action that type ii pneumocytes may have in dad . in our cases , stanley described morphological atypias , such as increased nuclear - cytoplasmic ratio , in type ii pneumocytes found in bals during ards . these atypical pneumocytes are usually aggregated in clusters and are supposed to have a reparative role . all our patients developed ards and we observed pneumocytes aggregated in clusters in the bals performed earlier ( cases numbers 2 and 3 ) . according to this first hypothesis , the atypical cells we observed could be described as reactive immature type ii pneumocytes . type ii pneumocytes have been described as the main target of influenza a / h1n1 2009 infection . nakajima et al . in 2012 described four autopsy cases of influenza a / h1n1 with a histopathological pattern of acute dad who presented with influenza virus antigen - positive type ii pneumocytes , perhaps indicating a direct role of the virus - infected cells in the acute alveolar damage . no specific cytophatic effect or viral inclusion has been described so far at lm in lung tissue specimens during influenza pneumonia . several observations have been performed via em on autopsy specimens . during the recent influenza pandemic , mauad et al . found type ii pneumocytes with vesicles , approximately 100  nm in diameter , with an electron - dense center . bal and colleagues described cytoplasmic inclusions in pneumocytes , which ranged in diameter from 74 to 82  nm and showed surface spikes characteristic of influenza virus . em observations of our specimens revealed some atypical pneumocytes showing both small round cytoplasmic inclusions with an irregular surface and small vesicles ( similar to those described by bal and mauad ) . according to this second hypothesis , atypical type ii pneumocytes could be a specific morphological marker of influenza virus infection . from the data collected so far , we can not favor one explanation ; in fact , they could coexist in cases of severe influenza a / h1n1 pneumonia associated with ards . limitations of the present study include the following : first , the paucity of cases analyzed ; second , we did not perform immunohistochemical staining for h1n1 antigens on bal samples . the description of bal in a control group of patients with ards not associated with h1n1 pneumonia is beyond the scope of this case series . however , other authors described bal cytology in non - h1n1 ards , and although they reported morphological atypias in type ii pneumocytes , they did not describe these peculiar plasmoblastic / plasmocytoid - like cells [ 10 , 12 , 13 ] . the collection of the airway specimens of patients number 2 and number 3 , in which the nosocomial pathogens were isolated , took place after bal was performed . therefore , these pathogens most likely did not affect the cellularity in bal . in conclusion , plasmoblastic / plasmocytoid - like type ii pneumocytes characterize the bal of our patients with influenza a / h1n1 2009 pneumonia associated with ards . they could represent a pathognomonic marker of influenza virus pneumonia as well as reparative cellular activation after dad . more observations of bal cytology in patients with influenza pneumonia are needed to understand their characteristics and role .
we present the results of bronchoalveolar lavage ( bal ) performed in three patients with severe influenza a / h1n1 pneumonia complicated by acute respiratory distress syndrome ( ards ) . 
 light microscopy analysis of bal cytocentrifugates showed the presence of characteristic large , mononuclear , plasmoblastic / plasmocytoid - like cells never described before . via transmission electron microscopy , these cells were classified as atypical type ii pneumocytes and some of them showed cytoplasmic vesicles and inclusions . 
 we concluded that plasmoblastic / plasmocytoid - like type ii pneumocytes might represent a morphologic marker of a / h1n1 influenza virus infection as well as reparative cellular activation after diffuse alveolar damage .