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      Detection of Hemosiderin-Laden Macrophages in Bronchoalveolar Lavage Fluid of COVID-19 Patients: Is Perls Stain a Potential Indicator of Oxidative Alveolar Damage?


      Acta Cytologica

      S. Karger AG

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          Dear Editor, An interesting special issue was recently published in your journal Acta Cytologica (January–February 2020) concerning the ancillary techniques in cytopathological specimens, supervised by Baloch and Gupta as guest editors [1]. In this issue, the manuscript of Zhou and Moreira stressed the importance of auxiliary techniques and special staining in pulmonary cytopathology, not only in the differential diagnosis and predictive testing of lung tumors but also in the detection of pathogenic agents of infectious disease [2]. The last few days have seen an important revelation regarding the pathophysiology of the pandemic COVID-19 infection caused by SARS-CoV-2 for severe acute respiratory syndrome coronavirus 2. This novel RNA virus is composed of RNA-dependent RNA polymerase, structural proteins (spike protein, envelope protein, membrane protein, and nucleocapsid phosphoprotein), and a set of nonstructural proteins (ORFs) [3]. The typical chest computed tomography scan features of emerging COVID-19 pneumonia included bilateral ground-glass opacities with a predominantly peripheral distribution [4]. First, a proinflammatory syndrome with notably increased levels of cytokines and chemokines (cytokine storm) or macrophage activation syndrome has been noted in hospitalized COVID-19 patients [5, 6]. More recently, bioinformatics analysis reveals that the virus causes prolonged and progressive hypoxia by binding to the heme groups of hemoglobin in red blood cells (RBCs) and inhibiting heme metabolism [7]. Consequently, the pulmonary lesions described on chest computed tomography scan are thought to be the result of the inability to exchange carbon dioxide and oxygen and the release of oxidative iron from the hemes, which overwhelm the natural defenses against pulmonary oxidative stress and may eventually result in bilateral ground-glass-like opacities in COVID-19 patients. It is well known that RBCs carry oxygen from the lungs to other organs. They can do this with the help of hemoglobin, which is an assembly of 4 globular protein subunits called hemes. A heme group consists of an iron atom (Fe) held in a heterocyclic ring, known as porphyrin acting as its container. The Fe may be either in the ferrous (Fe2+) or in the ferric (Fe3+) state, but Fe3+ cannot bind oxygen. Oxygenation changes the electronic state of the Fe2+-heme complex. When RBCs are exposed to oxidizing agents, the heme iron in hemoglobin is oxidized from Fe2+ to Fe3+ state to form methemoglobin, which is unable to bind oxygen [8]. Thus, iron must exist in the Fe2+ state to bind oxygen. In this way, the iron ion can be safely transported by hemoglobin, but used to bind to oxygen when it reaches the pulmonary alveoli, where all the gas exchanges take place, and then goes to deliver oxygen to the other organs. In the case of COVID-19 infection, the surface glycoprotein of the virus binds to the porphyrin of the heme. At the same time, nonstructural proteins of SARS-CoV-2 coordinate attack the heme on the 1-beta chain of hemoglobin to dissociate the iron from the porphyrin [7], and in doing so, dissociated oxidizing iron ion moves freely. Without the iron ion, hemoglobin can no longer bind to oxygen. In theory, once all hemoglobin is altered, the RBC becomes unable to carry oxygen and simply runs with the SARS-CoV-2 attached to its porphyrin. This means, on the one hand, a lack of oxygen for all the organs, and on the other hand, that released iron floats freely causing oxidative damage to these organs. This hypothesis may explain in part extrapulmonary lesions caused by COVID-19. However, the lungs have a primary defense mechanism to maintain iron homeostasis, known as iron sequestration. The initial players in this mechanism are the alveolar macrophages that collect free radicals such as iron [9]. In COVID-19 patients, this mechanism seems to be overwhelmed by the excess of oxidizing iron and so begins the process of pulmonary oxidative stress, which leads to inflammation that is usually bilateral with COVID-19 infection. In practice, ferric iron could be easily identified in cells and tissue samples in cytologpathology and histopathology laboratories using the routine Perls Prussian blue stain under light microscope [10]. Perls Prussian blue stain, also called as Perls stain, was described in 1867 by the pathologist Max Perls. It allows detecting the presence of iron in cells by conversion of iron to Prussian blue as shown in the following chemical formula [11]: Ferric chloride + Potassium ferrocyanide → Ferric ferrocyanide + Potassium chloride (Prussian blue), 4FeCl3 + 3K4Fe(CN)6 → Fe4[Fe(CN)6]3 + 12KCl. Perls stain is used to color cellular nonheme iron such as ferritin and hemosiderin but does not stain iron that is bound to porphyrin such as hemoglobin and myoglobin [12]. A combined Perls-hematoxylin-eosin stain was also proposed to easily check the presence of ferric iron in tissue sections [13]. Moreover, an immunohistochemical technique can be used to detect the presence of ferritin in tissue samples with results equivalent to Perls stain [14]. Therefore, Perls stain may be used to identify excess iron deposits caused by oxidative damage mechanism in COVID-19 patients. The bronchoalveolar lavage (BAL) fluid is a good technique to explore alveolar macrophages, allowing for determining their percentage, size and shape, and their cytoplasm content. It also allows for making a cellular formula and searching for pathogens. Thus, LBA is considered as an important tool in the diagnosis of inflammatory, autoimmune, and infectious diseases. In BAL fluids, hemosiderin-laden alveolar macrophages can be scored by the cytopathologist according to the hemosiderin content and the semi-quantitative method described by Golde (Golde score). Initially, the Golde score is established to assess alveolar hemorrhage in the event of capillary bed hyperpressure or alveolar wall injury; RBCs pass from the capillaries into the alveolar lumen, resulting in erythrophagocytosis. The Golde score requires a count of 100 macrophages and the establishment of a value from 0 to 4, depending on the iron density (in blue) in their cytoplasm (0 = no color in the cytoplasm, 1 = weak blue in a minor portion of cytoplasm, 2 = dark blue in a minor portion of cytoplasm or intermediate color throughout the cytoplasm, 3 = dark blue in most areas of cytoplasm, and 4 = dark blue throughout the macrophages). The result of this score depends on the sum of the number of macrophages X the value corresponding to the iron load (if counting 100 macrophages) to obtain a numerical score (Golde score: 0–20 normal, 20–70 intermediate resorption, >70 high resorption, and >100 occult alveolar hemorrhage) [15]. According to the hypothesis developed above, the alveolar macrophages collect free iron ions following heme attack by COVID-19 which separates iron from porphyrin. Consistent with this hypothesis, it can be assumed that the higher the Golde score, the more severe the hypoxia and oxidative damage. In addition, cytological examination of BAL may help prove the alveolar damage. For example, some signs of pulmonary parenchyma aggression causing early alveolar damage such as hyaline membranes, inflammation, and desquamation of bronchiolar pneumocytes can be seen by cytological examination using standard staining protocols (Papanicolaou and May-Grünwald Giemsa stains). These pathological findings were recently described in COVID-19 patients' tissue and autopsy reports [16, 17]. BAL fluids may reveal possible viral cytopathogenic effect not obviously shown so far for COVID-19. Moreover, the BAL when clinically indicated allows looking for coinfection by the presence of second pathogen using special stains (Gram, Giemsa, Grocott-Gomori, periodic acid-Schiff, and Ziehl-Neelsen satins) [2], which could worsen the health status of COVID-19 patients, especially in higher infectious risk patients such as HIV and diabetes patients [18, 19]. It is evident that confirmation of the utility of Golde score using Perls stain or immunocytochemical technique to detect ferric iron as an indicator of pulmonary damage in COVID-19 patients requires validation by a series of cytological examination of BAL, while taking the necessary technical precautions as fresh BAL of COVID-19 patients is considered a high-risk infectious fluid for the laboratory team. If this hypothesis is confirmed in practice, the score may need to be adapted at a later date to assess the severity of COVID-19's damage. Obviously, careful examination of cytological specimens by using routine and special staining or ancillary technique can provide important diagnostic and prognostic information that may impact the management of COVID-19 patients. Disclosure Statement The author declares that he has no competing interests. Funding Sources The author did not receive any funding.

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          Most cited references 9

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          Characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (NCP)

           S WAN,  Q YI,  S Fan (2020)
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            Is Open Access

            Increased Iron Sequestration in Alveolar Macrophages in Chronic Obtructive Pulmonary Disease

            Free iron in lung can cause the generation of reactive oxygen species, an important factor in chronic obstructive pulmonary disease (COPD) pathogenesis. Iron accumulation has been implicated in oxidative stress in other diseases, such as Alzheimer’s and Parkinson’s diseases, but little is known about iron accumulation in COPD. We sought to determine if iron content and the expression of iron transport and/or storage genes in lung differ between controls and COPD subjects, and whether changes in these correlate with airway obstruction. Explanted lung tissue was obtained from transplant donors, GOLD 2–3 COPD subjects, and GOLD 4 lung transplant recipients, and bronchoalveolar lavage (BAL) cells were obtained from non-smokers, healthy smokers, and GOLD 1–3 COPD subjects. Iron-positive cells were quantified histologically, and the expression of iron uptake (transferrin and transferrin receptor), storage (ferritin) and export (ferroportin) genes was examined by real-time RT-PCR assay. Percentage of iron-positive cells and expression levels of iron metabolism genes were examined for correlations with airflow limitation indices (forced expiratory volume in the first second (FEV1) and the ratio between FEV1 and forced vital capacity (FEV1/FVC)). The alveolar macrophage was identified as the predominant iron-positive cell type in lung tissues. Futhermore, the quantity of iron deposit and the percentage of iron positive macrophages were increased with COPD and emphysema severity. The mRNA expression of iron uptake and storage genes transferrin and ferritin were significantly increased in GOLD 4 COPD lungs compared to donors (6.9 and 3.22 fold increase, respectively). In BAL cells, the mRNA expression of transferrin, transferrin receptor and ferritin correlated with airway obstruction. These results support activation of an iron sequestration mechanism by alveolar macrophages in COPD, which we postulate is a protective mechanism against iron induced oxidative stress.
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              Tuberculosis and HIV responses threatened by COVID-19

               Paul Adepoju (2020)
              As the first cases of COVID-19 affect Nigeria's health-care workers, will the country's HIV and tuberculosis responses weather the pandemic? Paul Adepoju reports. March 24, 2020, was World Tuberculosis Day 2020, but this year tuberculosis was overshadowed by the COVID-19 pandemic. By the end of that week, COVID-19 had already exceeded 600 000 confirmed cases and about 30 000 deaths worldwide. Before the emergence of COVID-19 as a public health emergency of international concern, coinfection with tuberculosis was probably the priority for HIV/AIDS control efforts in Africa. “Tuberculosis is an issue throughout the world. But in Africa, the tuberculosis epidemic is driven by HIV, and HIV-tuberculosis coinfection has clear impact on mortality”, said Matteo Zignol (WHO Global Tuberculosis Programme). The immune status that makes people with HIV vulnerable to tuberculosis could also make them susceptible to coronavirus infection. COVID-19 is already affecting control measures for tuberculosis and HIV, while WHO and UNAIDS are compiling data and evidence to guide recommendations for the management of COVID-19 in the context of HIV-tuberculosis coinfection. To flatten the curve of the COVID-19 pandemic, governments across the world are shutting cities down and restricting movements; they are also encouraging residents to stay indoors. But capacities of health-care facilities are being stretched, and there are increasing demands for more hospital materials such as personal protective equipment (PPE) and ventilators. With a large proportion of resources committed to stemming the spread of COVID-19, services across all sectors are affected Tereza Kasaeva (Director of the WHO Global Tuberculosis Programme) told The Lancet HIV that WHO wants to ensure that the response to the COVID-19 pandemic does not affect the continuity of essential services for people affected by tuberculosis. The organisation is aiming to maximise joint support to tackle both diseases. For 2020, WHO's tuberculosis focus is to scale up access to tuberculosis treatment by deploying a number of strategies targeting risk groups, such as close contacts of a known case and individuals with underlying health conditions, and risk factors, such as HIV infection. WHO also recently recommended shorter tuberculosis prevention regimens with a 1 month daily regimen of rifapentine and isoniazid. WHO wants to assist national tuberculosis programmes and health personnel to maintain continuity of essential services during the COVID-19 pandemic, through innovative people-centred approaches and maximising joint support to tackle both diseases, but this may not be a realistic aim. Kasaeva told The Lancet HIV that the supply and transportation of tuberculosis drugs may be disrupted by flight cancellations and imposed travel restrictions. “Flights are being cancelled and airlines are closing down. This is going to be a big issue for drug supply and is a concern for WHO. Countries should not run out of stock of tuberculosis drugs because patients must have access to treatment. They need to complete the treatment because if they don't, the worst outcome is drug resistance which is a great threat to the global tuerculosis control.” Nigeria has a substantial tuberculosis disease burden, and the gap between estimated and reported cases of the disease is also large. 5 days after the first case of COVID-19 was confirmed in Ibadan, one of Nigeria's major cities, health-care workers already had heightened concern about personal safety. Friday March 27 was the last working day before a city-wide shutdown of Ibadan went into effect on Sunday, but the impacts of COVID-19 on the health sector were already being felt. Over the weekend, Jess Otegbayo, head of University College Hospital Ibadan and Oluwabunmi Olapade-Olaopa, provost of University of Ibadan's College of Medicine, tested positive for COVID-19. Health officials working in Nigeria's primary health centres expressed worry about the paucity of protective materials such as hand sanitisers, making it impossible for them to provide regular health-care services to patients including those who might have tuberculosis. Before the advent of COVID-19, the primary health centre at Iyana Church in Ibadan was serving as an active tuberculosis sample collection centre, from where samples are taken to a central government laboratory for testing. But since the confirmation of cases of COVID-19 in Ibadan, Wale Adeosun, the tuberculosis officer at the health centre, said he had not been touching the samples. “We have 25 samples that ought to be processed. But they will be discarded because no one wants to touch them in this season of coronavirus. When everything returns to normal, the patients will bring fresh samples for tuberculosis testing”, Adeosun told The Lancet HIV. Health workers are reluctant to handle samples for tuberculosis testing because of similarities in the symptoms of tuberculosis and COVID-19—coughing, fever, and difficulty in breathing. “They both attack the lungs and are both transmitted mainly by close contacts. But they are quite different. tuberculosis is a chronic disease with patients coughing for a minimum of 2 weeks but COVID-19 has a rapid onset. Simple differential diagnosis by well trained service providers will be able to distinguish them”, Zignol said. But health-care professionals in Nigeria are not taking chances. “We lack the resources to protect everyone before getting the opportunity to figure out what a patient may likely have which is why we are taking extreme measures”, Matron Oni Aluko (head of the Iyana Church health centre) told The Lancet HIV. At Oyo state's central tuberculosis screening and treatment centre, the Jericho Chest Hospital, nurses and support staff shared similar concerns. Although the hospital has been designated a quarantine facility for patients with confirmed COVID-19, health workers interacting with people with suspected tuberculosis who might actually have COVID-19 were yet to be properly trained on the viral disease. Unlike the health centre at Iyana Church, Ibadan North East Local Government has a tuberculosis clinic within its secretariat in Ibadan. The clinic is popular in the metropolis. Aside from screening for new cases, the clinic provides treatment. On one of the shelves in the office section of the clinic, dozens of boxes containing drugs for patients with tuberculosis being managed at the clinic were arranged, and each box had the name of the patient written on it with a blue marker. “All of these patients ought to have come today to receive their drugs but they've not shown up to do so. Eight of them are coinfected with HIV and I am very worried about their safety and health status”, Bankole Adewumi, an officer at the facility, told The Lancet HIV. As disappointing as the development was, Adewumi said it was not surprising. Patients with tuberculosis can suffer discrimination if others think they are infected with COVID-19. “This is a wrong time to be coughing and commuting via the public transportation system. Presently it is embarrassing but as COVID-19 persists and new cases are recorded, the situation might become particularly dangerous for our patients as people may become more restless, aggressive and impatient about ending the outbreak”, he added. Adewumi revealed that the centre handles up to 50 patients daily, but on the day that The Lancet HIV visited, only one person showed up to receive drugs that will last only a couple of weeks. “We are scared that the health conditions of our patients will deteriorate during this period because, even though the government asked us to still continue to run the clinic during the shutdown, many of our patients rely on public transportation to commute to the clinic”, he said. One of the available options is to give patients sufficient doses of their drugs to last for months. But the unanticipated nature of COVID-19 onset did not allow for adequate restocking of the drug store to accommodate such logistical changes. Individuals living with tuberculosis and HIV coinfection are not the only ones that will be affected; the broader HIV control mechanisms in African countries implementing lockdowns and struggling with fragile health systems are also likely to be compromised. Onyekachi Onumara (Senior Programme Officer at Rural Health Foundation) said that in Nigeria the “major blow will be on prevention because field staff who engage in peer sessions have to rejig strategies”. Health workers caring for people living with HIV are being asked to give antiretroviral drugs that will last for 3 months. Funds for HIV intervention initiatives could also be depleted as a result of previously unincluded expenses such as procurement of sanitisers and PPE for field staff. “The panic will also see ad-hoc staff pull out of programmes for fear. The lockdown is the biggest blow to fieldwork as prevention services and field testings may be halted”, Onumara told The Lancet HIV. Whereas the Nigerian government said its top priority is to stem the spread of COVID-19 and to ensure that HIV and tuberculosis clinics remain open, UNAIDS proposed implementing differentiated service delivery for HIV. UNAIDS proposals include multimonth dispensing, community antiretroviral distribution, and self-testing to empower clients, to limit unnecessary exposures to COVID-19 at clinics and to reduce pressure on facility-based health-care systems. Acknowledging that heath systems will be stretched in some places, UNAIDS calls governments not to relax tuberculosis diagnosis and treatment. “Messaging needs to be clarified, so that people with tuberculois symptoms including prolonged fever and cough do not avoid health facilities or delay assessment due to COVID”, said Shannon Hader (UNAIDS Deputy Executive Director). Hospital visits need to be minimised to reduce exposure for COVID-19 and to reserve them for people requiring hospitalisation. UNAIDS also encouraged countries to consider multimonth scripting and virtual communication platforms to assist patients. UNAIDS says that hospital visits need to be minimised to reduce exposure for COVID-19 and to reserve them for serious conditions requiring hospitalisation. UNAIDS also encour-aged countries to consider virtual communication platforms to assist patients. “UNAIDS is working with people living with HIV and key population networks to understand needs for information, COVID-19 prevention supports, and HIV service continuity needs to help respond”, Hader told The Lancet HIV. “But what is also needed is an intensity of COVID testing, surveillance, and prevention services concentrated in some of the highest risk settings from slums to informal settlements to settings of incarceration, if the most vulnerable people are to be protected. Empowerment of communities of people living with HIV and tuberculosis across these responses is essential as is resilience of health systems.”

                Author and article information

                Acta Cytol
                Acta Cytol
                Acta Cytologica
                S. Karger AG (Allschwilerstrasse 10, P.O. Box · Postfach · Case postale, CH–4009, Basel, Switzerland · Schweiz · Suisse, Phone: +41 61 306 11 11, Fax: +41 61 306 12 34, )
                5 June 2020
                : 1-3
                Department of Anatomic Pathology and Cytology, Cayenne Hospital Center, Cayenne, French Guiana
                Author notes
                *Kinan Drak Alsibai, Department of Anatomic Pathology and Cytology, Cayenne Hospital Center, Av. des flamboyants, BP 6006, Cayenne 97300 (French Guiana), kdrak.alsibai@
                Copyright © 2020 by S. Karger AG, Basel

                This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections.

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                References: 19, Pages: 3
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