Biobank integration of large-scale clinical and histopathology melanoma studies within the European Cancer Moonshot Lund Center
2018

Johan Malm
Yutaka Sugihara
Marcell Szasz
Ho Jeong Kwon
Henrik Lindberg
Roger Appelqvist
György Marko-Varga
Biobank integration of large-scale clinical and histopathology melanoma studies within the European Cancer Moonshot Lund Center

Clin Trans Med
728201810.1186/s40169-018-0203-8RESEARCH
We present the Cancer Moonshot clinical project located at the European center in Lund. Here, tissue and blood samples have been collected and stored in a large-scale biobank. Multiple clinical centers around the world are participating and tissue and blood samples are sent to the European Cancer Moonshot Lund Center that acts as the clinical hub. Our center has been developed to generate and build large-scale biostorage archives of patient melanoma samples, which is then combined with a histopathological capability to characterize the patient tumours. Such a large-scale clinical sample processing initiative has begun with the aim of creating high-end histopathology indexing with database computational power and including proteogenomic analysis. The biobank at Lund has become an important resource in clinical research worldwide. Following suite, several national health programs are being initiated with the aim of also building large-scale biobank storages with a wealth of high-quality patient samples. In our Cancer Moonshot R&D activities, samples in the biobanks and the data derived from these samples are being used to build an understanding of disease presentation and using this information to move towards 'Big Data' proteogenomic and mass spectrometry imaging studies. Additionally, we report here a sample processing workflow that has been adapted to a fully-automated biobank processing strategy for large-scale studies.© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Background

Today, cancer impact by optimal treatment is already at the front line worldwide with the latest immuno-and cell therapies. To reach many of these milestone achievements, researchers have been storing hundreds of millions of specimens in various biobanks. Recent reports have indicated that the rate of sample collection is in the order of 20 million samples per year [1,2]. In Europe, the council of the European Union has approved the policy on human biological specimens in biobanks. By relating the number of biobank samples to the number of cancer patients, there is an estimate of 14,738,719 people living with cancer of any type in the United States (2014). Based on 2012-2014 data, it is predicted that approximately 38.5% of men and women will be diagnosed with cancer of any type at some point during their lifetime [3]. By 2020, the estimated cost for cancer care in the US alone is estimated to be $158 billion. This value was predicted by the NIH, where changes within the US population and forthcoming cancer trends were taken into consideration.

In Sweden, a relatively small country (population approx. 10.1 Mill.), approximately 60.000 new cancer diagnoses are made every year and every third person will be diagnosed with cancer at some time during their life time. The incidence of cancer has been increasing for many years and the total annual cost of cancer care has been estimated to more than four billion euro. Every year 3-4 Million new tissue samples (incl. blood) are added to the existing + 150 Mill. samples.

Amongst all cancers, malignant melanoma is the fifth most common cancer type. In 2017, there was an estimated increase of 87,110 new cases, and an estimated 9730 deaths. As such, melanoma of the skin represents 5.2% of all new cancers in the US only [4]. For comparison, the annual number of new melanoma cases in Sweden are approx. 5000 and the incidence is now more than four times higher than 25 years ago. Globally, there is an increasing interest and need to support research areas that can help solve disease understanding and improve patient care. This includes approaches such as 'precision medicine' , alternative treatment technologies and early indication of disease diagnosis utilizing both imaging techniques and biomarker diagnostics [5,6]. Ultimately, it is the patients who suffer and experience the current limitations in treatment. From an international perspective, the biobanking developments within the Cancer Moonshot activities in Lund have thus been given top priority to provide high-quality and well-defined tissue and biofluid specimens [7][8][9]. The center includes pathological characterization, where the tumour tissues are evaluated with high integrity and in great detail, with annotations that relate to the types of cells presented in the tumour tissues, their nature, and the morphology captured by histology images. Associated clinical data are also included into the RedCap database. In real time, the mapping and monitoring of the study progress occurs with our clinical partners worldwide [10,11].

Malignant melanoma

Malignant melanoma is the most dangerous type of skin cancer, and develops from pigment-containing cells known as melanocytes. These cells occur primarily in the skin, however, such cells also exist on the genitalia, in the mouth and the eye. Additionally, men often have a higher occurrence on the back; whilst with women, the most common occurrence is on the legs. It is known that the primary cause of melanoma is exposure to UV light. This risk increases when combined with low levels of skin pigment, a compromised immune system, and other genetic factors [3,4].

Most commonly, the diagnosis of a melanoma is determined from any suspicious skin lesion. Summarizing the basic melanoma pathology, patients are grouped into: superficial spreading melanoma (SSM), nodular melanoma (NM), lentigo maligna melanoma (LMM) and acral lentiginous melanoma (ALM) sub-groups, as presented in Table 1.

The usual treatments for these patient groups are presented in Fig. 1. Here, surgery is still perhaps the most pre-emptive and efficient method with a good outcome for the patient. As melanoma tumours usually intensively metastasize throughout the body, it can be challenging to manage with surgical intervention alone. This is particularly the case if the tumour has metastasized to multiple organs and sites. When this occurs, alternative treatment is usually necessary. These include chemotherapy, traditional, or targeted treatments using precision or personalised medicine; and can be combined with or without radiation (see Fig. 1).

Typically, surgical removal of a tumour is the most efficient approach. Assessment of nearby lymph nodes for metastases is also often performed; and in general, most people do not relapse if metastases has not occurred. With treatment, the 5-year survival rate in the United States for those with a localised disease, and metastatic spread, is 98 and 17%, respectively. The likelihood of reoccurrence or metastases is dependent on the thickness of the original tumour and/or how rapidly the cells are dividing.

The new generation of omic-integrative medicine includes genomic and proteomic expression data analysis in the final diagnosis and decision on the best treatment for patients. Genomic changes, however, are not always present at the protein level. This suggests that the additional level of proteomic analysis will lead to an enhanced functional understanding of tumour resistance and/or toxicity to therapy. Ultimately, this may lead to the ability to predict treatment response by examining drug response or toxicity. One of the major objectives of the Cancer Moonshot program is the belief that integrating genomic and proteomic data can provide more information and insight into the development and growth of cancer; and the most efficient treatment, preferably with precision medicine.

Considering the ever-increasing number of cancer patients worldwide, we are facing a real challenge in society both in terms of healthcare efficiency and scientific delivery of new principles. Particularly for the latter, an understanding of the mechanisms of cancer development is imperative both in terms of drug treatment and disease diagnosis. An overview of European hospital discharge rates for in-patients with neoplasms is depicted in Fig. 2. A substantial variation is seen with the highest rates in Austria, Germany and Hungary.

Biobank impact on cancer healthcare

To build clinical value in the future by combining patient care and expanding large-scale sample collections, there are three major groups that need to work together. Overall healthcare costs are increasing and according to the Centers for Medicare and Medicaid Services, healthcare expenditure accounted for 17.8% of the U.S. gross domestic product in 2015. There are a variety of factors that contribute to the increased cost and varying opinions on how resources should be allocated to account for the cost gap. One thing is certain: the United States spends an inordinate amount of money on healthcare. The strain on the economy is obvious, and healthcare professionals are attempting new and innovative measures to contain costs [11,13].

Recently, there was a report on the economic aspects of biobanks, particularly those facilitated by the state [14]. It has been noted that national biobanks are often funded by public/private partnerships, with finance provided by a combination of national research councils, medical charities, pharmaceutical company investment and biotech venture capital [15]. In this way, national biobanks enable an economic relationship mediated between states, national populations, and commercial entities. It has been illustrated that there is a strong commercial incentive underlying the systematic collection of tissue material. This is particularly evident in the field of genomic research, where population-sized studies lend themselves more easily towards diagnostic technologies rather than basic etiological studies [16].

Also legal aspects of biobanking patient samples have to be considered, not only for establishing the physical biobank but also for each study that uses the biobank for storage. The Lund biobank has been designed according to Swedish law and recommendations and for each study ethical approval by the regional ethical committee is required-both for storing samples and for using samples stored in the biobank.

In 2017 at the Lund biobank, approximately 300,000 sample tubes of blood fractions have been processed. Using a dedicated protocol and workflow, most of these were processed within a maximum of 2 h. The patient blood sample tubes samples were then transported to the BMC biobank facility. Here, the blood samples were fractionated by automated liquid handling using the Hamilton work station (see Figs. 3 and 4). When surgery was performed, the tissue samples were processed and stored in the same way as the blood fractions in the automated biobank. The biobank stores all samples with dedicated barcodes and the accompanying data set on the server.

The Cancer Moonshot biobank logistics and program has been outline for the operations in Lund that has been developed to generate and build large-scale biostorage archives of melanoma patient samples for proteogenomic analysis. Within Region Skåne, the Southern Swedish healthcare, within the melanoma patient treatment, we are following the progression and treatment of each respective patient. In practice, this means that whenever a melanoma patient enters the hospital in Lund or other hospitals in the region, sampling and clinical follow up is being made that our study can follow up. In most cases, blood can be taken from the patients. If surgical intervention is required, part of the patient tissue is routinely linked into the workflow we have built. The team work between our proteomic unit and the clinicians at the hospital is very good. Senior scientists, post-doctoral fellows and Ph.D. students are trained, and moved into this important biobanking activity.

The integrity of the biobank is of key importance, as the benefiters will be provided with high quality clinical material and associated clinical data.

The three major stakeholder groups that are involved in the daily biobank operations are:

Patients that contribute by sample donation, where proteogenomic profiling information concerning the tumour is undertaken. Given the genomic profile of the tumour combined with other relevant information, such as clinical data, it will be possible to learn which treatments may provide the best outcome for the patient.

Researchers can identify possible targets for the development of new treatments and preventive interventions. This will include early indication of disease onset, determining how to optimally treat a patient, and how to avoid or counter drug resistance. Access to samples stored in the biobank and associated clinical information can be given in collaborative research projects with the European Cancer Moonshot Lund team.

Medical doctors will have access to information that predicts better treatment outcomes and helps control the symptoms and side effects in the patient. The earlier cancer treatment is initiated, the better the outcome for the patient [17][18][19].

Characterising cancer tissue heterogeneity

Currently, there are a number of clinical studies that show that tumour heterogeneity is linked to the survival of cancer patients, i.e., lower heterogeneity correlates with longer survival. Throughout 2017 in a collaboration study with the Semmelweis Oncological Dermatology team in Budapest, Hungary, surgically-isolated melanoma tumours have been processed. A workflow to handle and process surgically-removed tumour tissue has been built.

Once the histopathological evaluation was performed, the areas of the different tissue compartments were manually outlined to create a mask plane for each cell type. These mask planes were then used to query specific mass spectrometry-generated ion signatures that are correlated or anti-correlated with a given histological structure or cell type.

By mapping the compartments within the tissue by imaging mass spectrometry, the sections of the tumours were divided into: (i) melanoma cells; (ii) lymphocytes; and (iii) macrophages. Figure 5 illustrates the heterogeneity of the tumour with different cell types dominating in different compartments of a melanoma tissue sample. The corresponding mass spectrum is also given (lower panel). The coverage over the tumour was shown to vary substantially. Entire coverage was apparent for metabolites with m/z values of 184.073 and 222.030; whilst the metabolite at m/z 296 only showed expression in the upper epithelial part of the tumour (data not shown).

A corresponding mass spectrum is depicted in Fig. 6, where all metabolites are shown as singly-or doublycharged ions within the tumour tissue. Depending on the instrumentation used, the technique also offers high spatial resolution (to the cellular level). At present MSI is performed only on selected cases to demonstrate tumour heterogeneity. Different ionisation methods are available, but matrix-assisted laser desorption/ionisation (MALDI) is perhaps the most widespread in mass spectrometry imaging (MSI). In MALDI, a so-called matrix compound is applied to the samples. The matrix absorbs the energy from the laser which is transferred to the analyte via a process referred to as 'soft' ionisation. MALDI-MSI is widely-used to characterise drug distribution in various tissue types. The method is also used to investigate various endogenous molecules, such as lipids, carbohydrates, peptides and proteins. MSI has gained significant interest over the past few decades from the pharmaceutical community. As a result of continued technical development,  After H&E staining, the same tissue section was histologically characterized. The pathologists identified several tissue compartments and cell types in the analysed tissue samples, i.e., cancer cells, lymphocytes, macrophages, connective tissue and other tissue compartments. The H&E stained image section of a melanoma proliferative lesion and attached subcutaneous tissue is shown in the images above. This is a representative illustration of a patient tumour sample that was processed and analysed within the European Cancer Moonshot Center Lund laboratories.

The H&E stained section showed a melanoma proliferative lesion with attached subcutaneous tissue. Dermal involvement of atypical melanocytes with cytologic atypia was observed. The tumour lesion was divided into two areas by morphological features of melanocytes. One area contained large melanocytes with abundant cytoplasm and polygonal nuclei (Fig. 5, melanoma cells). Additionally, another area contained small melanocytes with poor cytoplasm and small nuclei composed of dense chromatin (Fig. 5, lymphocyte). Also, an infiltration area of brown pigment-laden macrophages was observed in the tumour proliferative lesion and attached subcutaneous tissue (Fig. 5, macrophages).

MALDI MSI demonstrated specific ions from each area. In addition, what is reported here reflects that the vast majority of the signal defined as a metabolite is clearly located within recognisable structures within the tissue, illustrated in a counterplot image as shown in Fig. 7.

In conclusion, the European Cancer Moonshot Lund Center, an infrastructure that is associated with one Fig. 5 Tissue compartment presentation in a melanoma tissue sample-different cell types dominate in different parts of the section. The corresponding mass spectrum is also given (lower panel). The coverage over the tumour was shown to vary substantially. Entire coverage was apparent for metabolites with m/z values of 184.073 and 222.030; whilst the metabolite at m/z 296 only showed expression in the upper epithelial part of the tumour (data not shown). Phosphocholine is used as a reference Fig. 6 Resultant mass spectrum from a melanoma tumour. TIC-total ion current, CHCA-α-cyano-4-hydroxycinnamic acid (matrix). All metabolites are shown as singly-or doubly-charged ions within the tumour tissue Fig. 7 MALDI mass spectrometry image overlay with histology from a patient cancer tissue sample isolated after surgery demonstrating tumour heterogeneity and metabolite localization. Spatial resolution indicated of the world's largest cancer projects, has now been in operation since June 2017 and we hope it will initiate many collaborative studies.

Fig. 1
1Several types of treatment are used for melanoma patients; surgery, radiation, drug therapy, and immunotherapy. These treatments are often used in combination-especially for patients with metastatic disease

Fig. 2
2Hospital discharge rates for in-patients with neoplasms, 2014 (per 100,000 inhabitants). Note the substantial variation between different European countries (source: Eurostat)

Fig. 3
3Schematic illustration of the biobank workflow that has been developed and implemented in Lund. Blood and tissue samples (primary tumour and metastases) are stored in the automated biobank and can be used in research projects, e.g. aiming at finding new biomarkers

Fig. 4
4The fully-automated robotics of the biobank is shown; the heart of the robotic entry point is shown in the upper image; whilst the upper view of the robot is given in the central image. The lower image shows the automated biobank storage with the robotic 384-sample tube plate entering the − 80 °C compartment MSI will undoubtedly become increasingly important in pathology and in the clinic.

Table 1
1Melanoma Pathology (Proposed by Mool and Krausz)SSM
NM
LMM
ALM

Peak incidence
Middle adult life
Middle adult life
Late adult life
Late adult life

Race
Usually whites
Usually whites
Only whites
All races

Localization
All skin
All skin
Sun-damaged skin
Palms, Soles, Subungual

Main cell type in epidermis
Epithelioid
Epithelioid
Dendritic
Dendritic

Pagetoid lateral spread
Usual
Absent and minimal
Rare
Rare

Lentiginous lateral spread
Uncommon
Absent
Usual
Usual

Epidermal hyperplasia
Often
Sometimes
Absent
Present

Epidermal atrophy
Sometimes
Sometimes
Present
Absent

Actinic damage of skin
Sometimes
Sometimes
Present
Absent

Relative frequencies (Clark et al. [12])
unclassifiable: 10%

67%
10%
9%
4%

FundingThis study was supported by a joint grant from the fru Berta Kamprad Foundation (Melanoma Cancer Moonshot) and was also supported by grants from the National Research Foundation of Korea (NRF-2015K1A1A2028365, 2016K2A9A1A03904900) and the Brain Korea 21 Plus Project, Republic of Korea.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Competing interestsThe authors declare that they have no competing interests.Availability of data and materialsRaw data (e.g. scans) can be obtained from the authors.Ethics approval and consent to participateThe study followed the recommendations of the Declaration of Helsinki. Oral and written information was provided to the patients enrolled in the study and confirmed consent in writing was received before inclusion. In addition, ethical approval for this study was granted by the Institutional Review Board at Lund University Hospital; Approval Number: DNR 191/2007, 101/2013.
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BMC Pediatrics Opportunistic screening for iron-deficiency in 6-36 month old children presenting to the paediatric emergency department

Martin V Pusic email@example.com
Dept of Pediatrics
University of B.C
VancouverBC Canada

Brenda J Dawyduk email@example.com
University of Manitoba
WinnipegManitobaCanada

David Mitchell email@example.com
Dept. of Pediatrics
McGill University
MontrealQuebecCanada

BMC Pediatrics Opportunistic screening for iron-deficiency in 6-36 month old children presenting to the paediatric emergency department
10.1186/1471-2431-5-42BioMed Central Page 1 of 6 (page number not for citation purposes) Research article * Corresponding author
Background:The Complete Blood Count (CBC) is a test frequently performed on children presenting to the Pediatric Emergency Department (PED), usually for the evaluation of an infectious illness. The CBC also allows for screening for Iron-deficiency Anemia. This study aims to determine the prevalence of a low Mean Cell Volume (MCV) in children having a CBC performed during a PED visit and whether physicians acted upon the abnormal value.Methods:We present a retrospective cohort study. We reviewed the PED charts of all children 6-36 months of age who had a CBC performed during a 4-month period and the red blood cell mean cell volume was low. Our main outcome variable was whether or not the possible iron deficiency was addressed through documentation of either iron therapy or further investigation.Results: 938 children had a CBC performed during the two periods. Of these, 78 (8%) had an abnormal MCV or Hemoglobin with no previously identified explanation. Physicians documented either treatment or follow-up investigations in 27 cases (35%, 95% CI: 24-46%). Factors associated with the physician documenting either treatment or investigation plan were the following: hemoglobin level (OR 12.6; 95%CI: 4.0, 39) and age ≤ 18 months (OR 4.2; 95%CI: 1.4, 13).Conclusion:Children who have had a CBC in the PED can be screened for iron deficiency at no additional cost. Physicians may be under-utilizing this information.

Background

Iron deficiency anemia in infants and young children can have serious adverse effects on their development including irreversible cognitive impairment and behaviour problems [1][2][3][4][5]. The negative sequelae can be prevented with simple treatment with iron-fortified infant formula or oral iron supplementation. High risk groups include children from low socioeconomic status families, Chinese children, aboriginal children, infants of low birth weight, children whose mothers were iron-deficient during preg-nancy, and children who consume cow's milk [6,7]. Despite an overall decline in the incidence over the past 20 to 30 years there is still a significant proportion of children at risk. The prevalence of iron-deficiency anemia among a random group of Canadian 9 month olds was 7%, and 24% had low iron stores, despite the introduction of iron-fortified cereals by six months of age [7]. Among African-American children aged 9 to 36 months the prevalence of iron deficiency anemia was 8% [8].

The Canadian Task Force on Preventive Health Care provides a grade B recommendation (fair evidence to support) for routine hemoglobin measurement for infants at high risk [6]. The U.S. Preventive Services Task Force recommends that screening should be offered once to all infants, and the American Academy of Pediatrics recommends at least one measurement of hemoglobin or hematocrit in infancy, and at least during the ages of 1 to 4 years and again in adolescence [9].

Basic screening for iron-deficiency anemia consists of a complete blood count (CBC) looking for hemoglobin less than the age-adjusted normal range, with a sensitivity of 86% and a specificity of 97% [6]. The diagnosis is supported by a low mean corpuscular volume (MCV), and increased red cell distribution width (RDW). Confirmation of the diagnosis is commonly obtained with a trial of iron supplementation and measurement of the response [10]. More complex measures of iron stores including serum ferritin, transferring saturation and erythrocyte protoporphyrin activity are used to more accurately determine iron status [11].

It is unclear what proportion of infants and children are currently being screened as recommended. Ideally every child would be screened but this likely does not happen for reasons of cost and the pain associated with the blood test.

A large number of infants and children who are assessed in emergency departments have a CBC done. This presents an opportunity to screen for iron-deficiency anemia. However, in this setting the physician is likely to be focused on the white cell count as most of these patients are being assessed for the possibility of infection. As a result, CBC indices that suggest iron-deficiency anemia may be overlooked. Our hypothesis was that the CBC information that pertains to iron-deficiency is underutilized given that this is not usually the primary indication for the test. If confirmed, we proposed to raise physician awareness of this method of secondary screening.

Methods

Setting

The Montreal Children's Hospital is a 181-bed tertiarycare referral center affiliated with McGill University. The Pediatric Emergency Department (PED) has an annual census of 85,000 visits and is staffed by 6 full-time Pediatric Emergency Medicine physicians. A large group of community-based pediatricians also contribute from 1 to 6 shifts per month. Medical students, paediatric, family medicine and emergency medicine residents and fellows all do training rotations in the PED.

Study design

This is a retrospective before-after cohort study. We identified all children who had a Complete Blood Count (CBC) performed in the PED during a 2-month period (February-March 1999), implemented an educational intervention and then collected data during the same two months a year later. The Institutional Review Board approved the study.

Subjects

Using our laboratory information system, we were able to identify all children who had a CBC done in the PED during the two study periods. We then selected children aged 6 to 36 months based on their age at the time the CBC was done. We excluded children with a known history of the following: anemia on treatment, sickle cell disease or trait, thalassaemia or trait, chronic illness likely to cause anemia. Patients who presented twice during a study period were handled as follows: if, on the first visit iron-deficiency was recognized, they were excluded on the next visit. If, on the first visit, iron-deficiency was not identified, then they were included on the second visit.

Laboratory techniques

CBCs were determined using a Bayer Advia 120 hematology analyzer (Bayer AG, Leverkusen, Germany). We defined the lower limit of normal for MCV as 70 fl while 110 g/L was the cutoff for normal Hemoglobin.

Definitions

MCV addressed

We identified children as having had their iron status addressed if either treatment or further investigations was documented on the PED encounter sheet. Treatment could include any iron preparation initiated by the PED physician. Further assessment could include specific referral to their family doctor for assessment of iron deficiency anemia or arrangement of confirmatory laboratory tests (any of: Hgb Electrophoresis, Serum Fe, Ferritin, Free Erythrocyte Protoporphyrin, Transferrin Level).

MCV not addressed

Children with a low MCV who did not have either treatment or arrangements for further assessment, as defined above, documented on either their PED encounter sheet or, if admitted, on their discharge summary.

Data collection

From the Laboratory Information System we collected the hemoglobin measurement, all standard CBC indices, ordering physician name, patient date of birth and hospital chart number. We performed chart reviews on all children with low MCV. We limited the chart review to the PED encounter sheet of the CBC date as well as the inpatient chart for those patients who were admitted at the time of the initial encounter. From the charts we collected information concerning the reason for the visit, any past history relevant to anemia, a list of medications, and whether any note had been made regarding possible irondeficiency.

Educational intervention

We collected the data in two sessions. In April 1999 we identified the first cohort (Feb-Mar 1999). These initial data were analyzed and showed that a significant number of patients were not having possible iron deficiency addressed. We presented this information (that we pediatricians were missing up to 75% of children with abnormal MCV's in this age group) in a one-hour PED Grand Round in June 2000. To reinforce the message, we mailed a one-page abstract to them with their monthly work schedules in January 2000. This abstract stressed the importance of examining the MCV results on all CBC's done. We collected the second set of data after these interventions in Feb-March 2000. We hoped that this intervention would improve the rate of recognition of abnormal MCVs.

Data analysis

We analyzed the data using descriptive statistics and used chi-squared tests to look for factors that differed between groups where the possible iron-deficiency had been addressed. We calculated 95% confidence intervals around proportions using the binomial distribution. We also developed a univariate logistic regression model to see which factors predict proper identification and documentation of iron deficiency. Our sample size was determined based on the following assumptions: with 25 subjects in each group, we could detect an improvement from 10% identification to 50% with a power of 80% at alpha = 0.05.

Results

PED patients with CBCs

During the four months studied, a total of 7571 patients aged 6-36 months were seen in the PED. Of these, 934 (12%) had CBC's performed which is 29% of the 3206 CBCs performed for all children seen in the PED during the study periods.

The MCV was less than 70 fl in 94 patients (10%) while 184 (19.7%) of the children had a hemoglobin < 110 g/L. Fifty-seven had both low MCV and low hemoglobin. The relationship between MCV and HgB is shown graphically in Figure 1.

Patients with possible iron deficiency

Of the patients with low MCV, 16 were excluded from the analysis for the following reasons: sickle cell disease (1), thalassemia (3), previously identified iron deficiency (8) and chronic disease (2 -autoimmune hepatitis and liver mass). Two charts were missing. Discharge diagnosis and some form of discharge plan had been documented on every chart. For the 78 included patients, 45 (57%) had a hemoglobin value less than 110 g/L.

Patients' discharge diagnoses were available for all included patients. The most frequent diagnoses were: Gastroenteritis (19; 24%), Respiratory Illnesses (18; 23%), Nonspecific Viral Illness (13; 17%), and Febrile Seizures (4; 5%). No other diagnosis made up more than three of the cases.

Patients with low MCV's had an iron medication prescribed in 21 cases (27%). In another 6 (8%) cases, explicit follow-up was arranged so that, in total 27 of 78 patients had their low MCV addressed (35%; 95% CI: 24-46%). Even markedly decreased MCV's (≤65 fl) were addressed only 12 of 28 times (43%; 95% CI: 24-63%). Iron was addressed in 18/25 patients whose Hgb < 100.

Regression model

In our univariate logistic regression model (Table 2), patient age and the value of the Hemoglobin measurement predicted that the iron status would be addressed. The following variables were not significantly associated: patient has a primary care physician, presence of fever, intravenous placement, admission to hospital, full-time versus part-time status of ordering Emergency Physician. In addition, there was no improvement in our rate of identifying children after our educational interventions. Figure 1 MCV versus Hemoglobin for all children having a CBC in the PED.

MCV versus Hemoglobin for all children having a CBC in the PED

Discussion

We report the results of a cohort study examining the prevalence of an index of iron deficiency (MCV) in children who have had a blood test in the PED. We found that PED physicians did not reliably document either a diagnostic or therapeutic plan to deal with the possibility of iron deficiency suggesting that this opportunity for secondary screening is being missed.

The risk of iron deficiency in U.S. children aged 12-35 months is approximately 9% [11]. Major national organizations such the American Academy of Pediatrics suggest that all children be screened with a blood test at age 12 months [9]. This is not commonly done possibly due to the perception that the risk of problems due to iron deficiency, for any individual child, are low especially when the diet includes iron-fortified foods. In addition, the cost, both financial and in terms of pain and inconvenience, is a barrier to routine screening.

The population of patients that present to urban PEDs is likely to be of low socio-economic status and skewed towards the preschool age group [12]. Both of these factors (low socio-economic status, age 3-24 months) are also predictive of iron deficiency suggesting that a PEDbased secondary screening program could be successful.

For the PED patient population, we were unable to identify a published estimate of the predictive value of hemoglobin value or CBC indices compared with gold standard measures of iron status. Both low MCV and low Hemoglobin are imperfect predictors of iron deficiency. Using Hemoglobin cutoff 110 g/L; MCV (mean cell volume) cutoff 70 fL the National Health and Nutrition Examination Survey III (1988)(1989)(1990)(1991)(1992)(1993)(1994) data, White showed that the positive predictive value of a low Hemoglobin value (<110 g/L) for iron deficiency is only 29% (95%CI: 20-38%) in children 12-35 months [11]. Iron deficiency was defined as the presence of ≥2 of the following: low ferritin, low transferrin saturation or high erythrocyte protoporphyrin.

Oski suggested a staging classification of iron status in which a period of iron-deficient erythropoiesis, characterized by low MCV and high RDW but normal hemoglobin, preceded frank anemia [3]. We felt that by identifying children at that stage, potential iron-deficiency could be intercepted at an early stage with simple interventions such as administration of an oral iron preparation [10]. Hemoglobin levels may be low due to recent viral illnesses or they may be elevated in dehydrated patients [10]. Interestingly, in our population, we found that more children had low hemoglobin values than low MCV's. We can only speculate that this might be due to the effects of intercurrent illness since we had no laboratory studies of iron stores available to us.

The health impact of mild iron deficiency is controversial. Anemia due to iron deficiency may be associated with behavioural changes, impaired psychomotor development and impaired cognitive function [13]. A frequently cited study found that children who were frankly anemic scored significantly lower on a test of cognitive function than controls [14]. The difference would not be perceptible in an individual child but would have important implications at the population level. Children with abnormal indices but normal hemoglobin did not differ from controls. This study and one other randomized controlled trial suggest that iron therapy can reverse the developmental effects after at least two months of therapy [13][14][15].

Widespread screening for thalassemia traits has not been routine likely because it is clinically benign, not of immediate clinical importance for young pediatric patients, and because they are concentrated within certain genetic backgrounds; however, discovering a thalassemia trait in a patient provides an opportunity for genetic counseling.

This is opportunistic secondary screening. The cost has already been paid and there is no need to change existing workflow to take advantage of this information in contrast to screening at the time of the well-child visit [16]. While it will likely only benefit those patients who have had a CBC done for another reason, heightened awareness of this issue might predispose PED physicians to address this issue more frequently in their patients. For example, increased screening of dietary histories in children at risk might be effective [17].

Our retrospective study is susceptible to a documentation bias. Physicians may well have considered iron deficiency but not documented it on the chart. This is all the more likely given that a health maintenance issue would rarely have been the primary indication for the visit to the PED. However, in informal discussions, our staff related that they were likely to be missing this diagnosis. Also, the absence of a Hawthorne effect in the publicized second phase of data collection suggests that the physicians do not consistently consider this diagnosis. We note that even if the documentation bias caused us to under-report appropriate therapy by a factor to two, we would still be missing close to half of the opportunities for closer assessment of these children.

Why do our physicians, many of whom are primary care pediatricians, not use this flagged information? The answer may lie in cognitive psychology. We speculate that a normally adaptive heuristic may be working against the physician in this particular situation. Heuristics are shortcuts that humans use to process information more quickly [18]. The CBC report presents the time-pressured PED physician a list of 12 numbers in a densely-typed column, all in the same font size. Some of these numbers are clearly more important than others: specifically, the White Blood Count, Hemoglobin and Platelet Counts. It may be that the physicians have subconsciously made a decision not to process all the numbers. Instead they have trained themselves to scan the list for only the three most important values. In essence, they see the list of numbers through an opaque mask that has only three holes cut out of it. This idea is supported by better performance of the physicians in detecting abnormal hemoglobin levels than abnormal MCVs.

Another heuristic is also likely working against the physician in this case. In the search satisfying heuristic, the physician who ordered a lab test for one reason (e.g. fever) will likely most closely attend to the feature of the result (e.g. White Blood Count) that is directly related to the reason for ordering the test. This may lead them to miss unanticipated results (e.g. anemia). What is not clear is why the physicians did not attend to the asterisks used to flag abnormal results (another adaptive heuristic is to scan a long list of lab reports for asterisks and then specifically attend to those results). Careful attention to the display of the information could increase physician attention to abnormal results without decreasing their efficiency [19].

We were disappointed that our educational interventions did not improve the situation. From the PED charts it appears that the rate at which clinicians arranged assessment or treated patients with evidence of iron deficiency did not change after we had presented a round to our Division and sent a one-page memo to every physician in the PED. These standard interventions have been shown to be relatively ineffective in changing physician behaviour in many settings [20]. While we did observe a trend towards improvment, it is likely instead that we will need to reanalyze and change our process of care [21]. For example, we could establish a protocol whereby all CBC samples that meet certain criteria for iron deficiency would automatically have a Free Erythrocyte Protoporphyrin test run on the same sample. The result would then be reported to the ordering physician. This strategy would have the advantage that, besides confirming the diagnosis, the added report would draw the physician's attention to the potential problem.

Conclusion

In summary, we found that Pediatric Emergency Dept. physicians are not using all of the information available to them when they consider Complete Blood Count results. Careful attention to the Hemoglobin and MCV can suggest an opportunity for secondary prevention of iron-deficiency in children 6-36 months of age presenting to the ED. This information comes at no cost to the patient and can help avoid cognitive impairment in some children.

Children 18-36 months of age did not have significantly different MCV (79.4 fl) or hemoglobin (122 g/L) values compared with children 6-17 months (78.5 fl, 124 g/L respectively; both p = NS).

Table 1 :
1Assessment And Treatment Of Children Who Had A CBC Performed In The PED That Showed MCV < 70 fl.N
Iron Therapy
Prescribed

No Iron Therapy but Explicit
Follow-Up Arranged

Neither Iron Therapy nor
Follow-up Documented

Hemoglobin Normal
MCV Low

33
2
1
30

Hemoglobin Low
MCV Low

45
19
5
21

All with low MCV
78
21
6
51

Table 2 :
2Factors Potentially Associated with Identification of Potential Iron Deficiency in Children with MCV <70Total N
Number in
whom Iron
Prescribed or
Tests Arranged

Odds Ratio
95% Confidence
Interval

(page number not for citation purposes)
AcknowledgementsThe authors gratefully acknowledge the cheerful assistance of Nathalie Gauthier who queried the Laboratory Information System database to identify patients for us and Suzanne MacDonald who did several of the chart reviews. We are also grateful to Gudrun Aubertin who helped write the background section of this manuscript.Competing interestsThe author(s) declare that they have no competing interests.Authors' contributionsAll authors participated in the design of the study. BD designed the data collection instrument, and with MP, did the chart reviews; DM and MP analyzed the data; MP drafted the manuscript with considerable revision by DM; All authors read and approved the final manuscript.
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Towards a genetic AIDS vaccine

Antonia V Bordería
Department of Medical Microbiology
Center for Infectious Diseases and Immunology Amsterdam (CINIMA)
Academic Medical Center (AMC)
Laboratory of Experimental Virology
University of Amsterdam
the Netherlands

Ben Berkhout email@example.com
Department of Medical Microbiology
Center for Infectious Diseases and Immunology Amsterdam (CINIMA)
Academic Medical Center (AMC)
Laboratory of Experimental Virology
University of Amsterdam
the Netherlands

Towards a genetic AIDS vaccine
10.1186/1742-4690-6-93BioMed Central Page 1 of 4 (page number not for citation purposes) Retrovirology Commentary * Corresponding author
We discuss a recent Nature Medicine publication by Philip Johnson and co-workers (Vectormediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys. Nat. Med. 2009, 15: 901-906)  in which an effective HIV-1 vaccine was designed that is based on gene therapy. The introduced gene produces an antibody-like immunoadhesin in the blood that neutralizes the virus.

Commentary

The current status of HIV vaccines

The quest for a vaccine against the human immunodeficiency virus type 1 (HIV-1) has recently been given a bad prognosis [1]. Most attempts at developing an HIV-1 vaccine have used substances (immunogens) aimed at stimulating the body's own immune system to produce antibodies or killer T cells that would either neutralize the virus during transmission from one to the other host (protective vaccine) or control the level of virus replication in an individual who is already infected by HIV-1 (therapeutic vaccine). Such traditional HIV vaccines have not elicited protective immune responses, and it is apparent that out-of-the-box thinking is needed to generate some truly innovative ideas that should help us in this HIV-1 vaccine crisis.

A genetic twist to HIV-1 vaccines

The study by Philip Johnson and his colleagues of the Children's Hospital in Pennsylvania published in a recent issue of Nature Medicine presents an unorthodox, yet surprisingly simple approach. These authors used gene transfer technology to produce antibody-like molecules in the blood that effectively block viral infection [2]. First, they created artificial antibody-like proteins called immunoadhesins that were specifically designed to bind to the simian immunodeficiency virus (SIV) that infects macaques to cause an AIDS-like disease. Second, a gene therapy approach was used to deliver the antiviral immunoadhesin gene into the macaques. This immunoadhesin gene therapy approach bypasses the immune system altogether, and promising results were reported in the preclinical macaque model. The insights gained from how to achieve protection against SIV infection by a gene therapy approach could possibly be translated to the control of HIV-1 infection in humans.

Antiviral immunoadhesins

The antibody genes chosen in the study are immunoadhesins, artificial chimeric antibody-like molecules that were pre-selected to bind the Envelope protein (Env gp120) on the SIV virions. The immunoadhesins are based on two previously characterized gp120-specific Fab clones from SIV-infected macaques. These Fabs show potent in vitro neutralizing capacity against the SIVmac316 isolate, and also against other SIV strains. The immunoadhesins were designed by coupling the variable heavy (V H ) and variable light (V L ) chains from the Fabs via a linker to an IgG2 Fc fragment of the rhesus macaque (constructs 4L6 and 5L7). In a separate construct N4, the IgG2 domain was fused to the rhesus macaque CD4 molecule (domain 1 and 2), the primary receptor for SIV entry into cells. The purpose of both the Fab and the CD4 molecules is to block the viral Envelope protein and thus abrogate the capability of the virus to bind and infect target cells. Based on the binding affinity, it could be expected that CD4 would be less efficient in virus neutralization than the Fabs. In addition, the CD4 molecules may cause unwanted side effects through interaction with other molecules of the immune system, e.g. the T cell receptor complex.

AAV gene therapy vector

A harmless adeno-associated virus (AAV) vector was used to deliver the antiviral immunoadhesin or CD4 gene. The AAV vector has some clear advantages over the traditional use of adenoviruses or other vector systems in gene therapy. First, AAV has a relatively low immunogenicity and only triggers the synthesis of neutralizing antibodies, but no cytotoxic T cell activity. Second, AAV vectors can provide a durable therapeutic effect because of the high transduction efficiency and the accumulation of episomal concatamers. A disadvantage of AAV vectors is the limited cloning capacity, which complicates the usage of large genes. The traditional AAV vector was used for insertion of the CD4 gene. As pointed out by the authors, by cloning the expression cassettes between the AAV inverted terminal repeats (ITR), two genomes can anneal to double the capacity of the vector. This novel vector arrangement was used for the immunoadhesin genes. The authors confirmed that the designed immunoadhesin-IgG2 genes were functionally expressed. Initial tests were performed in mice [3], and based on promising results, the authors moved to the macaque model.

Macaque immunization

Three groups of 3 rhesus macaques were immunized by intramuscular AAV injection. The animals received either the 4L6 or 5L7 vector encoding an anti-SIV immunoadhesin or the N4 vector that encodes the rhesus macaque CD4 molecule. All macaques showed a detectable level of transgene protein expression in the sera. The immunoadhesin concentration in the blood was similarly high for the 4L6 and 5L7 vectors, and persisted for over a year. A somewhat reduced protein level was measured for the N4 vector, which could be due to differences in the AAV vector used. We note that both the AAV vector and the SIVmac316 challenge virus stock were prepared in the human 293-HEK cell line, which may cause cross-species reactions that were not addressed in the study design that used unvaccinated control animals.

The SIV challenge experiment

The 9 immunized macaques and 6 control non-immunized macaques were challenged one month after immunization by intravenous injection of the live, AIDScausing SIVmac316 strain. This challenge was repeated to ensure virus transmission, and indeed all control macaques became infected. Four of these control animals had to be sacrificed due to AIDS-related complications, and only a single animal was able to suppress spontaneously viral replication. In contrast, 6 of the 9 immunized macaques remained uninfected after challenge. Furthermore, none of the 9 immunized animals suffered any AIDS-related complications for up to 85 weeks. The protection induced by the immunoadhesin expressing 4L6 vector seemed most effective (3 of 3 animals tested), followed by the CD4-IgG2 expressing vector N4. The single 5L7 animal that was protected from infection showed a pattern of immunoadhesin abundance and neutralization activity similar to the 4L6 monkeys.

All control macaques developed SIV-specific gp120 and Gag antibodies due to the successful infection. The 3 immunized macaques that appeared not to be protected against SIV challenge also developed antibodies against gp120, which could in fact be distinguished from the anti-gp120 immunadhesin expressed from the AAV vector. The 6 protected macaques exhibited different neutralization patterns. The 4L6 and 5L7 groups showed a higher level of neutralizing titers than the N4 group. The macaques that failed to be protected by AAV-immunization, 2 animals in the 5L7 group and 1 in the N4 group, showed a strong immunoadhesin-specific antibody response in comparison with the protected animals that did not develop any immune reaction against the immunoadhesins. The positive role of the IgG2 Fc domain in the effector functions of the immunoadhesin warrants further studies.

Towards an HIV-1 vaccine

Based on the important proof of concept in the SIVmacaque model, the Johnson team is gearing up for clinical trials with potential "superantibodies" from people who spontaneously resist HIV-1 infection. At the same time, the authors cautioned that many hurdles remain before the technique used in this animal study might be translated into an HIV-1 vaccine for humans. The approach is still in the early stages of animal testing, and there is much to do in order to prove that this approach could yield a successful and safe vaccine that protects against HIV-1 infection. For instance, the monkeys were challenged by direct SIV injection in the blood, exactly the location where the antiviral immunoadhesins reside. Additional tests will be needed to show that the vaccine also protects against sexually acquired virus, as the virus may not encounter the antiviral immunoadhesins in this natural transmission route across the mucosa during the first days of virus replication.

Any test of vector-mediated delivery of protective antibody genes will require extensive tests with a range of antibodies and subsequent optimization tests before human safety studies can take place. For instance, the best protection was observed in those animals that did not mount an immune response against the immunoadhesin vaccine. This finding would argue for the development of improved "humanized" versions of the vaccine so that the human body does not produce vaccine-inactivating antibodies, which then would also allow repeated treatments as booster vaccinations. The final choice of antibody will depend on its ability to protect against the different HIV-1 subtypes. A combination of antibodies could be developed to widen the breadth of the antiviral response. Alternatively, it may be better to develop several subtypespecific gene vaccines that can be used in matching geographic regions, e.g. subtype A in West Africa and subtype C in East Africa. Repeated and massive viral challenges should be done in long-term follow-up experiments to test the longevity of protection and to study the potential for the evolution of vaccine-escape virus variants that could theoretically start to spread in an unrestricted manner in the "gene vaccine protected" population.

Such an unorthodox gene-based vaccine will likely be tested first in small groups of volunteers with a high risk of infection, e.g. discordant couples where one partner is HIV-positive and the other could be vaccinated. As the proposed vaccine will not likely be given to the general population, vector production issues seem less of an urgent issue. These issues would include the stability of the AAV vectors and the ability to manufacture large-scale quantities of these vectors in a cost-effective manner. Finally, this gene vaccination technology may also have potential use in the prevention of other infectious diseases that resist orthodox vaccination strategies.

Alternative vaccine approaches

One could argue that the only hope of controlling the HIV pandemic is to develop a protective vaccine. The urgency to do so has not really been tempered. HIV-1 has killed 25 million people since the early 1980s and currently infects 33 million individuals. Even if the gene therapy technique leads to an effective HIV vaccine, such a vaccine may be years away from practical application. Thus, it seems wise to develop more unconventional vaccination strategies in order to spread our chances on winning the battle against HIV-1. This includes the use of replication-competent viral delivery systems [4] and possibly even the use of safe versions of a live-attenuated HIV-1 vaccine [5]. In addition, one should speed up approaches to remodel the viral Env protein such that it becomes an effective immunogen that elicits neutralizing antibodies [6]. It cannot be predicted which method will turn out to be the best in terms of vaccine efficacy and breadth of protection. A renewed focus on live-attenuated virus variants seems warranted to define the correlates of protection, a pivotal issue that cannot be learned from the premade immunoadhesin strategy.

Alternative gene therapy approaches

Future gene therapy has been proposed for individuals who are already infected by HIV-1. Several gene therapy trials are ongoing with antiviral molecules such as ribozymes [7] or inhibitors based on the mechanism of RNA interference (RNAi) [8]. The RNAi approaches have yielded especially impressive results when targeting the viral RNA genome [9], but one could also think of targeting an important cellular co-factor. The CCR5 receptor is an obvious and attractive target. HIV-1 infected people who carry a defective CCR5 gene, CCR5-Δ32, show delayed disease progression, and people homozygous for CCR5-Δ32 are healthy and largely protected from HIV-1 infection [10]. Targeting of CCR5, resulting in perhaps only a partial knock-down, is expected to provide a therapeutic benefit for HIV-infected patients. This potential was recently highlighted in a proof of principle study in non-human primates [11]. Primates received blood stem cells treated with an SIV lentiviral vector expressing a shRNA against CCR5. CCR5 expression was knocked down and T cells from these primates were found to be less susceptible to SIV infection as compared to the appropriate control cells. The primates exhibited normal hematopoietic reconstitution, an important indication that the treatment is safe.

The potential of such an anti-CCR5 gene therapy is further supported by the functional cure of an HIV-infected patient, who had leukemia in addition to AIDS, and received a special bone marrow transplant [12]. The patient's medical condition warranted the high risk blood stem cell transplant, although 10% to 30% of people who receive bone marrow transplants die. Even though the odds are extremely small, a donor was identified who was both a good tissue match and a carrier of the CCR5-inactivating mutation. Prior to the transplant, a standard regimen of drugs and radiation was administered to kill the patient's bone marrow cells and many cells of the immune system. Antiretroviral treatment was stopped when the donor cells were transfused because of concerns about their survival ability. While the plan was to resume the antiretroviral regimen once HIV-1 re-emerged in the patient's blood, standard tests have not detected HIV-1 in his blood for more than 600 days. As stated above, this treatment is unthinkable for the millions of people living with HIV/AIDS. Nevertheless, the results form a "virtual
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Evaluation of peak cough flow in Brazilian healthy adults

Fernanda Ef Cardoso
Luiz Carlos De Abreu
Rodrigo Daminello Raimundo
Natália Anm
Faustino
Suellen F Araújo
Vitor E Valenti
Monica Akemi Sato
Silvia Rg Martins
Jamili A Torquato
Evaluation of peak cough flow in Brazilian healthy adults
S H O R T R E P O R T Open Access
Introduction: In this study we aimed to evaluate the peak cough flow (PCF) in healthy Brazilian subjects. Methods: We evaluated 484 healthy subjects between 18 and 40 years old. Subjects were seated and oriented were asked to perform a maximal inspiration followed by a quick, short and explosive expiration on the peak flow meter. Three measures were carried out and recorded the average of the three results for each individual.Results:The PCF values ranged between 240 and 500 L/min. The PCF values were lower in females than in males. The PCF was inversely proportional to age. Conclusion: The values for Brazilian adult healthy subjects regarding PCF were between 240 and 500 L/min.

Introduction

Among the methods used to evaluate pulmonary function, we may include some parameters such as forced vital capacity [1], total lung capacity [2] and peak expiratory flow (PEF) [3]. PEF is a maximum flow generated during a forced expiration after lung inflation, i.e., starting from total lung capacity (TLC) [3][4][5].

Respiratory disorders impairs quality of life in several ages [6][7][8]. Along with PEF, peak cough flow (PCF) has provided clinical data to identify common variations in the respiratory muscles function [3][4][5]. Respiratory and neuromuscular diseases induce respiratory muscles weakness, resulting in accumulation of secretions in the airways, leading to pneumonia development, tracheal intubation and tracheostomy [9].

Due to respiratory changes, which results in ineffective cough, it is believed that the development of a PCF normative values in normal individuals from Brazil is important to early detect values below the reference from normal and intervene in respiratory muscle training and cough. Therefore, we aimed to evaluate the PCF in Brazilian healthy adult subjects.

Methods

We selected 484 healthy Caucasian Brazilian individuals according to spirometric values from public institutions in São Paulo city between March 2009 and August 2009, which were submitted to the PCF test. All subjects presented normal respiratory function according to the American Thoracic Society [10]. We considered the following exclusion criteria: smokers, cardiopulmonary disorders, neurological and other impairments that prevent the subject known to perform the procedures, treatment with drugs that influence respiratory function and body mass index (BMI) below 20 kg/m 2 and above 25 kg/m 2 . Participants in this study signed a consent form. The study was approved by the Ethics in Research of the School of Medicine of ABC (No. 009/2009).

Among the subjects evaluated, 232 (47.9%) were males and 252 (52.1%) were females, they aged between 18 and 40 years old. We included 230 men (50%) and 230 women (50%) for the preparation of the values, distributed according to age as shown in Table 1. The exclusion of 24 individuals was based on inadequate comprehension of the PCF test, installation of lung disease, smokers, values not in accordance with the American Thoracic Society [10] and recent surgeries.

Subjects were seated and asked to perform a voluntary couch. The volunteers performed a maximal inspiration, followed by a quick, short and explosive expiration on the peak flow meter. The difference from the peak expiratory flow is the higher glottis pressure and the higher resistance induced by the closed glottis, which characterizes a forced cough. Three measures were carried out and we considered the average of three results for each individual. It was performed a total of 1380 PCF measures in 460 subjects (230 men).

The peak flow meter used was the Mini-Wright Peak Flow Meter Clement Clarke International Ltd., portable, easy to handle and made of plastic material. It was used in order to assess the strength and speed exerted by the expiration in liters per minute (L/min). During the period we used our mechanical tool we did not realize any damage of the instrument [5,11].

We applied the Kolmorogov-Smirnov normality test in order to evaluate the distributions. Considering that all distributions were parametric, we applied the Student t test to compare PFC between men and women and the Pearson correlation coefficient to verify the correlation between age and PFC. Differences were considered significant when the probability of a type I error was less than 5% (p < 0.05). We used the software SPSS 11.5 for Windows.

Results

The PCF values ranged from 240 to 500 L/min in relation to the whole group. In males mean PCF values ranged from 316 L/min to 499 L/min and in females it ranged from 242 L/min to 355 L/min. Figure 1 displays data regarding comparison of PCF between males and females. We noted that males presented higher values than females.

In Table 1 we observed that there was a significant decrease in the PCF in men according to age. In women we found that there was a decrease in PCF without major modifications. The female data are homogeneous compared to males, which presented a major difference between ages.

The both groups presented a relative decrease of the PEF according to the age. There was a significant correlation between PEF and age for both groups (r = −0.851; p = 0.001 for men and r = −0.941; p = 0.001 for women).

Discussion

Based on our data, the values for PCF in Brazilian healthy adults ranged between 240 and 500 L/min. The PCF correlates with respiratory muscle strength, especially with inspiratory muscle strength. In the inspiratory phase of cough, inhalation of high volumes promotes a relationship of time and tension of the muscles and increases the elastic recoil of the respiratory system, optimizing expiratory pressures. The muscle strength to inhale appropriate volumes lead to chest expansion and the muscle force to increase intrathoracic pressure are important to generate effective cough and high flows [12].

One of the limitations of the voluntary evaluation of PCF is the understanding and cooperation of the  subjects. Also, the cough reflex mechanism would be triggered by a reasonable way to replacing the method of evaluation. The differences in voluntary and reflex cough go beyond the way they are triggered. However, there are differences in how muscle action is triggered [13]. Our data showed that there are minimal changes between ages, and we observed in our sample that it may not be changed due to the no comprehension of the subject, since we excluded those who did not comprehend the test. During the voluntary cough the expiratory and accessories muscles action only generates one PCF. On the other hand, during the reflex cough the muscle action is harmonious and simultaneous, generating two or more peak expiratory flow of lower amplitude [13].

According to our results, the PCF values ranged from 240 to 500 L/min. According to the literature, in healthy individuals, the average PCF is higher than 300 L/min in Caucasian European subjects. Additionally, the PCF must be higher than 160 L/min for an effective cough according to the literature [14].

Our findings were obtained from healthy subjects without decrease in muscle strength. The consequence of an inspiratory muscle weakness is the loss of ability to perform a deep inspiration, which is necessary to maintain peripheral alveolar ventilation in addition to affecting the first phase of cough (deep breath). The weakness of the expiratory muscles may be seen in a reduction in maximal expiratory pressure, reduced ability to perform a deep expiration and cough [15].

We found some difficulties such as lack of reference to studies in relation to similar themes or methodologies on the PCF values. Furthermore, there were difficulties in the selection of the subjects, which limited the study sample.

The intent of this study was to evaluate PCF in a cohort of healthy Brazilian subjects. However, age range was not large enough; therefore, it is hard to develop prediction equations from the values we obtained. We suggest future studies to provide prediction equations for this variable. This equation would provide a reference against which measurements from patients with respiratory muscle weakness could be compared in order to identify early those at risk of problems with cough and secretion clearance. While normative values for other aspects of respiratory function such as PEF, TLC and maximum inspiratory and expiratory muscle strength are available, this is not the case for PCF, and therefore, normative data would be very important. This information could be used in developing prevention programs by identifying and intervening in patients at risk.

Conclusion

In this study we investigated PCF in Brazilian healthy adults (between 240 and 500 L/min). The PCF was inversely proportional to age, the higher the age the lower the PCF in both genders.

Figure 1
1Maximal peak cough flow according to gender. *p < 0.05: Different from women.

Table 1
1Average peak cough flow values (L/min) in men and women according to ageAge (years)
Men -Mean ± SD (n)
Women -Mean ± SD (n)

18
499 ± 51 (10)
355 ± 31 (10)

19
486 ± 48 (10)
320 ± 29 (10)

20
484 ± 47 (10)
327 ± 34 (10)

21
435 ± 49 (10)
314 ± 28 (10)

22
428 ± 36 (10)
313 ± 25 (10)

23
407 ± 39 (10)
313 ± 34 (10)

24
406 ± 43 (10)
302 ± 33 (10)

25
494 ± 34 (10)
302 ± 31 (10)

26
486 ± 37 (10)
310 ± 30 (10)

27
484 ± 45 (10)
304 ± 24 (10)

28
439 ± 35 (10)
298 ± 23 (10)

29
430 ± 41 (10)
297 ± 31 (10)

30
426 ± 44 (10)
296 ± 38 (10)

31
410 ± 39 (10)
286 ± 25 (10)

32
399 ± 32 (10)
295 ± 36 (10)

33
380 ± 41 (10)
285 ± 21 (10)

34
380 ± 33 (10)
282 ± 29 (10)

35
376 ± 32 (10)
280 ± 34 (10)

36
381 ± 43 (10)
278 ± 35 (10)

37
360 ± 33 (10)
279 ± 21 (10)

38
347 ± 49 (10)
262 ± 30 (10)

39
337 ± 39 (10)
258 ± 23 (10)

40
316 ± 38 (10)
242 ± 34 (10)

N=number of people; l/min=litters per minute; SD=standard deviation.

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AcknowledgementsThis study received financial support from Universidade Cruzeiro do Sul. The manuscript publication charges were paid by UNESP.Competing interestThe authors declare that they have no competing interest.Authors' contributions FEFC, LCA, RDR, NANMF, SFA, VEV, MAS, SRGM, JAT participated in the acquisition of data and revision of the manuscript. All authors conceived of the study, determined the design, performed the statistical analysis, interpreted the data and drafted the manuscript. All authors read and gave final approval for the version submitted for publication.Submit your next manuscript to BioMed
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