Blood counts in adult and elderly individuals: defining the norms over eight decades of life

The blood count is one of the most common tests used for health assessment. In elderly individuals, selection of a ‘healthy’ reference population for laboratory assessment is difficult due to the high prevalence of chronic morbidities, leading to uncertainty regarding appropriate reference intervals. In particular, age-specific lower haemoglobin reference limits to define anaemia are controversial. Here, we applied a data mining approach to a large dataset of 3 029 904 clinical routine samples to establish blood count reference intervals. We excluded samples from units/specialists with a high proportion of abnormal blood counts, samples from patients with an unknown or decreased estimated glomerular filtration rate, and samples with abnormal test results in selected other analytes. After sample exclusion, 566 775–572 060 samples from different individuals aged 20–100 years were available for analysis. We then used an established statistical algorithm to determine the distribution of physiological test results and calculated age- and sex-specific reference intervals. Our results show substantial trends with age in haematology analytes' reference intervals. Most notably, haemoglobin and red cell counts decline in men with advanced age, accompanied by increases in red cell volume in both sexes. These findings were confirmed in an independent dataset, and suggest an at least partly physiologic cause.     

Establishment of haematological and immunological reference values for healthy Tanzanian children in Kilimanjaro Region

Objective To determine the normal haematological and immunological reference intervals for healthy Tanzanian children. Methods We analysed data from 655 HIV‐seronegative, healthy children from 1 month to 18 years of age from the Kilimanjaro Region of Tanzania for this cross‐sectional study. Median and 95% reference ranges were determined for haematological and immunological parameters and analysed by age cohorts, and by gender for adolescents. Results Median haemoglobin (Hb) and haematocrit (Hct) for all age groups were higher than established East African reference intervals. Compared to U.S. intervals, reference ranges encompassed lower values for Hb, Hct, mean corpuscular volume, and platelets. Applying the U.S. National Institute of Health Division of AIDS (DAIDS) adverse event grading criteria commonly used in clinical trials to the reference range participants, 128 (21%) of 619 children would be classified as having an adverse event related to Hb level. CD4‐positive T‐lymphocyte absolute counts declined significantly with increasing age (P < 0.0001). For those aged under five years, CD4‐positive T‐lymphocyte percentages are lower than established developed country medians. Conclusions Country‐specific reference ranges are needed for defining normal laboratory parameters among children in Africa. Knowledge of appropriate reference intervals is critical not only for providing optimal clinical care, but also for enrolling children in medical research. Knowledge of normal CD4‐positive T‐lymphocyte parameters in this population is especially important for guiding the practice of HIV medicine in Tanzania.     

Hemochrome parameters during the first two years of life in children with perinatal HIV-1 infection

Monthly hemochrome parameters were obtained during the first 2 years of age in 22 children with perinatal human immunodeficiency virus type-1 (HIV-1) infection and in 58 exposed seroreverted children. Timing and predictive value of hemochrome modifications were investigated. Exclusion criteria were hemoglobinopathies and zidovudine (AZT) treatment in pregnancy. When AZT treatment was undertaken children were eliminated from the study. From the second month of life red blood cell (RBC) counts, hemoglobin (Hb) concentrations, and hematocrit values were significantly lower in infected than in uninfected children. RBC counts progressively diverged in infected and uninfected children, and mean values in the former group never reached 4.10(12)/L. No difference was observed in Hb content ratios and RBC size parameters. At 2 months RBC counts, Hb concentrations, and hematocrit values below reference values were associated with a 15.8 (95% confidence limits [CL]: 5.5-48.8) relative risk of being infected. In infected infants aged 5 months a decrease in these parameters was associated with an 11.2 (95% CL: 1.6-77.8) relative risk of developing eventual severe clinical outcome. Low RBC counts, Hb concentrations, and hematocrit values may be included among predictive criteria in infants of HIV-1 infected mothers.     

Ethnic variation in certain hematological and biochemical reference intervals in a south Indian healthy adult population

BACKGROUND: We established the biochemical and hematological reference intervals among a south Indian healthy adult population attending an HIV referral centre in Chennai, southern India. METHODS: In a cross sectional study, 213 study subjects (129 male and 84 female) were studied between March and August 2005. All of the parameters were analyzed using standard hematological and biochemical techniques. RESULTS: Certain biochemical (viz. total bilirubin, alanine transaminase, albumin, creatinine, total protein, lipid profile, creatine phosphokinase, uric acid and lactate) and hematological (mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and lymphocyte levels) parameters presented higher upper limits. In addition, the upper limits of white blood cell count, platelet count, hematocrit, red blood cell count and hemoglobin level were low in comparison to the currently reported ranges. CONCLUSION: Ethnic variation in reference intervals was observed in certain biochemical and hematological analytes in a south Indian adult population.     

Haematological reference values in Spanish adolescents: the AVENA study

Objectives: To provide reference values for haematological indices in Spanish adolescents according to age and gender. Methods: A cross sectional study conducted in five Spanish cities was performed. Blood was drawn from a representative sample of 581 adolescents with age ranging from 13 to 17–18.5 yr. Age‐ and gender‐specific means, standard deviations and percentiles were determined for the following parameters: total red blood cell counts (RBC), haemoglobin concentration (Hb), haematocrit percentage (Hct), mean corpuscular volume (MCV), mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distribution width and total white blood cell (WBC) counts as well as counts and percentage of neutrophils, lymphocytes, monocytes, eosinophils and basophils; platelet count (PLT), mean platelet volume and plateletcrit percentage. Results: Younger male subjects presented lower RBC, Hb, Hct and MCV means that their older counterpart. By contrast these differences were not observed in female subjects. As expected, RBC, Hb and Hct mean values in males were found significantly higher than in girls for all studied age groups. No significant differences were observed in WBC by age and gender. PLT values gradually decreased with age, except for females aged 17–18.5 yr. Conclusion: The present study provides reference data on the distribution of haematological indices of Spanish adolescents. These data can be useful biomarkers of the nutritional status in adolescents.     

Haematological reference ranges for schoolchildren

There are few reports of reference ranges for haematological values in school age children and most studies extend over a small age range or have excluded a considerable proportion of the study population in an effort to omit those with haemoglobinopathies or anaemia. Blood samples from 2135 children aged 4–19 years, from randomly selected schools, were analysed by automated counter. Reference ranges for red cell, white cell and platelet indices are provided from the results. Median haemoglobin and red blood cell count values for girls and boys rose together with increasing age, up to 12 years, but then diverged. Girls had a higher platelet count than boys. Mean platelet volume rose with age and was inversely related to the platelet count. Plateletcrit fell with age but in girls there was a peri-pubertal peak. Total leucocyte count fell with age. The upper limits for total leucocyte count in this study are approximately 2×10⁹ lower than those quoted in modern haematology textbooks. Lymphocyte, eosinophil and basophil counts fell with age with little difference between the sexes. Neutrophil and monocyte counts were similar for younger girls and boys but diverged in the older children with the older girls having higher values than boys.     

Total and differential leucocyte counts in infants at 2, 5 and 13 months of age

Reference ranges for the total and differential leucocyte counts were determined from venous blood collected at 2, 5 and 13 months of age from a cohort of 112 healthy children of north European ancestry. At 2, 5 and 13 months, the ranges for neutrophils were found to be 0.7-4.7, 1.1-5.6 and 1.0-7.6 x 109/l, and for lymphocytes 3.3-10.5, 3.4-11.3 and 3.5-10.4 x 109/l, respectively. The upper limits for monocytes at each age were 1.2, 1.2 and 0.91 x 109/l, and for eosinophils 0.84, 1.0 and 0.88 x 109/l, respectively. Mean counts for all cell types, except monocytes, increased between 2 and 5 months of age. There was little change in mean counts between 5 and 13 months. Statistically significant correlations existed between the numbers of each cell type at 2 months of age, and were still present at 13 months between monocytes and each of the granulocyte series and between basophils and all other cell types. By comparison with older data these findings indicate a lower reference limit for neutrophils at 2 months of age, and a narrower range for this cell type at both 2 and 5 months of age. Reference ranges for lymphocytes and eosinophils are wider than indicated by some previous studies.     

慢性HBV感染儿童外周全血细胞及肝功能特点分析

目的探讨慢性HBV感染儿童外周全血细胞指标、肝功能与HBV感染状态关系,为临床诊治儿童慢性HBV感染提供参考。方法回顾性调查127例慢性HBV感染（分为免疫耐受期和低复制期）儿童外周全血细胞指标、肝功能指标及HBVDNA水平。将调查对象按照年龄段分为低龄儿童组（年龄〈5岁）和大龄儿童组（年龄≥5岁）,在各自年龄段内比较上述指标在免疫耐受组和低复制组问差异,并与49例健康儿童（健康对照组）对照。结果在低龄儿童中,低复制组的RBC计数明显低于免疫耐受组和健康对照组（P均〈0．05）,但是后2组间差异无统计学意义（P〉0．05）。在大龄儿童中,免疫耐受组和低复制组的淋巴细胞绝对值显著低于健康对照组（P〈0．05）,但是前2组之间的淋巴细胞绝对值差异无统计学意义（P〉O．05）。在所有年龄段儿童中,外周全血其他指标在3组之间差异无统计学意义。在低龄儿童中,免疫耐受组和低复制组ALB低于健康对照组。所有慢性HBV感染患儿的ALT、AST都显著高于健康对照组（P均〈0．05）。结论在慢性HBV感染的低龄儿童中,低复制组外周血红细胞数降低;大龄儿童的淋巴细胞数减少;低龄患儿ALB、TP下降;所有患儿ALT、AST升高。     

Are different reference intervals for methylmalonic acid and total homocysteine necessary in elderly people?

Abstract: To determine whether age-adjusted reference intervals for methymalonic acid (MMA) and total homocysteine (tHcy) are necessary, data from 2 previous studies were used. First, the reference intervals for serum vitamin B₁₂, folate, methylmalonic acid and total homocysteine were determined in 2 reference sample groups: 99 healthy adults and 64 selected healthy elderly subjects living at home. Secondly, health-related reference limits were estimated in 143 elderly subjects after supplementing the nutritional status with a combination of vitamin B₁₂, vitamin B₆ and folate during a 3-wk period. In the latter group, the adjusted reference intervals for MMA (46–288 nmol/L) as well as for tHey (5–13.6 μmol/L) are different with considerably lower upper reference limits than that estimated with the original reference values (72–476 nmol/L for MMA; 6.8–21 μmol/L for tHcy) in the elderly subjects. Our data suggest that the MMA and tHcy levels increase with age. Moreover, the upper reference limit for MMA and tHcy, conventionally defined as the mean +2 SD in a healthy elderly reference sample group without supplementing the nutritional status, might result in falsely high values due to a high prevalence of an inadequate vitamin B₁₂, vitamin B₆ and folate status.     

Biomarkers of splenic function in infants with sickle cell anemia: baseline data from the BABY HUG Trial

We evaluated spleen function in 193 children with sickle cell anemia 8 to 18 months of age by (99m)Tc sulfur-colloid liver-spleen scan and correlated results with clinical and laboratory parameters, including 2 splenic biomarkers: pitted cell counts (PIT) and quantitative Howell-Jolly bodies (HJB) enumerated by flow cytometry. Loss of splenic function began before 12 months of age in 86% of infants in association with lower total or fetal hemoglobin and higher white blood cell or reticulocyte counts, reinforcing the need for early diagnosis and diligent preventive care. PIT and HJB correlated well with each other and liver-spleen scan results. Previously described biomarker threshold values did define patients with abnormal splenic function, but our data suggest that normal spleen function is better predicted by PIT of ≤1.2% or HJB ≤55/10(6) red blood cells and absent function by PIT ≥4.5% or HJB ≥665/10(6). HJB is methodologically advantageous compared with PIT, but both are valid biomarkers of splenic function. This trial was registered at www.clinicaltrials.gov as #NCT00006400.     

Comprehensive pediatric reference intervals for 79 hematology markers in the CALIPER cohort of healthy children and adolescents using the Mindray BC-6800Plus system

Background

Hematological parameters vary significantly throughout growth and development due to physiological processes such as fetal-to-adult erythropoiesis and puberty. Pediatric age- and sex-specific reference intervals (RIs) are thus essential for appropriate clinical decision-making. The current study aimed to establish RIs for both common and novel hematology parameters on the Mindray BC-6800Plus system.

Methods

Six hundred and eighty-seven healthy children and adolescents (30 days to 18 years) were enrolled. Participants were recruited as part of the Canadian Laboratory Initiative on Pediatric Reference Intervals Program upon informed consent or identified from apparently healthy outpatient clinics. Whole blood was collected and assayed for 79 hematology parameters on the BC-6800Plus system (Mindray). Age- and sex-specific RIs were established as per Clinical and Laboratory Standards Institute EP28-A3c guidelines.

Results

Dynamic reference value distributions were observed for several hematology parameters, including erythrocytes, leukocytes, platelets, reticulocytes, and research-use-only markers. Age partitioning was required for 52 parameters, demonstrating changes in infancy and puberty. Sex partitioning was required for 11 erythrocyte parameters (i.e., red blood cell (RBC), hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, RBC distribution width coefficient of variation, hemoglobin distribution width, macrocyte count, macrocyte percentage, RBC (optical), and reticulocyte production index). Few parameters had undetectable levels in our healthy cohort (i.e., nucleated RBC count and immature granulocyte count).

Conclusions

The current study completed hematological profiling for 79 parameters on the BC-6800Plus system in a healthy cohort of Canadian children and adolescents. These data emphasize the complex biological patterns of hematology parameters in childhood, particularly at the onset of puberty, and support the need for age- and sex-specific RIs for clinical interpretation.     

Haematological Abnormalities and Reference Intervals in the Elderly

Abstract. We compared three representative population samples aged 70 (n=312), 75 (n=486) and 81 (n=404) years. Anaemia defined either as blood haemoglobin concentration below the corresponding health-related lower reference limits or according to WHO was, in the total study groups, more common at age 81. Anaemia of unknown cause was rare, but somewhat more common at age 81. Mean blood haemoglobin concentrations in subsamples without definable disorders were significantly lower at age 81 than at ages 70 and 75, whereas the lower health-related reference limits did not differ significantly. White blood cell counts tended to be lower and iron deficiency was somewhat more common at age 81. No significant differences were found in platelet counts or in the prevalence of low plasma cobalamin concentrations. These results indicate a fall in blood haemoglobin with advancing age. The arbitrary WHO criteria for anaemia are clinically applicable at age 70–81.     

Reference intervals for common laboratory tests in Melanesian children

Pediatric reference intervals for biochemical tests are often derived from studies in Western countries and may not be applicable to the developing world. No such intervals exist for Melanesian populations. The aim of this study was to provide specific reference intervals for children from Papua New Guinea (PNG). We assayed plasma from 327 healthy Melanesian children living in Madang Province for common biochemical and hematological analytes. We used well-validated commercially available assay methodology. Compared with reference intervals from children from Western countries and/or African children, there were substantial differences in hemoglobin, soluble transferrin receptor, ferritin, calcium, phosphate, and C-reactive protein. Differences in the upper limits of reference intervals for bilirubin and alanine aminotransferase were also observed. Available reference intervals from Western and African countries may be inappropriate in PNG and other Melanesian countries. This has implications for clinical care and safety monitoring in pharmaceutical intervention trials and vaccine studies.     

Evaluation of whole blood filterability with increasing age in healthy men and women

Whole blood filterability, expressed as volume of red blood cells filtered per minute (VRBC), was evaluated in 53 healthy men and 56 healthy women; in each sex, subjects were divided into 4 groups according to their age: adult age (20-50 yr.), advanced adult age (51-60 yr); presenile age (61-70 yr.) and senile age (greater than 70 yr.). In addition, plasma fibrinogen, PCV, white cell and platelet total counts were measured and correlated with VRBC. Whole blood filterability and plasma fibrinogen levels were also correlated with age. VRBC and PCV decreased, whereas fibrinogen levels increased with increasing age. VRBC values showed a significant negative correlation with age and plasma fibrinogen values; a significant positive correlation was found between fibrinogen levels and age. No other examined parameter changed with increasing age nor correlated with VRBC values. These findings indicate that with aging there are modifications of some rheological parameters, such as an increase in plasma fibrinogen levels and a decrease in whole blood filterability.     

The reference range of CD4+ and CD8+ T-lymphocytes in healthy non-infected infants born to HIV-1 seropositive mothers; a preliminary study at Siriraj Hospital.

The results of CD4+, CD8+ T-lymphocyte values as percentage, number, and ratio were studied in infants aged 1 to 29 months. The 283 subsequent blood samples from 89 infants born to HIV-1 seropositive mothers were investigated. From 208 sequential samples of 70 healthy non-infected infants, the reference values of CD4+ and CD8+ T-lymphocytes have been established and compared to Caucasian reference values. The results were analysed in 4 difference age groups (1-5, 6-11, 12-17 and > or = 18 months). At age 12 months, CD4 number and percentage declined significantly while CD8 percent increased. At age 6 months CD4/CD8 ratio decreased. Of 19 infected infants CD4+ percentage and number as well as CD4/CD8 ratio declined at age 6 months and showed significant differences from uninfected infants. A significantly elevated CD8 percentage was demonstrated in infected infants at age of 12 months. In 9 infants who showed symptoms at age 6-18 months, the CD4 and CD8 values were different from the reference range and 6 of 9 patients showed lower CD4 percentage, CD4 number and reversed CD4/CD8 ratio before the symptoms appeared. In 10 infants who were asymptomatic at age 18 months, there was no evidence of immunosuppression at age 6 months or before. After age 6 months, lymphocyte subset values of some asymptomatic infected children were beyond the reference range. These preliminary findings should be very useful for monitoring children born to HIV infected mothers. The results of CD4+ and CD8+ T-lymphocytes in uninfected infants could be used as reference values for the Thai and other Southeast Asian pediatric populations.     

Processing cord blood from premature infants into autologous red-blood-cell products for transfusion

Background and Objectives The use of umbilical cord blood (UCB) for transfusion purposes has gained interest the past years. UCB transfusion could serve premature infants, who often need transfusions early in life. Material and Methods We investigated the suitability of different storage media. UCB was collected after 25 ⁰/₇–35 ⁶/₇ gestational weeks and centrifuged to concentrate red cells subsequently stored in saline–adenine–glucose–mannitol (SAGM), or in additive solution‐3 (AS‐3), or stored as whole blood in citrate–phosphate–dextrose–adenine‐1. Quality parameters were measured at 7 day intervals during 35 days and compared to the standard RBC product. Results White‐blood‐cell‐ and platelet counts were higher in the UCB products. In the fractionated units, haemolysis remained below 1·0% in 64% after 14 days, and in 30% after 21 days. Storage in SAGM or AS‐3 showed similar quality. Whole blood UCB showed better pH and haemolysis rates after 21 days. Conclusion UCB can be processed into autologous products for premature infants. Shelf‐life is limited to 14–21 days and compares unfavourably to stored whole blood. Considering the early transfusion needs in these infants, a short shelf‐life would not be a practical objection.     

Changes in the blood cell counts with aging

We analyzed the blood cell counts and serum levels of total protein (TP), total cholesterol (TC) and triglyceride (TG) of 2,231 healthy subjects (1,295 men and 936 women) between age 20 and 99 years in order to clarify the following two subjects. (1) In the approximately 10 years since the report of Shirakura et al in 1978, eating habits have improved and the average life expectancy has extended in Japan. Is there any effect of such betterment on blood cell counts of the aged? (2) It has been pointed out that quality of everyday life, such as staying at home but not in an old-age home, working, traveling, and so forth, had an influence on the blood cell counts of aged. Is there any difference between the blood cell counts of people under 60 years and those of people older than 60 years who have a good quality of life as mentioned above? The hemoglobin concentration, red blood cell count, and hematocrit value began to decrease in men in their sixth decade and in women in their seventh decade and the change was more prominent with advancing age, especially in men. The white blood cell count and platelet count tended to decrease with advancing age. The serum levels of TP, TC, and TG also declined with age in those over 60 years of age. These results confirmed that the hemoglobin concentration, red blood cell count, and hematocrit value decrease in the elderly subjects as they grow older and it may be considered that reduced ingestion of protein is one of the causes of the phenomenon.     

Serum IGF-1 and IGFBP-3 levels in healthy children between 0 and 6 years of age

Objective: Along with growth hormone (GH) levels, measurements of serum insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) are used in the diagnosis of GH deficiency and in monitoring the efficacy and safety of long-term GH treatment. The purpose of the present study was to establish reference values for serum IGF-1 and IGFBP-3 in healthy Turkish children less than 6 years of age.Methods: This study was designed as a multicenter project. Five hundred sixty-seven healthy children younger than 6 years of age from different geographical regions of Turkey, with weight and height values between the 10th and 90th percentiles according to the national standards were included in the study. In addition to anthropometric parameters, serum IGF-1 and IGFBP-3 levels were measured in all subjects.Results: Although not statistically significant, the serum IGF-1 levels in infants at age 6 months were lower than those in infants at age 3 months. The IGF-1 levels showed a slow increase with age. Serum IGF-1 levels were lower in girls as compared to boys only at age 6 months. No correlation was found between either serum IGFBP-3 levels and body mass index (BMI) or serum IGFBP-3 and weight and height standard deviation scores (SDS). A weak correlation was observed between serum IGF-1 and IGFBP-3 concentrations.Conclusions: The age- and gender-specific reference values for serum IGF-1 and IGFBP-3 reported in this study will aid in the diagnosis of GH deficiency and in the monitoring of children receiving GH treatment.Conflict of interest:None declared.     

Hematological indices reference intervals for a healthy Arab population in Qatar: Effect of age,gender, and geographic location

Hematologic reference intervals vary with gender, age, ethnicity, and geographic area. Therefore, local or national laboratory reference ranges are essential to enhance the accuracy when diagnosing health conditions. Still, no comprehensive list of reference ranges tailored to the Arab population living in Qatar. Accordingly, this study aims at establishing a hematology reference guide for Arabs in Qatar.This is a retrospective study where 750 healthy volunteers (18–69 years) from 2015 to 2019 were included, analyzed by an automated hematology analyzer. Arab adults were divided into African (Egypt, Libya, Tunisia, Morocco) and Asian (Syria, Lebanon, Jordon, Palestine, Qatar). The Cell-Dyn and Sysmex were used for measuring hematological parameters.The mean +/- 2SD were established for all the study groups. Arab males had significantly higher Hb, Hct, red cell distribution width, absolute neutrophil count, lymphocytes, and monocyte counts than females. Asian-Arab males had significantly higher Hb concentration and higher WBC, lymphocytes, and eosinophils than African Arabs. Asian-Arab young (>18: < 40 years) males had significantly higher Hb and lymphocytes and lower monocytes than older males (>40 years). African-Arab young males had significantly higher lymphocytes and lower monocytes than older males. Asian-Arab young females had higher WBC and absolute neutrophil count than older Asian Arabs.The findings of this study will help in establishing specific reference intervals in the Arab world. The differences in hematology reference intervals considering age, gender, and geographical location highlight the importance of establishing blood reference intervals in each country considering the ethnic diversity of each country.     

Reference intervals of complete blood count constituents are highly correlated to waist circumference: Should obese patients have their own “normal values?”

Body mass index (BMI), the prevalent indicator of obesity, is not easily grasped by patients nor physicians. Waist circumference (WC) is correlated to obesity, is better understood and has a stronger relationship to the metabolic syndrome. We compiled WC, complete blood count (CBC) parameters as well as other pertinent data of 6766 25–55‐year‐old US volunteers sampled in the US National Health and Nutrition Examination Survey, in the years 2005–2010. To determine reference intervals of typical US patients visiting their clinician, we used minimal exclusion criteria. We compiled hemoglobin, red blood cell count, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration, mean cell hemoglobin (MCH), red cell distribution width (RDW), platelet count, mean platelet volume, and counts of white blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils, and basophils. In addition, we also compiled serum C reactive protein and serum iron. The three major US races were studied and reference interval diagrams were constructed for each CBC parameter plotted against WC. WBC count, RDW, lymphocyte, neutrophil, and red blood cell count increase with WC. Conversely, serum iron and MCH and MCV decrease. These relationships may be related to insulin resistance and chronic activation of the immune system and the resulting low‐grade inflammatory state. WC is a strong predictor for many CBC parameters, suggesting that WC should be taken into account when evaluating blood count results. Clinicians who take care of obese patients should be aware of altered hematology and investigate and treat accordingly. Am. J. Hematol. 89:671–677, 2014. © 2014 Wiley Periodicals, Inc.