The clinical utility of discriminant functions for the differential diagnosis of microcytic anemias

Several groups of authors have derived discriminant functions (DFs) based on red cell indices (primarily MCH, MCV, and RDW) that can be used to differentiate iron deficiency from thalassemia minor. The Technicon H*1 analyzer provides a direct MCHC measurement (termed the CHCM), in addition to the conventional computed value (Hgb/PCV). To evaluate the clinical utility of red cell discriminant analysis, chart review was performed in 176 cases for which hemoglobin characterization and quantitation studies had been requested. Six published discriminants were evaluated for cases of clearly defined iron deficiency anemia and thalassemia minor. Overall diagnostic efficiency ranged from 50%-82%, and the diagnostic performance of three of the discriminants failed to achieve statistical significance. Mean values for both MCHC and CHCM were significantly lower in patients with iron deficiency than in patients with other causes of microcytic anemia. It was also observed that MCHC was significantly greater than CHCM in patients with iron deficiency anemia, but not in patients with other causes of microcytic anemia. Both MCHC and the difference between MCHC and CHCM showed potential value as parameters for the differential diagnosis of iron deficiency from other causes of microcytic anemia. It was noted, however, that in 67% of the cases studied, the use of a DF could not have resolved the diagnosis to the extent that hemoglobin characterization and quantitation studies were no longer indicated.     

A diagnostic expert system

The introduction of new technologies in the field of electronics has influenced the development of technical equipment over the last few years. The progressive miniaturization of integrated circuits makes possible an expansion of the spectrum of functions offered by this equipment. This also applies to medical technology. These more complex units call for new methods of fault detection and diagnosis. In addition to analytical redundancy, tools developed by the artificial intelligence research community, such as expert systems, are becoming more and more important for fault diagnosis. On the basis of a realized diagnosis expert system the possibilities as well as the limits of such system are discussed. Also, possible future developments of artificial intelligence, like machine learning, are considered.     

Porotic hyperostosis: a new perspective.

Porotic hyperostosis is a paleopathologic condition that has intrigued researchers for over a century and a half. It is now generally accepted that anemia, most probably an iron deficiency anemia, is the etiologic factor responsible for lesion production. Although there can be a number of factors involved in the development of iron deficiency anemia, a dietary explanation has often been invoked to explain the occurrence of porotic hyperostosis in past human skeletal populations. In fact, porotic hyperostosis has been referred to as a "nutritional" stress indicator. Traditionally those groups with a higher incidence of porotic hyperostosis have been considered to be less successful in adapting to their environment or more nutritionally disadvantaged than other groups. A new perspective is emerging that is challenging previous views of the role of iron in health and disease, thus having profound implications for the understanding of porotic hyperostosis. There is a new appreciation of the adaptability and flexibility of iron metabolism; as a result it has become apparent that diet plays a very minor role in the development of iron deficiency anemia. It is now understood that, rather than being detrimental, hypoferremia (deficiency of iron in the blood) is actually an adaptation to disease and microorganism invasion. When faced with chronic and/or heavy pathogen loads individuals become hypoferremic as part of their defense against these pathogens, thus increasing their susceptibility to iron deficiency anemia. Within the context of this new perspective porotic hyperostosis is seen not as a nutritional stress indicator, but as a indication that a population is attempting to adapt to the pathogen load in its environment.     

Physicochemical properties and inhibition effect on iron deficiency anemia of a novel polysaccharide-iron complex (LPPC)

Porphyran (P) was extracted from red algae Porphyra by boiling water. A novel polysaccharide-iron complex (LPPC) was prepared under the alkaline condition by adding a ferric chloride solution to the low molecular weight porphyran (LP) solution. Physicochemical properties and inhibition effect on iron deficiency anemia of this complex were studied. The content of iron(III) in the complex is 21.57% determined with iodometry. The results indicate that LPPC was product required. The complex can increase red blood cell count (RBC), hemoglobin (Hb), Serum iron (SI), spleen index, spleen mass and mass of mice with iron deficiency anemia (IDA). Although the structure and deeper mechanisms on hemolytic anemia of LPPC should be further studied, LPPC is hoped to be developed as a late-model iron supplement which has a synergism on anemia.     

Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia

Oxidant stress has been shown to play an important role in the pathogenesis of iron deficiency anemia. The aim of this study was to investigate the association between lymphocyte DNA damage, total antioxidant capacity and the degree of anemia in patients with iron deficiency anemia. Twenty-two female with iron deficiency anemia and 22 healthy females were enrolled in the study. Peripheral DNA damage was assessed using alkaline comet assay and plasma total antioxidant capacity was determined using an automated measurement method. Lymphocyte DNA damage of patients with iron deficiency anemia was significantly higher than controls (p<0.05), while total antioxidant capacity was significantly lower (p<0.001). While there was a positive correlation between total antioxidant capacity and hemoglobin levels (r=0.706, p<0.001), both total antioxidant capacity and hemoglobin levels were negatively correlated with DNA damage (r=-0.330, p<0.05 and r=-0.323, p<0.05, respectively). In conclusion, both oxidative stress and DNA damage are increased in IDA patients. Increased oxidative stress seems as an important factor that inducing DNA damage in those IDA patients. The relationships of oxidative stress and DNA damage with the severity of anemia suggest that both oxidative stress and DNA damage may, in part, have a role in the pathogenesis of IDA.     

Iron Fortification of Banana by the Expression of Soybean Ferritin

Iron deficiency anemia is one of the serious ailments related to nutrition in the developing countries. Fruit and vegetable crops favor the bioavailability of iron. Banana is consumed as a staple food in the tropics. Iron-fortified bananas provide an effective means of controlling the iron deficiency. Embryogenic cells of banana cv. Rasthali (AAB) were transformed with soybean ferritin cDNA using two different expression cassettes pSF and pEFE-SF to express ferritin. Transgenic nature of the regenerated plants was confirmed by PCR. Transgenic plants were regenerated and analyzed through PCR and PCR-Southern analysis. The expression of ferritin was confirmed by RT-PCR. Iron and zinc levels in the transgenic and control plants were estimated by atomic absorption spectroscopy. A 6.32-fold increase in iron accumulation and a 4.58-fold increase in the zinc levels were noted in the leaves of transgenic plants. Thus, iron- and zinc-fortified bananas could be developed as a functional food to overcome the malnutrition-related iron deficiency. This is the first report on the iron and zinc fortification of banana.     

Development of an expert system for preoperative assessment of female urinary incontinence

We developed a simple expert system for preoperative assessment of women complaining of involuntary loss of urine and scheduled to undergo surgery for incontinence. The aim of the system was to use the parameters obtained at urodynamic investigation to arrive at the correct diagnosis. We used an IBM-PC with two disk drives and 256k RAM, and the expert system shell EXSYS, a rule based system with the possibility of assigning probabilities to the different solutions. To write the rules forming the knowledge base we used a two-fold approach: we constructed tree diagrams for each diagnosis and calculated the corresponding predictive values (statistical approach), and we added rules based on our experience (heuristic approach). The expert system has been found reliable in a clinical setting and is useful for teaching purposes.     

Effects of daily iron supplementation in primary-school–aged children: systematic review and meta-analysis of randomized controlled trials

Background:

Anemia is an important public health and clinical problem. Observational studies have linked iron deficiency and anemia in children with many poor outcomes, including impaired cognitive development; however, iron supplementation, a widely used preventive and therapeutic strategy, is associated with adverse effects. Primary-school–aged children are at a critical stage in intellectual development, and optimization of their cognitive performance could have long-lasting individual and population benefits. In this study, we summarize the evidence for the benefits and safety of daily iron supplementation in primary-school–aged children.

Methods:

We searched electronic databases (including MEDLINE and Embase) and other sources (July 2013) for randomized and quasi-randomized controlled trials involving daily iron supplementation in children aged 5–12 years. We combined the data using random effects meta-analysis.

Results:

We identified 16 501 studies; of these, we evaluated 76 full-text papers and included 32 studies including 7089 children. Of the included studies, 31 were conducted in low- or middle-income settings. Iron supplementation improved global cognitive scores (standardized mean difference 0.50, 95% confidence interval [CI] 0.11 to 0.90, p = 0.01), intelligence quotient among anemic children (mean difference 4.55, 95% CI 0.16 to 8.94, p = 0.04) and measures of attention and concentration. Iron supplementation also improved age-adjusted height among all children and age-adjusted weight among anemic children. Iron supplementation reduced the risk of anemia by 50% and the risk of iron deficiency by 79%. Adherence in the trial settings was generally high. Safety data were limited.

Interpretation:

Our analysis suggests that iron supplementation safely improves hematologic and nonhematologic outcomes among primary-school–aged children in low- or middle-income settings and is well-tolerated.An estimated 25% of school-aged children worldwide are anemic.¹ Iron deficiency is thought to account for about half of the global cases of anemia² and is associated with inadequate dietary iron and, in developing settings, hookworm and schistosomiasis.³ In developed settings, anemia is prevalent among disadvantaged populations, including newly arrived refugees, indigenous people⁴ and some ethnic groups (e.g., Hispanic people in the United States).^5,6 About 3% of primary-school–aged children in Canada are anemic.⁷ Programs to address anemia are constrained by concerns that iron supplements cause adverse effects, including an increased risk of infections such as malaria in endemic areas.⁸In observational studies, iron deficiency has been associated with impaired cognitive and physical development. It has been estimated that each 10 g/L decrement in hemoglobin reduces future intelligence quotient (IQ) by 1.73 points.⁹ However, observational data are susceptible to confounding,¹⁰ and a causal relation between iron deficiency and cognitive impairment has not been confirmed.¹¹ Randomized controlled trials should overcome confounding, but results of trials examining this question have not agreed.Optimizing cognitive and physical development in primary-school–aged children could have life-long benefits.¹² However, anemia-control recommendations must balance safety and efficacy. We performed a systematic review of the effects of daily iron supplementation, a commonly used strategy to combat anemia,² in primary-school–aged children. We examined cognitive, growth and hematologic outcomes and adverse effects across all settings.     

Discriminant functions

Discriminant Functions (DFs), first described by Fisher in 1936, have been applied to the classification of microcytic disorders such as iron deficiency and heterozygous thalassemia. Mathematically DFs are weighted linear combinations of variables. If the underlying assumption of multivariate normality is valid DFs provide the best possible classification. Variables may need to be transformed before the DF is derived. When two groups have to be classified it is easy to visualise the DF. With one variable the DF is represented by the point which provides the best separation. In the bivariate situation the two groups form ellipses and the DF is the best line of separation whilst in the trivariate case the two groups are ellipsoids and a plane forms the best separation. Ratios and power functions are equivalent to DFs but they are less efficient and less rigorously derived. To apply DFs in hematological practice it is necessary to carefully select the measurements to be included and to define the case selection criteria. Once the DF has been derived it should be tested on a new data set and its transferability assessed. Like any single test the DF will have sensitivity and specificity which may need to be adjusted by changing the "cut-off" if the DF is used for screening rather than for differential diagnosis.     

Confidence intervals of effect size for paired studies

To construct a confidence interval of effect size in paired studies, we propose four approximate methods--Wald method, variance-stabilizing transformation method, and signed and modified signed log-likelihood ratio methods. We compare these methods using simulation to determine those that have good performance in terms of coverage probability. In particular, simulations show that the modified signed log-likelihood ratio method produces a confidence interval with a nearly exact coverage probability and highly accurate and symmetric error probabilities even for very small samples. We apply the methods to data from an iron deficiency anemia study.     

A Novel Approach to Evaluating the Iron and Folate Status of Women of Reproductive Age in Uzbekistan after 3 Years of Flour Fortification with Micronutrients

Background

The Uzbekistan 1996 Demographic Health Survey reported 60.4% of women of reproductive age (WRA) had low hemoglobin concentrations (<120 g/L), and anemia was an important public health problem. Fortification of wheat flour was identified as an appropriate intervention to address anemia due to the ubiquitous consumption of wheat flour. A National Flour Fortification Program (NFFP) was implemented in 2005.

Methodology/Principal Findings

After 3-years of the NFFP, a national survey using large country-lot quality assurance sampling was carried out to assess iron, folate, hemoglobin and inflammation status of WRA; the coverage and knowledge of the fortified first grade UzDonMakhsulot (UDM) flour/grey loaf program; and consumption habits of women to investigate the dietary factors associated with anemia. Estimated anemia prevalence was 34.4% (95% CI: 32.0, 36.7), iron depletion 47.5% (95% CI: 45.1, 49.9) and folate deficiency 28.8% (95% CI: 26.8, 30.8); the effect of inflammation was minimal (4% with CRP >5 mg/L). Severe anemia was more prevalent among folate deficient than iron depleted WRA. Presence of UDM first grade flour or the grey loaf was reported in 71.3% of households. Among WRA, 32.1% were aware of UDM fortification; only 3.7% mentioned the benefits of fortification and 12.5% understood causes of anemia. Consumption of heme iron-containing food (91%) and iron absorption enhancers (97%) was high, as was the consumption of iron absorption inhibitors (95%).

Conclusions/Significance

The NFFP coincided with a substantial decline in the prevalence of anemia. Folate deficiency was a stronger predictor of severe anemia than iron depletion. However, the prevalence of iron depletion was high, suggesting that women are not eating enough iron or iron absorption is inhibited. Fortified products were prevalent throughout Uzbekistan, though UDM flour must be adequately fortified and monitored in the future. Knowledge of fortification and anemia was low, suggesting consumer education should be prioritized.     

A new red cell discriminant incorporating volume dispersion for differentiating iron deficiency anemia from thalassemia minor

A novel red cell discriminant function [MCV2 x RDW/(Hgb x 100)] was compared to six other discriminants in 102 patients with established mild iron deficiency anemia and 33 patients with beta-thalassemia minor. The discriminant incorporates the two key measurements of erythrocyte cell volume distribution, namely the mean (MCV) and standard deviation (RDW), which are known to be helpful for distinguishing between these two frequent causes of microcytic hypochromic anemia. Data used for the learning set to develop the new discriminant were obtained using an electrical impedance automated whole blood analyzer (Coulter S + IV) and were applied as a validation set for six other discriminants. The discriminant was also tested on smaller subsets of the patients groups using data obtained on either an alternate electrical impedance instrument (Sysmex E-5000) or a laser light scattering based system (Technicon H*1). From the comparison it was concluded that use of a discriminant function that incorporates a measurement of red cell volume dispersion results in enhanced accuracy for distinguishing iron deficiency anemia from thalassemia minor.     

Serum erythropoietin titers in the anemia of chronic renal failure and other hematological states

Erythropoietin (Epo) titers in various hematological states were determined by a radioimmunoassay. Epo titers in patients with uremic anemia and iron deficiency anemia were inversely correlated with their respective hemoglobin concentrations. Epo titers in patients with uremic anemia were significantly lower than those in patients with iron deficiency anemia with comparable hemoglobin concentrations.     

Copper Deficiency Leads to Anemia,Duodenal Hypoxia,Upregulation of HIF-2α and Altered Expression of Iron Absorption Genes in Mice

Iron and copper are essential trace metals, actively absorbed from the proximal gut in a regulated fashion. Depletion of either metal can lead to anemia. In the gut, copper deficiency can affect iron absorption through modulating the activity of hephaestin - a multi-copper oxidase required for optimal iron export from enterocytes. How systemic copper status regulates iron absorption is unknown. Mice were subjected to a nutritional copper deficiency-induced anemia regime from birth and injected with copper sulphate intraperitoneally to correct the anemia. Copper deficiency resulted in anemia, increased duodenal hypoxia and Hypoxia inducible factor 2α (HIF-2α) levels, a regulator of iron absorption. HIF-2α upregulation in copper deficiency appeared to be independent of duodenal iron or copper levels and correlated with the expression of iron transporters (Ferroportin - Fpn, Divalent Metal transporter – Dmt1) and ferric reductase – Dcytb. Alleviation of copper-dependent anemia with intraperitoneal copper injection resulted in down regulation of HIF-2α-regulated iron absorption genes in the gut. Our work identifies HIF-2α as an important regulator of iron transport machinery in copper deficiency.     

Artificial neural networks and their use in quantitative pathology

A brief general introduction to artificial neural networks is presented, examining in detail the structure and operation of a prototype net developed for the solution of a simple pattern recognition problem in quantitative pathology. The process by which a neural network learns through example and gradually embodies its knowledge as a distributed representation is discussed, using this example. The application of neurocomputer technology to problems in quantitative pathology is explored, using real-world and illustrative examples. Included are examples of the use of artificial neural networks for pattern recognition, database analysis and machine vision. In the context of these examples, characteristics of neural nets, such as their ability to tolerate ambiguous, noisy and spurious data and spontaneously generalize from known examples to handle unfamiliar cases, are examined. Finally, the strengths and deficiencies of a connectionist approach are compared to those of traditional symbolic expert system methodology. It is concluded that artificial neural networks, used in conjunction with other nonalgorithmic artificial intelligence techniques and traditional algorithmic processing, may provide useful software engineering tools for the development of systems in quantitative pathology.     

Treatment of nutritional anemia in infants

While iron deficiency is a common cause of anemia in infants, requiring specific treatment by administration of iron, there are other causes which require treatment equally specific. Anemia due to a nutritional deficiency can be improved only by providing the needed nutriment in sufficient quantity. Two case reports are presented. One is of megaloblastic anemia in an infant, which was treated with a variety of vitamin preparations before administration of folic acid produced improvement. The other report is of anemia due to iron deficiency treated successfully with ferrous sulfate in a dosage twenty times that previously used prophylactically. In prescribing an antianemic preparation the physician should assure himself that the dosage is adequate (for example, 0.5 gm. daily of ferrous sulfate for iron deficiency). "Broad-spectrum" preparations may be lacking in specific hematinics while containing a variety of vitamins which have no specific hematologic value.     

Real-time PCR demonstrates Ancylostoma duodenale is a key factor in the etiology of severe anemia and iron deficiency in Malawian pre-school children

Background

Hookworm infections are an important cause of (severe) anemia and iron deficiency in children in the tropics. Type of hookworm species (Ancylostoma duodenale or Necator americanus) and infection load are considered associated with disease burden, although these parameters are rarely assessed due to limitations of currently used diagnostic methods. Using multiplex real-time PCR, we evaluated hookworm species-specific prevalence, infection load and their contribution towards severe anemia and iron deficiency in pre-school children in Malawi.

Methodology and Findings

A. duodenale and N. americanus DNA loads were determined in 830 fecal samples of pre-school children participating in a case control study investigating severe anemia. Using multiplex real-time PCR, hookworm infections were found in 34.1% of the severely anemic cases and in 27.0% of the non-severely anemic controls (p<0.05) whereas a 5.6% hookworm prevalence was detected by microscopy. Prevalence of A. duodenale and N. americanus was 26.1% and 4.9% respectively. Moderate and high load A. duodenale infections were positively associated with severe anemia (adjusted odds ratio: 2.49 (95%CI 1.16–5.33) and 9.04 (95%CI 2.52–32.47) respectively). Iron deficiency (assessed through bone marrow examination) was positively associated with intensity of A. duodenale infection (adjusted odds ratio: 3.63 (95%CI 1.18–11.20); 16.98 (95%CI 3.88–74.35) and 44.91 (95%CI 5.23–385.77) for low, moderate and high load respectively).

Conclusions/Significance

This is the first report assessing the association of hookworm load and species differentiation with severe anemia and bone marrow iron deficiency. By revealing a much higher than expected prevalence of A. duodenale and its significant and load-dependent association with severe anemia and iron deficiency in pre-school children in Malawi, we demonstrated the need for quantitative and species-specific screening of hookworm infections. Multiplex real-time PCR is a powerful diagnostic tool for public health research to combat (severe) anemia and iron deficiency in children living in resource poor settings.     

Anemia, iron deficiency, and stress fractures in female combatants during 16 months

Yanovich, R, Merkel, D, Israeli, E, Evans, RK, Erlich, T, and Moran, DS. Anemia, iron deficiency, and stress fractures in female combatants during 16 months. J Strength Cond Res 25(12): 3412-3421, 2011-The purpose of this study is to evaluate the hematological profile of military recruits in different settings and training programs and to investigate the link between anemia and iron deficiency with stress fracture (SF) occurrence. We surveyed 3 groups of recruits for 16 months: 221 women (F) and 78 men (M) from 3 different platoons of a gender-integrated combat battalion and a control group (CF) of 121 female soldiers from a noncombat unit. Data were fully collected upon induction and at 4 and 16 months from 48F, 21M, and 31CF. Blood tests, anthropometry, physical aerobic fitness, and SF occurrence were evaluated. On induction day, 18.0 and 19.0% of F and CF were found to be anemic, and 61.4 and 50.9%, respectively, were found to have iron deficiency, whereas 7.7% of M were found to be anemic and 10.2% iron deficient. During the 4 months of army basic training (ABT), anemia and iron deficiency prevalence did not change significantly in any group. After 16-months, anemia prevalence decreased by 8% among F and CF and abated in M. Iron deficiency was prevalent in 50.0, 59.4, and 18.8% of F, CF, and M, respectively. Stress fractures were diagnosed in 14 F during ABT, and they had a significantly higher prevalence (p < 0.05) of anemia and iron deficiency anemia compared to F without SFs. The observed link between anemia and iron deficiency on recruitment day and SFs suggests the importance of screening female combat recruits for these deficiencies. To minimize the health impact of army service on female soldiers, preventative measures related to anemia and iron deficiency should be administered. Further research is needed for evaluating the influence of low iron in kosher meat as a possible explanation for the high prevalence of iron deficiency among young Israeli recruits.     

Macrophage iron, hepcidin, and atherosclerotic plaque stability

Hepcidin has emerged as the key hormone in the regulation of iron balance and recycling. Elevated levels increase iron in macrophages and inhibit gastrointestinal iron uptake. The physiology of hepcidin suggests an additional mechanism by which iron depletion could protect against atherosclerotic lesion progression. Without hepcidin, macrophages retain less iron. Very low hepcidin levels occur in iron deficiency anemia and also in homozygous hemochromatosis. There is defective retention of iron in macrophages in hemochromatosis and also evidently no increase in atherosclerosis in this disorder. In normal subjects with intact hepcidin responses, atherosclerotic plaque has been reported to have roughly an order of magnitude higher iron concentration than that in healthy arterial wall. Hepcidin may promote plaque destabilization by preventing iron mobilization from macrophages within atherosclerotic lesions; the absence of this mobilization may result in increased cellular iron loads, lipid peroxidation, and progression to foam cells. Marked downregulation of hepcidin (e.g., by induction of iron deficiency anemia) could accelerate iron loss from intralesional macrophages. It is proposed that the minimally proatherogenic level of hepcidin is near the low levels associated with iron deficiency anemia or homozygous hemochromatosis. Induced iron deficiency anemia intensely mobilizes macrophage iron throughout the body to support erythropoiesis. Macrophage iron in the interior of atherosclerotic plaques is not exempt from this process. Decreases in both intralesional iron and lesion size by systemic iron reduction have been shown in animal studies. It remains to be confirmed in humans that a period of systemic iron depletion can decrease lesion size and increase lesion stability as demonstrated in animal studies. The proposed effects of hepcidin and iron in plaque progression offer an explanation of the paradox of no increase in atherosclerosis in patients with hemochromatosis despite a key role of iron in atherogenesis in normal subjects.