Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2023;13(3):e2023202 1 Micronutrient Deficiencies and Digital Computerized Phototrichogram Analysis in Telogen Effluvium: a Retrospective Correlation Study in a Tertiary Medical Center Hakan Arslan1, Özgür Gündüz2 1 Department of Dermatology, University of Health Sciences Dr Sami Ulus Maternity and Children’s Hospital, Ankara, Turkey 2 Department of Dermatology, University of Kırıkkale, Kırıkkale, Turkey Key words: telogen effluvium, trichoscan, automated digital photothrichogram, vitamin deficiencies, mineral deficiencies Citation: Arslan H, Gündüz Ö. Micronutrient Deficiencies And Digital Computerized Phototrichogram Analysis in Telogen Effluvium: A Retrospective Correlation Study in a Tertiary Medical Center. Dermatol Pract Concept. 2023;13(3):e2023202. DOI: https://doi .org/10.5826/dpc.1303a202 Accepted: March 11, 2023; Published: July 2023 Copyright: ©2023 Arslan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. Funding: None. Competing Interests: None. Authorship: All authors have contributed significantly to this publication. Corresponding Author: Hakan Arslan, Department of Dermatology, University of Health Sciences Dr Sami Ulus Maternity and Children Training and Research Hospital, 06080, Ankara, Turkey. Orcid ID: 0000-0002-4880-7335 Telephone Number: +905436036105 E-mail: drarslanhakan@gmail.com Abbreviations: ADCP: Automated digital computerized phototrichogram, AHR: Anagen hair ratio, HGB: Hemoglobin, TE: Telogen effluvium, Vit-B12: Vitamin B12, Vit-D: Vitamin D Introduction: Telogen effluvium (TE) is a common form of non-scarring alopecia that may manifest as acute or chronic hair shedding. Several studies evaluated a possible relationship between various vitamin and mineral deficiencies and TE, but it is still a controversial topic. Objectives: This study aimed to investigate the status of vitamin and mineral deficiencies in patients diagnosed with TE and to evaluate their correlation with anagen hair ratios (AHR) calculated with an automated digital phototrichogram (ADCP). Methods: Electronic records of 973 TE patients were retrospectively analyzed. Demographic, clini- cal data, parameters such as ferritin, vitamin B12 (Vit-B12), vitamin D (Vit-D), folic acid, zinc and hemoglobin (HGB) serum levels were evaluated. Anagen to telogen hair ratios were also assessed in forty-two patients via ADCP. Results: The rates of anemia, low ferritin level, and Vit-B12, folate, Vit-D, and zinc deficiencies were 11.9% (N = 109), 44% (N = 332), 1.5% (N = 13), 2.5% (N = 14), 87% (N = 51), and 4.5% (N  = 2), respectively. A positive correlation was found between HGB levels and AHR in female patients ABSTRACT 2 Original Article | Dermatol Pract Concept. 2023;13(3):e2023202 Introduction The hair is an ectoderm-originated skin appendage with high cosmetic and psychological importance. It helps individuals maintain their self-esteem, which encourages healthy social interactions. Telogen effluvium (TE) is the most common cause of diffuse hair shedding [1]. The actual prevalence of TE is un- known because of its subclinical nature. Both sexes may be affected; however, women tend to seek more medical advice and be over-represented [2]. Objectives Although vitamin and mineral deficiencies have been associated with TE, this is still a controversial topic. This study aimed to investigate demographic features, define the detectable vitamin and mineral deficiencies in patients with acute and chronic TE, and associate their levels with the anagen hair ratio (AHR) on an automated digital computerized phototrichogram (ADCP). Methods The study was approved by the local ethics committee (de- cision no. 15/31, 01.10.2018). Laboratory results, electronic medical records, and ADCP findings of patients who were diagnosed with TE between March 2015 and March 2018 were retrospectively obtained from the medical electronic and dermoscopy unit databases of our hospital. The following demographic and clinical features were extracted from the electronic medical records: age, sex, application month, and biochemical test results, including hemoglobin (HGB), ferritin, vitamin B12 (Vit-B12), folate, vitamin D (Vit-D), zinc, and AHR. Raw data were ano- nymized. Patients were categorized into four groups accord- ing to age: <20, 20–29, 30–50, and >50 years. Normal values of variables were determined as follows: HGB, 14–18 g/dL for men and 12–16 g/dL for women; ferritin, >40 μg/mL; Vit-D, >25 ng/mL; Vit-B12, >200 pg/mL; folate, >4 ng/mL; zinc, >70 μg/dL; and AHR, 80%–100%. Hair examination data on ADCP were collected from our clinic dermoscopy unit. The procedure was as follows: on day 1, the plate with a 1 cm2 circular incision was placed on the highest point of the patient’s right pinna. Subsequently, the hair in the selected area was trimmed to a length of 0.5 mm using a hair trimmer Rowenta® TN9160 (Rowenta Werke GmbH, Erbach) and photographed with 20× magnification via FotoFinderdermoscope® (FotoFinder System GmbH). The patients were instructed not to bathe for three days and then to revisit our clinic. On the second visit, the trimmed area was stained with black dye (Wella®Koleston, Darmstadt). The dye was cleaned with 80% alcohol solution 15 minutes later, and another set of images was taken at 20× magnification again. The most suitable image was selected, and the results were evaluated using automatic computerized phototrichogram software (Tricholog GmbH), which is based on the principle of analyzing hair length on each hair strand, and then differ- entiating and categorizing growing hairs (≥ 0.35 mm/day) as anagen and non-growing hairs (<0.35 mm/day) as telogen. Statistical analysis was performed using IBM SPSS Sta- tistics for Windows, version 20.0 (IBM Corp). Descriptive statistics were presented as a number, percentage, mean, standard deviation, median, minimum, and maximum. The Kolmogorov–Smirnov test and graphics were used to inves- tigate whether the numerical data fit the normal distribu- tion. The independent t-test was performed for two groups, whereas the one-way analysis of variance (ANOVA) test was performed for more than two groups with normal distribu- tions. Pearson correlation coefficient was used to analyze the relationship between two numeric variables when the data set showed normal distribution, whereas Spearman correla- tion was used for non-normally distributed ones. A P value < 0.05 was considered statistically significant. Results A total of 973 patients diagnosed with TE who applied between March 2015 and March 2018 were included in this retrospec- tive study. Of these patients, 86.3% (N = 840) were female, and 13.7% (N = 133) were male, with a female/male ratio of 6.31. The mean age was 27.54 ± 9.42 (minimum, 13; maximum, 72) years. Of the patients, 18.1% (N = 176) were < 20 years old; 51.8% (N = 504), 21–29 years old; 27.1% (N  = 264), 30–50 years old; and 3% (N = 29), ≥ 51 years (Table 1). (Spearman rank, r = 0.417, P = 0.008). No statistically significant relationship was found between ferritin, Vit-B12, folate, zinc serum levels and AHR. The relationship between Vit-D and AHR could not be assessed due to the insufficient number of patients with Vit-D data. Conclusions: HGB value is the only marker that is positively correlated with the AHR of patients with TE. Ordering HGB can be used as an initial test for managing TE patients cost-effectively. Original Article | Dermatol Pract Concept. 2023;13(3):e2023202 3 Table 1. Sociodemographic characteristics of patients with telogen effluvium. N % Sex Male Female 133 840 13.7 86.3 Age ≤ 20 21–29 30–50 ≥ 51 176 504 264 29 18.1 51.8 27.1 3.0 0 20 40 60 80 100 120 140 160 180 200 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Dec Number of Patients Figure 1. Number of the patients according to the month of admission. Patients were also distributed according to the months of admission: January, 7.9% (N = 77); February, 7.1% (N = 69); March, 5.5% (N = 54); April, 3.9% (N = 38); May, 6.3% (N = 61); June, 4.3% (N = 42); July, 6.4% (N = 62); August, 8.2% (N = 80); September, 8.7% (N = 85); October, 12.7% (N = 124); November, 18.5% (N = 180); and Decem- ber, 10.4% (N = 101). November and April had the highest and lowest number of admissions, respectively (Figure 1). The following biochemical tests were evaluated: 94.1% (N = 916), 77.5% (N = 755), 86.4% (N = 841), 58.5% (N = 570), 5.9% (N = 58), and 4.5% (N = 44) of the patients had available HGB, ferritin, Vit-B12, folate, Vit-D, and zinc data, respectively. The rates of anemia, low ferritin level, and Vit-B12, folate, Vit-D, and zinc deficiencies were 11.9% (N = 109), 44% (N = 332), 1.5% (N = 13), 2.5% (N = 14), 87% (N = 51), and 4.5% (N = 2), respectively. AHR obtained in the dermoscopy unit was available in 4.3% (N = 42) of the patients. The mean AHR value of the female patients was 68.57 ± 8.88%, and that of male pa- tients was 74.73% ± 5.36%. No significant difference was found between sex and AHR (independent t-test, P = 0.247). Additionally, no significant difference was found between age groups (≤ 20, 21–30, and 31–50) and AHR (ANOVA, P = 0.197). The age group > 50 years was not included in the analysis because there were not enough subjects. The correlation analysis between biochemical tests and AHR was conducted only for female patients (N = 39) be- cause there were not enough male patients for the correlation analysis (N = 3). For female patients, no significant relation- ship was found between ferritin, Vit-B12, and folate levels and AHR (Spearman rank, r = −0.112, P = 0.523; r = 0.149, P = 0.386; r = 0.186 and p = 0.346, respectively). No signif- icant relationship was found between zinc levels and AHR (r = −0.423, P = 0.577). The relationship between Vit-D and AHR was not assessed because there were insufficient pa- tients with Vit-D data. A positive correlation was available between the HGB levels and AHR of female patients (r = 0.417, P = 0.008, Table 2 and Figure 2). Conclusions This study focused on the deficiency rates of HGB, ferritin, Vit-B12, folic acid, Vit-D, and zinc levels in patients diag- nosed with TE and correlation between the micronutrient levels and AHR. Hair follicle cells have a high-turnover rate. Thus, they need organic and inorganic molecules, minerals, and oxygen for their metabolic activities. Congenital or acquired defi- ciencies of these metabolites due to low intake, insufficient absorption, or pathological losses may result in abnormalities 4 Original Article | Dermatol Pract Concept. 2023;13(3):e2023202 laboratories use different ferritin levels, ranging from 20 to 70 μg/mL; however, a cutoff below 41 μg/mL yields sensitiv- ity and specificity of 98% [7]. Deloche et alinvestigated the relationship between iron deficiency and hair loss in more than 5000 women aged 35–60 [8]. They were divided into three groups according to their hair loss status, namely, ab- sence of hair loss, moderate, and excessive. Moreover, 59% of the patients with ferritin levels < 40 µg/L were placed in the excessive hair loss group, whereas this rate was 48% in other participants. Their regression analysis revealed that the probability of excessive hair loss increased by 28% with a 30 µg/L decrease in ferritin levels in women; in other words, the severity of hair loss increases as the ferritin level decreases. Moeinvaziri et al reported that ferritin levels were signifi- cantly lower in their 30 patients with diffuse telogen hair loss than in their 30 healthy volunteers without hair loss [8]. However, some researchers did not find a direct relationship between iron level and hair loss [9,10]. In their study, Sin- clair investigated the relationship between iron deficiency and chronic telogen hair loss, and five of their patients were treated with iron to increase serum ferritin levels >20 µg/L; however, hair loss did not improve during follow-up [10]. Another study investigated serum ferritin levels and telogen hair ratio in 181 female patients, who were divided into three groups according to their ferritin levels: ≤10 µg/L, 10–30 µg/L, and >30 µg/L. The telogen hair ratios of these groups were compared, and no significant statistical correlation was found [11]. In the present study, 755 patients had ferritin data, and 44% of these patients had low ferritin levels. How- ever, no significant correlation was found between AHR and ferritin levels in patients with TE (r = −0.112, P = 0.523). Vit-B12 and folate have very important functions on both hematologic and nervous systems. Vit-B12 is a cofactor for methylmalonyl-CoA mutase and homocysteine methyl- transferase, a folate-dependent pathway [12]. The critical importance of homocysteine methyltransferase is that the reaction of the conversion of 5-methyltetrahydrofolate to tetrahydrofolate takes place at this stage, and it catalyzes a reaction associated with the synthesis of purine and pyrimi- dine bases and thus DNA synthesis [12,13]. This makes both in the structure of the hair, pigmentation changes, and hair loss [3,4]. In this study, 86.3% of the patients with TE who applied to our dermatology department were female. These patients were mostly between 20 and 30 years old (51.8%), followed by patients aged 30–50 (27.1%). The most frequent applica- tion month was November with 18.5% (N = 180), followed by 12.7% (N = 124) in October. The incidence of TE changes with seasonal transitions [5]. However, our hospital is lo- cated within the university campus, and the population of the district where it is located doubles as other departments of the university start education in October–November. The true incidence and seasonal fluctuations in TE are not well established due to the lack of data regarding particu- larly subclinical cases. Furthermore, male patients are sig- nificantly less likely to consult a physician than their female counterparts. Therefore, large-scale field studies are needed to determine the true incidence of TE. Iron deficiency is the world’s most common nutritional deficiency and is considered one of the most prevalent causes of hair loss [6]. To define iron deficiency, different Table 2. Distribution of the anagen hair ratios, zinc, and vitamin levels of the patients. N Mean Median SD Min Max AHR, % 42 69.01 70 8.78 50.7 84.2 Ferritin, μg/mL 755 33.56 22 32.75 2 293 Vit-B12, pg/mL 841 325.88 299.60 149.72 61.4 2000 Folate, ng/mL 570 8.21 7.75 3.45 1.73 20 Vit-D, ng/mL 45 15.58 13.97 8.68 3.37 45.10 Zinc, μg/dL 44 92.35 91.40 16.47 64 131.1 AHR = anagen hair ratios; SD = standard deviation. A na ge n H ai r R at io , % Hemoglobin, g/dl 50,0 10,0 11,0 12,0 13,0 14,0 15,0 16,0 60,0 70,0 80,0 90,0 Figure 2. Positive correlation between the hemoglobin level and an- agen hair percentage of female patients. Original Article | Dermatol Pract Concept. 2023;13(3):e2023202 5 In our study, large-scale TE patients’ biochemical tests were evaluated in a tertiary care center. Also, correlation anal- ysis was performed to detect essential ones which was only HGB level. In order to manage TE patients cost-effectively, a complete blood count can be used as an initial test. References 1. İbiş S, Aksoy Saraç G, Akdağ T. Evaluation of MCV/RDW Ratio and Correlations With Ferritin in Telogen Effluvium Patients. Dermatol Pract Concept. 2022;12(3):e2022151. DOI: 10.5826/dpc.1203a151. PMID: 36159144. PMCID: PMC9464528. 2. Mysore V, Parthasaradhi A, Kharkar RD, et al. Expert consensus on the management of Telogen Effluvium in India. Int J Trichol- ogy. 2019;11(3):107-112. DOI: 10.4103/ijt.ijt_23_19. PMID: 31360038. PMCID: PMC6580807. 3. Cheung EJ, Sink JR, English Iii JC. Vitamin and Mineral De- ficiencies in Patients With Telogen Effluvium: A Retrospec- tive Cross-Sectional Study. J Drugs Dermatol. 2016;15(10): 1235-1237. PMID: 27741341. 4. Yorulmaz A, Hayran Y, Ozdemir AK, et al. Telogen effluvium in daily practice: Patient characteristics, laboratory parameters, and treatment modalities of 3028 patients with telogen effluvium. J Cosmet Dermatol. 2022;21(6):2610-2617. DOI: 10.1111 /jocd.14413. Epub 2021 Aug 27. PMID: 34449961. 5. Liyanage D, Sinclair R. Telogen Effluvium. Cosmetics. 2016;3(2):13. DOI:10.3390/cosmetics3020013. 6. Trost LB, Bergfeld WF, Calogeras E. The diagnosis and treat- ment of iron deficiency and its potential relationship to hair loss. J Am Acad Dermatol. 2006;54(5):824-844. DOI: 10.1016/j .jaad.2005.11.1104. PMID: 16635664. 7. Shrivastava SB. Diffuse hair loss in an adult female: approach to diagnosis and management. Indian J Dermatol Venereol Lep- rol. 2009;75(1):20-72; DOI: 10.4103/0378-6323.45215. PMID: 19172026. 8. Deloche C, Bastien P, Chadoutaud S, et al. Low iron stores: a risk factor for excessive hair loss in non-menopausal women. Eur J Dermatol. 2007;17(6):507-512. DOI: 10.1684/ejd.2007.0265. PMID: 17951130. 9. Olsen EA, Reed KB, Cacchio PB, Caudill L. Iron deficiency in female pattern hair loss, chronic telogen effluvium, and con- trol groups. J Am Acad Dermatol. 2010;63(6):991-999. DOI: 10.1016/j.jaad.2009.12.006. PMID: 20947203. 10. Sinclair R. There is no clear association between low se- rum ferritin and chronic diffuse telogen hair loss. Br J Der- matol. 2002;147(5):982-984. DOI: 10.1046/j.1365-2133 .2002.04997.x. PMID: 12410711. Vit-B12 and folate important for the high-turnover rates in hair follicle cells. However, no sufficient evidence confirms the association between Vit-B12 and folate deficiencies and hair loss. In this study, 841 patients had Vit-B12 data, of which 1.5% had Vit-B12 deficiency, whereas 2.5% of 570 patients with folate data had folate deficiencies. No signifi- cant correlation was found between AHR and levels of these vitamins (r = 0.149, P = 0.386). Vit-D modulates the growth and differentiation of hair follicle keratinocytes via nuclear Vit-D receptor (VDR) with the highest activity in the anagen stage [4]. VDR mutations cause Vit-D-dependent rickets type 2, which equates to full body hair loss and present evidence for the relationship between hair loss and Vit-D deficiency [15,16]. In this study, 87.9% of 58 patients with Vit-D data had low Vit-D levels. The correlation analysis be- tween Vit-D levels and AHR could not be performed be- cause there were not enough subjects with Vit-D results and ADCP analysis. Zinc is an essential trace element involved in protein and nucleic acid synthesis and various metabolic pathways. Transient zinc deficiency in acrodermatitis enteropathica, resulting in hair loss, demonstrated effects on hair. Kil et al reported that zinc levels were significantly lower in patients with alopecia areata, TE, and androgenetic alopecia (three main hair loss diseases) compared with healthy volunteers [17]. In our study, 4.5% of the 44 patients with serum zinc data had zinc deficiency. Correlation analysis with AHR was not significant (r = −0.423, P = 0.577). In our study, 916 patients had HGB data, of which 12.3% of the female and 9.1% of the male patients were anemic. In addition, a significant positive correlation was found between HGB level and AHR in female patients (r = 0.417 and p = 0.008). This suggests that deficiencies in mi- cronutrients such as Vit-B12, folate, and iron can indirectly alter the severity of TE if they decrease HGB level. The main limitation of our study was its retrospective nature and that some analyses could not be performed due to missing data, such as Vit-D level and AHR correlation and male patient’s correlation analysis. Also, our city is a college town. Its population increases in the winter months. This may affect the monthly admission rates and cause an increase in patient numbers in certain months. Table 3. Correlation study between biochemical tests and anagen hair ratio. Ferritin Vit-B12 Folate Zinc Vit-D HGB Anagen hair ratio ra P 0.149 0.386 b 0.149 0.386 b 0.186 0.346 b −0.423 0.577 c 0.149 0.386 b 0.417 0.008 b HGB = hemoglobin. aCorrelation coefficient bSpearman rank correlation coefficient test cPearson correlation coefficient test 6 Original Article | Dermatol Pract Concept. 2023;13(3):e2023202 15. Vupperla D, Lunge SB, Elaprolu P. Vitamin D-Dependent Rickets Type II with Alopecia: A Rare Case Report. Indian J Derma- tol. 2018;63(2):176-179. DOI: 10.4103/ijd.IJD_434_17. PMID: 29692463. PMCID: PMC5903051. 16. Santos R, Neves S, Gomes C, Neves F, Correia AJ. Raquitismo vitamina D dependente tipo II [Rickets vitamin-D-dependent type 2]. Acta Med Port. 2009;22(6):861-866. PMID: 20350471. 17. Kil MS, Kim CW, Kim SS. Analysis of serum zinc and copper concentrations in hair loss. Ann Dermatol. 2013;25(4):405-409. DOI: 10.5021/ad.2013.25.4.405. PMID: 24371385. PMCID: PMC3870206. 11. Bregy A, Trueb RM. No association between serum ferritin lev- els >10 microg/l and hair loss activity in women. Dermatology. 2008;217(1):1-6. DOI: 10.1159/000118505. PMID: 18309237. 12. Brescoll J, Daveluy S. A review of vitamin B12 in dermatology. Am J Clin Dermatol. 2015;16(1):27-33. DOI: 10.1007/s40257- 014-0107-3. PMID: 25559140. 13. Stabler SP. Clinical practice. Vitamin B12 deficiency. N Engl J Med. 2013;368(2):149-160. DOI: 10.1056/NEJMcp1113996. PMID: 23301732. 14. Almohanna HM, Ahmed AA, Tsatalis JP, Tosti A. The Role of Vitamins and Minerals in Hair Loss: A Review. Dermatol Ther (Heidelb). 2019;9(1):51-70. DOI: 10.1007/s13555-018-0278-6. PMID: 30547302. PMCID: PMC6380979.