A Rare Alpha Globin Chain Variant Hemoglobin Fontainebleau Poses Diagnostic Challenges by Yielding Different Results in Two Different HPLC Systems

A Rare Alpha Globin Chain Variant Hemoglobin Fontainebleau Poses Diagnostic Challenges by Yielding Different Results in Two Different HPLC Systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report A Rare Alpha Globin Chain Variant Hemoglobin Fontainebleau Poses Diagnostic Challenges by Yielding Different Results in Two Different HPLC Systems Amar Dasgupta, Sachin Patil, Manoj Sawadkar, Akshay Dhotre, Manish Karekar, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6628570/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Alpha globin chain variants constitute a very small fraction of the total hemoglobinopathy cases. Their rarity and the often-unfamiliar elution times/patterns in high performance liquid chromatography (HPLC) that mimic those of normal hemoglobin (Hb) fractions and/or known beta globin variants, render their primary diagnosis difficult. We describe here Hb Fontainebleau, a rare alpha globin chain variant, in an adult female that eluted in the HbA2 window in a less commonly used HPLC platform, Lifotronic H8, thereby causing difficulties in the diagnosis. The matter was resolved by running the sample on a more commonly used platform wherein the abnormal Hb yielded a familiar elution time and pattern known to be associated with Hb Fontainebleau. This was further confirmed by gene sequencing which showed a Codon 21 (G◊C); HBA2:c.64G > C (or HBA1) mutation. A literature search failed to reveal any published case of the unique elution pattern of Hb Fontainebleau on Lifotronic H8 HPLC platform and the associated diagnostic challenges. An increasing use of the newer HPLC platforms calls for greater awareness of these issues. Hb Fontainebleau Alpha globin chain variant High performance liquid chromatography Lifotronic H8 Tosoh G8 Figures Figure 1 Figure 2 Figure 3 Introduction Alpha globin chain variants are significantly less common than beta chain variants [ 1 ]. Most of the alpha chain variants are also clinically silent. Hence, their detection is incidental - usually during investigation of anemia due to other causes, during antenatal screening or when seen in combination with pathogenic beta chain variants [ 2 , 3 ]. Diagnosis of these cases poses challenges when the alpha chain variant is present alone or in combination with beta chain variants [ 2 , 3 ]. Hemoglobin (Hb) Fontainebleau is a rare alpha globin chain variant that typifies this issue as reported earlier [ 4 , 5 ]. The primary screening of blood samples for hemoglobinopathies is commonly performed using high performance liquid chromatography (HPLC). Well validated and popular platforms for this purpose are only a few and experience with the less commonly used systems is limited [ 6 ]. The elution times and patterns of abnormal Hb peaks are sometimes different in the latter systems [ 7 ], thereby posing problems in diagnosis. Here we describe a case of Hb Fontainebleau in an adult female that showed a different and unfamiliar elution pattern in a less commonly used HPLC platform, leading to confusion in the primary diagnosis of the variant Hb. Clinical history DS, a 29 yrs old primigravida, had a spontaneous abortion after 4 months’ pregnancy. Her anemia predated her pregnancy and progressed further during the pregnancy. Therefore, she was investigated for the same. There was no history of jaundice or organomegaly or of blood transfusion. The parents of the patient and the siblings could not be investigated due to their nonavailability for testing. Materials and methods A complete blood count (CBC) was performed in K2 EDTA anticoagulated blood sample on Yumizen H550 automated hematology analyzer (Horiba Medicals, Japan). Reticulocytes were enumerated manually after staining with new methylene blue. Hemoglobin analysis was carried out on two different HPLC systems – first on Lifotronic H8 (Shenzhen Lifotronic Technology Co. Ltd, China.) and later on Tosoh G8 (Tosoh Corporation, Japan). DNA was extracted from the leucocytes using the Qiagen FlexiGene DNA extraction kit (QIAGEN, Germany) following the manufacturer’s instructions. Mutational analysis of the alpha globin gene was performed by DNA sequencing using ABI 3730xl Gene Analyzer (Thermo Fisher Scientific, USA). Results The patient’s Hb was 9.1 g/dl, hematocrit 27.5%, red cell (RBC) count 4.01 million/cumm, mean corpuscular volume (MCV) 68.4 fl, mean corpuscular Hb concentration (MCHC) 33.2 g/dl, mean corpuscular Hb (MCH) 22.7 pg and RBC distribution width (RDW) (CV) 18.6%, a picture suggesting iron deficiency anemia. However, iron studies were not performed. Hb analysis on Lifotronic H8 HPLC system (Fig. 1 a; Table 1 ) showed the following results: HbA 73.5% and HbF 0.1% with an abnormal Hb peak (11.4%) in HbA2 window having a retention time (RT) of 193.3 seconds, similar to the RT of HbA2. HbA2 level was not available due to masking the HbA2 peak by the unknown (abnormal) Hb peak. Since HbE peak elutes separately and at a different RT from HbA2 peak in Lifotronic H8 HPLC system, the possibility of the unknown Hb peak representing HbE was not considered. The RT in Lifotronic HPLC system is measured in seconds unlike the other commonly used systems. In view of this fact and that the abnormal Hb peak on the former platform did not conform to any commonly encountered abnormal Hb known to elute in HbA2 window, the same sample was also analysed on Tosoh HPLC system. Interestingly, in Tosoh HPLC system the abnormal Hb eluted as a hump (12%) at the base of the descending limb of HbA peak with an RT of 2.78 mins (Fig. 1 b), a pattern seen with Hb Fontainebleau as reported earlier [ 2 , 3 , 5 ]. Table 1 depicts the HPLC findings of this patient on Tosoh system alongside those on Lifotronic system. Sequencing of alpha globin gene showed G->C substitution in codon 21 (HBA2:c.64G > C (or HBA1) confirming that the variant was indeed Hb Fontainebleau (Fig. 3 ). Table 1 Red cell parameters and HPLC findings in the patient. Parameter Result (unit) Reference interval Remarks RBC parameters Hemoglobin 9.1 g/dl 12–15 Hematocrit 27.5% 36–46 RBC Count 4.01 X 10 12 /l 3.8–4.8 MCV 68.4 fl 83–101 MCH 22.7 pg 27–32 MCHC 33.2 g/dl 31.5–34.5 RDW 18.6% 11.6–14 HPLC findings Lifotronic Tosoh HbA 73.5% 71.8% HbA2 11.4%* 1.8–3.5 0.9% HbF 0.1% < 0.8 0.8% HbX (unknown) * Abnormal Hb peak eluted in HbA2 window - 12% Discussion Hb Fontainebleau is a rare alpha globin chain variant that elutes as a hump at the base of the descending limb of HbA peak in commonly used HPLC platforms such as Bio Rad Variant II [ 2 , 3 , 5 ], Tosoh G8 etc. It results from Codon 21 (G◊C); HBA2:c.64G > C mutation [ 4 , 5 ]. Although it is clinically silent, it can pose diagnostic dilemma in the interpretation of the chromatograms especially when present in combination with other globin chain variants [ 3 ]. As highlighted in this communication, a new dimension to these diagnostic challenges is added by the difficulty in interpretation of the chromatographic pattern of Hb Fontainebleau seen in less commonly used HPLC systems that follow a different principle for separation of normal and abnormal Hb fractions [ 6 ]. The Hb Fontainebleau peak in Lifotronic H8 HPLC system at the first level of Hb analysis in this case posed a diagnostic challenge since (a) it eluted in HbA2 window and (2) the level of the variant Hb peak was abnormally high (11.4%) for it to be HbA2 (Fig. 1 ). As HbE and HbA2 elute separately on Lifotronic H8, and HbE peak elutes at a later time point compared to HbA2 (~ 230 seconds for HbE vs. ~200 seconds for HbA2), the possibility of the abnormal Hb peak representing HbE in this case was ruled out. At the same time, a peak of 11.4% in HbA2 window was unlikely to represent HbA2 on account of its high level. Hence, the possibility of a non-HbA2, non-HbE variant Hb, possibly an alpha chain variant, was considered at this point and the sample was analysed on a more commonly used platform - Tosoh G8 HPLC system. As shown in Fig. 2 , the elution pattern of the abnormal Hb was different on this platform but conformed to the known pattern of Hb Fontainebleau described above [ 3 , 5 ]. That the abnormal Hb was indeed Hb Fontainebleau was confirmed subsequently by sequencing studies [Figure 3 ]. This case highlights the need for a good knowledge of the elution times and patterns of less common Hb variants in different HPLC systems, an awareness of the variables introduced by different HPLC platforms in these parameters and an access to more than one screening platforms working on different principles, if required. Declarations Compliance and ethical standards Funding: This study was not supported by any funding. Conflict of Interest: The authors declare that they have no conflict of interest. Ethical approval:All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent: Informed consent was obtained from all individual participants included in the study. Consent for publication: Consent for publication was obtained for every individual person’s data included in the study. Author contribution All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by AD, PT and SP. The first draft of the manuscript was written by AD and all authors commented on previous versions of the manuscript. All authors read and approved the finalmanuscript. Ethics approval: The study was approved by the appropriate institutional research ethics committee and the committee certifies that the study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards . Consent to participate: Informed consent was obtained from the patient for participation in the study. Consent for publication: Consent was obtained from the patient for publication of test results. Conflict of interest: The authors declare no competing interests. References Patrinos GP, Giardine B, Riemer C, Miller W, Chui DHK, Anagnou NP, Wajcman H, Hardison RC (2004) Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies. Nucleic Acids Res 132(Database issue):D537–D541. 10.1093/nar/gkh006 Nair S, Nadkarni A, Warang P, Bhave A, Ghosh K, Colah R (2010) Five alpha globin chain variants identified during screening for haemoglobinopathies. Eur J Clin Invest 40:226–232 Upadhye DS, Jain D, Nair SB, Nadkarni AH, Ghosh K, Colah RB et al (2012) First case of Hb Fontainebleau with sickle haemoglobin and other nondeletional alphagene variants identified in neonates during newborn screening for sickle cell disorders. J Clin Pathol 65:654–659 Wajcman H, Blouquit Y, GombaudSaintonge G, Riou J, Galacteros F (1989) Hb Fontainebleau [alpha21(B2)Ala→Pro], a new silent mutant hemoglobin. Hemoglobin 13:421–428 Sidhwa K, Daruwalla MR, Pawar R, Nadkarni A, Hariharan P, Mehta P, Das Gupta A (2019) Diagnostic challenges posed by a rare alpha globin chain variant Hb Fontainebleau in a pregnant female and its potential effects in her children in view of multiple globin gene defects in her husband. IndIan J Pathol MIcrobIol 62:323–325 Karadağ ME, Akbulut ED, Avcı E, Oğuz EF, Kader S, Abuşoğlu G, Serdar M, Yılmaz FM (2020) Evaluation of four different HPLC devices for hemoglobinopathy screening. Turk J Biochem. 10.1515/tjb-2019-0484 Li R, Huamei H, Kan L, Xiong D, Cheng X, Xu Y, Liu X, Zhang X (2020) Evaluation on the separated effect of 13 hemoglobin variants by a new automatic Hba1c analyzer. J Clin Lab Anal. 2020;34:e23446 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 Jun, 2025 Reviews received at journal 24 Jun, 2025 Reviewers agreed at journal 11 Jun, 2025 Reviewers invited by journal 16 May, 2025 Editor assigned by journal 12 May, 2025 Submission checks completed at journal 12 May, 2025 First submitted to journal 09 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6628570","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":457679201,"identity":"a5d1d3ce-00cd-4a82-9023-fed714a5506a","order_by":0,"name":"Amar Dasgupta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYNACNhDBfICBsQEqkECUFja2BJK18BggtOAD/NMOP/vAUFZnt12+55vEzx02cgzshw8wPNyBW4vE7TTjGQznDifvbOPdJtl7Js2YgSctgSHxDB5rbicYMzC2HUg2OMa7TYK37XBigwTQhYltuHXI307/DNRSB9TC80zyLzFaDG7ngGxhtgNqYZMmyhbD2znFDEC/JFi2pRlbywIJNqBfDuDTInc7fTMDMMTszZkPP7z5ts1Gjp/98MGHP/FoAQHmPwwMiRsYGFgkQDxQHB3ArwEC7A2AWj8Qo3IUjIJRMApGHgAA2vhMvLgcpvsAAAAASUVORK5CYII=","orcid":"","institution":"Lupin Diagnostics","correspondingAuthor":true,"prefix":"","firstName":"Amar","middleName":"","lastName":"Dasgupta","suffix":""},{"id":457679203,"identity":"15ac3688-c0ce-4d75-852a-64545a2253ff","order_by":1,"name":"Sachin Patil","email":"","orcid":"","institution":"Lupin Diagnostics","correspondingAuthor":false,"prefix":"","firstName":"Sachin","middleName":"","lastName":"Patil","suffix":""},{"id":457679212,"identity":"eeb8b4c7-5aa6-4d16-891c-6b77ccce13ee","order_by":2,"name":"Manoj Sawadkar","email":"","orcid":"","institution":"Lupin Diagnostics","correspondingAuthor":false,"prefix":"","firstName":"Manoj","middleName":"","lastName":"Sawadkar","suffix":""},{"id":457679213,"identity":"afbd0aa5-6f74-40bc-afcb-a66f483075a8","order_by":3,"name":"Akshay Dhotre","email":"","orcid":"","institution":"Lupin Diagnostics","correspondingAuthor":false,"prefix":"","firstName":"Akshay","middleName":"","lastName":"Dhotre","suffix":""},{"id":457679214,"identity":"49bbb700-1f0b-4c31-8e01-10265a42de72","order_by":4,"name":"Manish Karekar","email":"","orcid":"","institution":"Lupin Diagnostics","correspondingAuthor":false,"prefix":"","firstName":"Manish","middleName":"","lastName":"Karekar","suffix":""},{"id":457679216,"identity":"ba70e36b-a11c-4ce9-a1f6-e89a9dda07f6","order_by":5,"name":"Aniruddha Belsare","email":"","orcid":"","institution":"Lupin Diagnostics","correspondingAuthor":false,"prefix":"","firstName":"Aniruddha","middleName":"","lastName":"Belsare","suffix":""},{"id":457679217,"identity":"a2377230-2f9f-4c81-97ed-9b804157be1e","order_by":6,"name":"Shubhangi Mohanty","email":"","orcid":"","institution":"Lupin Diagnostics","correspondingAuthor":false,"prefix":"","firstName":"Shubhangi","middleName":"","lastName":"Mohanty","suffix":""},{"id":457679221,"identity":"79d65047-0593-4c86-8c42-2c03a83c6ba5","order_by":7,"name":"Pallavi Thakar","email":"","orcid":"","institution":"National Institute of Immunohaematology","correspondingAuthor":false,"prefix":"","firstName":"Pallavi","middleName":"","lastName":"Thakar","suffix":""},{"id":457679222,"identity":"656dfb79-57e8-4d45-9ba3-7ed5f563493d","order_by":8,"name":"Prabhakar S. Kedar","email":"","orcid":"","institution":"National Institute of Immunohaematology","correspondingAuthor":false,"prefix":"","firstName":"Prabhakar","middleName":"S.","lastName":"Kedar","suffix":""}],"badges":[],"createdAt":"2025-05-09 12:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6628570/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6628570/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83200015,"identity":"04c02175-5779-4dd5-a3d8-5b3b736628e1","added_by":"auto","created_at":"2025-05-21 06:09:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66571,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram on Lifotronic H8 HPLC platform shows an abnormal Hb peak (11.4%) in HbA2 window (arrow). The suspicion of an alpha globin chain variant was raised because the level of the peak was too high for HbA2 and HbE elutes at a later time point than HbA2 in this platform.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6628570/v1/5d584396c43f7af886123ae2.jpg"},{"id":83198361,"identity":"0eddbfda-41f9-4d81-bce7-228d34b75ea0","added_by":"auto","created_at":"2025-05-21 06:01:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25372,"visible":true,"origin":"","legend":"\u003cp\u003eThe same sample run on Tosoh HPLC system showed a different retention time and eluted as a hump in the descending arm of the HbA peak, (arrow), a picture different from the Lifotronic chromatogram (Figure 1). This pattern is typically observed with Hb Fontainebleau in other HPLC systems ( ).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6628570/v1/21a904c9c6a6ce588ad6d272.jpg"},{"id":83198363,"identity":"937090d0-c3a6-43c3-a7b0-9689c99f977b","added_by":"auto","created_at":"2025-05-21 06:01:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":36029,"visible":true,"origin":"","legend":"\u003cp\u003eSanger sequencing of the sample showing an α globin gene mutation: Codon 21 (G\u003cstrong\u003e→\u003c/strong\u003eC); HBA2:c.64G\u0026gt;C (or HBA1) \u003cem\u003e(arrow)\u003c/em\u003e characteristic of Hb Fontainebleau.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6628570/v1/769052498b8f6cd9dc66d61f.jpg"},{"id":83200735,"identity":"edd2f492-9c31-4563-8ae6-d69b041f933b","added_by":"auto","created_at":"2025-05-21 06:17:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":550551,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6628570/v1/8e4cf68a-0913-459a-a6e3-74272b229d84.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Rare Alpha Globin Chain Variant Hemoglobin Fontainebleau Poses Diagnostic Challenges by Yielding Different Results in Two Different HPLC Systems","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlpha globin chain variants are significantly less common than beta chain variants [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Most of the alpha chain variants are also clinically silent. Hence, their detection is incidental - usually during investigation of anemia due to other causes, during antenatal screening or when seen in combination with pathogenic beta chain variants [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Diagnosis of these cases poses challenges when the alpha chain variant is present alone or in combination with beta chain variants [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Hemoglobin (Hb) Fontainebleau is a rare alpha globin chain variant that typifies this issue as reported earlier [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe primary screening of blood samples for hemoglobinopathies is commonly performed using high performance liquid chromatography (HPLC). Well validated and popular platforms for this purpose are only a few and experience with the less commonly used systems is limited [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The elution times and patterns of abnormal Hb peaks are sometimes different in the latter systems [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], thereby posing problems in diagnosis. Here we describe a case of Hb Fontainebleau in an adult female that showed a different and unfamiliar elution pattern in a less commonly used HPLC platform, leading to confusion in the primary diagnosis of the variant Hb.\u003c/p\u003e\n\u003ch3\u003eClinical history\u003c/h3\u003e\n\u003cp\u003eDS, a 29 yrs old primigravida, had a spontaneous abortion after 4 months\u0026rsquo; pregnancy. Her anemia predated her pregnancy and progressed further during the pregnancy. Therefore, she was investigated for the same. There was no history of jaundice or organomegaly or of blood transfusion.\u003c/p\u003e \u003cp\u003eThe parents of the patient and the siblings could not be investigated due to their nonavailability for testing.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eA complete blood count (CBC) was performed in K2 EDTA anticoagulated blood sample on Yumizen H550 automated hematology analyzer (Horiba Medicals, Japan). Reticulocytes were enumerated manually after staining with new methylene blue. Hemoglobin analysis was carried out on two different HPLC systems \u0026ndash; first on Lifotronic H8 (Shenzhen Lifotronic Technology Co. Ltd, China.) and later on Tosoh G8 (Tosoh Corporation, Japan). DNA was extracted from the leucocytes using the Qiagen FlexiGene DNA extraction kit (QIAGEN, Germany) following the manufacturer\u0026rsquo;s instructions. Mutational analysis of the alpha globin gene was performed by DNA sequencing using ABI 3730xl Gene Analyzer (Thermo Fisher Scientific, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe patient\u0026rsquo;s Hb was 9.1 g/dl, hematocrit 27.5%, red cell (RBC) count 4.01\u0026nbsp;million/cumm, mean corpuscular volume (MCV) 68.4 fl, mean corpuscular Hb concentration (MCHC) 33.2 g/dl, mean corpuscular Hb (MCH) 22.7 pg and RBC distribution width (RDW) (CV) 18.6%, a picture suggesting iron deficiency anemia. However, iron studies were not performed.\u003c/p\u003e \u003cp\u003eHb analysis on Lifotronic H8 HPLC system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea; Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed the following results: HbA 73.5% and HbF 0.1% with an abnormal Hb peak (11.4%) in HbA2 window having a retention time (RT) of 193.3 seconds, similar to the RT of HbA2. HbA2 level was not available due to masking the HbA2 peak by the unknown (abnormal) Hb peak. Since HbE peak elutes separately and at a different RT from HbA2 peak in Lifotronic H8 HPLC system, the possibility of the unknown Hb peak representing HbE was not considered. The RT in Lifotronic HPLC system is measured in seconds unlike the other commonly used systems. In view of this fact and that the abnormal Hb peak on the former platform did not conform to any commonly encountered abnormal Hb known to elute in HbA2 window, the same sample was also analysed on Tosoh HPLC system. Interestingly, in Tosoh HPLC system the abnormal Hb eluted as a hump (12%) at the base of the descending limb of HbA peak with an RT of 2.78 mins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), a pattern seen with Hb Fontainebleau as reported earlier [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e depicts the HPLC findings of this patient on Tosoh system alongside those on Lifotronic system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSequencing of alpha globin gene showed G-\u0026gt;C substitution in codon 21 (HBA2:c.64G\u0026thinsp;\u0026gt;\u0026thinsp;C (or HBA1) confirming that the variant was indeed Hb Fontainebleau (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRed cell parameters and HPLC findings in the patient.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResult (unit)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference interval\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRemarks\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRBC parameters\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemoglobin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.1 g/dl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12\u0026ndash;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHematocrit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36\u0026ndash;46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRBC Count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.01 X 10\u003csup\u003e12\u003c/sup\u003e/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.8\u0026ndash;4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.4 fl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e83\u0026ndash;101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.7 pg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u0026ndash;32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCHC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.2 g/dl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.5\u0026ndash;34.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRDW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.6\u0026ndash;14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHPLC findings\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLifotronic\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTosoh\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHbA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.8%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHbA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e11.4%*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.8\u0026ndash;3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHbF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHbX (unknown)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e* Abnormal Hb peak eluted in HbA2 window\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHb Fontainebleau is a rare alpha globin chain variant that elutes as a hump at the base of the descending limb of HbA peak in commonly used HPLC platforms such as Bio Rad Variant II [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], Tosoh G8 etc. It results from Codon 21 (G\u0026loz;C); HBA2:c.64G\u0026thinsp;\u0026gt;\u0026thinsp;C mutation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although it is clinically silent, it can pose diagnostic dilemma in the interpretation of the chromatograms especially when present in combination with other globin chain variants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As highlighted in this communication, a new dimension to these diagnostic challenges is added by the difficulty in interpretation of the chromatographic pattern of Hb Fontainebleau seen in less commonly used HPLC systems that follow a different principle for separation of normal and abnormal Hb fractions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Hb Fontainebleau peak in Lifotronic H8 HPLC system at the first level of Hb analysis in this case posed a diagnostic challenge since (a) it eluted in HbA2 window and (2) the level of the variant Hb peak was abnormally high (11.4%) for it to be HbA2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As HbE and HbA2 elute separately on Lifotronic H8, and HbE peak elutes at a later time point compared to HbA2 (~\u0026thinsp;230 seconds for HbE vs. ~200 seconds for HbA2), the possibility of the abnormal Hb peak representing HbE in this case was ruled out. At the same time, a peak of 11.4% in HbA2 window was unlikely to represent HbA2 on account of its high level. Hence, the possibility of a non-HbA2, non-HbE variant Hb, possibly an alpha chain variant, was considered at this point and the sample was analysed on a more commonly used platform - Tosoh G8 HPLC system. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the elution pattern of the abnormal Hb was different on this platform but conformed to the known pattern of Hb Fontainebleau described above [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. That the abnormal Hb was indeed Hb Fontainebleau was confirmed subsequently by sequencing studies [Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis case highlights the need for a good knowledge of the elution times and patterns of less common Hb variants in different HPLC systems, an awareness of the variables introduced by different HPLC platforms in these parameters and an access to more than one screening platforms working on different principles, if required.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance and ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eFunding: This study was not supported by any funding.\u003c/li\u003e\n \u003cli\u003eConflict of Interest: The authors declare that they have no conflict of interest.\u003c/li\u003e\n \u003cli\u003eEthical approval:All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.\u003c/li\u003e\n \u003cli\u003eInformed consent: Informed consent was obtained from all individual participants included in the study.\u003c/li\u003e\n \u003cli\u003eConsent for publication: Consent for publication was obtained for every individual person\u0026rsquo;s data included in the study.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by AD, PT and SP. The first draft of the manuscript was written by AD and all authors commented on previous versions of the manuscript. All authors read and approved the finalmanuscript.\u003c/p\u003e\n\u003cp\u003eEthics approval: The study was approved by the appropriate institutional research ethics committee and the committee certifies that the study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards\u003cstrong\u003e\u003cu\u003e.\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent to participate: Informed consent was obtained from the patient for participation in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for publication: Consent was obtained from the patient for publication of test results.\u003c/p\u003e\n\u003cp\u003eConflict of interest: The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePatrinos GP, Giardine B, Riemer C, Miller W, Chui DHK, Anagnou NP, Wajcman H, Hardison RC (2004) Improvements in the HbVar database of human hemoglobin variants and thalassemia mutations for population and sequence variation studies. Nucleic Acids Res 132(Database issue):D537\u0026ndash;D541. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkh006\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkh006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNair S, Nadkarni A, Warang P, Bhave A, Ghosh K, Colah R (2010) Five alpha globin chain variants identified during screening for haemoglobinopathies. Eur J Clin Invest 40:226\u0026ndash;232\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUpadhye DS, Jain D, Nair SB, Nadkarni AH, Ghosh K, Colah RB et al (2012) First case of Hb Fontainebleau with sickle haemoglobin and other nondeletional alphagene variants identified in neonates during newborn screening for sickle cell disorders. J Clin Pathol 65:654\u0026ndash;659\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWajcman H, Blouquit Y, GombaudSaintonge G, Riou J, Galacteros F (1989) Hb Fontainebleau [alpha21(B2)Ala\u0026rarr;Pro], a new silent mutant hemoglobin. Hemoglobin 13:421\u0026ndash;428\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSidhwa K, Daruwalla MR, Pawar R, Nadkarni A, Hariharan P, Mehta P, Das Gupta A (2019) Diagnostic challenges posed by a rare alpha globin chain variant Hb Fontainebleau in a pregnant female and its potential effects in her children in view of multiple globin gene defects in her husband. IndIan J Pathol MIcrobIol 62:323\u0026ndash;325\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaradağ ME, Akbulut ED, Avcı E, Oğuz EF, Kader S, Abuşoğlu G, Serdar M, Yılmaz FM (2020) Evaluation of four different HPLC devices for hemoglobinopathy screening. Turk J Biochem. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1515/tjb-2019-0484\u003c/span\u003e\u003cspan address=\"10.1515/tjb-2019-0484\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi R, Huamei H, Kan L, Xiong D, Cheng X, Xu Y, Liu X, Zhang X (2020) Evaluation on the separated effect of 13 hemoglobin variants by a new automatic Hba1c analyzer. J Clin Lab Anal. 2020;34:e23446\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-hematopathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Journal of Hematopathology](https://link.springer.com/journal/12308)","snPcode":"12308","submissionUrl":"https://submission.springernature.com/new-submission/12308/3","title":"Journal of Hematopathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Hb Fontainebleau, Alpha globin chain variant, High performance liquid chromatography, Lifotronic H8, Tosoh G8","lastPublishedDoi":"10.21203/rs.3.rs-6628570/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6628570/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlpha globin chain variants constitute a very small fraction of the total hemoglobinopathy cases. Their rarity and the often-unfamiliar elution times/patterns in high performance liquid chromatography (HPLC) that mimic those of normal hemoglobin (Hb) fractions and/or known beta globin variants, render their primary diagnosis difficult. We describe here Hb Fontainebleau, a rare alpha globin chain variant, in an adult female that eluted in the HbA2 window in a less commonly used HPLC platform, Lifotronic H8, thereby causing difficulties in the diagnosis. The matter was resolved by running the sample on a more commonly used platform wherein the abnormal Hb yielded a familiar elution time and pattern known to be associated with Hb Fontainebleau. This was further confirmed by gene sequencing which showed a Codon 21 (G\u0026loz;C); HBA2:c.64G\u0026thinsp;\u0026gt;\u0026thinsp;C (or HBA1) mutation. A literature search failed to reveal any published case of the unique elution pattern of Hb Fontainebleau on Lifotronic H8 HPLC platform and the associated diagnostic challenges. An increasing use of the newer HPLC platforms calls for greater awareness of these issues.\u003c/p\u003e","manuscriptTitle":"A Rare Alpha Globin Chain Variant Hemoglobin Fontainebleau Poses Diagnostic Challenges by Yielding Different Results in Two Different HPLC Systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-21 06:01:04","doi":"10.21203/rs.3.rs-6628570/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-26T13:37:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-24T20:27:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274203997505836139554980784010004570610","date":"2025-06-11T18:38:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-16T14:11:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-12T23:27:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-12T23:27:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Hematopathology","date":"2025-05-09T12:10:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-hematopathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Journal of Hematopathology](https://link.springer.com/journal/12308)","snPcode":"12308","submissionUrl":"https://submission.springernature.com/new-submission/12308/3","title":"Journal of Hematopathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c94e5ae3-f769-458a-90e5-08a668376a68","owner":[],"postedDate":"May 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-26T02:08:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-21 06:01:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6628570","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6628570","identity":"rs-6628570","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}