Infantile Pyknocytosis: A Rare but Not So Rare Condition. A new Case Report

Infantile Pyknocytosis: A Rare but Not So Rare Condition. A new Case Report | 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 Infantile Pyknocytosis: A Rare but Not So Rare Condition. A new Case Report Ribersani Michela, Rubeo Alice, Roberta Colletti, Regoli Daniela, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6001245/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Infantile pyknocytosis is a rare cause of neonatal jaundice associated with transient hemolytic and usually severe anemia, with a favorable outcome. Its etiology remains undetermined. The diagnosis is mainly based on the presence of abnormal erithrocytes (pyknocytes) on the peripheral blood smears. We report the description of a new case recently occurring in a 12-day old caucasian male. The newborn was submitted, together with his parents, to a complete diagnostic process including erithrocytes tests for osmotic resistance, membrane fragility and lysis, and the study of hemoglobin stucture. All the studies resulted compatible with the physiological state of the little patients and his parents. The aim of this paper, is to underline the importance of including this condition among the differential diagnostic hypotheses when dealing with a picture of neonatal hemolytic anemia. Pyknocytosis hemolytic anemia newborn Figures Figure 1 Figure 2 Figure 3 Introduction Infantile Pyknocytosis (IP) is a rare, probably misestimated cause of non-immune neonatal hemolytic anemia, first described by Tuffy in 1959[1]. The diagnosis is based on a blood smear finding of abnormal erythrocytes (pyknocytes) that are either hyper-dense, or contracted and/or with a spiny surface, associated with rapid hemolysis and formation of Heinz bodies[1–3]. The disease usually presents itself around the newborn’s second week of life, is frequently severe with jaundice secondary to hyper-bilirubinemia, and hemolytic anemia of undetermined aetiology. The severity of the disorder may require intensive phototherapy, blood or exchange transfusions. However, IP is self-limited; the episode tends to resolve spontaneously within a few weeks or months, without recurrences[2,3]. We report a recently occurred new case of IP in a newborn that led us to review the more recent medical literature about this disorder. Case presentation A 12-day-old caucasian male was transferred from a peripheral birth center to the neonatal intensive care unit (NICU) of our University Hospital, to carry out further diagnostic tests following the finding of prolonged jaundice and anemia. The child was born at 38 weeks gestation of a normal pregnancy with a eutocic delivery and of an adequate weight for gestational age (3,150 g). The APGAR score was 9 and 10, respectively, at the first and fifth minute of life. The family history was negative for any blood disorders, and there was no consanguinity between the parents. Furthermore, there were no history of parental jaundice or neonatal anemia. The infant was exclusively breast-fed and showed good weight gain. The neonate became jaundiced in the first 24 hours after delivery and had received a first course of phototherapy at the peripheral birth center. The anti-human globulin/Coombs test was negative and the Glucose-6-phosphate dehydrogenase (G6PD) levels were normal. Upon NICU entry, the newborn was found to be in good clinical conditions but for pallor and scleral icterus. Vital signs were normal and no fever, respiratory distress, hepatosplenomegaly or neural deficits were noted. Laboratory tests showed normocytic anemia (red blood cell count 3.110x10 9 /L, hematocrit-HCT 27.8%, Hb level 9.6 g/dl, mean red blood cell volume-MCV 89.4 fl); white blood cell and platelets counts were in the normal range (15.37x10 9 /L, and 493x10⁹/L, respectively). The morphological examination of peripheral blood smear (May Grunwald –Giemsa staining), showed the presence of rather distorted, irregular, densely stained erythrocytes (pyknocytes), which also appeared having various spiny projections and were, when counted, around 10% as per instrumental count (Fig. 1 ). The reticulocyte percentage was increased, (3.58%), haptoglobin decreased (100 mg/L) and the lactic dehydrogenase level increased (LDH 371 U/L ). Total bilirubin was 16.92 mg/dL, with UCB level of 15.25 mg/dL. The neonate was submitted to a new phototherapy course resulting in UCB levels gradually decrease. During hospitalisation, the Hb values ​​progressively decreased, reaching 7.1 g/dl at 18 days of life, which made a transfusion of concentrated red blood cells necessary (15 ml/kg). The negative Coombs test raised a strong suspicion of hemolytic anemia due to intra-erythrocytic causes, excluding a possible G6PDH deficiency because it had already been studied at the onset. Therefore, the evaluation studies for suspected hemolytic anemia due to intra-globular causes were carried out before blood transfusion. The diagnostic process included the erythrocyte osmotic resistance tests to evaluate a possible erythrocyte membrane fragility with acidified glycerol tests (homemade reagents), to study the kinetics of erythrocyte lysis and, with Osmored B (Eurospital) test, to study the erythrocyte lysis in end point. The High Performance Liquid Chromatography (HPLC) separation method (VARIANT II β-Thalassemia Short Program BIORAD) was used to study the hemoglobin structure; this allows to separate and quantify the hemoglobin fractions present. All tests resulted compatible with the physiological state of the little patient (detailed results are available in the supplementary information). The complete blood count test with the ADVIA2120 dual laser hematology system (Siemens), in addition to the quantitative information expressed by the low Hb concentration (7.1 g/dL) and reticulocytosis (6.13%), provided suggestive qualitative information: the red blood cells-RBC V/HC cytogram demonstrated a share of hyperchromic elements that could be quantified at around 10% with the "RBC Matrix" (Fig. 2 ). The RBC/HC histogram showed asymmetry in the distribution curve of the Hb corpuscular concentration with a further high-density shoulder. A diagnosis of IP was made on the basis of instrumental data typical for pyknocytosis, microscopic observation of the peripheral blood smear together with the child’s objective clinical examination that did not reveal hepatosplenomegaly. All the studies were then extended to the parents, finding a completely normal picture in both of them (detailed results are reported in supplementary information). The study of the maternal Hb structure, using the HPLC separation method (BIORAD), showed an HbA2 of 1.6%, a value compatible, in case of a normal iron picture, with Delta-thalassemia. In conclusion, these studies did not highlight any hereditary problem that could justify such a severe hemolytic anemia in the newborn, thus further supporting the suspicion of pyknocytosis (Fig. 3 ). The neonate was discharged with HCT value ​​of 27.3% and Hb and total bilirubin levels of 9.2 mg/dl and 11.26 mg/dl, respectively and then followed-up at our Haematology Institute outpatient unit. In the subsequent checks, a progressive increase in Hb values ​​and a reduction in the number of pyknocytes were observed; in particular, at 3 months of life, Hb was 10.3 g/dl, HCT 32%, red blood cells 3.910x10 9 /L, white blood cells 9.35x10 9 /L, and platelets count, 381.0x10 9 /L; the reticulocyte percent was 2.1% with absolute reticulocyte count of 81.500/mcl. Total bilirubin was further reduced to normal values (0.24 mg/dl), with UCB level of 0.14 mg/dl; pyknocytes had dropped to 2%. Discussion IP, despite representing 9.4% of unexplained neonatal hemolytic anemias[4], is still considered a relatively rare and poorly described pathology, often under-diagnosed due to the non-specific clinical picture and therefore based on exclusion criteria. Its etiology remains undetermined. Some contributing extrinsic factors, such as oxidative stress, have been suggested. This hypothesis has been supported by experiments demonstrating the transformation of donor erythrocytes, labeled with chromium, into pyknocytes when transfused into patients with IP[5] and in studies that have demonstrated the association between IP and G6PD deficiency, that is strictly related to a greater susceptibility of the red blood cell to oxidative stress[6]. Treatment includes phototherapy, exchange transfusions to maintain bilirubin levels within safety limits, and/or concentrated red blood cells transfusions, necessary to correct anemia secondary to hemolysis. In cases where, after the blood transfusion, hemolysis and pyknocytosis persist with progressive decrease of Hb levels and inappropriate reticulocyte count, treatment with recombinant human erythropoietin (rHu-Epo), subcutaneously administered, has been successfully used to increase Hb values ​​and avoid further blood transfusions[7]. However, the utilisation of rHu-Epo as an adjunct therapy is still not currently the standard of care and should only be considered after complete investigations careful assessment. Since 1959, patients with IP have been reported; a PubMed search for IP, in 2020[2], identified 71/75 cases. More recently, other 26 IP cases have been described; all patients presented persistent jaundice and anemia at 1–3 weeks of age, requiring, in the majority of cases, phototherapy and blood transfusions[2,3,8–14]. Most patients showed blood film with pyknocytes. No genetic mutations have been identified as being causative; in a neonate, a next-generation sequencing (NGS) panel examining 28 genes involved in neonatal hemolytic disorders was performed and the UTG1A1*6 variant was identified, even if this result was no definite[9]. As previously reported, also in the Drouilly study[3] that describes 16 IP cases, more than half of them had African or Mediterranean descent. This association could be linked to genetic predisposition or to increased sensitivity to vitamin deficiencies which may exacerbate initial oxidative stress. IP therefore remains a rare and underestimated clinical entity. We continue to emphasise that the IP diagnosis is primarily driven by red blood cell morphology and, consequently, any hemolytic anemia in newborns should raise the suspicion of IP, thus requiring a careful review of the blood smear. The presence of a highly specialised laboratory with qualified personnel has allowed, in our case, to reach an exact diagnosis. In this report, we wanted to not only highlight the real diagnostic possibility but also provide the tools and laboratory data that can give the right indications for a correct diagnosis. Declarations Funding : the authors declare no funidng received or requested Data availability : No datasets were generated or analysed during the current study Consent for pubblication. Patient’s parents provided consent for the pubblication of this case Competing interests : The authors declare no competing interests Author Contribution R.M. wrote the main text and reviewed the literature and was involved in the management of the child; RA was involved in the management of the child; CR performed the analysis of the diagnostic process; RD was involved in the management of the child at NICU and critically reviewed the manuscript; NMG performed the peripheral blood morphological examination and prepared the figures; BW and PG critically reviewed the manuscript; TAM reviewed the literature and and contributed to the writing of the manuscript. All authors revised and approved the lat version of the manuscript References Tuffy P, Brown AK, Zuelzer WW (1959) Infantile pyknocytosis: a common erytrocyte abnormality in the first trimester. AMA J Dis Child. 98(2):227-241. Castillo J, Ness TE, Mehta PS (2020) Infantile Pyknocytosis: An Uncommon cause of newborn Hemolytic Anemia. J Pediatr Hematol Oncol. 42(4):e251-e253. doi: 10.1097/MPH.0000000000001461. Drouilly M, Jourdan L, Gèrard D, Russello J, Bobèe V, Audouy A, Phulpin A, Perrin J (2024) Infantile pyknocytosis, a neonatal hemolytic anemia with Heinz bodies: A cohort study. Pediatr Blood Cancer. 71(8):e31078. doi: 10.1002/pbc.31078. Epub 2024 May 24. https://doi.org/10.1002/pbc.31078 Eysette-Guerreau S, Bader-Mennier B, Garcon L, Guitton C, Cynober T (2006) Infantile pyknocytosis: a cause of hemolytic anemia of the newborn. Br J Haematol. 133(4):439-442. doi: 10.1111/j.1365-2141.2006.06033.x. https://doi.org/10.1111/j.1365-2141.2006.06033.x Keimowitz R, Desforges JF (1962) Infantile pyknocytosis. N Engl J Med. 273(21):1152-1154. Zannos-Mariolea L, Kattamis C, Paidoucis M. Br J Haematol. 1962;8:258-265. doi: 10.1056/NEJM196511182732109. Amendola G, Di Concilio R, D’Urzo G, Danise P, Parisi G, della Ragione F, Rossi F, Nobili B, Perrotta S (2008) Erythropoietin treatment can prevent blood transfusion in infantile pyknocytosis. Br J Haematol. 143(4):593-595. doi: 10.1111/j.1365-2141.2008.07344.x. Epub 2008 Sep 9.https://doi.org/10.1111/j.1365-2141.2008.07344.x Bobèe V, Lahary A (2020) Numerous Heinz bodies in a case of infantile pyknocytosis. Blood. 135(9):701. doi: 10.1182/blood.2019004019. https://imagebank.hematology.org Bahr TM, Knudsen MC, Lozano-Chinga M, Agarwal AM, Meznarich JA, Ohls RK, Christensen RD (2020) Infantile Pyknocytosis: End-Tidal CO, %Micro-R Measurements, Next-Generation Sequencing, and Transfusion Avoidance with Darbepoetin. Biomed Hub. 5(3):227-234. doi: 10.1159/000511388. eCollection 2020 Sep-Dec.https://doi.org/10.1159/000511388 Català L, Salazar JJ, Pujol M, Garcìa MJ, Escribano P, Torrent M, Moliner E (2025) Infantile Pyknocytosis as a Cause of Neonatal Hemolytic Anemia. Indian J Pediatr. 92(2):201. doi: 10.1007/s12098-024-05333-5. Epub 2024 Nov 19.https://doi.org/10.1007/s12098-024-05333-5 Fraga A, Conde AR, Araùjo B, Silva A (2023) Pyknocytosis, a rare form of neonatal hemolytic anemia. An Pediatr (Engl Ed). 99(6):455-456. doi: 10.1016/j.anpede.2023.11.003. Epub 2023 Nov 18.https://doi.org/10.1016/j.anpede.2023.11.003 Namba T, Ochi M, Ogura H, Kanno H, Higuchi Y (2021) Infantile pyknocytosis with marked hemolytic anemia. Pediatr Neonatol. 62(5):563-564. doi: 10.1016/j.pedneo.2021.05.015. https://doi.org/10.1016/j.pedneo.2021.05.015 Rees C, Lund K, Bain BJ. (2019). Infantile pyknocytosis. Am J Hematol. 94(4):489-490. https://doi.org/10.1002/ajh.25358. Spooremberg ME, Wachters-Hagedoorn RE, van Wijk R, de Kok JB (2021) Infantile Pyknocytosis in a Premature Dichorionic Diammiotic Twin. J Pediatr Hematol Oncol.43(7):e1037-e1039. doi: 10.1097/MPH.0000000000002004. https://pubmed.ncbi.nlm.nih.gov/3323513/ Additional Declarations No competing interests reported. Supplementary Files supplementaryinformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 Apr, 2025 Reviewers agreed at journal 18 Apr, 2025 Reviewers agreed at journal 17 Apr, 2025 Reviewers invited by journal 17 Apr, 2025 Submission checks completed at journal 17 Apr, 2025 First submitted to journal 01 Apr, 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. 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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-6001245","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":445013121,"identity":"8795a12c-1098-4010-961b-6b3628b22714","order_by":0,"name":"Ribersani Michela","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDACZh4IzcbeA2WASQMGHsJaeM4gaylg4MGtByYjkYMs+oEBpzXm7bwHH1f8scvjk3x78MMHhsN5fNKHj274YcAgY49Di8xhvmTDs23JxWzSecmSMxgOF7PxpaXd7DHA7TAJZh4zycYG5sQ26RwzoL8OJ7bx8Jjd4MGvxfxnw5/6xDbJM2bMf6Babv4hYAtjAxtQpQSPGTMDVMtt/LbwJUs2th0HqgT6pccgHchgS7stYyDBw3MAhxb+swc/NvypTpzffvbghx8V1onze5iP3Xzzx8aevQGHNajAAGEWUepHwSgYBaNgFGAHAAgzSryewwv1AAAAAElFTkSuQmCC","orcid":"","institution":"Policlinico Umberto I University Hospital, Sapienza University of Rome","correspondingAuthor":true,"prefix":"","firstName":"Ribersani","middleName":"","lastName":"Michela","suffix":""},{"id":445013122,"identity":"8b27f819-dbf1-441a-84fb-c0ed9177fd43","order_by":1,"name":"Rubeo Alice","email":"","orcid":"","institution":"Sant’Andrea University Hospital, Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Rubeo","middleName":"","lastName":"Alice","suffix":""},{"id":445013123,"identity":"8e44250f-1f20-496b-9486-7164f48d4989","order_by":2,"name":"Roberta Colletti","email":"","orcid":"","institution":"Policlinico Umberto I University Hospital, Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"","lastName":"Colletti","suffix":""},{"id":445013124,"identity":"14361d7a-50e5-4641-a192-b6f641b7e7d4","order_by":3,"name":"Regoli Daniela","email":"","orcid":"","institution":"Policlinico Umberto I University Hospital, Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Regoli","middleName":"","lastName":"Daniela","suffix":""},{"id":445013126,"identity":"475b4a38-24ad-42cd-b93d-c6d2e9d5b44e","order_by":4,"name":"Nardacci Maria Grazia","email":"","orcid":"","institution":"Policlinico Umberto I University Hospital, Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Nardacci","middleName":"Maria","lastName":"Grazia","suffix":""},{"id":445013128,"identity":"895dc2dc-d983-4ab3-8c73-efee48cb2d53","order_by":5,"name":"Barberi Walter","email":"","orcid":"","institution":"Policlinico Umberto I University Hospital, Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Barberi","middleName":"","lastName":"Walter","suffix":""},{"id":445013129,"identity":"99eaf62d-24d0-42db-af87-ae3759f74bb3","order_by":6,"name":"Palumbo Giovanna","email":"","orcid":"","institution":"Policlinico Umberto I University Hospital, Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Palumbo","middleName":"","lastName":"Giovanna","suffix":""},{"id":445013130,"identity":"84b69e13-57ee-45dc-b98f-8e2169fd62a0","order_by":7,"name":"Testi Anna Maria","email":"","orcid":"","institution":"Policlinico Umberto I University Hospital, Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Testi","middleName":"Anna","lastName":"Maria","suffix":""}],"badges":[],"createdAt":"2025-02-10 17:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6001245/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6001245/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82065232,"identity":"229cb266-f379-4799-b1a0-c7cdaeadcce6","added_by":"auto","created_at":"2025-05-06 12:29:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":366905,"visible":true,"origin":"","legend":"\u003cp\u003ePeripheral smears from our case with the characteristic pyknocytes\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6001245/v1/f119b391e0b859f3f7eb883c.png"},{"id":82065239,"identity":"33ce71db-550f-49bd-84ce-67faf9e613d1","added_by":"auto","created_at":"2025-05-06 12:29:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":125433,"visible":true,"origin":"","legend":"\u003cp\u003eArrow a\u003cstrong\u003e) C\u003c/strong\u003eytogram demonstrated a share of hyperchromic normocytic red cells that are increased at around 10% with the \"RBC Matrix\" (normal count \u0026lt; 1%); arrow b) the hyperchromic microcyte red cells are only \u0026lt;1%); arrow c) the RBC/HC histogram showed asymmetry in the distribution curve of the Hb corpuscular concentration with a further high-density right shoulder; arrow d) the same aspect is showed in the cytogram V/HC.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6001245/v1/c4d17da60e40cb3d7232da0c.png"},{"id":82065235,"identity":"52a9f3b8-4cd9-48d2-a305-f168ae7ffb21","added_by":"auto","created_at":"2025-05-06 12:29:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":36138,"visible":true,"origin":"","legend":"\u003cp\u003eFlow diagram summarizing the diagnostic process\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6001245/v1/6e17735d11c2cf92473cc3ff.png"},{"id":82067383,"identity":"72bcfb2d-7e1e-41b9-84f1-8875e4751ed5","added_by":"auto","created_at":"2025-05-06 12:45:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":842960,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6001245/v1/680754c3-41f3-49ec-990d-362b638d60e6.pdf"},{"id":82065236,"identity":"7de23fa7-db7c-4c6d-9432-67b2455ed321","added_by":"auto","created_at":"2025-05-06 12:29:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12889,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6001245/v1/29e79425e43d75089cb0534f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eInfantile Pyknocytosis: A Rare but Not So Rare Condition. A new Case Report\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfantile Pyknocytosis (IP) is a rare, probably misestimated cause of non-immune neonatal hemolytic anemia, first described by Tuffy in 1959[1]. The diagnosis is based on a blood smear finding of abnormal erythrocytes (pyknocytes) that are either hyper-dense, or contracted and/or with a spiny surface, associated with rapid hemolysis and formation of Heinz bodies[1\u0026ndash;3]. The disease usually presents itself around the newborn\u0026rsquo;s second week of life, is frequently severe with jaundice secondary to hyper-bilirubinemia, and hemolytic anemia of undetermined aetiology. The severity of the disorder may require intensive phototherapy, blood or exchange transfusions. However, IP is self-limited; the episode tends to resolve spontaneously within a few weeks or months, without recurrences[2,3].\u003c/p\u003e \u003cp\u003eWe report a recently occurred new case of IP in a newborn that led us to review the more recent medical literature about this disorder.\u003c/p\u003e"},{"header":"Case presentation","content":"\u003cp\u003eA 12-day-old caucasian male was transferred from a peripheral birth center to the neonatal intensive care unit (NICU) of our University Hospital, to carry out further diagnostic tests following the finding of prolonged jaundice and anemia. The child was born at 38 weeks gestation of a normal pregnancy with a eutocic delivery and of an adequate weight for gestational age (3,150 g). The APGAR score was 9 and 10, respectively, at the first and fifth minute of life. The family history was negative for any blood disorders, and there was no consanguinity between the parents. Furthermore, there were no history of parental jaundice or neonatal anemia. The infant was exclusively breast-fed and showed good weight gain. The neonate became jaundiced in the first 24 hours after delivery and had received a first course of phototherapy at the peripheral birth center. The anti-human globulin/Coombs test was negative and the Glucose-6-phosphate dehydrogenase (G6PD) levels were normal.\u003c/p\u003e \u003cp\u003eUpon NICU entry, the newborn was found to be in good clinical conditions but for pallor and scleral icterus. Vital signs were normal and no fever, respiratory distress, hepatosplenomegaly or neural deficits were noted. Laboratory tests showed normocytic anemia (red blood cell count 3.110x10\u003csup\u003e9\u003c/sup\u003e/L, hematocrit-HCT 27.8%, Hb level 9.6 g/dl, mean red blood cell volume-MCV 89.4 fl); white blood cell and platelets counts were in the normal range (15.37x10\u003csup\u003e9\u003c/sup\u003e/L, and 493x10⁹/L, respectively). The morphological examination of peripheral blood smear (May Grunwald \u0026ndash;Giemsa staining), showed the presence of rather distorted, irregular, densely stained erythrocytes (pyknocytes), which also appeared having various spiny projections and were, when counted, around 10% as per instrumental count (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe reticulocyte percentage was increased, (3.58%), haptoglobin decreased (100 mg/L) and the lactic dehydrogenase level increased (LDH 371 U/L\u003cb\u003e).\u003c/b\u003e Total bilirubin was 16.92 mg/dL, with UCB level of 15.25 mg/dL. The neonate was submitted to a new phototherapy course resulting in UCB levels gradually decrease. During hospitalisation, the Hb values ​​progressively decreased, reaching 7.1 g/dl at 18 days of life, which made a transfusion of concentrated red blood cells necessary (15 ml/kg).\u003c/p\u003e \u003cp\u003eThe negative Coombs test raised a strong suspicion of hemolytic anemia due to intra-erythrocytic causes, excluding a possible G6PDH deficiency because it had already been studied at the onset. Therefore, the evaluation studies for suspected hemolytic anemia due to intra-globular causes were carried out before blood transfusion. The diagnostic process included the erythrocyte osmotic resistance tests to evaluate a possible erythrocyte membrane fragility with acidified glycerol tests (homemade reagents), to study the kinetics of erythrocyte lysis and, with Osmored B (Eurospital) test, to study the erythrocyte lysis in end point. The High Performance Liquid Chromatography (HPLC) separation method (VARIANT II β-Thalassemia Short Program BIORAD) was used to study the hemoglobin structure; this allows to separate and quantify the hemoglobin fractions present. All tests resulted compatible with the physiological state of the little patient (detailed results are available in the supplementary information).\u003c/p\u003e \u003cp\u003e The complete blood count test with the ADVIA2120 dual laser hematology system (Siemens), in addition to the quantitative information expressed by the low Hb concentration (7.1 g/dL) and reticulocytosis (6.13%), provided suggestive qualitative information: the red blood cells-RBC V/HC cytogram demonstrated a share of hyperchromic elements that could be quantified at around 10% with the \"RBC Matrix\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The RBC/HC histogram showed asymmetry in the distribution curve of the Hb corpuscular concentration with a further high-density shoulder.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA diagnosis of IP was made on the basis of instrumental data typical for pyknocytosis, microscopic observation of the peripheral blood smear together with the child\u0026rsquo;s objective clinical examination that did not reveal hepatosplenomegaly.\u003c/p\u003e \u003cp\u003eAll the studies were then extended to the parents, finding a completely normal picture in both of them (detailed results are reported in supplementary information). The study of the maternal Hb structure, using the HPLC separation method (BIORAD), showed an HbA2 of 1.6%, a value compatible, in case of a normal iron picture, with Delta-thalassemia. In conclusion, these studies did not highlight any hereditary problem that could justify such a severe hemolytic anemia in the newborn, thus further supporting the suspicion of pyknocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe neonate was discharged with HCT value ​​of 27.3% and Hb and total bilirubin levels of 9.2 mg/dl and 11.26 mg/dl, respectively and then followed-up at our Haematology Institute outpatient unit. In the subsequent checks, a progressive increase in Hb values ​​and a reduction in the number of pyknocytes were observed; in particular, at 3 months of life, Hb was 10.3 g/dl, HCT 32%, red blood cells 3.910x10\u003csup\u003e9\u003c/sup\u003e/L, white blood cells 9.35x10\u003csup\u003e9\u003c/sup\u003e/L, and platelets count, 381.0x10\u003csup\u003e9\u003c/sup\u003e/L; the reticulocyte percent was 2.1% with absolute reticulocyte count of 81.500/mcl. Total bilirubin was further reduced to normal values (0.24 mg/dl), with UCB level of 0.14 mg/dl; pyknocytes had dropped to 2%.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIP, despite representing 9.4% of unexplained neonatal hemolytic anemias[4], is still considered a relatively rare and poorly described pathology, often under-diagnosed due to the non-specific clinical picture and therefore based on exclusion criteria. Its etiology remains undetermined. Some contributing extrinsic factors, such as oxidative stress, have been suggested. This hypothesis has been supported by experiments demonstrating the transformation of donor erythrocytes, labeled with chromium, into pyknocytes when transfused into patients with IP[5] and in studies that have demonstrated the association between IP and G6PD deficiency, that is strictly related to a greater susceptibility of the red blood cell to oxidative stress[6].\u003c/p\u003e \u003cp\u003eTreatment includes phototherapy, exchange transfusions to maintain bilirubin levels within safety limits, and/or concentrated red blood cells transfusions, necessary to correct anemia secondary to hemolysis. In cases where, after the blood transfusion, hemolysis and pyknocytosis persist with progressive decrease of Hb levels and inappropriate reticulocyte count, treatment with recombinant human erythropoietin (rHu-Epo), subcutaneously administered, has been successfully used to increase Hb values ​​and avoid further blood transfusions[7]. However, the utilisation of rHu-Epo as an adjunct therapy is still not currently the standard of care and should only be considered after complete investigations careful assessment.\u003c/p\u003e \u003cp\u003eSince 1959, patients with IP have been reported; a PubMed search for IP, in 2020[2], identified 71/75 cases. More recently, other 26 IP cases have been described; all patients presented persistent jaundice and anemia at 1\u0026ndash;3 weeks of age, requiring, in the majority of cases, phototherapy and blood transfusions[2,3,8\u0026ndash;14]. Most patients showed blood film with pyknocytes. No genetic mutations have been identified as being causative; in a neonate, a next-generation sequencing (NGS) panel examining 28 genes involved in neonatal hemolytic disorders was performed and the UTG1A1*6 variant was identified, even if this result was no definite[9]. As previously reported, also in the Drouilly study[3] that describes 16 IP cases, more than half of them had African or Mediterranean descent. This association could be linked to genetic predisposition or to increased sensitivity to vitamin deficiencies which may exacerbate initial oxidative stress.\u003c/p\u003e \u003cp\u003eIP therefore remains a rare and underestimated clinical entity. We continue to emphasise that the IP diagnosis is primarily driven by red blood cell morphology and, consequently, any hemolytic anemia in newborns should raise the suspicion of IP, thus requiring a careful review of the blood smear. The presence of a highly specialised laboratory with qualified personnel has allowed, in our case, to reach an exact diagnosis. In this report, we wanted to not only highlight the real diagnostic possibility but also provide the tools and laboratory data that can give the right indications for a correct diagnosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: the authors declare no funidng received or requested\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: No datasets were generated or analysed during the current study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for pubblication.\u003c/strong\u003e Patient\u0026rsquo;s parents provided consent for the pubblication of this case\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.M. wrote the main text and reviewed the literature and was involved in the management of the child; RA was involved in the management of the child; CR performed the analysis of the diagnostic process; RD was involved in the management of the child at NICU and critically reviewed the manuscript; NMG performed the peripheral blood morphological examination and prepared the figures; BW and PG critically reviewed the manuscript; TAM reviewed the literature and and contributed to the writing of the manuscript. All authors revised and approved the lat version of the manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTuffy P, Brown AK, Zuelzer WW (1959) Infantile pyknocytosis: a common erytrocyte abnormality in the first trimester. AMA J Dis Child. \u0026nbsp;98(2):227-241.\u003c/li\u003e\n \u003cli\u003eCastillo J, Ness TE, Mehta PS (2020) Infantile Pyknocytosis: An Uncommon cause of newborn Hemolytic Anemia. J Pediatr Hematol Oncol. 42(4):e251-e253. doi: 10.1097/MPH.0000000000001461.\u003c/li\u003e\n \u003cli\u003eDrouilly M, Jourdan L, G\u0026egrave;rard D, Russello J, Bob\u0026egrave;e V, Audouy A, Phulpin A, Perrin J (2024) Infantile pyknocytosis, a neonatal hemolytic anemia with Heinz bodies: A cohort study. Pediatr Blood Cancer. 71(8):e31078. doi: 10.1002/pbc.31078.\u0026nbsp;Epub 2024 May 24. https://doi.org/10.1002/pbc.31078\u003c/li\u003e\n \u003cli\u003eEysette-Guerreau S, Bader-Mennier B, Garcon L, Guitton C, Cynober T (2006) Infantile pyknocytosis: a cause of hemolytic anemia of the newborn. Br J Haematol. 133(4):439-442. doi: 10.1111/j.1365-2141.2006.06033.x. https://doi.org/10.1111/j.1365-2141.2006.06033.x\u003c/li\u003e\n \u003cli\u003eKeimowitz R, Desforges JF (1962) Infantile pyknocytosis. N Engl J Med. 273(21):1152-1154.\u003c/li\u003e\n \u003cli\u003eZannos-Mariolea L, Kattamis C, Paidoucis M. Br J Haematol. 1962;8:258-265. doi: 10.1056/NEJM196511182732109.\u003c/li\u003e\n \u003cli\u003eAmendola G, Di Concilio R, D\u0026rsquo;Urzo G, Danise P, Parisi G, della Ragione F, Rossi F, Nobili B, Perrotta S (2008) Erythropoietin treatment can prevent blood transfusion in infantile pyknocytosis. Br J Haematol. 143(4):593-595. doi: 10.1111/j.1365-2141.2008.07344.x.\u0026nbsp;Epub 2008 Sep 9.https://doi.org/10.1111/j.1365-2141.2008.07344.x\u003c/li\u003e\n \u003cli\u003eBob\u0026egrave;e V, Lahary A (2020) Numerous Heinz bodies in a case of infantile pyknocytosis. Blood. 135(9):701. doi: 10.1182/blood.2019004019. https://imagebank.hematology.org\u003c/li\u003e\n \u003cli\u003eBahr TM, Knudsen MC, Lozano-Chinga M, Agarwal AM, Meznarich JA, Ohls RK, Christensen RD (2020) Infantile Pyknocytosis: End-Tidal CO, %Micro-R Measurements, Next-Generation Sequencing, and Transfusion Avoidance with Darbepoetin. Biomed Hub. 5(3):227-234. doi: 10.1159/000511388.\u0026nbsp;eCollection 2020 Sep-Dec.https://doi.org/10.1159/000511388\u003c/li\u003e\n \u003cli\u003eCatal\u0026agrave; L, Salazar JJ, Pujol M, Garc\u0026igrave;a MJ, Escribano P, Torrent M, Moliner E (2025) Infantile Pyknocytosis as a Cause of Neonatal Hemolytic Anemia. Indian J Pediatr. 92(2):201. doi: 10.1007/s12098-024-05333-5.\u0026nbsp;Epub 2024 Nov 19.https://doi.org/10.1007/s12098-024-05333-5\u003c/li\u003e\n \u003cli\u003eFraga A, Conde AR, Ara\u0026ugrave;jo B, Silva A (2023) Pyknocytosis, a rare form of neonatal hemolytic anemia. An Pediatr (Engl Ed). 99(6):455-456.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edoi: 10.1016/j.anpede.2023.11.003.\u0026nbsp;Epub 2023 Nov 18.https://doi.org/10.1016/j.anpede.2023.11.003\u003c/li\u003e\n \u003cli\u003eNamba T, Ochi M, Ogura H, Kanno H, Higuchi Y (2021) Infantile pyknocytosis with marked hemolytic anemia. Pediatr Neonatol. 62(5):563-564.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edoi: 10.1016/j.pedneo.2021.05.015.\u0026nbsp;https://doi.org/10.1016/j.pedneo.2021.05.015\u003c/li\u003e\n \u003cli\u003eRees C, Lund K, Bain BJ. (2019). Infantile pyknocytosis. Am J Hematol. 94(4):489-490. https://doi.org/10.1002/ajh.25358.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSpooremberg ME, Wachters-Hagedoorn RE, van Wijk R, de Kok JB (2021) Infantile Pyknocytosis in a Premature Dichorionic Diammiotic Twin. J Pediatr Hematol Oncol.43(7):e1037-e1039. doi: 10.1097/MPH.0000000000002004. https://pubmed.ncbi.nlm.nih.gov/3323513/\u003c/li\u003e\n\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":"annals-of-hematology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aohe","sideBox":"Learn more about [Annals of Hematology](http://link.springer.com/journal/277)","snPcode":"277","submissionUrl":"https://submission.nature.com/new-submission/277/3","title":"Annals of Hematology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pyknocytosis, hemolytic anemia, newborn","lastPublishedDoi":"10.21203/rs.3.rs-6001245/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6001245/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInfantile pyknocytosis is a rare cause of neonatal jaundice associated with transient hemolytic and usually severe anemia, with a favorable outcome. Its etiology remains undetermined. The diagnosis is mainly based on the presence of abnormal erithrocytes (pyknocytes) on the peripheral blood smears. We report the description of a new case recently occurring in a 12-day old caucasian male. The newborn was submitted, together with his parents, to a complete diagnostic process including erithrocytes tests for osmotic resistance, membrane fragility and lysis, and the study of hemoglobin stucture. All the studies resulted compatible with the physiological state of the little patients and his parents. The aim of this paper, is to underline the importance of including this condition among the differential diagnostic hypotheses when dealing with a picture of neonatal hemolytic anemia.\u003c/p\u003e","manuscriptTitle":"Infantile Pyknocytosis: A Rare but Not So Rare Condition. A new Case Report","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 12:29:10","doi":"10.21203/rs.3.rs-6001245/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-04-19T03:11:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124858216518055566724155904078502887120","date":"2025-04-18T07:37:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310240652991315063935945756319147296665","date":"2025-04-18T01:16:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-17T18:03:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-17T11:41:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Annals of Hematology","date":"2025-04-01T16:35:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"annals-of-hematology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aohe","sideBox":"Learn more about [Annals of Hematology](http://link.springer.com/journal/277)","snPcode":"277","submissionUrl":"https://submission.nature.com/new-submission/277/3","title":"Annals of Hematology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c744803c-5a2b-4d3f-8ecd-e13ad75d81a4","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-06T12:29:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 12:29:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6001245","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6001245","identity":"rs-6001245","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}