Blueberry Muffin Syndrome and Hyperleukocytosis in a Newborn: A Diagnostic Challenge.

Blueberry muffin syndrome (BMS) in neonates, characterized by widespread nodular lesions, presents diagnostic challenges due to its diverse etiologies. Hyperleukocytosis, with leukocyte counts exceeding 100,000/µL, is a rare phenomenon associated with severe complications in neonates. Congenital leukemia (CL), a rare diagnosis within the first month of life, is linked to high mortality. This case report presents a unique case of BMS with hyperleukocytosis as the initial presentation of CL. A full-term male newborn, born after an uncomplicated pregnancy, except for Kell isoimmunization, with an Apgar score of 9/10, and an irrelevant family history, showed widespread purple nodules consistent with BMS at birth. Laboratory workup revealed mild anemia, hyperleukocytosis with immature granulocytes on peripheral blood (PB) smear, positive direct antiglobulin test, and elevated alanine aminotransferase and lactate dehydrogenase, without hyperbilirubinemia. Empirical antibiotics and hyperhydration were started, and the neonate was transferred to a level 3 neonatal intensive care unit for further evaluation. A comprehensive etiological investigation was conducted, comprising infectious, immunological, metabolic, and neoplastic factors. A skin nodule biopsy revealed an infiltrate of blast cells, indicative of leukemia cutis, and a bone marrow aspirate confirmed acute myeloid leukemia (AML). The patient successfully completed the NOPHO-DBH-2012 chemotherapy protocol at five months and remains in complete remission at nine months. This case report contributes to the literature by highlighting the diagnostic approach and management strategies for CL presenting with BMS and hyperleukocytosis. This case aims to enhance awareness and understanding of BMS as an initial manifestation of CL. Additionally, the challenges of treating leukemia in neonates, coupled with the lack of specific guidelines for this age group, further underscore the complexities in managing such patients.

A new case of platelet-type von Willebrand disease supports the recent findings of gain-of-function GP1BA variants outside the C-terminal disulphide loop enhances affinity for von Willebrand factor.

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder characterized by an increased ristocetin-induced platelet aggregation (RIPA) and enhanced affinity of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF). To date, only seven variants have been described with this gain-of-function effect, most of them located in the C-terminal disulphide loop of the VWF-binding domain of GPIbα. We herein describe a patient with moderate bleeding symptoms, mild thrombocytopenia and increased RIPA. By direct sequencing of GP1BA, a novel leucine-rich repeat heterozygous variant was identified (c.580C>T; predictably p.Leu194Phe), strongly suggestive as being the underlying cause for the PT-VWD phenotype of our patient.

CalDAG-GEFI Deficiency in a Family with Symptomatic Heterozygous and Homozygous Carriers of a Likely Pathogenic Variant in RASGRP2.

RASGRP2 encodes the calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) identified as a Rap1-activating molecule. Pathogenic variants previously identified in RASGRP2 allowed the characterization of CalDAG-GEFI deficiency as a non-syndromic, autosomal recessive platelet function disease. We report on the clinical manifestations and laboratory features of a Portuguese family with a likely pathogenic variant in RASGRP2 (c.999G>C leading to a p.Lys333Asn change in the CDC25 catalytic domain of CalDAG-GEFI) and discuss the contribution of this variant to the disease manifestations. Based on the study of this family with one homozygous patient and five heterozygous carriers and on a critical analysis of the literature, we challenge previous knowledge that CalDAG-GEFI deficiency only manifests in homozygous patients. Our data suggest that at least for the RASGRP2 variant reported herein, there is a phenotypic expression, albeit milder, in heterozygous carriers.

IL-31 and IL-8 in Cutaneous T-Cell Lymphoma: Looking for Their Role in Itch.

The itch associated with cutaneous T-cell lymphoma (CTCL), including Mycosis Fungoides (MF) and Sézary syndrome (SS), is often severe and poorly responsive to treatment with antihistamines. Recent studies have highlighted the possible role of interleukins in nonhistaminergic itch. We investigated the role of IL-31 and IL-8 in CTCL, concerning disease severity and associated itch. Serum samples of 27 patients with CTCL (17 MF and 10 SS) and 29 controls (blood donors) were analyzed for interleukin- (IL-) 31 and IL-8; correlations with disease and itch severity were evaluated. IL-31 serum levels were higher in CTCL patients than in controls and higher in SS than in MF. Also, serum IL-31 levels were higher in patients with advanced disease compared to those with early disease, and they correlated positively with lactate dehydrogenase and beta 2-microglobulin levels, as well as with the Sézary cell count. Itch affected 67% of CTCL patients (MF: 47%; SS: 100%). Serum IL-31 levels were higher in itching patients than in controls and in patients without itching. There was no association between serum IL-8 and disease severity, nor with itching. Serum IL-8 levels correlated positively with peripheral blood leukocyte and neutrophil counts in CTCL patients. Our study suggests a role for IL-31 in CTCL-associated itch, especially in advanced disease and SS, offering a rational target for new therapeutic approaches. Increased serum IL-8 observed in some patients may be related to concomitant infections, and its role in exacerbating itch by recruiting neutrophils and promoting the release of neutrophil proteases deserves further investigation.

αIIbß3 variants in ten families with autosomal dominant macrothrombocytopenia: Expanding the mutational and clinical spectrum.

BACKGROUND: Rare pathogenic variants in either the ITGA2B or ITGB3 genes have been linked to autosomal dominant macrothrombocytopenia associated with abnormal platelet production and function, deserving the designation of Glanzmann Thrombasthenia-Like Syndrome (GTLS) or ITGA2B/ITGB3-related thrombocytopenia. OBJECTIVES: To describe a series of patients with familial macrothrombocytopenia and decreased expression of αIIbß3 integrin due to defects in the ITGA2B or ITGB3 genes. METHODS: We reviewed the clinical and laboratory records of 10 Portuguese families with GTLS (33 patients and 11 unaffected relatives), including the functional and genetic defects. RESULTS: Patients had absent to moderate bleeding, macrothrombocytopenia, low αIIbß3 expression, impaired platelet aggregation/ATP release to physiological agonists and low expression of activation-induced binding sites on αIIbß3 (PAC-1) and receptor-induced binding sites on its ligand (bound fibrinogen), upon stimulation with TRAP-6 and ADP. Evidence for constitutive αIIbß3 activation, occurred in 2 out of 9 patients from 8 families studied, but also in 2 out of 12 healthy controls. We identified 7 missense variants: 3 in ITGA2B (5 families), and 4 in ITGB3 (5 families). Three variants (αIIb: p.Arg1026Trp and p.Arg1026Gln and ß3: p.Asp749His) were previously reported. The remaining (αIIb: p.Gly1007Val and ß3: p.Thr746Pro, p.His748Pro and p.Arg760Cys) are new, expanding the αIIbß3 defects associated with GTLS. The integration of the clinical and laboratory data allowed the identification of two GTLS subgroups, with distinct disease severity. CONCLUSIONS: Previously reported ITGA2B and ITGB3 variants related to thrombocytopenia were clustered in a confined region of the membrane-proximal cytoplasmic domains, the inner membrane clasp. For the first time, variants are reported at the outer membrane clasp, at the transmembrane domain of αIIb, and at the membrane distal cytoplasmic domains of ß3. This is the largest single-center series of inherited macrothrombocytopenia associated with αIIbß3 variants published to date.

Human Peripheral Blood Gamma Delta T Cells: Report on a Series of Healthy Caucasian Portuguese Adults and Comprehensive Review of the Literature.

Gamma delta T cells (Tc) are divided according to the type of Vδ and Vγ chains they express, with two major γδ Tc subsets being recognized in humans: Vδ2Vγ9 and Vδ1. Despite many studies in pathological conditions, only a few have quantified the γδ Tc subsets in healthy adults, and a comprehensive review of the factors influencing its representation in the blood is missing. Here we quantified the total γδ Tc and the Vδ2/Vγ9 and Vδ1 Tc subsets in the blood from 30 healthy, Caucasian, Portuguese adults, we characterized their immunophenotype by 8-color flow cytometry, focusing in a few relevant Tc markers (CD3/TCR-γδ, CD5, CD8), and costimulatory (CD28), cytotoxic (CD16) and adhesion (CD56) molecules, and we examined the impacts of age and gender. Additionally, we reviewed the literature on the influences of race/ethnicity, age, gender, special periods of life, past infections, diet, medications and concomitant diseases on γδ Tc and their subsets. Given the multitude of factors influencing the γδ Tc repertoire and immunophenotype and the high variation observed, caution should be taken in interpreting "abnormal" γδ Tc values and repertoire deviations, and the clinical significance of small populations of "phenotypically abnormal" γδ Tc in the blood.

Identification of novel variants in ten patients with Hermansky-Pudlak syndrome by high-throughput sequencing.

Background: Hermansky-Pudlak syndrome (HPS) is a rare inherited platelet disorder characterized by bleeding diathesis, oculocutaneous albinism (OCA) and a myriad of often-serious clinical complications. Methods: We established the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS; including platelet aggregation, flow cytometry, platelet dense granule content, electron microscopy and high-throughput sequencing (HTS). Results: The clinical presentation showed significant heterogeneity and no clear phenotype-genotype correlations. HTS revealed two known and three novel disease-causing variants. The Spanish patients carried a homozygous p.Pro685Leufs17* deletion (n = 2) in HPS4, or the novel p.Arg822* homozygous variant (n = 1) in HPS3. In the case of two Turkish sisters, a novel missense homozygous HPS4 variant (p.Leu91Pro) was found. In two Portuguese families, genetic studies confirmed a previously reported nonsense variant (p.Gln103*) in DTNBP1 in three patients and a novel duplication (p.Leu22Argfs*33) in HPS6 in two unrelated patients. Conclusions: Our findings expand the mutational spectrum of HPS, which may help in investigating phenotype-genotype relationships and assist genetic counselling for affected individuals. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS. Key messages We established the relationships between the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS. Molecular analysis is useful in confirming the diagnosis and may offer some prognostic information that will aid in optimizing monitoring and surveillance for early detection of end-organ damage. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS.

KIT D816V Positive Acute Mast Cell Leukemia Associated with Normal Karyotype Acute Myeloid Leukemia.

INTRODUCTION: Mast cell (MC) leukemia (MCL) is extremely rare. We present a case of MCL diagnosed concomitantly with acute myeloblastic leukemia (AML). CASE REPORT: A 41-year-old woman presented with asthenia, anorexia, fever, epigastralgia, and diarrhea. She had a maculopapular skin rash, hepatosplenomegaly, retroperitoneal adenopathies, pancytopenia, 6% blast cells (BC) and 20% MC in the peripheral blood, elevated lactate dehydrogenase, cholestasis, hypoalbuminemia, hypogammaglobulinemia, and increased serum tryptase (184 µg/L). The bone marrow (BM) smears showed 24% myeloblasts, 17% promyelocytes, and 16% abnormal toluidine blue positive MC, and flow cytometry revealed 12% myeloid BC, 34% aberrant promyelocytes, a maturation blockage at the myeloblast/promyelocyte level, and 16% abnormal CD2-CD25+ MC. The BM karyotype was normal, and the KIT D816V mutation was positive in BM cells. The diagnosis of MCL associated with AML was assumed. The patient received corticosteroids, disodium cromoglycate, cladribine, idarubicin and cytosine arabinoside, high-dose cytosine arabinoside, and hematopoietic stem cell transplantation (HSCT). The outcome was favorable, with complete hematological remission two years after diagnosis and one year after HSCT. CONCLUSIONS: This case emphasizes the need of an exhaustive laboratory evaluation for the concomitant diagnosis of MCL and AML, and the therapeutic options.

A pilot study on the usefulness of peripheral blood flow cytometry for the diagnosis of lower risk myelodysplastic syndromes: the "MDS thermometer".

BACKGROUND: Immunophenotypic analysis of the bone marrow (BM) cells has proven to be helpful in the diagnosis of Myelodysplastic Syndromes (MDS). However, the usefulness of flow cytometry (FCM) for the detection of myelodysplasia in the peripheral blood (PB) still needs to be investigated. The aim of this pilot study was to evaluate the value of FCM-based PB neutrophil and monocyte immunophenotyping for the diagnosis of lower risk MDS (LR-MDS). METHODS: We evaluated by 8-color FCM the expression of multiple cell surface molecules (CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD34, CD45, CD56, CD64 and HLA-DR) in PB neutrophils and monocytes from a series of 14 adult LR-MDS patients versus 14 normal individuals. RESULTS: Peripheral blood neutrophils from patients with LR-MDS frequently had low forward scatter (FSC) and side scatter (SSC) values and low levels of CD11b, CD11c, CD10, CD16, CD13 and CD45 expression, in that order, as compared to normal neutrophils. In addition, patients with LR-MDS commonly display a higher fraction of CD14+CD56+ and a lower fraction of CD14+CD16+ monocytes in the PB. Based on these results, we proposed an immunophenotyping score based on which PB samples from patients with LR-MDS could be distinguished from normal PB samples with a sensitivity 93% and a specificity of 100%. In addition, we used this score to construct the MDS Thermometer, a screening tool for detection and monitoring of MDS in the PB in clinical practice. CONCLUSIONS: Peripheral blood neutrophil and monocyte immunophenotyping provide useful information for the diagnosis of LR-MDS, as a complement to cytomorphology. If validated by subsequent studies in larger series of MDS patients and extended to non-MDS patients with cytopenias, our findings may improve the diagnostic assessment and avoid invasive procedures in selected groups of MDS patients.

Chemokine Receptor Expression on Normal Blood CD56(+) NK-Cells Elucidates Cell Partners That Comigrate during the Innate and Adaptive Immune Responses and Identifies a Transitional NK-Cell Population.

Studies of chemokine receptors (CKR) in natural killer- (NK-) cells have already been published, but only a few gave detailed information on its differential expression on blood NK-cell subsets. We report on the expression of the inflammatory and homeostatic CKR on normal blood CD56(+low) CD16(+) and CD56(+high) CD16(-/+low) NK-cells. Conventional CD56(+low) and CD56(+high) NK-cells present in the normal PB do express CKR for inflammatory cytokines, although with different patterns CD56(+low) NK-cells are mainly CXCR1/CXCR2(+) and CXCR3/CCR5(-/+), whereas mostly CD56(+high) NK-cells are CXCR1/CXCR2(-) and CXCR3/CCR5(+). Both NK-cell subsets have variable CXCR4 expression and are CCR4(-) and CCR6(-). The CKR repertoire of the CD56(+low) NK-cells approaches to that of neutrophils, whereas the CKR repertoire of the CD56(+high) NK-cells mimics that of Th1(+) T cells, suggesting that these cells are prepared to migrate into inflamed tissues at different phases of the immune response. In addition, we describe a subpopulation of NK-cells with intermediate levels of CD56 expression, which we named CD56(+int) NK-cells. These NK-cells are CXCR3/CCR5(+), they have intermediate levels of expression of CD16, CD62L, CD94, and CD122, and they are CD57(-) and CD158a(-). In view of their phenotypic features, we hypothesize that they correspond to a transitional stage, between the well-known CD56(+high) and CD56(+low) NK-cells populations.

Aggressive mature natural killer cell neoplasms: report on a series of 12 European patients with emphasis on flow cytometry based immunophenotype and DNA content of neoplastic natural killer cells.

We report 12 cases of aggressive natural killer (NK) cell neoplasms diagnosed in Portugal, with emphasis on flow cytometry. Ten patients had extranodal NK/T cell lymphoma, nasal type and two had aggressive NK cell leukemia, and seven were men and five were women, with a median age of 50 years. NK cells brightly expressed the CD56 adhesion molecule and CD94 lectin type killer receptor and had an activation-related HLA-DR+ CD45RA+ CD45RO+ immunophenotype, in most cases. In contrast, dim CD16 expression was found in a minor proportion of cases, whereas CD57 and the CD158a and CD158e1 killer immunoglobulin-like receptors were negative. One-third of cases showed a hyperploid DNA content and nearly all had a very high S-phase proliferative rate. The phenotypic features of the neoplastic NK cells would suggest that they represent the transformed counterpart of the CD56 + bright NK cells that circulate in normal blood.