T-large granular lymphocyte frequencies and correlates in disease states detected by multiparameter flow cytometry in pediatric and young adult population.

T-large granular lymphocytes (T-LGL) characterized by dim CD5 staining, although not completely understood, have unique roles in the immune system. Expansion of peripheral blood (PB) clonal T-LGL populations is associated with various entities in adults. We have previously demonstrated clonal T-LGL proliferations in pediatric immune dysregulation/inflammatory/proliferative conditions. However, T-LGL populations have not been studied in broader spectrum pathologies. In this study we evaluated sizes and correlates of T-LGL populations in the pediatric and young adult populations with various disease states. Lymphocytes including T-LGL were investigated retrospectively by reviewing PB multiparameter flow cytometric data with various indications over a 4-year period. Associations with clinical, laboratory findings, and T-LGL population sizes were sought. Among 520 cases reviewed, 240 were females and 280 males with a mean age of 9 years (0-33 years); mean T-LGL population constituted 14% (1-67%) in PB T cells. There were significant differences between T-LGL and CD5-bright, regular T cells. T-LGL correlated with CD8 + /DR + (R = 0.570; P < 0.01) and CD8 + /CD11b + (R = 0.597; P < 0.01) expression, indicating activated cytotoxic phenotype. The highest average T-LGL were seen in bone marrow transplant recipients (23.7%), Evans syndrome (23.7%), lymphoma (20.6%), and acute EBV infection (20.4%) cases, all with underlying immune dysregulation pathologies. In pediatric and young adult patients with different clinical conditions, PB T-LGL constitute an average of 14% of the T cells and have a predominantly activated cytotoxic T cell phenotype. Higher relative presence was seen in cases with an immune dysregulation background. These results may serve as a reference for T-LGL research efforts.

Aberrant myelomonocytic CD56 expression in Down syndrome is frequent and not associated with leukemogenesis.

Children with Down syndrome (DS) are at an increased risk of developing transient abnormal myelopoiesis (TAM) and acute leukemia. Aberrant expression of CD56 has been observed on myeloid leukemic blasts in DS patients. In general, CD56 expression in acute myeloid leukemia (AML) is considered a promoter of leukemogenesis. We did a retrospective flow cytometric study to investigate mature myelomonocytic cell CD56 expression patterns in TAM, non-TAM, and leukemia cases with DS. Flow cytometric analysis showed that granulocyte and monocyte aberrant/dysplastic CD56 expression is an inherent characteristic of most DS patients irrespective of the presence of TAM or leukemia. Increased CD56 expression in monocyte and granulocyte populations in DS could be multifactorial; greater expression of RUNX1 secondary to the gene dose effect of trisomy 21 along with the maturational state of the cells are the potential contributors. Unlike AML seen in non-DS patients, CD56 overexpression in DS AML cases does not appear to play a role in leukemogenesis.

Distinctive phenotypes in two children with novel germline RUNX1 mutations - one with myeloid malignancy and increased fetal hemoglobin.

RUNX1 associated familial platelet disorder (FPD) is a rare autosomal dominant hematologic disorder characterized by thrombocytopenia and/or altered platelet function. There is an increased propensity to develop myeloid malignancy (MM) - acute myeloid leukemia, myeloproliferative neoplasms or myelodysplastic syndrome often in association with secondary somatic variants in other genes. To date, 23 FPD-MM pediatric cases have been reported worldwide. Here, we present two new kindreds with novel RUNX1 pathogenic variants in which children are probands. The first family is a daughter/mother diad, sharing a heterozygous frameshift variant in RUNX1 gene (c.501delT p.Ser167Argfs*9). The daughter, age 13 years, presented with features resembling juvenile myelomonocytic leukemia - severe anemia, thrombocytopenia, high white cell count with blast cells, monocytosis, increased nucleated red cells and had somatic mutations with high allele burden in CUX1, PHF6, and SH2B3 genes. She also had increased fetal hemoglobin and increased LIN28B expression. The mother, who had a long history of hypoplastic anemia, had different somatic mutations- a non-coding mutation in CUX1 but none in PHF6 or SH2B3. Her fetal hemoglobin and LIN28B expression were normal. In the second kindred, the proband, now 4 years old with thrombocytopenia alone, was investigated at 3 months of age for persistent neonatal thrombocytopenia with large platelets. Molecular testing identified a heterozygous intragenic deletion in RUNX1 encompassing exon 5. His father is known to have increased bruising for several years but is unavailable for testing. These two cases illustrate the significance of secondary mutations in the development and progression of RUNX1-FPD to MM.

CD14/16 monocyte profiling in juvenile myelomonocytic leukemia.

Monocyte subset analysis by flow cytometry has been shown to be a useful diagnostic tool in chronic myelomonocytic leukemia in adults. An increase in the classical monocyte fraction (CD14++/CD16-) greater than 94.0% of total monocytes is considered highly sensitive and specific in distinguishing chronic myelomonocytic leukemia from other myeloproliferative disorders. In a pilot study of juvenile myelomonocytic leukemia cases, we noted that CD14++/CD16- monocyte fraction was >95% in de novo juvenile myelomonocytic leukemia (JMML) with somatic PTPN11 mutations but normal in those with monosomy 7 or Noonan syndrome. Monocyte subgroup profiling by itself is not diagnostic of JMML but may distinguish molecular subgroups within JMML.

Clonal T-cell large granular lymphocyte proliferations in childhood and young adult immune dysregulation conditions.

BACKGROUND: Proliferation of large granular lymphocytes (LGL) and T-cell LGL (T-LGL) in peripheral blood along with demonstration of clonality are the hallmarks of a heterogeneous group of disorders, including T-LGL leukemia or T-LGL lymphocytosis. They are often associated with neutropenia and responsive to immunosuppression. The true nature of this entity is not well understood. Some cases are reported as reactive phenomena with very limited experience in pediatric population. METHODS: Hematology/Oncology Flow Cytometry Laboratory database has been reviewed retrospectively. Patients with identifiable distinct CD5-dim T-cell population and positive clonal T-cell receptor rearrangement were included in the analysis. Clinical and laboratory data were then reviewed. RESULTS: Sixteen cases of children and young adults with increased peripheral blood clonal T-LGL population characterized by dim CD5 expression with wide range of underlying immune dysregulation/stimulation disorders were reviewed. Extended follow up with repeat testing suggested the reactive nature of persistent clonal T-LGL proliferations in this group. CONCLUSIONS: Our observations indicate that clonal T-LGL proliferations in children and young adults are reactive in nature and some can be persistent with an indolent course with unknown consequentiality. Clonal T-LGL cells could be targeting the most prominent immunogenic stressor(s) involved as a control mechanism.

Acquired Pure Red Cell Aplasia and Acquired Amegakaryocytic Thrombocytopenia Associated With Clonal Expansion of T-Cell Large Granular Lymphocytes in a Patient With Lipopolysaccharide-responsive Beige-like Anchor (LRBA) Protein Deficiency.

Acquired pure red cell aplasia and acquired amegakaryocytic thrombocytopenic purpura are rare in children. Similarly, clonal expansion of T-cell large granular lymphocytes is infrequently seen in pediatrics. Lipopolysaccharide-responsive beige-like anchor (LRBA) protein deficiency is a recently described immunodeficiency syndrome that has been associated with inflammatory bowel disease and autoimmune phenomena such as Evans syndrome. Here, we describe a patient with LRBA deficiency who developed acquired pure red cell aplasia and acquired amegakaryocytic thrombocytopenic purpura associated with expansion of clonal T-cell large granular lymphocytes. This has not been described in the literature previously and adds to the knowledge on the spectrum of manifestations of LRBA deficiency.

Mild erythrocytosis as a presenting manifestation of PIEZO1 associated erythrocyte volume disorders.

Piezo1, encoded by the gene PIEZO1, is an erythrocytic cellular membrane mechanoactivated cation channel. Mutations have been implicated in erythrocyte volume disorders (EVDs)-especially hereditary xerocytosis (HX)/dehydrated stomatocytosis (DHS). We identified three patients, all with novel PIEZO1 mutations, but only one displaying the HX/DHS phenotype. Retrospective review of three cases. Osmotic gradient red cell deformability (Osmoscan) was assessed via the Technicon Ektacytometer. Red cell band 3 content was estimated using Eosin-5'-Maleimide staining. Patient 1 was evaluated for polycythemia. Osmoscans suggested mild spherocytosis; a novel PIEZO1 mutation (p.Thr1589Ile, exon 35) was identified, causing mild erythrocytosis without hemolytic anemia. Patient 2 was evaluated for macrocytosis/reticulocytosis, normal-to-high hemoglobin, and indirect hyperbilirubinemia. Osmoscans suggested increased cellular hydration; a second novel PIEZO1 mutation (p.Arg1728Cys, exon 37) was identified, resulting in overhydrated stomatocytosis with well-compensated hemolysis. Patient 3 was evaluated for indirect hyperbilirubinemia only. Osmoscans suggested dehydrated stomatocytosis (DHS, xerocytosis); a third novel PIEZO1 mutation (p.Arg2279Cys, exon 47) was identified. All three patients' blood smears demonstrated stomatocytes and spherocytes. EVDs may be underdiagnosed due to the lack of "expected" anemia in a hemolytic disorder; two of three patients had high hemoglobin and red cell counts and one had high normal values for both parameters and the presence of stomatocytes/dehydrated cells lead to identification of causative PIEZO1 mutations. PIEZO1-associated EVDs may be more common than previously suspected and should be included in the diagnostic algorithms for mild erythrocytosis/unexplained jaundice.

Flow cytometry for assessment of the tumor microenvironment in pediatric Hodgkin lymphoma.

BACKGROUND: The role of flow cytometry in diagnosis and management of Hodgkin lymphoma (HL) remains limited. As knowledge emerges of the tumor microenvironment in this disease, various methods are being evaluated in its study. This study examines the microenvironment using flow cytometry to assess differences between subtypes and clinicopathologic correlates. PROCEDURE: A retrospective cross-sectional study was performed analyzing the tumor immunophenotype, by flow cytometry, for 31 children with classical HL. Correlation was made with patient information, including outcome. RESULTS: The makeup of the tumor microenvironment varies across subtype of HL, with T cells predominating in nodular sclerosis (NS), and similar proportions of B and T cells in mixed cellularity (MC). CD4 cells predominate in NS, whereas CD8 more so in MC subtype. The rate of continuous complete remission is significantly higher in the MC subgroup. Last, the proportion of HLA-DR/CD38 copositive lymphocytes was an independent prognostic factor for relapse/refractoriness. CONCLUSIONS: This study indicates that flow cytometry can be used to examine the tumor microenvironment in HL and that percentage of HLA-DR/CD38 copositive lymphocytes may be a biomarker for relapse and refractoriness in pediatric HL.

KLF1 E325K-associated Congenital Dyserythropoietic Anemia Type IV: Insights Into the Variable Clinical Severity.

We identified a child with KLF1-E325K congenital dyserythropoietic anemia type IV who experienced a severe clinical course, fetal anemia, hydrops fetalis, and postnatal transfusion dependence only partially responsive to splenectomy. The child also had complete sex reversal, the cause which remains undetermined. To gain insights into our patient's severe hematologic phenotype, detailed analyses were performed. Erythrocytes from the patient and parents demonstrated functional abnormalities of the erythrocyte membrane, attributed to variants in the α-spectrin gene. Hypomorphic alleles in SEC23B and YARS2 were also identified. We hypothesize that coinheritance of variants in relevant erythrocyte genes contribute to the clinical course in our patient and other E325K-linked congenital dyserythropoietic anemia IV patients with severe clinical phenotypes.

Contrast-dependent red-green hue shift.

On bright surrounds, red-green-balanced yellow targets become greenish brown with decreased target luminance, and red-green-balanced brown targets become reddish yellow with increased target luminance. These effects imply luminance- and/or contrast-dependent weighting of M- and L-cone signals in post-receptoral pathways. We show psychophysically that luminance contrast between the surround and the target is the primary determinant of the magnitude of red-green hue shift, requiring surround luminance at least twice the target luminance and increasing with further increases of surround/target contrast. There is a much smaller effect of absolute stimulus luminance, with dimmer stimuli showing slightly larger hue shifts. To evaluate a possible retinal origin of the changes in cone-signal weightings underlying the hue shift, we recorded spike responses from both ON- and OFF-center midget ganglion cells in peripheral primate retina. We found no evidence that the relative strength of L- and M-cone post-receptoral responses changed systematically with change of surround irradiance. Nor was there any systematic difference between ON- and OFF-subtypes. This suggests that the change in cone signal weighting occurs later in the visual system.

Contrast-dependent red-green balance shifts depend on S-cone activity.

Previous research from our lab has established that red-green-balanced yellow targets become greenish-brown as surround luminance increases, while red-green-balanced brown targets become reddish-yellow as surround luminance decreases. To help assess the generality and underlying processes of this contrast-dependent red-green hue shift, we investigated red-green hue shifts for target stimuli that appeared achromatic or blue as well as yellow/brown. Results confirmed that the red-green hue shift was largest for yellow/brown targets and was progressively reduced for achromatic and blue targets as target excitation of S cones increased. The magnitude of the hue shift could be predicted by the S/(L+M) excitation of the target when bright white surrounds are used. The hue shift also requires that the target and surround are presented to the same eye, consistent with processing in monocular pathways. Increased S-cone excitation by the surround was associated with red-green hue shifts for all targets equally. Thus, S-cone signals from bright white surrounds might play a role in the contrast-dependent red-green hue shift, but the source of the variation of the magnitude of the hue shift with variations in target S-cone excitation when presented on those surrounds is unknown.

Color Vision 2018: Introduction by the feature editors.

This feature issue of the Journal of the Optical Society of America A (JOSA A) reflects the basic and applied research interests of members of the color vision community. Most of the articles stem from presentations at the 24th Biennial Symposium of the International Colour Vision Society (ICVS).

Evidence for Increased Response to Induced Endoplasmic Reticulum Stress in Myeloid Cells in Acquired Aplastic Anemia.

Autoimmune response targeting the hematopoietic stem cells highlights the current understanding of acquired aplastic anemia (AAA) pathogenesis. Upregulation of the unfolded protein response is the cell's rejoinder to a variety of stresses, which either result in restoring homeostasis or cell death by increased expression of the transcription factor C/EBP homologous protein. We hypothesized that there is an inherent increased sensitivity to various cellular stressors, including the ones that target endoplasmic reticulum (ER) in AAA leading to a decreased proliferation and potentially contributing to susceptibility to autologous cytotoxicity. Using archived bone marrow aspirate samples, we demonstrate that the culture-expanded AAA myeloid cells have an increased response to ER stress induced by tunicamycin leading to decreased cell proliferation. Within the AAA myeloid samples, we show that the disease status, active versus response to therapy at the time of sampling does not alter the ER stress response. This is the first report, which provides evidence for an inherent defective stress control in the myeloid cells as a possible mechanism of evolution of the disease process in AAA.

Glucose phosphate isomerase (GPI) Tadikonda: Characterization of a novel Pro340Ser mutation.

After a thirty-year lag, we serendipitously reestablished contact with a patient with glucose phosphate isomerase deficiency and hydrops fetalis first reported in 1987. We now provide a clinical update and provide results of mutation analysis in this patient, from Southern India. The patient now an adult female of 36 years of age has moderate anemia but requires no transfusions except with some intercurrent illnesses. Exome sequencing studies showed a homozygous c.1018C>T (Pro340Ser) mutation in exon 12 of the glucose phosphate isomerase gene and later confirmed by direct sequencing. This mutation has not been previously described. To our knowledge, this is also the first known homozygous mutation in the hydrophobic core of the protein and is a highly deleterious mutation by in silico analysis and by clinical history in the family. Flow cytometry studies of band 3 content with eosin maleimide showed a unique tail of red cells on histograms, reflecting the dense red cells (presumably ATP depleted) seen on blood smears; similar findings were seen in patients with pyruvate kinase and phosphoglycerate kinase deficiency.

Dichoptic perception of brown.

Two experiments assessed mechanisms underlying brown induction by presenting a foveal target disk and concentric annular surround stimuli that varied in contrast relative to larger backgrounds. Stimuli were presented under monocular, binocular, and dichoptic viewing conditions. Observers adjusted the luminance of the target disk to a criterion brown level. We found evidence for at least two separate mechanisms for brown induction: one mechanism that is dependent on physically contiguous contrast and operates in monocular pathways and another mechanism that responds to high luminance contrast anywhere in the visual field and can operate after convergence of signals from the two eyes.

Luminance-dependent long-term chromatic adaptation.

There is theoretical and empirical support for long-term adaptation of human vision to chromatic regularities in the environment. The current study investigates whether relationships of luminance and chromaticity in the natural environment could drive chromatic adaptation independently and differently for bright and dark colors. This is motivated by psychophysical evidence of systematic difference shifts in red-green chromatic sensitivities between contextually bright- versus dark-colored stimuli. For some broad classes of scene content, consistent shifts in chromaticity are found between high and low light levels within images. Especially in those images in which sky and terrain are juxtaposed, this shift has direction and magnitude consistent with the observed psychophysical shifts in the red-green balance between bright and dark colors. Taken together, these findings suggest that relative weighting of M- and L-cone signals could be adapted, in a luminance-dependent fashion, to regularities in the natural environment.

No effects of surround complexity on brown induction.

A yellow stimulus turns brown when it is made sufficiently darker than its surroundings. Most previous studies have used simple contiguous surround stimuli to induce brown, so we know little about how brown induction may be controlled by more distant and more complex surround features. We begin to address this issue by varying the complexity of two configurations of achromatic surround stimuli. It was shown that the area most immediately contiguous to the test stimulus has strong effects on brown induction. More importantly, we found that neither the number of surround features nor the distribution of light in the surround region had an effect on brown induction, as long as the overall size of the surround region remained constant. Instead, we found that brown induction depended on the total amount of light in the constant-size surround region, regardless of how that light was distributed. This potentially distinguishes the mechanisms of brown induction from those of brightness induction.

Influence of surround proximity on induction of brown and darkness.

A bright white surround makes a yellow long-wavelength target look both browner and darker. We explored the parallel between these two types of induction by examining their dependence on the proximity of the bright surround to the target at two different time scales with 27 ms and 1 s stimulus durations. We assessed (a) brown induction by adjustment of target luminance to perceptual brown and yellow boundaries and (b) darkness induction by a successive matching procedure. We found that brown induction is a quick process that is robust even for 27 ms stimuli. For darkness induction, there was a strong, spatially localized surround proximity effect for the 27 ms stimuli and much weaker proximity effect for the 1 s stimuli. For brown induction, proximity effects were generally weaker but still showed relatively stronger localized proximity effects for 27 ms stimuli than for 1 s stimuli. For these stimuli, darkness induction predicts the relative pattern but not the magnitudes of brown induction. Both brown and darkness inductions show the operation of quick, spatially localized processes that are apparently superseded by other processes for extended stimulus presentations.

Color vision: introduction by the feature editors.

This feature issue of the Journal of the Optical Society of America A (JOSA A) reflects the basic and applied research interests of members of the color vision community. Most of the articles stem from presentations at the 23rd Biennial Symposium of the International Colour Vision Society (ICVS).