EDTA-anticoagulated Blood Research Articles

T-helper cell polarisation following severe polytrauma

Introduction Severe polytrauma induces an immunosuppressive response and is associated with a very high incidence of nosocomial infections. Previous studies have inferred that this detrimental immune response results from polarisation of the T helper (Th) response towards an anti-inflammatory, TH2 dominated, response at the expense of a bactericidal, Th1 response [1]. Objectives 1) To define alterations in TH cell subsets following severe blunt polytrauma. Methods Patients presenting to the emergency department within 2 hours of severe polytrauma were eligible if intubated either at the scene or in ED. Isolated head injuries and those not expected to survive 24 hours were excluded. EDTA anti-coagulated blood was drawn at 0hr (within 2 hours of injury), at 24 and 72hrs. Samples were immediately lysed, washed, stained and analysed using a standardised human 8-colour TH 1, 2 & 17 panel [2] on an LSR II flow cytometer. A paired white cell count differential was obtained at each sampling point. Patients were followed until discharge or death. Data were analysed using non-parametric statistics, with results presented as median and IQR. Results 15 consecutive severe polytrauma patients requiring Intensive Care Unit (ICU) admission were recruited. Demographic and clinical data are outlined in Figure 1. Twelve (80%) lymphocytosis (3.3x109/L, 2.5 - 4.4x109/L) (Figyre 2A). At 72 hours leukocytes had fallen (P < 0.01, figure 2A) such that 6 (54%) of those surviving were lymphopenic (0.9x109/L, 0.6 - 1.2x109/L). Circulating CD4+ (P = 0.01; Figure 2B) and CD4+CD25+ (P < 0.05) lymphocytes increased over 72 hours. When expressed as a percentage of total circulating lymphocytes no significant change in the proportions of the TH 1, 2 & 17 subpopulations was detected (Figure 2C-E). Conclusions Severe polytrauma patients swiftly become lymphopenic. Although a failure to normalise this during the ICU stay correlates with higher mortality [3] our study of TH cell subtypes demonstrates no evidence of a switch to a detrimental anti-inflammatory TH2 subtype at the expense of the potentially protective bactericidal TH1 subtype.

Abstract 5374: Development of a method to measure acetylated histone H4 in the nuclei of circulating myeloid cells as a surrogate tissue for the pharmacodynamics of HDAC inhibitors in the treatment of solid tumors

Abstract Valproate has been used clinically as an anticonvulsant for the last 50 years and is one of the major drugs used in the management of epilepsy. More recently it has been observed that valproate also functions as a histone deacetylase (HDAC) inhibitor, and this has kindled interest in the drug as an anti-cancer agent. We have developed and validated an assay to measure the proportion of myeloid cells in peripheral blood with nuclei localised acetylated Histone H4 detectable by immunofluorescence and imaging flow cytometry. Whole blood samples from healthy volunteers were incubated for 4 hours with 0, 50, 150 or 300 μg/ml of sodium valproate at 37°C with agitation. Following ex vivo exposure the samples were transferred to Transfix stabilising tubes and stored at 4°C to mimic collection from the clinic. Leukocytes were isolated from the fixed and EDTA anti-coagulated blood by red blood cell lysis and centrifugation prior to permeabilisation with ice cold methanol and staining with PE conjugated anti-acH4 and DAPI. The stained cells were run through an Imagestream MkII imaging flow cytometer with excitation at 488 and 405nm. Images of myeloid and lymphoid cells were resolved by area of the cell and side scatter. An x/y plot of acH4 staining intensity over bright detail similarity between acH4 and DAPI staining was used to identify myeloid cells with nuclear acH4 staining. Ex vivo incubation of whole blood samples with pharmacologically relevant concentrations of valproate resulted in a measurable increase in the proportion of myeloid cells with acH4 positive nuclei in 4/5 samples. The percentage of myeloid cells with acH4 positive nuclei increased from 0.01-0.29% in untreated samples to 0.75-7.0% following exposure to 300 μg/ml valproate. Validation experiments were carried out to assess variance due to staining and imaging of single incubations with 0 and 300 μg/ml valproate when processed independently. Coefficient of variation values were high, 110% (0.6 ± 0.67%) and 42% (3.1 ± 1.3%), respectively. However, the 5 fold increase in positive myeloid cells retained statistical significance (p &amp;lt; 0.05, t-test). We have developed a semi-quantitative cell based imaging method to identify increased histone H4 acetylation of myeloid cells following exposure to valproate. This assay will be used to detect valproate HDAC inhibition in myeloid cells as a surrogate tissue in children being treated as part of the European SIOP Ependymoma 2012 trial. Citation Format: Jamieson David, Wai Wong, Gareth Veal. Development of a method to measure acetylated histone H4 in the nuclei of circulating myeloid cells as a surrogate tissue for the pharmacodynamics of HDAC inhibitors in the treatment of solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5374. doi:10.1158/1538-7445.AM2015-5374

OMIP-028: activation panel for Rhesus macaque NK cell subsets.

OMIP-028: activation panel for Rhesus macaque NK cell subsets.

Activation of the NOTCH signaling pathway stimulates migration of human b-lymphocytes

The NOTCH signaling pathway is required for embryonic development, cell differentiation, and for maintenance of tissue homeostasis and stem cell function. Consequently, dysfunction is associated with several genetic diseases or cancer development. Engagement in inflammatory disorders is under investigation. We explored whether the NOTCH ligands Jagged-1 and Delta-like-4 might affect migration of human B-lymphocytes in vitro. B-lymphocytes were isolated from EDTA-anticoagulated venous blood of healthy volunteers. For migration assays 5µm pore sized nitrocellulose membrane filters and a modified 48well microchemotaxis chamber were used. After an incubation period of 90 min., the distances cells migrated were measured microscopically. The “Chemotactic Index” was defined as the ratio between the distance cells migrated towards the NOTCH ligands and that towards control medium. Additionally, migration behavior was investigated after preincubation of cells with the γ-secretase inhibitor DAPT and ADAM17. We report for the first time that the NOTCH ligands Jagged-1 and Delta-like-4 significantly stimulate the migration of human B-lymphocytes in vitro. The observed effects were comparable to the maximal effects elicited by the chemoattractant fMLP. Furthermore, preincubation of cells with DAPT and ADAM17 diminished migration. Checkerboard assays showed that B-lymphocytes migrate both, in a chemokinetic and chemotactic manner in response to the NOTCH ligands tested. In conclusion this study shows that activation of the NOTCH signaling pathway can stimulate migration of B-lymphocytes and thus may serve as a new target in inflammatory pulmonary diseases.

Relation between the Level of Soluble Endothelial Protein C Receptor and the Risk of Deep Venous Thrombosis in Sudanese

Background: Deep vein thrombosis (DVT) may lead to serious complication; the pulmonary embolism; one of the most serious conditions that cause morbidity and mortality worldwide. sEPCR circulates in the plasma and has high affinity to bind protein C and activated protein C (APC). This binding interferes with the anticoagulant function of APC and results in increased risk for DVT. The aim of this study is to explore the role of sEPCR as a risk factor for DVT in Sudanese individuals. Methods: A total of 100 Sudanese DVT patients and 100 apparently healthy individuals were recruited for this study. EDTA-anticoagulated venous blood samples were collected from all participants. Plasma sEPCR levels were measured by enzyme linked immunosorbent Assay (ELISA). All results were analyzed using SPSS. Results: The plasma level of sEPCR was higher in DVT group than in healthy individuals, while male patients showed higher level when compared to females. Age correlates positively with sEPCR, whilst BMI and ethnicity showed no effect on the level of sEPCR. Individuals in the top quartiles of showed to increased risk of DVT when compared to those in the lower quartile. Conclusion: It can be concluded that elevated sEPCR level increases the risk to develop DVT in Sudanese. The level of the soluble receptor is influenced by the gender of individual more than by his/her ethnicity or body mass index. Results are also indicative of a much higher risk in those with sEPCR level more than 120 ng/ml when compared to those with lower levels.

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene.

Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.

Screening for Paroxysmal Nocturnal Hemoglobinurea with Cytodiff Analysis

Introduction. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidylinositolglycan complementation Class A (PIG-A) gene. PIGA mutations in PNH patients lead to a glycosylphosphatidylinositol (GPI)-linked membrane proteins expression deficiency.In PNH, there is a partial or absolute inability to make GPI-anchored proteins including complement-defense structures such as CD55 and CD59 on RBCs and WBCs. Clinical features of PNH include intravascular hemolysis, bone marrow failure, and thrombosis, all major causes of morbidity and mortality. Flow cytometry (FCM) plays a key role in the laboratory investigation of PNH, and rapid diagnosis of this condition is highly desirable. A definitive diagnosis of PNH can be established by demonstrating the absence of cell membraneGPI-anchored proteins from granulocytes or red blood cells (RBC) according to ICCS Guidelines for the diagnosis and monitoring of PNH by flow cytometry. It has been also described that the expression of CD16 can be decreased on PNH-affected granulocytes.Recently new method for extended flow WBC differential was introduced by Beckman Coulter. This method uses flow cytometric analysis with CytoDiff™** reagents which is a 5-color/6-marker reagent that provides a 10-part cytometric differential from whole blood specimens and comprises CD36-FITC, (CD2+CD294)-PE, CD19-ECD, CD16-PC5, and CD45-PC7 (Beckman Coulter).The protocol allows detection of mature neutrophils, total lymphocytes, total monocytes, eosinophils, basophils, immature granulocytes, B lymphocytes, CD16-negative T/NK lymphocytes, CD16-positive T/NK lymphocytes, CD16 positive and CD16 negative monocytes, and blasts cells with lineage orientation. This method also allows the detection of the abnormal antigen expression on WBC, for example low CD16 expression on neutrophils. In case of abnormal low CD16 expression on segmented neutrophils they will be classified as Immature Granulocytes (Imm Gran). The aim of the study was to evaluate the efficacy of CytoDiff** analysis of peripheral blood for PNH screening detecting low CD16 expression on neutrophils.Methods: EDTA-anticoagulated blood samples from 53 patients with PHN suspicion were prospectively included in the study. Analysis of the PNH clones was conducted in according with international protocol, using CD235a for RBC gating, CD15-PE/CD45-PC7 for granulocyte gating, CD64-PC5/CD45-PC7 for monocyte gating and GPI-anchored proteins CD59-PE, FLAER-FITC/CD24-PC5 and FLAER-FITC/CD14-PE for RBCs, granulocytes and monocytes accordingly.For extended flow WBC differential analysis the blood samples were stained with the CytoDiff** panel, lyzed with Versalyse (Beckman Coulter) and 20 000 leucocytes were analyzed on a FC500 Flow Cytometer (Beckman Coulter) using CytoDiff** CXP software.Results:Totally 53 patients with PHN suspicion were prospectively included in the study. All these patients were characterized by anemia, thrombocytopenia and/or leucopenia. PNH diagnosis was confirmed in 6 patients and in other 7 patients the final diagnosis was aplastic anemia with PNH clone.For all 13 patients (4 males, 9 females, with median age of 41.5 years) with confirmed presence of PNH clone CytoDiff** reported increased number of Imm Gran (range 3-45%). Microscopy analysis did not detect the presence of Imm Grans in the slide, so we concluded that falsely reported increased Imm Gran count was due to the decreased expression of CD16 on Neutrophils. Good correlation (r=0.9257) was observed between Imm Gran count and the size of granulocytic PNH clone.Conclusion: Our data demonstrate that CytoDiff** analysis, which is able to detect a wide spectrum of normal and pathological cells in peripheral blood allows determination of CD16-low expression on neutrophils and thus provide efficient screening for suspicious of PNH in patients with anemic syndrome.** Not available in the United States and other geographies. DisclosuresLugovskaya:Beckman Coulter: Research Funding.

Evaluation of EDTA induced pseudothrombocytopenia and the effect of alternative anticoagulants

Background: Artifactual Thrombocytopenia is a condition in which there is falsely lowered platelet in patients who have thrombocytopenia but the absence of petechiae or echymoses. Pseudothrombocytopenia is also an artifactual thrombocytopenia caused by anticoagulant dependent agglutinins. The aim of this study was to compare the platelet count in pseudothrombocytopenia in EDTA anticoagulated samples and other alternative anticoagulants.Materials and methods: This study was performed in the department of hemotology hematology, Institute of medicine. All cases during study period were evaluated by EDTA-anticoagulated whole blood samples but criteria for selecting pseudothrombocytopenia patients was unexpectedly low platelet counts with clumping/aggregate on peripheral blood smear. Additional samples were collected in sodium citrate and heparin for examined.Results: A total of 50 patients aged between 18 to 90 years were found to have pseudothrombocytopenia. Platelet counts in samples anticoagulated with EDTA ranged from 20x109/l to 149x109/l and samples from same patients anticoagulated with citrate ranged from 41x109 /l to 312x109 /l and heparin showed platelet count ranging from 29x10 9 /l to 210x109 /l. The mean platelet count in EDTA- anticoagulated blood of individuals with pseudothrombocytopenia was 104x109/l whereas the mean platelet count in citrate and heparin-anticoagulated samples was 151x109/land123x109/l respectively. Platelet counts decreased dramatically in the EDTA samples in contrast to the samples anticoagulated with citrate or heparin post four hours of collection.Conclusion: Peripheral blood smears should be examined for platelet clumping/aggregates in cases with low platelet count not correlating with clinical presentation or in isolated thrombocytopenia flagged in hematology analyser. Alternative anticoagulants should be used for correct estimation of platelet count.DOI: http://dx.doi.org/10.3126/jpn.v4i8.11498 Journal of Pathology of Nepal; Vol.4,No. 8 (2014) 626-629

A novel point mutation in the β1-tubulin gene in asymptomatic macrothrombocytopenic Norfolk and Cairn Terriers.

An asymptomatic macrothrombocytopenia, phenotypically similar to asymptomatic inherited macrothrombocytopenia in Cavalier King Charles Spaniels, was described in a group of Norfolk Terriers (NT) from Northern Italy, and isolated cases were also reported in Cairn Terriers (CT). The purpose of this work was to evaluate for the presence of a genetic defect in the β1-tubulin gene in macrothrombocytopenic NT and CT. Samples from 20 healthy dogs (13 NT and 7 CT) were collected at different institutions in Italy (n=8), United Kingdom (n=3), and United States (n=9). Genomic DNA was harvested from EDTA-anticoagulated blood and all coding areas and exon-intron splice sites in the gene encoding β1-tubulin were amplified and sequenced. Twelve dogs (9 NT and 3 CT) showed a single nucleotide polymorphism (SNP) in exon 1 at nucleotide position 5 (G5A) that would result in the change of an arginine to a histidine at amino acid position 2 (R2H). Four dogs (3 NT and one Cairn Terrier) were heterozygous for the SNP, and 4 dogs (one Norfolk Terrier and 3 CT) matched the normal canine genome. Homozygous dogs for the SNP were macrothrombocytopenic with platelet counts ranging from 19,000 to 110,000/μL. Heterozygous and normal dogs had normal platelet counts and morphology. None had the CKCS point mutation. The β1-tubulin N-terminal amino acids form the nucleotide-binding domain and thus this mutation could affect GTP binding enough to influence platelet formation in homozygous but not in heterozygous dogs. The presence of macrothrombocytopenia only in homozygous affected dogs reveals an association between the SNP and the phenotype.

Effects of pH changes of stock normal saline solution on 5 percent red cell suspension

Abstract The red cell suspension (RCS) is a universally used indicator system to demonstrate antigen and antibody reactions in vitro. Saline solutions that are used in its preparation are preferred to be fresh to avoid changes in pH that may affect the results. Thus, buffered saline such as phosphate-buffered saline (PBS) is the ideal diluent because its pH is maintained for a certain period. However, normal saline solution (NSS) is more commonly used because it is inexpensive and easy to make. pH changes in the saline solutions and the RCSs were monitored for 1 week. Macroscopic examination of changes in degree of redness of RCS was also observed. Red blood cell (RBC) indices of the ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood used in the preparation of the RCS were measured in the performance of an automated complete blood count. Qualitative examination of the crenation of RBCs was done on the prepared blood smears and graded by three registered medical technologists. Percentage crenation was then determined using an improved Neubauer counting chamber. Three trials were performed, and results were averaged. Statistical analysis showed that there were significant differences in the average pH of PBS and NSS and the average pH of RCS-PBS and RCS-NSS over 1 week. RBC indices measured in EDTA-anticoagulated donor blood showed no significant difference. Macroscopic examination of changes in degree of redness of the RCS showed that color darkened over 1 week but only by a small degree. Qualitative and quantitative examination of crenation of RBCs in RCS-PBS and RCS-NSS both showed no significant differences over 1 week. The experimental group (RCS-NSS) continuously showed a higher grade of crenation than the control group (RCS-PBS). Crenation of RBCs still manifests microscopically despite the lack of a significant relationship between the pH of the saline solutions and the degree and percentage of crenation. Crenation, therefore, cannot be attributed to pH alone but occurs as a result of other factors. Immunohematology 2014;30:126–134.

Comparative microbiological features of Bartonella henselae infection in a dog with fever of unknown origin and granulomatous lymphadenitis

We report the first documented case of Bartonella henselae infection in a dog from France and the first isolation of B. henselae from a dog with fever of unknown origin. This observation contributes to the "One Health" concept focusing on zoonotic pathogens emerging from companion animals. A 1-year-old female German shepherd dog was referred for evaluation of fever of unknown origin of 1month duration. Diagnostic investigations confirmed diffuse pyogranulomatous lymphadenitis. The dog became afebrile, and lymph node size normalized in response to a 6-week course of doxycycline. Retrospectively, Bartonella DNA was amplified from an EDTA-anticoagulated blood sample obtained before antimicrobial therapy, with the gtlA fragment sharing 99% identity with the 350-bp gtlA fragment of the B. henselae Houston-1 strain. The same strain was isolated in the blood of three healthy cats from the household. Two months after discontinuation of doxycycline, the dog experienced a febrile relapse. Bartonella DNA was again amplified from blood prior to and immediately after administration of a 6-week course azithromycin therapy. However, without administration of additional medications, PCR was negative 9months after azithromycin therapy and the dog remains clinically healthy 12months following the second course of antibiotics. The medical management of this case raises several clinically relevant comparative infectious disease issues, including the extent to which Bartonella spp. contribute to fever of unknown origin and pyogranulomatous inflammatory diseases in dogs and humans, and the potential of doxycycline and azithromycin treatment failures. The possibility that dogs could constitute an underestimated reservoir for B. henselae transmission to people is also discussed.

Hemoglobin Variants That Produce Abnormal Elevations In Hb A1c In The Clinical Laboratory: Electrophoretic Patterns, HPLC Analysis and Globin Gene Sequencing

Introduction There are two commonly-used methods for measuring Hemoglobin A1c (Hb A1c) – chromatographic and immunochemical. Immunochemical analysis depends on a specific antibody directed towards an epitope that consists of the linked glucose as well as the N-terminal betachain. High-performance liquid chromatography (HPLC)-determined Hb A1c concentrations that are unusually high (above 18%) may be a consequence of a hemoglobinopathy. Standard alkaline and acid gel electrophoresis and even an extended HPLC chromatogram may not fully resolve the abnormal hemoglobin. Globin gene analysis is an invaluable aid in such a situation. Over the course of a 12-month period, 4 individuals were identified in whom the HbA1c fraction exceeded 20%. All cases were encountered as part of our routine clinical chemistry hemoglobin A1c service. All cases were shown to be a result of heterozygosity for an uncommon or rare hemoglobin variant. This resulted in an early-eluting variant that was close to, or coincident with, the Hb A1c peak. Only one of these 4 subjects had anemia and microcytosis, apparently related to iron deficiency and chronic kidney disease. Methods &amp; Materials Hb A1c quantitation was carried out using EDTA-anticoagulated blood on a Bio-Rad Variant II HPLC analyzer (Bio-Rad Laboratories, Hercules CA). Gel electrophoresis was performed on a SPIFE 3000 system (Helena Laboratories, Beaumont TX) using spun, washed red cell pellets. Extended-run HPLC analysis utilized a BioRad D-10™ Hemoglobin Analyzer in the A2/F mode. Globin gene analysis was carried out at Boston University Medical Center using the residual washed whole blood pellet. The globin genes were amplified by PCR, the amplicons were purified, and nucleotide sequencing was performed. Case Descriptions Case 1. Female 58 y. Hb A1c23.8%. o Hb 13.6 g/dL, Hct 41.2%, MCV 94 fL, RBC 4.39 x 106 per mm3, RDW 13.4% o Alkaline gel electrophoresis: Very fast variant band, 23% of total Hb. o Acid Gel electrophoresis: Normal. o Globin gene analysis: Heterozygous for Hb I (α2 codon 16 AAG&gt;GAG or Lys16Glu). Case 2. Female 66 y. Hb A1c46%. o Hb 7.3 g/dL, Hct 24.9 %, MCV 70 fL, RBC 3.5 x 106 per mm3, RDW 16.5%. Iron deficient with low serum ferritin of 8.3. The patient also had a history of diabetes and chronic kidney disease. o Alkaline gel electrophoresis: “Fast” hemoglobin, but migrating very close to &amp; slightly anodal of Hb A. o Acid Gel electrophoresis: Variant hemoglobin migrates with Hb F o D-10 HPLC A2/F Mode: 45% A1c o Globin gene analysis: Heterozygous for Hb Hope, β chain codon 136 GGT&gt;GAT or Gly136Asp Case 3. Female 47 y. Hb A1c 43%. o Hb 12.6 g/dL, Hct 41.7%, MCV 96.2 fL, RBC 4.33x 106 per mm3, RDW 22.9% o Alkaline gel electrophoresis: Normal. o D-10 HPLC A2/F Mode: Variant peak, 46% eluting between Hb A1c and A0 o Globin gene analysis: Heterozygous for Hb Moriguchi, β codon 97 CAC&gt;TAC or His97Tyr. Case 4. Female 83 y. Hb A1c 41% as “labile Hb A1c” or 51% as Hb A1c by extended run in A2/F mode. o Hb 13.8 g/dL, Hct 43.4%, MCV 88.5 fL, RBC 4.9 x 106 per mm3, RDW 14% o Alkaline gel electrophoresis: Normal. o Acid Gel electrophoresis: Variant band, slightly anodal to Hb F o D-10 HPLC A2/F Mode: 51% Hb A1c o Globin gene analysis: Heterozygous for a novel variant hemoglobin, β codon 2 CAC&gt;AAC or Histidine to Asparagine Conclusions Hb A1c analysis by HPLC frequently reveals the presence of a variant hemoglobin in asymptomatic individuals who have the S, C or E trait. In such cases, Hb A1c can be accurately determined despite the presence of the abnormal hemoglobin. We describe 4 individuals with marked elevation of Hb A1c. All these individuals were heterozygous for uncommon globin chain variants due to single point mutations. One subject had a Hb variant that had not been previously recognized. It is unclear whether the immunochemical assays might experience interference from these unusual variants. Disclosures: No relevant conflicts of interest to declare.

Infection with Panola Mountain Ehrlichia sp. in a Dog with Atypical Lymphocytes and Clonal T‐Cell Expansion

Infection with Panola Mountain <i><scp>E</scp>hrlichia</i> sp. in a Dog with Atypical Lymphocytes and Clonal T‐Cell Expansion

Monocytes circulate in constant reversible interaction with platelets in a [Ca2+]-dependent manner

We aimed to provide evidence that blood monocytes belonging to all subsets predominantly circulate in constant and usually reversible interactions with platelets, which are predominantly [Ca2+] dependent. The proportions of monocyte–platelet aggregates (MPAs) attributable to individual monocyte subsets in fresh and promptly processed heparin-anticoagulated blood from 10 healthy subjects (median age 35 years, 50% male) were analysed by flow cytometry and compared to samples anticoagulated with a potent [Ca2+] chelator, ethylenediaminetetraacetic acid (EDTA). Additional experiments with [Ca2+] depletion or supplementation were also performed. Monocytes subsets were defined as CD14++CD16–CCR2+ cells (Mon1), CD14++CD16+CCR2+ cells (Mon2) and CD14+CD16++CCR2− cells (Mon3). Vast majority of monocytes showed aggregation with platelets in heparinised samples, but most monocytes were free of platelets when EDTA was used (p < 0.001 for all subsets). Addition of the heparinised blood to EDTA-containing vacutainers reduced the proportion of MPAs to values seen in the directly EDTA-anticoagulated blood (p = 0.005 for all subsets). Supplementation with CaCl2 resulted in dose-dependent increase in MPAs (p < 0.001 for all subsets). Although the overall trend for the monocyte–platelet interactions was applicable to all monocyte subsets, the proportion of MPAs in heparinised samples was lowest for Mon3 (p < 0.0001). In contrast, Mon3 showed the highest proportion of MPAs in EDTA-anticoagulated samples (p = 0.004). In healthy subjects monocytes circulate in constant, but predominantly reversible and [Ca2+]-dependent aggregation with platelets. These observations may reflect a complex involvement of platelets in regulation of monocyte activity.

Prevalence and diversity of Hepatozoon canis in naturally infected dogs in Japanese islands and peninsulas

Canine hepatozoonosis is a worldwide protozoal disease caused by Hepatozoon canis and Hepatozoon americanum and is transmitted by ixodid ticks, Rhipicephalus and Amblyomma spp., respectively. H. canis infection is widespread in Africa, Europe, South America, and Asia, including Japan. The objective of this study was to study the distribution pattern and diversity of H. canis in naturally infected dogs in nine Japanese islands and peninsulas. Therefore, 196 hunting dogs were randomly sampled during the period from March to September 2011 and the ages and sexes were identified. Direct microscopy using Giemsa-stained blood smears revealed H. canis gametocytes in the peripheral blood of 45 (23.6%) dogs. Polymerase chain reaction (PCR) was performed on EDTA-anticoagulated blood, initially with the common primer set (B18S-F and B18S-R) amplifying the 1,665-bp portion of the 18S rRNA gene, and then with the specific primer set (HepF and HepR) amplifying about 660 bp fragments of the same gene. Based on PCR, 84 (42.9%) dogs were positive using the common primer and 81 (41.3%) were positive using the specific primer. The current investigation indicated that all screened areas, except for Sado Island and Atsumi Peninsula, were infected. Yaku Island had the highest infection rate (84.6% in males and 100.0% in females), while Ishigaki Island showed the lowest infection rates (8.3% in males and 17.7% in females). Both sexes were infected with no significant difference. However, diversity of infection among the surveyed islands and peninsulas was significantly different (P < 0.05). Although H. canis has previously been reported in dogs in Japan, the higher infection rate described in the current study and the diversity of infection in a wide range of islands strongly encourage prospective studies dealing with the prevention and treatment of the infection in dogs, as well as control of ticks.

Botryoid nuclei in the peripheral blood of a dog with heatstroke

An EDTA-anticoagulated blood sample collected from a 1.5-year-old, intact male, English Bulldog was submitted for a CBC. The CBC data and blood smear evaluation revealed borderline high hematocrit (54%, reference interval 37-55%), inappropriate rubricytosis, moderate leukopenia due to both mature neutropenia and lymphopenia, and mild thrombocytopenia. Numerous leukocytes showed evidence of karyolysis, pyknosis, and karyorhexis, and apoptotic bodies were frequent in the background. Many neutrophils had botryoid nuclei characterized by increased numbers of nuclear segments radially arranged with spoke-like, delicate chromatin filaments connecting the segments centrally. The finding of botryoid nuclei and inappropriate rubricytosis was indicative of severe hyperthermia, such as heatstroke. The dog had been exercised a long time during conditions of high temperature and humidity until he collapsed. The dog was diagnosed with severe heatstroke, hypovolemic shock, disseminated intravascular coagulation, and multiorgan dysfunction syndrome. Despite aggressive treatment, the patient died of cardiopulmonary arrest. Botryoid nuclei are frequent in people with heatstroke. In the authors' experience, botryoid nuclei are seen commonly in dogs with heatstroke, but they have never been reported in veterinary medicine. The presence of petechiation with only mild thrombocytopenia and inappropriate rubricytosis also is suggestive of heatstroke and manifests ongoing life-threatening vascular derangement.

Role of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Refractory Anemias

AbstractAbstract 4406 Background:The presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in the setting of aplastic anemia (AA) and myelodysplastic syndrome (MDS) highlights the pathogenetic link between these disorders. The awareness that small clones of PNH cells could be detected by flow cytometry in aplastic anaemia patients who had negative Ham test has led to classifying PNH into two broad groups such as Haemolytic PNH which can have 50% presenting with thrombosis and a hypo plastic group which behaves like aplastic anaemia. Even if there is a small PNH clone in the setting of aplastic anaemia, then the patients respond better to immunosuppressive therapy.Flowcytometry is a relatively new advanced technique in many parts of India and there is no widespread consensus among methodology. Further, the clinical and prognostic significance of monitoring PNH clone size by serial flow cytometry is yet to be established here. Objective:To validate the role of flowcytometry in the diagnosis of PNH, in the setting of aplastic anaemia, myelodysplastic syndrome or abdominal or cerebral vein thrombosis in the setting of PNH which may help to prognosticate the course and treatment. Material and methods:The study was both retrospective and prospective. The prospective arm of the study was conducted from September 2009 to April 2010. Ham's test and Sucrose lysis test were done as screening tests and were reported as positive or negative. Other laboratory studies including hemogram and bone marrow analysis were done as part of standard care. In the retrospective group there were 32 patients who were diagnosed as PNH based on Ham's and Sucrose lysis test followed up from 1990.Four color flowcytometry immunophenotypic analysis with CD55 and CD59 was used to detect PNH clones in RBCs. EDTA-anticoagulated whole blood was stained with anti–CD 55-Phycoerythrin (PE) and anti–CD 59-PE.A minimum of 10,000 events were acquired. For analysis, RBCs were identified by light-scatter properties. For the purpose of the study, 3% of type II and/or type III cells were considered as threshold to diagnose PNH clones in both CD55 and CD 59 gated population. The results of laboratory studies including transfusion history and flow cytometry were correlated. Institutional ethical committee approval was obtained for the conduct of the study. Results:33 patients were included as part of the prospective group. The number of males was more compared to females (19:14). 78% of patients had hemoglobin of less than 7gm%. The presentation varied from unexplained anemia to thrombosis. Based on flow cytometry, 3 patients were found to have PNH clones in the RBC lineage by both CD 55 and CD 59. CD 55 alone picked up one more patient. In the retrospective group there were 32 patients who were followed up from 1990. Interestingly, two of them who had earlier presented with aplastic anemia had transformed into PNH in a span of 3years and 6 months. All patients were treated with Stanazolol and cyclosporine. The median survival was 12.14. months with Kaplan-Meier survival estimates of 30 and 20 percent at 121 months, and 143 months after diagnosis, respectively. The patients in the prospective group are being followed up. Discussion:The Ham test, sucrose lysis test, and modified Ham tests rely on the differential sensitivity of PNH red cells to haemolysis. These tests, although suitable for haemolytic PNH, cannot reliably detect small populations of PNH red cells nor differentiate partially and completely deficient cells. One patient who presented with DVT in whom increase in clone size was demonstrated by serial flowcytometry. Testing for the presence of PNH RBC clones provides additional diagnostic information and the thrombotic event may probably directly related to the size of the PNH clone.Conclusions: Our data demonstrate the utility of RBC assay in flow cytometry in the primary screening of PNH, AA, and MDS patients. The clinical and prognostic significance of monitoring PNH clone size by serial flow cytometry is relatively new and could be demonstrated in one patient. Disclosures:No relevant conflicts of interest to declare.

Assessment of Minimal Residual Disease Levels in B-CLL After Front-Line Treatment with FC-R and Rituximab Maintenance

AbstractAbstract 4599 Objective:To prospectively analyze change over time in minimal residual disease (MRD) in a cohort of 84 untreated patients diagnosed with CD20+ B-CLL who received from October 2007 to December 2010 six cycles of R-FC (Rituximab 375 mg/m2 IV in cycle 1 and 500 mg/m2 in cycles 2–6; F: 25 mg/m2, and C: 250 mg/m2 on days 1–3; every 28 days). Patients who achieved response were treated with rituximab 375 mg/m2 every 2 months for 3 years. Materials and methods:EDTA-anticoagulated peripheral blood (PB) and bone marrow (BM) samples were taken at diagnosis, after 3 and 6 cycles of R-FC, and every 6 months during the maintenance phase. MRD was centrally assessed by four-color multiparameter flow cytometry using the following antibody combinations: CD22/CD23/CD19/CD5, CD81/CD22/CD19/CD5, CD20/CD38/CD19/CD5, and CD20/CD79b/CD19/CD5. An acquisition was performed by selection of CD19+ cells/SSC including at least 200,000 events, reaching a sensitivity level of 0.01% (10-4). Results:PB/BM samples for assessing MRD were available from 28 of the 84 patients after 3 cycles of R-FC and from 79 patients after the 6 induction cycles, and 43 (58%) of the 74 patients on maintenance reached the interim study at 18 months of maintenance (9 cycles). Correlation between PB and BM was 57%, 68%, and 86% at the three study time points. After three R-FC cycles, 16 patients (56.8%) had a MRD- in PB, but only 4 patients (14%) also had MRD- in BM. After six R-FC cycles, 58 patients (73.3%) had a negative study in PB, and 41.7% also in BM. Among the 24 patients analyzed after the third cycle with BM+, 11 achieved negativization after 6 cycles, and MRD was negative in PB in 10 of 12 patients. After 18 months of maintenance, 11 patients (26.2%) converted to MRD- in PB/BM, and a single patient (2.4%) converted to MRD+. Conclusion:Six cycles of R-FC achieved better MRD- rates in PB and BM as compared to 3 cycles. Addition of rituximab as maintenance after R-FC increased the number of MRD-negative in BM, and in consequence the MRD-negative CRs. A good correlation was not found between MRD values in blood and bone marrow in the induction phase. However, these levels improved in the maintenance phase due to an increase in the number of cases with RMD-negative in BM witch were negatives in PB during induction phase. Disclosures:Off Label Use: R in Maintenance.

Monocyte Shift to a Non-Classical CD14dim/CD16+ Phenotype Correlates with Fetal Hemoglobin Levels in Sickle Cell Anemia Patients Treated with Hydroxyurea

AbstractAbstract 817Vaso-occlusion in sickle cell anemia (SCA) involves inflammation and cell activation; fetal hemoglobin (HbF) elevation by hydroxyurea (HU) remains the mainstay of SCA treatment. Monocytes are activated in SCA, and their contribution to the chronic inflammatory state includes the production of cytokines and reactive oxygen species (ROS). Monocytes are a heterogeneous group of leukocytes subdivided into distinct subsets: classical monocytes comprise over 80% of circulating monocytes, are highly positive for CD14 (CD14bright) and typically CD16-negative, while CD16-positive monocytes have been further subdivided into intermediate CD14bright/CD16+, and non-classical CD14dim/CD16+ monocytes. Intermediate monocytes are recognized as the main monocytic producers of ROS and are increased in inflammatory conditions such as atherosclerosis and sepsis. The less characterized non-classical subset is believed to have a patrolling behavior in blood vessels, does not produce ROS and constitutively produces IL-1 receptor antagonist (IL1-RA). Another relevant subgroup of monocytes expresses angiopoietin-2 receptor TIE2, and the role of TIE2-expressing monocytes (TEMs) has been investigated in angiogenesis in neoplastic diseases. TEMs usually correspond to intermediate monocytes, but their importance in inflammation is still unclear. We hypothesized that monocyte subsets in SCA patients would differ from controls, and that treatment with HU might also influence monocyte phenotypes, thus shedding light on the possible role of these subsets in an inflammatory condition not previously studied. EDTA-anticoagulated peripheral blood samples were collected upon written informed consent from 21 healthy controls (CON, ages 21–63 years) and 34 SCA patients (18 on HU, ages 16–58 years) in steady state, with no transfusion or acute sickling episode in the previous three months. Monocytes were immunophenotyped by flow cytometry on a multicolor FACSCalibur cytometer. Medical history of SCA-associated complications, HbF levels and dosage of HU in mg/kg/day were obtained from medical charts. Statistical analysis was performed on GraphPad Prism 5.0 software. As expected, we found that relative percentage and absolute count of CD16-positive monocytes were higher in SCA patients than in controls. Surprisingly, a significantly higher percentage of non-classical CD14dim/CD16+ monocytes, rather than intermediate cells, was found in SCA patients on HU (SCA-HU) treatment (mean±SEM: CON 2.06±0.43%, SCA 2.91±0.50%, SCA-HU 6.42±0.80%, P<0.0001). TEMs were also increased in SCA patients compared to controls (CON 2.64±0.72%, SCA 20.48±5.40%, SCA-HU 32.97±5.92%, P<0.0001), but HU treatment did not significantly influence TEM counts. Mean TIE2 expression did not vary among the groups, and there was no correlation between TEMs and presence of SCA complications pathophysiologically associated with disturbed angiogenesis, such as pulmonary hypertension, osteonecrosis, leg ulcers and retinopathy. Higher percentages of non-classical monocytes in HU-treated patients were initially interpreted as a possible toxic effect of HU on monocytopoiesis, but the lack of correlation of monocytes subsets with the degree of relative monocytopenia made this hypothesis unlikely. Moreover, we found a significant positive correlation between percentages of non-classical monocytes and HbF levels (rS=0.4763, P=0.0068, see figure). This suggests that successful HU treatment with higher HbF could correlate with the expansion of this particular monocyte subset. During the study period, only one patient was available for comparison before and after HU, but the increase in HbF from 4.2% to 11.6% and in non-classical monocytes from 1.82% to 9.48%, in this case, corroborates that HU therapy may explain this phenotype shift in monocytes. Whether non-classical monocytes expansion represents yet another pleiotropic effect of HU, if these cells are less likely to take part in the vaso-occlusive process and have an antiinflammatory role or, furthermore, if a bone marrow counterpart of this monocyte subset could be involved in increasing HbF production, remains to be investigated. The correlation of the expansion of non-classical monocytes with HbF levels could prove to be an interesting biomarker of response to HU, and future studies may address its clinical usefulness. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Point-of-Care Measurements of HbA1c: Simplicity Does Not Mean Laxity With Controls

Point-of-care HbA1c measurements (POC-A1Cs) have been adopted by many diabetes clinics to improve the quality of care provided to their patients (1). Herein, we show that reliability of this approach might be questioned. POC-A1Cs routinely used in the ambulatory section of our diabetes clinic was evaluated on 100 diabetic patients (type 1, n = 58; type 2, n = 42) attending the clinic from 1 October 2011 to 30 November 2011. Patients with abnormal hemoglobin traits or shortened erythrocyte life span were excluded. Blood-capillary samples were analyzed by POC-A1C (DCA Vantage; Siemens Medical Solutions Diagnostics, Cergy-Pontoise, France) and venous EDTA-anticoagulated blood specimens by the central laboratory high-performance liquid chromatography measurement (Tosoh HLC-723 GHb G8; BioSciences, Lyon, France). Both methods were certified (NGSP/Diabetes Control and Complications Trial [DCCT] and International Federation of Clinical …