Automated Quantitative Assessment of Bone Marrow in Normal Samples: Correlation with the Reference Method.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4192-4192
Author(s):  
Gina Zini ◽  
Mariagrazia Garzia ◽  
Antonella Di Mario ◽  
Elena Rossi ◽  
Giuliana Farina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Count ◽  
Blood Count ◽  
Hematopoietic Cells ◽  
Reference Method ◽  
Mean Value ◽  
White Blood Count ◽  
Quantitative Detection ◽  
Microscope Examination ◽  
Age Related

Abstract Bone marrow (BM) analysis is conventionally performed by microscope examination on films of about 0.3ml of aspirated bone marrow fluid, stained with Romanowsky dyes. Until 1996 the simple automated screening of marrow composition was made very difficult by a number of factors, mainly the lack of the erythroblasts quantitation and the fat interference. From 1996 last generation automated hematology analyzers provide accurate and precise erythroblasts counts; moreover same systems have improved their software reducing the problem of fat interference. We have analyzed data from 100 normal BM samples from patients submitted for diagnostic and/or follow up purposes in our Hematology Day Hospital. BM fluid was harvested from the superior posterior iliac crest. The first 0,3–0.5 ml were used for smears, while the next 1–2 ml of BM, collected into K3-EDTA, were analysed with Coulter LH 750, a fully automated hematology analyzer which provides Complete Blood Count, White Blood Count Differential included Nucleated Red Blood Cells (NRBC) and Reticulocytes count. We used the microscope examination conventionally performed on films stained with Romanowsky dyes as reference method. Quantitative detection BM cellularity was obtained by semi quantitative evaluation based on the evaluation of hematopoietic cells in several marrow particles: physiological differences age related were also taken in account. If hematopoietic cells occupy less than 25% or more than 85% the sample is defined respectively hypocellular or hypercellular (none of our sample was as). Differential cell count was usually performed on two different slides counting 500 cells (1000 when hypercellular, but none of our sample was as). We found a strict correlation between microscope semi-quantitative cellularity evaluation and the instrumental cell count as sum of WBC plus NRBC, the Total Nucleted Cell Count (TNCC). The mean value of the TNCC in normal PM samples was 29,48 x109/L with a range 25,9–54,9 x109/L. These results are in good agreement with normal BM cell count reported in the literature using a cytofluorimetric method, which is 34,5 x109/L (SD28.0). The instrumental mean percentage of BM granulocytes corrected for TNNC was 62% (range: 23,5–93,7) versus a mean microscope percentage of 58,42% (range: 40–72). The automated NRBC BM count corrected for TNCC was 11,38% (range: 2,7 – 39,17) versus a microscopic mean value of 28% (range: 9–45). These results, including the slight NRBC underestimation probably due to partial mature cell lysis, are in line with the data of the literature. This study confirms the feasibility of routine automated cell count using a hematology in normal BM fluid samples. Automated methods will support morphologists quickly providing accurate and precise quantitative information such as TNCC and myeloid/erythroid ratio.

scholarly journals Acute myeloid leukaemia: an unusual cause of biliary strictures

2019 ◽  
Vol 12 (3) ◽  
pp. e227821
Author(s):  
Adele Beck ◽  
Hannah Hunter ◽  
Simon Jackson ◽  
David Sheridan
Keyword(s):  
Bone Marrow ◽  
Obstructive Jaundice ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Blood Count ◽  
Extrahepatic Bile Duct ◽  
White Blood Count ◽  
Full Blood Count ◽  
Significant Past Medical History ◽  
Acute Myeloid

A 17-year-old man with no significant past medical history presented with a 2-week history of worsening jaundice, lethargy, anorexia and progressive right upper quadrant abdominal pain. There were no stigmata of chronic liver disease. Initial investigations were suggestive of cholangitis with large intrahepatic and extrahepatic bile duct strictures but otherwise normal hepatic and splenic appearances. A percutaneous transhepatic cholangiogram with the positioning of drains was performed to alleviate the obstructive jaundice. Within 2 weeks of the first presentation, full blood count revealed a significantly raised white blood count and a subsequent peripheral blood smear and bone marrow were consistent with a diagnosis of acute myeloid leukaemia. Chemotherapy was started after partial improvement of his obstructive jaundice. Complete morphological and cytogenetic remission was obtained 4 weeks after the first cycle of chemotherapy (half dose of daunorubicin and full dose of cytarabine, treated off trial) on control bone marrow. The patient remains in remission.

The Expression and Clinical Significance of PRAME Gene in Acute Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4225-4225
Author(s):  
Rong Fu ◽  
Kai Ding ◽  
Zonghong Shao
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Clinical Significance ◽  
Blood Count ◽  
Mononuclear Cells ◽  
Residual Disease ◽  
White Blood Count ◽  
Gene Expressions ◽  
Healthy Donors ◽  
Blast Cells

Abstract Objective To investigate the expression of PRAME (preferentially expressed antigen of melanoma) gene in acute leukemia and its clinical significance in monitoring prognosis, detecting minimal residual disease (MRD) and gene immunotherapy. Methods The expression of PRAME gene mRNA in bone marrow mononuclear cells is measured by reverse transcriptase polymerase chain reaction in 34 patients with acute leukemia and 12 bone marrow samples of health donors. The relationships between PRAME gene expressions and some clinical data, such as gender, age, white blood count, leukemic immunophenotype, the percentage of blast cells, and the karyotype of chromosome, were also estimated. Results PRAME gene was expressed in 38.2% of all the patients, 40.7% of all the AML patients, which was higher than the 28.6% of ALL patients (p >0.05). There was no expression of PRAME gene in healthy donors. In all the sub phenotypes of AML, the expressive rate of PRAME gene in M3 patients is 80%, which is higher than that in M2 (33.3%) and in M5 (28.6%). The expressive rate of PRAME gene was also positively correlated with the expression of CD15, CD33, and the abnormality in the karyotype of chromosome, but not correlated with age, gender, white blood count and percentage of blast cell in bone marrow. Conclusion PRAME gene is highly expressed in acute leukemia, and could be regarded as a useful tool for monitoring MRD. Differential expression in acute leukemia patients vs. healthy donors suggests that the immunogenic antigens PRAME are potential candidates for immunotherapy in acute leukemia.

P190-B RhoGAP Is Critical for Hematopoietic Stem Cell Niche Regulation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 221-221
Author(s):  
Rachna Raman ◽  
Ashwini Hinge ◽  
Rupali Kumar ◽  
Juying Xu ◽  
Kathleen Szczur ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Cultures ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Blood Count ◽  
Negative Regulator ◽  
White Blood Count ◽  
Hematopoietic Stem ◽  
Regulatory Pathways ◽  
Cell Functions

Abstract Abstract 221 Hematopoiesis is regulated by components of the stromal microenvironment, so-called niche. Although the concept of hematopoietic stem and progenitor cell (HSC/P) niche is well known, its molecular regulation remains ill-defined. Here, we provide evidence that p190-B GTPase Activating Protein (p190-B), a negative regulator of Rho activity, is a regulator of mesenchymal/stromal cell functions necessary for normal hematopoiesis during fetal development. Mice lacking p190-B die before birth. At day 14.5 post coitum, p190-B−/− embryos are paler than WT embryos with a 40% lower hematocrit. Cellularity and numbers of burst forming-unit (BFU) erythroid (E), colony forming-unit (CFU)-granulo-monocytic and CFU-erythroid per p190-B−/− fetal livers (FL) were 50% lower than WT. In addition, the frequency of LongTerm-HSC (LinnegScaposKitposCD150posCD48neg), ShortTerm-HSC (LSK-CD150posCD48pos), and progenitors (LK) appeared significantly reduced in the bone marrow of e17.5/18 embryos. These data suggest a defect in fetal hematopoiesis. However, p190-B−/− FL cells were able to fully reconstitute hematopoiesis of adult irradiated recipients (Xu, Blood 2009). Mice reconstituted with p190-B−/− FL cells exhibited bone marrow content, white blood count, red blood count and hematocrit similar to those reconstituted with WT cells. Furthermore, hematopoietic recovery of p190-B−/− reconstituted animals following 5-Fluorouracil or phenylhydrazine-induced stress was comparable to that of WT. Therefore, FL HSC/P retain hematopoietic potential, which suggests a non-cell autonomous defect in p190-B−/− embryos. In the present study, we examined in more detail this possibility. The numbers of CFU-fibroblast (CFU-F) were 2-fold lower in p190-B−/− FL compared to WT FL. We next examined the hematopoietic supportive capacity of p190-B−/− FL microenvironment using stromal cell cultures derived from e14.5 FL. No noticeable differences were observed during the establishment of the stromal cell cultures. p190-B−/− stromal cells exhibited a spindle-like shape and expressed CD90, CD44 and Sca-1 but not CD45, CD31 or CD11b similarly to WT stromal cells. Cobblestone Area Forming Cell assays (CAFC) was performed in co-culture between various number of WT BM cells and p190-B−/− and WT stromal cells. We examined 5 independent stromal cell cultures from both genotypes. Frequency of CAFC was 75% lower on p190-B−/− stroma than WT stroma at both 1 week (1 in 5 × 10 3 versus 1 in 21 × 10 3, n = 5 p < 0.01) and 2 weeks (1 in 21 × 10 3 versus 1 in 87 × 10 3, n = 4 p < 0.01) in culture. CFU with cells recovered from one week coculture was also performed. Cocultures with p190-B−/− stroma gave rise to 2-fold less CFU than with WT stroma (538+90 versus 255+81, p < 0.01, one representative experiment of 4). Using competitive repopulation assay, we then assessed the in vivo repopulation ability of CD45.2+ hematopoietic cells co-cultured for one week on each stroma. Fifteen weeks following transplantation, the frequency of CD45.2+ cells in the peripheral blood of mice that received cells cocultured on p190-B−/− stroma was dramatically reduced compared to mice that received cells cocultured on WT stroma (0.43% + 0.37 versus 32.4% + 7.5, n = 6, p < 0.01), equivalent to a 100-fold difference in calculated competitive repopulation unit. Therefore, p190-B−/− stroma exhibited defective hematopoietic supportive activity. Interestingly, p190-B−/− stromal cells, like WT, showed characteristic of mesenchymal stem cells (MSC), i.e. ability to give rise to CFU-F and to differentiate to adipocytes and osteoblasts. They express relatively high level of nestin and osteopontin. At a mechanistic level, gene expression analysis of molecules known to play a critical role in HSC maintenance in the niche indicated 200-fold downregulation of Kit-ligand and Wnt3a in p190-B−/− stroma compared to WT stroma (p<0.05). Surprisingly, BMP4, angiopoietin and CXCL12 were upregulated in p190-B−/− stroma, 35-fold, 14-fold and 16-fold, respectively (p<0.05). Hence, p190-B expression in MSC/stromal cell microenvironment fine-tunes niche regulatory pathways to maintain normal hematopoiesis. Therefore, p190-B may be a critical regulator of the mesenchymal stem/hematopoietic stem and progenitor niche. Disclosures: No relevant conflicts of interest to declare.

Plasma Level of Micro-RNA92-a in Acute Myeloidleukemia and Its Correlation with Prognostic Factors and Therapeutic Response

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4826-4826
Author(s):  
Ahmed Galal
Keyword(s):  
Bone Marrow ◽  
Prognostic Factors ◽  
Plasma Level ◽  
Mirna Expression ◽  
Blood Count ◽  
Therapeutic Response ◽  
Statistical Significance ◽  
White Blood Count ◽  
Control Group ◽  
Blast Cells

Abstract Abstract 4826 Background: Acute myelogenous leukemia (AML) is a clonal, malignant disease of hematopoietic tissues that is characterized by accumulation of abnormal blast cells, principally in the marrow, and impaired production of normal blood cells. MicroRNAs (miRNAs) are short non-coding RNAs of ∼ 21 to 23 nucleotides in length that post-transcriptionally regulate mRNA expression. High-throughput methodologies have shown deregulated miRNA expression in an increasing number of human cancers including colon, breast, lung, thyroid, cervical, ovarian and pancreatic cancers, and miRNA expression patterns have been found to distinguish tumors of different developmental origin, better even than traditional mRNA expression profiling. miR-92a is transcribed from miR-17–92 locus that encode the polycistronic precursor containing seven microRNAs:miR-17-5p, miR-17-3p, miR-18, miR-19a, miR-20, miR-19b andmiR-92a, and the human microRNA cluster miR-17–92 is amplified and/or overexpressed in cells of several cancer such as malignant lymphoma, lung cancer, thyroid cancer, and hepatocellular carcinoma. Aim: To assess plasma level of micro-RNA 92a in adult acute myeloid leukemia and to correlate it with prognostic factors and therapeutic response. Patients and Methods: This study was carried out on twenty five subjects with AML admitted to hematology unit in Alexandria university hospital as patients group as well as twenty five healthy subjects as control group. Conventional cytogenetics and FLT3/ITD gene mutation using PCR were done to patients group while measurement of plasma level of miR-92a using TaqMan quantitative RT-PCR and miR-638 as standardization was done to both groups. Therapeutic response was assessed in patients group by doing bone marrow aspirate at day 28 of induction chemotherapy. Results: The ratio of plasma miR-92a to miR-638 in patient group had a mean of 0.239±0.286 while in control group it had a mean of 0.969±0.490 that confirmed statistical significance (p=0.001*). Also there was significant negative correlation between RQ of miR-92a and white blood count in patient group (p= 0.001*). Patients who achieved complete response after induction chemotherapy had a mean RQ higher than non-responder (0.274±0.322 versus 0.073± 0.077 respectively) but with no statistical significance (p=0.212). Also there was no significant correlation between RQ of miR-92a and FLT3/ITD, cytogenetics and bone marrow blasts on admission. Summary/Conclusions: Plasma level of miR-92a is decreased in newly diagnosed AML patients than normal subjects, and it was related to high white blood count. If we correlate these findings with other studies that suggest that microRNAs are packaged inside exosomes that are secreted from cells to be delivered to other cells and function in a new location. Thus, it might be possible that blast cells specifically take in the exosomes that contain miR-92a and, as a result, miR-92a decreases from the plasma. This may justify the possibility of its usage in the prognosis of AML. Our data suggest the potential importance of the microRNAs as noninvasive cancer biomarkers helping in diagnosis, clinical prediction and therapeutic response. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Remethylation of Dnmt3a−/− hematopoietic cells is associated with partial correction of gene dysregulation and reduced myeloid skewing

2020 ◽  
Vol 117 (6) ◽  
pp. 3123-3134 ◽  
Author(s):  
Shamika Ketkar ◽  
Angela M. Verdoni ◽  
Amanda M. Smith ◽  
Celia V. Bangert ◽  
Elizabeth R. Leight ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Methyltransferase ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Dominant Negative ◽  
Older Individuals ◽  
Loss Of Function ◽  
Dna Hypomethylation ◽  
Age Related ◽  
Marrow Cells

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a−/− bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a−/− hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a−/− mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic “state” created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

scholarly journals Pediatric MDS and bone marrow failure-associated germline mutations in SAMD9 and SAMD9L impair multiple pathways in primary hematopoietic cells

Leukemia ◽  
2021 ◽  
Author(s):  
Melvin E. Thomas ◽  
Sherif Abdelhamed ◽  
Ryan Hiltenbrand ◽  
Jason R. Schwartz ◽  
Sadie Miki Sakurada ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Failure ◽  
Transcriptional Profiling ◽  
Hematopoietic Cells ◽  
Protein Translation ◽  
Germline Mutations ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Cellular Environment ◽  
Primary Mouse

AbstractPediatric myelodysplastic syndromes (MDS) are a heterogeneous disease group associated with impaired hematopoiesis, bone marrow hypocellularity, and frequently have deletions involving chromosome 7 (monosomy 7). We and others recently identified heterozygous germline mutations in SAMD9 and SAMD9L in children with monosomy 7 and MDS. We previously demonstrated an antiproliferative effect of these gene products in non-hematopoietic cells, which was exacerbated by their patient-associated mutations. Here, we used a lentiviral overexpression approach to assess the functional impact and underlying cellular processes of wild-type and mutant SAMD9 or SAMD9L in primary mouse or human hematopoietic stem and progenitor cells (HSPC). Using a combination of protein interactome analyses, transcriptional profiling, and functional validation, we show that SAMD9 and SAMD9L are multifunctional proteins that cause profound alterations in cell cycle, cell proliferation, and protein translation in HSPCs. Importantly, our molecular and functional studies also demonstrated that expression of these genes and their mutations leads to a cellular environment that promotes DNA damage repair defects and ultimately apoptosis in hematopoietic cells. This study provides novel functional insights into SAMD9 and SAMD9L and how their mutations can potentially alter hematopoietic function and lead to bone marrow hypocellularity, a hallmark of pediatric MDS.

Identifying Targets for the Prevention of Childhood Undernutrition in a Resource-Limited Peri-Urban Ecuadorian Community

2021 ◽  
pp. 037957212098250
Author(s):  
Suzanna L. Attia ◽  
Wolf-Peter Schmidt ◽  
Janeth Ceballos Osorio ◽  
Thomas Young ◽  
Aric Schadler ◽  
...  
Keyword(s):  
Vitamin A ◽  
Blood Count ◽  
Complete Blood Count ◽  
Dietary Diversity ◽  
Demographic Data ◽  
Serum Zinc ◽  
White Blood Count ◽  
Monthly Income ◽  
Vitamin Deficiencies ◽  
Dietary Diversity Score

Background: In middle-income countries, malnutrition concentrates in marginalized populations with a lack of effective preventive strategies. Objective: Identify risk factors for undernutrition in a peri-urban Ecuadorian community of children aged 12 to 59 months. Methods: Data from a cross-sectional survey in 2011 of children 1 to 5 years were analyzed including demographic data, medical history and examination, food frequency questionnaire (FFQ), anthropometric measurements, and blood for complete blood count, C-reactive protein, vitamin A, iron, and zinc levels. Dietary Diversity Score (DDS) was calculated from FFQ. Bivariate and multivariate analysis assessed effects on primary outcome of undernutrition by DDS, vitamin deficiencies, and demographic and nutritional data. Results: N = 67, 52.2% undernourished: 49.3% stunted, 25.4% underweight, and 3% wasted; 74.6% (n = 50) were anemic and 95.1% (n = 39) had low serum zinc. Dietary Diversity Score was universally low (mean 4.91 ± 1.36, max 12). Undernutrition was associated with lower vitamin A levels (20 306, IQR: 16605.25-23973.75 vs 23665, IQR: 19292-26474 ng/mL, P = .04); underweight was associated with less parental report of illness (43.8%, n = 7 vs 80% n = 40, P = .005) and higher white blood count (13.7, IQR: 11.95-15.8 vs 10.9, IQR: 7.8-14.23 × 109/L, P = .02). In multiple regression, risk of undernutrition decreased by 4% for every $10 monthly income increase (95 CI%: 0.5%-7.4%, P = .02, n = 23); risk of underweight decreased by 0.06 for every increased DDS point (adjusted odds ratio: 0.06; 95 CI%: 0.004-0.91, P = .04, n = 23). Conclusions: In this peri-urban limited resource, mostly Indigenous Ecuadorian community, stunting exceeds national prevalence, lower monthly income is the strongest predictor of undernutrition, lower DDS can predict some forms of undernutrition, and vitamin deficiencies are associated with but not predictive of undernutrition.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

Sign in / Sign up

Export Citation Format

Share Document