Human Induced Pluripotent Stem Cells: Clinical Significance and Applications in Neurologic Diseases

The generation of human induced pluripotent stem cells (iPSCs) from somatic cells using gene transfer opens new areas for precision medicine with personalized cell therapy and encourages the discovery of essential platforms for targeted drug development. iPSCs retain the genome of the donor, may regenerate indefinitely, and undergo differentiation into virtually any cell type of interest using a range of published protocols. There has been enormous interest among researchers regarding the application of iPSC technology to regenerative medicine and human disease modeling, in particular, modeling of neurologic diseases using patient-specific iPSCs. For instance, Parkinson’s disease, Alzheimer’s disease, and spinal cord injuries may be treated with iPSC therapy or replacement tissues obtained from iPSCs. In this review, we discuss the work so far on generation and characterization of iPSCs and focus on recent advances in the use of human iPSCs in clinical setting.

Human Induced Pluripotent Stem Cells: Clinical Significance and Applications in Neurologic Diseases

The generation of human induced pluripotent stem cells (iPSCs) from somatic cells using gene transfer opens new areas for precision medicine with personalized cell therapy and encourages the discovery of essential platforms for targeted drug development. iPSCs retain the genome of the donor, may regenerate indefinitely, and undergo differentiation into virtually any cell type of interest using a range of published protocols. There has been enormous interest among researchers regarding the application of iPSC technology to regenerative medicine and human disease modeling, in particular, modeling of neurologic diseases using patient-specific iPSCs. For instance, Parkinson’s disease, Alzheimer’s disease, and spinal cord injuries may be treated with iPSC therapy or replacement tissues obtained from iPSCs. In this review, we discuss the work so far on generation and characterization of iPSCs and focus on recent advances in the use of human iPSCs in clinical setting.

An Automated Draft Report Generator for Peripheral Blood Smear Examinations Based on Complete Blood Count Parameters

BACKGROUND: Complete blood count (CBC) results play an important role in peripheral blood smear (PBS) examinations. Many descriptions in PBS reports may simply be translated from CBC parameters. We developed a computer program that automatically generates a PBS draft report based on CBC parameters and age- and sex-matched reference ranges. METHODS: The Java programming language was used to develop a computer program that supports a graphical user interface. Four hematology analyzers from three different laboratories were tested: Sysmex XE-5000 (Sysmex, Kobe, Japan), Sysmex XN-9000 (Sysmex), DxH800 (Beckman Coulter, Brea, CA, USA), and ADVIA 2120i (Siemens Healthcare Diagnostics, Eschborn, Germany). Input data files containing 862 CBC results were generated from hematology analyzers, middlewares, or laboratory information systems. The draft reports were compared with the content of input data files. RESULTS: We developed a computer program that reads CBC results from a data file and automatically writes a draft PBS report. Age- and sex-matched reference ranges can be automatically applied. After examining PBS, users can modify the draft report based on microscopic findings. Recommendations such as suggestions for further evaluations are also provided based on morphological findings, and they can be modified by users. The program was compatible with all four hematology analyzers tested. CONCLUSIONS: Our program is expected to reduce the time required to manually incorporate CBC results into PBS reports. Systematic inclusion of CBC results could help improve the reliability and sensitivity of PBS examinations.

The Seroprevalence of IgM and IgG Antibodies to Helicobacter pylori Among Maternal, Neonatal, and Cord Bloods in Korea / 임상검사와정도관리

BACKGROUND: Helicobacter pylori (H. pylori) infection has been known closely related with gastritis, duodenal ulcer and gastric cancer and is prevalent among Koreans. However, the infection route and the time are unclear, especially during perinatal period. The aim of this study is to investigate the relationship of H. pylori IgG and IgM antibody prevalences and titers between maternal, neonatal, and cord blood. METHODS: We collected 45 simultaneous maternal, neonatal, and cord bloods and 150 single cord bloods during delivery. The specific H. pylori IgG and IgM antibody levels were measured by enzyme-linked immunosorbent assay (ELISA) method. RESULTS: The H. pylori IgG antibody-positive rate for maternal, neonatal, and cord bloods were equal as 35.6% (16/45). The H. pylori IgG antibody levels of neonatal and cord bloods were 52.7% and 70.7% of maternal blood level. The H. pylori IgG antibody levels between maternal and cord bloods (r2 = 0.9725, p<0.05), maternal and neonatal bloods (r2 = 0.8569, p<0.05), and neonatal and cord bloods (r2 = 0.9437, p<0.05) were well correlated. Only one case of maternal blood was H. pylori IgM antibody positive and it's antibody level was 52.3 U/mL. CONCLUSIONS: In this study, we provided the sero-prevalence of H. pylori IgG and IgM antibodies and the relationship of antibody level of H. pylori IgG in maternal, neonatal and cord bloods. To elucidate the exact route and time of H. pylori infection, further studies including serial measurement of H. pylori IgG and IgM level in neonates will be needed.

Suggestion of Maximum Surgical Blood Order Schedule for Major Elective Surgeries in Korea / 대한수혈학회지

BACKGROUND: There have been many efforts to determine maximum surgical blood order schedule (MSBOS) of elective surgery in Korea since 1980?. But most of these data were confined to single hospital, so it was somewhat irrelevant to suggest them as general data in Korea. In this study, we tried to establish MSBOS which can be suggested as substantial and objective data in our country and to increase the efficiency of limited blood products. METHODS: From September 2003 to February 2004, we analyzed average RBC utilization for elective surgery in four university hospitals (Soonchunhyang University Hospital, Soonchunhyang University Bucheon Hospital, Korea University Ansan Hospital, Ajou University Hospital) by referring to anesthesiologic records and blood delivery sheets. Also, we established MSBOS range in operations performed more than five times in three among four hospitals. RESULTS: Sixty-seven operations were analyzed in our study and 18 of them were performed in department of general surgery. Type and screen (T&S) was recommended in 69.8~83.6% and the percentage of operations recommended as T&S in all four hospitals was 52.2%. Operations of obstetric/gynecology, urology and otorhinolaryngology were mostly applicable to T&S. CONCLUSION: We suggested comprehensive and substantial MSBOS range by analyzing the operations performed in four hospitals whose operation numbers ranged from 470 to 1,200 per month. It was considered that MSBOS range, which could be applicable to various hospitals, should be reestablished and enforced regularly to decrease medical cost and improve blood utility

The Reduction Effect of Leukocyte Fragments by Leukodepletion Filter / 대한수혈학회지

BACKGROUND: The efficiency of leukocyte removal filter is influenced by many factors. But, filtration efficiency of leukocyte fragments was not well known. We performed this study to evaluate whether the filtration efficiency for packed red blood cells can be influenced by leukocyte fragments according to storage time. METHODS: Leukocyte fragments in packed red blood cells (three units) which were artificially made by incubation for 4 hrs at 56degrees C and each four units of packed red blood cells according to storage time (0 days, 10 days, 20 days, and 30 days) were filtered using Sepacell R-500A (Asahi medical Co, Japan). The leukocyte concentrations of the pre-leukodepleted samples were estimated using an automated hematology analyzer (XE-2100, Sysmex, Japan). The ratio between the number of normal leukocytes and leukocyte fragments on Wright Giemsa stained slide was used in the analysis. The leukocyte concentrations of the post-leukodepleted samples were performed by the conventional counting methods using Nageotte hemocytometer. RESULTS: The ratios of fragmented to total leukocytes in packed red blood cells at pre- and post leukoreduction according to storage times were 1.5% and 16.3% within 1 days, 4.5% and 30.0% at 10 days, 6.3% and 35.0% at 30 days, and 8.3% and 42.5% at 40 days, respectively. Leukoreduction efficiencies of normal leukocytes in packed red blood cells were 99.99 +/- 0.01%, 99.97 +/- 0.02%, 99.98 +/- 0.01%, and 99.86 +/- 0.09%, respectively. The 36.0% of leukocytes in packed red blood cells were changed to fragmented leukocytes, residual fragmented leukocytes ratio was 95.0% and filter efficiencies of normal leukocytes was low(99.28%, p<0.05). CONCLUSIONS: The leukodepleted efficiency for leukocyte fragments were lower than for normal leukocytes. Leukocytes fragments may be influenced to lower the leukodepleted efficiency of normal leukocytes with storage time elapse.

Two Cases of ABO Discrepancy due to Hypogammaglobulinemia / 대한수혈학회지

Two cases of ABO discrepancy were observed in thirty-year old woman with gall bladder abscess and fifty-five-year old woman with hepatocellular carcinoma. Their red cells were typed as group O and their serum had only anti-A antibody. Absence of A and B antigens on their RBCs were confirmed by adsorption elution test and saliva test. The B transferase activities were not demonstrated in their serum. Their ABO genotypes were O/O by sequence specific polymerase chain reaction. Their serum protein electrophoresis showed hypogammaglobulinemia pattern, and immunoglobulin levels (IgG, IgA, IgM) were decreased (39 mg/dL, 46 mg/dL, <5 mg/dL and 63 mg/dL, 65 mg/dL, 12 mg/dL, respectively).

Evaluation of the Ez step FOBTM for Fecal Occult Blood Test / 임상검사와정도관리

BACKGROUND: Fecal occult blood tests (FOBT) have been recommended for gastro- intestinal bleeding and colon cancer screening. This study compared the effectiveness of two fecal blood screening kit, OC-Hemodia II and Ez step FOB for fecal occult blood. METHODS: The detection limit was evaluated by using OC-control and serially diluted samples. The comparison study between OC-hemodia II and Ez step FOB were evaluated in 143 cases. RESULTS: The concordance rate between OC-Hemodia II and Ez step FOB was 85.6% and 50% of non-concordance cases have history related to gatrointestinal bleeding. Ez step FOB was possible to detect 35 ng/mL in serially diluted OC-control. CONCLUSIONS: The result of Ez step FOB satisfactory to clinical application and showed good concordance rate compared to OC-Hemodia II.

Evaluation of leukoreduction filter Bio R02 plusTM for packed RBC / 대한수혈학회지

BACKGROUND: As universal WBC filtration of RBCs prior to storage is currently under consideration, few data are available on the performance of WBC-reduction filtration in routine practice. The pre-leukodepletion is thought to minimize the incidence of transfusion associated adverese effects such as HLA alloimmunization, non-hemolytic febrile reactions, platelet refractoriness, transfusion associated graft versus host disease and transmission of infections. The aim of this study was to evaluate the one of pre-storage and bedside leukocyte removal filter, leukocytes Bio R02 plusTM (Fresenius HemoCare, Germany) for packed RBCs. METHODS: In order to evaluate leukocyte removal filter of leukocytes Bio R02 plusTM, thirteen units of red blood cells were prepared and filtered using Bio R02 plusTM, and were measured the WBC count, RBC count, volume, sodium, and pH before and after filtration, respectively and the reduction power of white blood cell and biochemical changes in red blood cell units were analysed. RESULTS: There were 99.99% reduction in WBC counts, residual leukocyte content of 0.06 +/- 0.08 106 per unit and 8.69% of RBC loss after filtration, and there was no difference in the sodium and pH. CONCLUSION: The leukocyte removal filter Bio R02 plusTM showed sufficient leukocyte removal and RBC recovery efficiency for RBC units.

A Case of Knee Joint Infection by Mycobacterium abscessus / 대한임상병리학회지

Rapid growing mycobacterium grows in less than 7 days on most types of solid media including the Ogawa media. Ninety percent of human diseases caused by rapid growing mycobacterium are due to Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium fortuitum. We report an isolated case of wound infection due to M. abscessus following total knee replacement arthroplasty surgery. The patient has presented arthralgia and fever for 3 weeks. From the joint fluid aspirates, pale gram-positive beaded rods were found but cultures were negative after 24 hours. After 48 hours, microorganisms grew on blood agar plates as tiny pinpoint colonies and they had odor of freshly-turned soil. They gave a positive reaction in a partial acid fast, an acid-fast stain and a heat-stable catalase but gave a negative reaction to PCR for IS6110. They were identified as the M. chelonae group biochemically and confirmed as M. abscessus through PCR-restriction fragment length polymorphism using restriction endonuclease, BstE II. Because rapid-growing mycobacterium can grow on a blood agar plate, an acid-fast stain should be selectively conducted in addition to a Gram stain in a microbiology laboratory.

A Case of Juvenile Myelomonocytic Leukemia in a Six Months Old Infant / 대한혈액학회지

Myelodysplastic syndrome (MDS) in childhood is a rare hematologic malignancy and its classification has been the subject of some controversy. Cases of pediatric MDS are subdivided into those with features of adult-type MDS and those with myeloproliferative features occasionally observed in infancy and early childhood. There appears to be an international consensus to rename the disease juvenile myelomonocytic leukemia (JMML), which includes all leukemias of childhood previously classed as chronic myelomonocytic leukemia (CMML), juvenile chronic myelogenous leukemia (JCML), and infantile monosomy 7 syndrome. We experienced a 6-month-old female infant with JMML who developed extensive extramedullary hematopoiesis. The patient developed abdominal distention, hepatosplenome-galy, anemia, thrombocytopenia, and leukocytosis with significant monocytosis and was found to have a high hemoglobin F level of 30%. Her bone marrow biopsy section and aspirate smears revealed normocellularity with no increment of blast cells and no dysplastic changes. Cytogenetic analysis revealed a normal 46, XX karyotype. Her liver, spleen, lymph nodes, and appendix were found to be heavily infiltrated by partially differentiated myelomonocytic cells. These findings supported the diagnosis of JMML with extensive extramedullary hematopoiesis.

Clinical Significance of p53, MDM2 and Cyclin D1 Protein Expression in Patients with Multiple Myeloma / 대한혈액학회지

BACKGROUND: Dysregulation of cell proliferation contributes to the pathogenesis of multiple myeloma (MM) and the number of S phase plasma cells is known to be one of the most important prognostic factors in MM. We analysed the cell cycle progression in MM using the expression of G1/S phase cell cycle regulators, such as p53, murine double minutes (MDM2) and cyclin D1. METHODS: The expressions of p53, MDM2 and cyclin D1 were evaluated by immunohistochemical staining with monoclonal antibodies, using bone marrow sections obtained from 48 patients with MM and 20 normal controls. RESULTS: The expressions of p53, MDM2 and cyclin D1 were demonstrated in 17 (35.4 %), 40 (83.3%) and 28 (58.3%) of 48 patients with MM, respectively. The expressions of cyclin D1 and p53 were positively correlated each other (P<0.05). However, no significant difference in MDM2 expression was found between the cyclin D1-positive and -negative groups. All of the control group showed negative expression. The expression of cyclin D1 and p53 in patients with MM correlated well with clinical and histologic stages (P<0.05). Even if MDM2 was upregulated in most patients with MM, no correlation was found with clinical or histologic stages. Serum beta2-microglobulin levels were reversely correlated with p53 expression, not with MDM2 or cyclin D1. After chemotherapy, all 5 patients with objective response showed decreased staining of these three proteins, comparing 10 of 13 patients with partial response or treatment failure showed no change or an increased degree of staining. No differences were observed in the survival rates between the groups with and without expression of each three proteins. CONCLUSIONS: These findings suggest that the expression of p53, MDM2 and cyclin D1 was increased in patients with MM and the expression rates of p53 and cyclin D1 were increased with the progression of the clinical and histological stages. It is considered that the detection of cell cycle regulatory proteins are important for understanding the biology of the malignant plasma cells, monitoring the results of treatment and determining the prognosis in MM.

Serum Eosinophil Cationic Protein Levels in Patients with Allergic Diseases / 대한임상병리학회지

BACKGROUND: Eosinophil cationic protein (ECP), one of the eosinophil granule proteins released during allergic reactions, may play a major role in the allergic inflammatory process. The measurement of ECP in serum may be a useful indicator of eosinophil activity in ongoing inflammatory processes. We investigated the clinical utility of ECP measurement in serum in patients with bronchial asthma, allergic rhinitis and atopic dermatitis, after standardizing sample processing. METHODS: We measured the serum ECP levels in patients with bronchial asthma (n=38), chronic obstructive pulmonary diseases (COPD) (n=13), respiratory symptoms (n=19), allergic rhinitis (n=26), non-allergic rhinitis (n=24), and atopic dermatitis (n=10) and in normal healthy controls (n=16) by the fluoroenzyme immunoassay using Pharmacia CAP System, and evaluated the correlation between ECP level and blood eosinophil number, or ECP and IgE levels. Blood eosinophil number was counted by the automated cell counter. RESULTS: Serum ECP levels were significantly higher in patients with bronchial asthma (15.6+/- 12.6 g/L), COPD (13.3+/-7.2 g/L), allergic rhinitis (23.8+/-13.2 g/L), and atopic dermatitis (20.6+/- 18.4 g/L) than in normal controls (7.5+/-4.2 g/L) (P <0.05). ECP levels were also significantly higher in patients with bronchial asthma and COPD than in patients with simple respiratory symptoms (6.9+/-4.7 g/L), whose ECP levels did not statistically differ from those in normal controls. ECP levels were also significantly higher in patients with allergic rhinitis than in patients with non-allergic rhinitis (9.5+/-5.1 g/L), whose ECP levels did not statistically differ from those in normal controls. Serum ECP level and eosinophil number in peripheral blood were correlated only in patients with bronchial asthma (r=0.53, P <0.01) and no correlation between ECP and IgE levels was found in all of the patients. CONCLUSIONS: ECP is the one of the secretory components released from the eosinophil granule and measurement of ECP in serum might be one of the noninvasive tool to assess the activity in relation to eosinophil involvement in various allergic diseases.

An Evaluation of Gel Test for Irregular Antibody Screening / 대한수혈학회지

BACKGROUND: The application of gel test to routine immunohematologic works brought on easier interpretation of results and better quality control over conventional tube method. Under the current Korean medical insurance system however, it is very difficult to apply gel test to all routine immunohematologic works because of high cost. We tried to assess its applicability to irregular antibody screening. MATERIALS AND METHODS: Antibody screenings of 2,005 sera from transfusion-scheduled patients were carried out using DiaMedTM LISS/Coombs gel card. Antibody identifications of screen-positive or screen-negative but incompatible cross matched sera were done by conventional tube method firstly, and then by DiaMedTM LISS/Coombs gel test or DiaMedTM NaCl/Enzyme gel test in the cases of negative results for conventional tube method. RESLUTS: Total 34 irregular antibodies (8 warm and 26 cold antibodies) were screened by gel test. For warm antibody screening, the reactivity of LISS/Coombs gel test was much higher than that of conventional tube method, and for cold antibodies, tube method or NaCl/Enzyme gel test revealed better reactivity. CONCLUSION: Antibody screening by LISS/Coombs gel test alone appears to be enough for detecting clinically significant warm antibodies.

Inhibition of Platelet Aggregation by Mononuclear Leukocytes / 대한혈액학회지

BACKGROUND: Although normal vascular endothelium prevents adhesion and aggregation of platelets by the release of nitric oxide (NO) and prostacyclin, circulating blood cells, such as polymorphonuclear leukocytes (PMNs) and mononuclear leukocytes (ML) may be considered to be also important in modulating platelet aggregation. Recently, nitric oxide synthase (NOS) activity was found in PMNs and ML, so these cells can also release NO to inhibit platelet aggregation. We studied platelet-ML interactions using an experimental model in which isolated ML were placed in the aggregometer in contact with human platelets, stimulated by collagen. METHODS: Platelet count in platelet-rich plasma (PRP) was adjusted to approximately 300x109/L. ML were separated using Ficoll-Hypaque (specific gravity 1.077) and finally resuspended at 1, 3 and 5x109/L, in Tyrode albumin buffer (TAB), respectively. Platelet aggregation was measured with Chrono-Log Aggregometer (USA) after adding variable numbers of the ML, stimulating with 2.5, 5 and 10 microgram/mL of collagen. Mechanisms of ML to inhibit the platelet aggregation were evaluated after incubating the ML with 10 micrometer indomethacin and 300 micrometer NG-monomethyl-L-arginine (L-NMMA). RESULTS: Non-stimulated ML (3x109/L) inhibited (43.2 +/- 19.6 versus TAB control 69.2 +/- 10.7% transmission) the platelet aggregation induced by 2.5 microgram/mL of collagen. The inhibition was not attenuated by increasing the concentration of collagen from 5.0 microgram/ mL (50.1 +/- 18.0% versus TAB control 75.5 +/- 13.1%, P<0.001) to 10 microgram/mL (62.9 +/- 17.3% versus TAB contol 82.3 +/- 12.6%, P<0.01). In addition, it was dependent on the number of ML and incubation time. While preincubation of the ML with indomethcin did not affect the antiaggregating capacity of the ML (63.4 +/- 11.1 versus TAB control 73.3 +/- 7.3%), preincubation of the ML with L-NMMA slightly inhibit the antiaggregating capacity of the ML (86.6 +/- 6.8 versus TAB control 73.3 +/- 7.3%). CONCLUSION: These findings suggest that blood ML inhibited the collagen-induced platelet aggregation, of which mechanism appears to be only partly dependent on NO and to be independent on prostaglandins. Release of other substances affecting platelet aggregation from ML requires to be clarified. Using our experimental model, it has been demonstrated that cell-cell contact may facilitate the exchange of a wide array of mediators between platelets and ML which may influence the cellular responses. This experimental model thus allows to study interactions between platelets and ML.

Determination of Reduced Glutathione in Red Blood Cells / 대한임상병리학회지

BACKGROUND: The product of oxygen-free radicals inf1ict oxidative injuries on healthy cells. Antioxidants such as superoxide dismutase(SOD), glutathione peroxidase, and reduced glutathione(GSH) are present in almost all cells and play important roles in metabolism, transport, and cellular protection. We measured blood GSH levels in healthy controls and patients with non insulin dependent diabetes mellitus(NIDDM) for evaluation of the clinical usefulness of GSH. METHODS: Erythrocyte GSH levels were measured in fifty healthy controls and thirty NIDDM patients with diabetic retinopathies by Beutler's method. We also tested within-run precision, between-run precision, linearity and recovery rate to evaluate this method measuring erythrocyte GSH levels. RESULTS: The GSH levels (mean +/-SD) of NIDDM patients (5.03+/-0.67mumo1/Hb) were significantly lower than those of healthy control group (6.46+/-0.85mumo1/Hb)(P<0.001). The results of within-run precision and between-run precision when stored at 4degrees Cwere excellent (coefficient of variation were 2.79% and 2.42%, respectively), however, when stored at the room temperature the GSH levels were sharply declined. The linearity and recovery rate were acceptable. CONCLUSIONS: The prescision, linearity, and recovery rate of GSH measurement were excellent. The GSH levels in NIDDM patient group were reduced, and this probably contributes to the defective defense mechanism against increased oxidative stress. Additional measurement of other antioxidants such as superoxide dismutase and glutathione Peroxidase may be required to clarify the pathologic significance of glutathione metabolism in various diseases.