Development of an Automated Image Analyzer for Microvessel Density Measurement in Bone Marrow Biopsies

Angiogenesis is important for the proliferation and survival of multiple myeloma (MM) cells. Bone marrow (BM) microvessel density (MVD) is a useful marker of angiogenesis and an increase in MVD can be used as a marker of poor prognosis in MM patients. We developed an automated image analyzer to assess MVD from images of BM biopsies stained with anti-CD34 antibodies using two color models. MVD was calculated by merging images from the red and hue channels after eliminating non-microvessels. The analyzer results were compared with those obtained by two experienced hematopathologists in a blinded manner using the 84 BM samples of MM patients. Manual assessment of the MVD by two hematopathologists yielded mean±SD values of 19.4±11.8 and 20.0±11.8. The analyzer generated a mean±SD of 19.5±11.2. The intraclass correlation coefficient (ICC) and Bland-Altman plot of the MVD results demonstrated very good agreement between the automated image analyzer and both hematopathologists (ICC=0.893 [0.840–0.929] and ICC=0.906 [0.859–0.938]). This automated analyzer can provide time- and labor-saving benefits with more objective results in hematology laboratories.

Preparation of Quality Control Slides and Establishment of an External Quality Assessment Program for Five Special Stains Routinely Used in Diagnostic Hematology

BACKGROUND: In general, internal/external quality control of special stains for diagnosis of hematological diseases may be unavailable in a clinical laboratory owing to the lack of an appropriate positive/negative control material. METHODS: We developed a protocol on positive/negative control materials for five special stains (iron, myeloperoxidase [MPO], periodic acid-Schiff [PAS], Sudan black B [SBB], and alpha-naphthyl acetate esterase [ANAE]) using a hematological malignant cell line. First, we compared stainability of seven cell lines (HL-60, THP-1, K562, Kasumi-1, KG-1, KO52, and NKM-1), then confirmed duration of stable stainability. A proficiency test using external quality control materials was conducted at eleven institutions, which participated voluntarily. RESULTS: HL-60 and THP-1 cell lines, which showed good stainability among the seven cancer cell lines, were selected as external quality control materials. The stainability of a prepared cell line fixed on control slides was stable for 3–4 weeks (MPO, SBB, and PAS) or 9–10 weeks (ANAE). The stainability of paraffin-embedded control material for iron stain was stable for 3 months. The results from 11 institutions were the same on iron, MPO, SBB, and ANAE. Nevertheless, two of 10 institutes showed discrepant results on PAS. CONCLUSIONS: In this study, we demonstrated that cell lines could serve as a standard quality control material for special stains. Most institutions showed representative results on special stains except for PAS. This protocol for special stain may be useful as an external or internal quality control in a haematology laboratory.

Chromothripsis in Treatment Resistance in Multiple Myeloma

Multiple myeloma (MM) is a malignant disease caused by an abnormal proliferation of plasma cells, of which the prognostic factors include chromosomal abnormality, β-2 microglobulin, and albumin. Recently, the term chromothripsis has emerged, which is the massive but highly localized chromosomal rearrangement in response to a one-step catastrophic event. Many studies have shown an association of chromothripsis with the prognosis in several cancers; however, few studies have investigated it in MM. Here, we studied the association between chromothripsis-like patterns and treatment resistance or prognosis. First, we analyzed nine MM cell lines (U266, MM.1S, RPMI8226, KMS-11, KMS-12-BM, KMS-12-PE, KMS-28-BM, KMS-28-PE, and NCI-H929) and bone marrow samples of four patients who were diagnosed with MM by next-generation sequencing-based copy number variation analysis. The frequency of the chromothripsis-like pattern was observed in seven cell lines. We analyzed the treatment-induced chromothripsis-like patterns in KMS-12-BM and KMS-12-PE cells. As a result, breakpoints and chromothripsis-like patterns were increased after drug treatment in the relatively resistant KMS-12-BM. We further analyzed the patients’ results according to the therapeutic response, which was divided into sensitive and resistant, as suggested by the International Myeloma Working Group. The chromothripsis-like pattern was more frequently observed in the resistant group. In the sensitive group, the frequency of the chromothripsis-like pattern decreased after treatment, whereas the resistant group showed increased chromothripsis-like patterns after the treatment. These results suggest that the chromothripsis-like pattern is associated with treatment response in MM.

Comparison of Two Apheresis Systems of COBE and Optia for Autologous Peripheral Blood Stem Cell Collection

Peripheral blood stem cell (PBSC) transplantation following myeloablative therapy is a mainstay of treatment for various types of malignancies. This study aimed to evaluate the differences between the Optia MNC and COBE Spectra MNC systems (Terumo BCT, Japan) according to apheresis procedures and the parameters of apheresis, products, and collection. The clinical data of 74 patients who underwent autologous PBSC collection from July 2012 to July 2015 were reviewed retrospectively. The patients comprised 48 (65%) men and 26 (35%) women with a median age of 56 yr (range, 23–66 yr). Of 216 procedures, 111 (51%) and 105 (49%) were processed by using COBE and Optia MNC, respectively. PBSC collection rates, throughput, numbers of stem cells retrieved, collection efficacy, and platelet loss were compared. There were no significant differences in the median CD34+ cell counts of collected products (0.61×10⁸ vs 0.94×10⁸), CD34 collection efficiency (43.5% vs 42.1%), and loss of platelets (40.1% vs 44.7%). The Spectra Optia MNC apheresis system was comparable to the COBE Spectra system in collecting autologous CD34+ hematopoietic stem cells and retention of platelets.

A Case of Washed Platelet Transfusion in a Patient with History of Recurrent Transfusion Reaction / 대한수혈학회지

Anaphylactic transfusion reaction is caused by deficiency of certain protein(s) in the recipient. We report on the experience of platelet count recovery using washed platelets for transfusion in a patient who developed an anaphylactic transfusion reaction. A 50-year old male diagnosed with angioimmunoblastic T-cell lymphoma was treated with chemotherapy followed by autologous hematopoietic stem cell transplantation. Immediately after starting transfusion of apheresis platelets, he began sweating and complained of visual impairment, chest discomfort, and abdominal pain. Both systolic and diastolic blood pressures and oxygen saturation monitored by pulse oximetry were decreased. Platelet transfusion was discontinued immediately and hydrocortisone was administered, and the symptoms and signs were resolved within two hours. Laboratory test using post-transfusion blood showed no apparent evidence of hemolysis. Platelet washing procedure using normal saline three times was newly set to prevent anaphylactic reaction in the patient. Transfusions of washed platelets were performed 20 times for 60 days, and the patient showed no anaphylactic reaction during this period. He showed no evidence of immunoglobulin A, haptoglobin, C3, or C4 deficiencies. We confirmed that washed platelet transfusion is highly effective for prevention of anaphylactic transfusion reaction.

Evaluation of Clinical Parameters and Symptoms in Healthy Donors for Granulocyte Collection / 대한수혈학회지

BACKGROUND: Donor granulocyte transfusion has been used as a salvage treatment for neutropenic patients with severe infection who did not respond to antibiotics. Here we investigated hematological parameters of granulocyte collection to evaluate its efficacy and safety. METHODS: The clinical data for 92 procedures of granulocyte collection performed on 82 healthy donors from April 2007 to July 2014 at National Cancer Center were reviewed retrospectively. Healthy donors were pre-medicated 12 hours before apheresis with subcutaneous injection of 600 microg of granulocyte-colony stimulating factor (G-CSF) with or without 8 mg of oral dexamethasone. Blood cell counts of donors at the time of pre- and post- granulocyte collection state of donors were investigated and any clinical symptoms and signs were monitored during the procedure. RESULTS: he median age was 29 years old (range, 18~52). The mean of collected granulocyte volume was 230 mL, and the granulocyte yield in apheresis products was 4.90x10(10) (1.46~7.86). Granulocyte yields showed significant correlation with volume of granulocyte collection, total processing volume, pre-WBC count, and pre-ANC. Granulocyte yields of dexamethasone and G-CSF administration were greater than with G-CSF administration alone. Pain (low back, hip, knee, and whole body), insomnia, fatigue, abdominal discomfort, and/or headache occurred in 21% of donors during mobilization. CONCLUSION: Granulocyte mobilization was safe and effective, and is well-tolerable in healthy donors because collection was possible in all donors without complications of G-CSF and dexamethasone administration and apheresis.

Adverse Prognostic Impact of Bone Marrow Microvessel Density in Multiple Myeloma

BACKGROUND: Angiogenesis is important for the proliferation and survival of multiple myeloma (MM) cells. Bone marrow (BM) microvessel density (MVD) is a useful marker of angiogenesis and is determined by immunohistochemical staining with anti-CD34 antibody. This study investigated the prognostic impact of MVD and demonstrated the relationship between MVD and previously mentioned prognostic factors in patients with MM. METHODS: The study included 107 patients with MM. MVD was assessed at initial diagnosis in a blinded manner by two hematopathologists who examined three CD34-positive hot spots per patient and counted the number of vessels in BM samples. Patients were divided into three groups according to MVD tertiles. Cumulative progression-free survival (PFS) and overall survival (OS) curves, calculated by using Kaplan-Meier method, were compared among the three groups. Prognostic impact of MVD was assessed by calculating Cox proportional hazard ratio (HR). RESULTS: Median MVDs in the three groups were 16.8, 33.9, and 54.7. MVDs were correlated with other prognostic factors, including beta2-microglobulin concentration, plasma cell percentage in the BM, and cancer stage according to the International Staging System. Multivariate Cox regression analysis showed that high MVD was an independent predictor of PFS (HR=2.57; 95% confidence interval, 1.22-5.42; P=0.013). PFS was significantly lower in the high MVD group than in the low MVD group (P=0.025). However, no difference was observed in the OS (P=0.428). CONCLUSIONS: Increased BM MVD is a marker of poor prognosis in patients newly diagnosed with MM. BM MVD should be assessed at the initial diagnosis of MM.

Ullrich Congenital Muscular Dystrophy Possibly Related With COL6A1 p.Gly302Arg Variant

Ullrich congenital muscular dystrophy (UCMD) is characterized by congenital weakness, proximal joint contractures, and hyperlaxity of distal joints. UCMD is basically due to a defect in extra cellular matrix protein, collagen type VI. A 37-year-old woman who cannot walk independently visited our outpatient clinic. She had orthopedic deformities (scoliosis, joint contractures, and distal joint hyperlaxity), difficulty of respiration, and many skin keloids. Her hip computed tomography showed diffuse fatty infiltration and the 'central shadow' sign in thigh muscles. From the clinical information suggesting collagen type VI related muscle disorder, UCMD was highly considered. COL6A1 gene sequencing confirmed this patient as UCMD with novel c.904G>A (p.Gly302Arg) variant. If musculoskeletal and dermatologic manifestations and radiologic findings imply abnormalities in collagen type VI network, COL6A related congenital muscular dystrophy was to be suspected.

Clinical Effects and Safety of Therapeutic Leukapheresis in Hyperleukocytosis / 대한수혈학회지

BACKGROUND: Therapeutic leukapheresis is the cytoreduction procedure performed before chemotherapy in patients with hyperleukocytosis for prevention of complication. However, there have been clinical concerns about bleeding tendency due to anticoagulant used during the procedure. The aim of our study was to compare the clinical characteristics and hematological parameters before and after therapeutic leukapheresis in order to evaluate its effect on bleeding tendency and to provide a guideline for treatment strategy. METHODS: The clinical data for 39 procedures of therapeutic leukapheresis performed on 17 patients with hyperleukocytosis from May 2005 to October 2013 at the National Cancer Center were reviewed retrospectively. RESULTS: The patients consisted of 11 males and six females. The mean age was 41 years old (range, 8~74). The mean number of therapeutic leukapheresis per patient was two (range, 1~4). Clinical symptoms improved in 14 patients (82%) after therapeutic leukapheresis and three patients (18%) were not yet to improve. The mean WBC count was significantly reduced by 32.6% (+/-17.4) after therapeutic leukapheresis, from 250,146/microL (+/-117,000) to 174,702/microL (+/-104,700) (P<0.001). The mean volume of single removal was 298 ml with 4.25x10(11)/L (+/-1.54) WBCs. After therapeutic leukapheresis, the mean platelet count showed a decline from 85x10(9)/L (+/-43) to 71x10(9)/L (+/-26). However, the prothrombin time (PT) and activated partial thromboplastin time (aPTT) did not show a significant increase (PT, P=0.637; aPTT, P=0.054). CONCLUSION: Therapeutic leukapheresis is demonstrated as an effective and safe treatment that can improve symptoms and reduce leukocytes in hyperleukocytosis.

Increasing the Efficiency of Laboratory Performance by Using the Onboard Dilution Algorithm of the Elecsys Hepatitis B Surface Antigen II Quantitative Assay / 임상검사와정도관리

BACKGROUND: The Elecsys hepatitis B surface antigen (HBsAg) II quantitative assay is a newly introduced electrochemiluminescence immunoassay incorporating an initial 1:400 onboard dilution and a simple algorithm to determine HBsAg levels in serum. We evaluated the performance of the Elecsys HBsAg II assay and determined the impact of its initial onboard dilution on laboratory efficiency. METHODS: HBsAg levels were determined using both Roche Elecsys and Abbott Architect HBsAg assays. Linearity and precision of the Elecsys HBsAg II assay and its correlation with the Architect HBsAg assay were evaluated. In particular, precision was verified at Samsung Medical Center, Severance Hospital, Seoul St. Mary's Hospital in Seoul, using the same pooled serum controls. The efficiency of the dilution algorithm for both methods was verified using data from 1,848 clinical samples. RESULTS: The Elecsys HBsAg II assay showed a good linearity from 0.1 to 48,000.0 IU/mL and a good correlation (r=0.9998) between expected and measured values. Precision analyses performed at Samsung Medical Center, Severance Hospital, Seoul St. Mary's Hospital showed excellent performance with coefficients of variation between 1.28% and 6.82%. The values of the Elecsys HBsAg II and Architect HBsAg assays were well correlated (n=506, r=0.987, P<0.001) and also reliably determined in hepatitis C virus- and hepatitis B virus-co-infected patient sera (n=27). In terms of efficiency, 64.0% of samples provided a final HBsAg result on the first run without the need for further dilution, when using the 1:400 onboard pre-dilution protocol of the Elecsys HBsAg II assay. CONCLUSIONS: Given the excellent precision and correlation with the Architect assay, the Elecsys HBsAg II assay showed a potential advantage for laboratory efficiency by significantly reducing the need for retesting samples with high HBsAg levels.

Network Computer Management System Development for Blood Transfusion in ABO-Incompatible Stem Cell Transplantation / 대한수혈학회지

BACKGROUND: The majority of patients undergoing stem cell transplantation (SCT) require a blood transfusion until the complete engraftment. Because blood transfusion rules for patients with ABO-incompatible SCT are complicated, we developed an ABO-incompatible transfusion management system (ABO-ITMS) for accurate blood transfusion and improved manageability. METHODS: A committee composed of medical doctors, technicians, and a programmer developed ABO-ITMS during the eight months from July 2013 to February 2014. The program has been linked with other databases, including clinical and laboratory databases and resulted in a new subsystem of the health information system. Server computer's operating system was Window Server 2008, and the database manager program was Oracle 11g. Programming language was ASP.Net (VBScript, C #), and the server and client computer were used to connect to the web server using a web browser. RESULTS: ABO-ITMS was designed to follow three main steps by hematologic oncology clinic, laboratory physician, and blood bank. In the first step, a hematologic-oncology clinic doctor inputs SCT recipients' data and appropriate ABO group for each phase of post-transplantation. Laboratory physician enters the isoagglutinin titer and ABO group at the second step. Finally, blood bank workers enter the results of type, screening, and antibody identification. The patient's SCT information and the previous immunohematologic test results are shown on the screen. CONCLUSION: ABO-ITMS can replace the existing complicated system and workflow. ABO-ITMS will contribute to reducing medical error and improving quality of SCT recipient care.

Isolation and Identification of Geosmithia argillacea from a Fungal Ball in the Lung of a Tuberculosis Patient

Geosmithia argillacea, an anamorph of Talaromyces eburneus, is a thermophilic filamentous fungus that has a phenotype similar to that of the Penicillium species, except for the creamy-white colonies and cylindrical conidia. Recently, a new genus called Rasamsonia has been proposed, which is to accommodate the Talaromyces and Geosmithia species. Here, we report the first Korean case of G. argillacea isolated from a patient with a fungal ball. The patient was a 44-yr-old Korean man with a history of pulmonary tuberculosis and aspergilloma. The newly developed fungal ball in his lung was removed and cultured to identify the fungus. The fungal colonies were white and slow-growing, and the filaments resembled those of Penicillium. Molecular identification was carried out by sequencing the internal transcribed spacer (ITS) region of the 28S rDNA and the beta-tubulin genes. A comparative sequence analysis using the GenBank (http://blast.ncbi.nlm.nih.gov/) database was performed with the basic local alignment search tool (BLAST) algorithm. The results revealed a 97-100% similarity with the G. argillacea ITS sequence. This case should increase awareness among physicians about the pathogenic potential of G. argillacea in humans and help them accurately identify this fungus, because it can be easily confused with Penicillium and Paecilomyces species owing to their similar phenotypic and microscopic characteristics. A molecular approach should be employed to enable accurate identification of G. argillacea.

Evaluation of the Xpert Flu for the Detection of Influenza A Virus and Influenza A/H1N1/2009 Strain / 대한임상미생물학회지

BACKGROUND: Xpert Flu (Cepheid, USA) allows for fully automated real-time RT-PCR using a single-use disposable cartridge. The aim of this study was to evaluate Xpert Flu for the detection of influenza A virus and subtype A/H1N1/2009 pandemic virus. METHODS: We conducted a prospective comparison study for Xpert Flu with the RealTime ready Influenza A/H1N1 Detection Set (Roche Diagnostics, Germany). Analytical specificities of the assays were determined by testing commonly encountered respiratory viral pathogens, including parainfluenza virus type 1/2/3, rhinovirus A, rhinovirus B, metapneumovirus, adenovirus, and coronavirus. The analytical sensitivities and workflow of both methods were also assessed. RESULTS: A total of 102 consecutive clinical specimens were tested by both methods. Total agreement between the two methods was estimated to be 99.0% (101/102): 11 A/H1N1/2009 and 3 seasonal influenza A by the RealTime ready Influenza A/H1N1 Detection Set; 10 and 3 by Xpert Flu. No cross-reactivity was observed between influenza A/H1N1/2009 and other respiratory viral pathogens in either method. The limits of detection of the RealTime ready Influenza A/H1N1 Detection Set and Xpert Flu were 500 TCID50/mL and 20 TCID50/mL, respectively. Xpert Flu required 85 minutes (10 minutes of hands-on time) for processing, while RealTime ready Influenza A/H1N1 Detection Set took 128 minutes (30 minutes of handson time). CONCLUSION: The results of Xpert Flu were comparable to those of the RealTime ready Influenza A/H1N1 Detection Set. It is of note that the fully automated and closed system of Xpert Flu could be advantageous for reducing hands-on time and for preventing cross-contamination during the testing process.