Flow Cytometric White Blood Cell Differential Using CytoDiff is Excellent for Counting Blasts

BACKGROUND: The usefulness of the CytoDiff flow cytometric system (Beckman Coulter, USA) has been studied in various conditions, but its performance including rapidity in detecting and counting blasts, the most significant abnormal cells in the peripheral blood, has not been well evaluated. The objective of this study was to evaluate the performance of the CytoDiff differential counting method in challenging samples with blasts. METHODS: In total, 815 blood samples were analyzed. Samples flagged as "blasts" or "variant lymphocytes" and showing 0.8) for neutrophils and lymphocytes but poor (r<0.8) for other cells. When the cutoff value of the CytoDiff blast count was set at 1%, the sensitivity was 94.4% (95% CI; 91.2-96.6) and specificity was 91.9% (95% CI; 89.0-94.1). The positive predictive value was 88.4% (95% CI; 84.4-91.5) (304/344 cases) and negative predictive value was 96.2% (95% CI; 93.9-97.7) (453/471 cases). The CytoDiff blast counts correlated well to the manual counts (r=0.9223). CONCLUSIONS: The CytoDiff method is a specific, sensitive, and rapid method for counting blasts. A cutoff value of 1% of at least 1 type of blast is recommended for positive CytoDiff blast counts.

A Novel Marker for Screening Paroxysmal Nocturnal Hemoglobinuria Using Routine Complete Blood Count and Cell Population Data

BACKGROUND: Final diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) may take years demanding a quick diagnosis measure. We used the facts that PNH cells are damaged in acid, and reagents for measuring reticulocytes in Coulter DxH800 (Beckman Coulter, USA) are weakly acidic and hypotonic, to create a new PNH screening marker. METHODS: We analyzed 979 complete blood counts (CBC) data from 963 patients including 57 data from 44 PNH patients. Standard criteria for PNH assay for population selection were followed: flow cytometry for CD55 and CD59 on red blood cells (RBCs) to a detection level of 1%; and fluorescent aerolysin, CD24 and CD15 in granulocytes to 0.1%. Twenty-four PNH minor clone-positive samples (minor-PNH+) were taken, in which the clone population was <5% of RBCs and/or granulocytes. Excluding PNH and minor-PNH+ patients, the population was divided into anemia, malignancy, infection, and normal groups. Parameters exhibiting a distinct demarcation between PNH and non-PNH groups were identified, and each parameter cutoff value was sought that includes the maximum [minimum] number of PNH [non-PNH] patients. RESULTS: Cutoff values for 5 selected CBC parameters (MRV, RDWR, MSCV, MN-AL2-NRET, and IRF) were determined. Positive rates were: PNH (86.0%), minor-PNH+ (33.3%), others (5.0%), anemia (13.4%), malignancy (5.3%), infection (3.7%), normal (0.0%); within anemia group, aplastic anemia (40.0%), immune hemolytic anemia (11.1%), iron deficiency anemia (1.6%). Sensitivity (86.0%), specificity (95.0%), PPV (52.1%), and NPV (99.1%) were achieved in PNH screening. CONCLUSION: A new PNH screening marker is proposed with 95% specificity and 86% sensitivity. The flag identifies PNH patients, reducing time to final diagnosis by flow cytometry.

Clinical Validation of AdvanSure GenoBlot Assay as Primary Screening and Test of Cure for Human Papillomavirus Infection

BACKGROUND: Clinical specificity and sensitivity are essential factors in the adoption of a human papillomavirus (HPV) test as a primary screening tool and test of cure after treatment of cervical cancer and precancerous lesions (High-Risk-Lesion). Using histologically-confirmed High-Risk-Lesion-patient specimens with postoperative follow-ups, we performed clinical validation of the AdvanSure GenoBlot Assay (GenoBlot; LG Life Sciences, Korea). METHODS: The study population included 100 cases with High-Risk-Lesion, 96 with high-risk genotype positive and cervical intraepithelial neoplasia (CIN) 1 or better, and 39 with HR-negative and better than CIN 1. Forty-eight High-Risk-Lesion cases received follow-up HPV exams after surgery. For validation as a test of cure, 48 preoperative specimens (PreOP) and 78 postoperative specimens (PostOP) from 48 subjects were separately analyzed. The results of HPV DNA chip tests (HPVDNAChip; BioMedLab Co., Korea) and sequencing were cross-compared. RESULTS: The concordance rates for each genotype between HPVDNAChip and GenoBlot were between 96.3-100%. The accuracy of HPVDNAChip and GenoBlot was 87.9% and 96.6%, respectively. Genotype-based specificity for High-Risk-Lesion detection was higher than 87% for both assays; genotype 16 showed the highest sensitivity. In the PostOP group, the positive rates for HPVDNAChip and GenoBlot were 30.8% and 47.4%, respectively. CONCLUSIONS: GenoBlot showed a higher positive rate than HPVDNAChip for each genotype, with concordance rate and accuracy being similar to previous reports. As a test of cure, GenoBlot performed better than the HPVDNAChip.

Novel Markers of Early Neutrophilic and Monocytic Engraftment after Hematopoietic Stem Cell Transplantation

BACKGROUND: Numerous studies tried to find new markers that after hematopoietic stem cell transplantation predict engraftment earlier than the conventional marker, absolute neutrophil count (ANC >500/microL). Early engraftment prediction can be achieved by a marker that reflects the release of neutrophils and monocytes into the leukopenic peripheral blood. METHODS: We analyzed blood cell parameters, including cell population data such as volume, conductivity, and light scatter in 77 patients who underwent HSCT (allogeneic, n=63; autologous, n=11) to detect possible markers. RESULTS: We identified 2 early engraftment markers of neutrophils (NEUTRO) and monocytes (MONO); a pair of mean-volume-neutrophils (MNV) and mean-conductivity-neutrophils (MNC) for NEUTRO; and a pair of mean-volume-monocytes (MMV) and mean-conductivity-monocytes (MMC) for MONO. The new markers showed distinct patterns for early engraftment wherein 1) on the engraftment day, MNV peaked as MNC notched simultaneously for every case, and 2) MMV peaked as MMC notched simultaneously in most cases. Engraftment was predicted 3.8+/-2.7 days earlier than by ANC in 74 successful engraftment cases by using NEUTRO and/or MONO: 1) 72 cases (97.3%), in which NEUTRO and/or MONO predicted earlier engraftment than ANC, 2) 1 case, in which the 3 markers predicted engraftment on the same day, and 3) 1 case, in which NEUTRO predicted engraftment on the same day as ANC and MONO failed to predict engraftment. CONCLUSIONS: By analyzing the data from daily complete blood counts, engraftment can be predicted approximately 4 days earlier than ANC >500/microL using NEUTRO as a base marker and MONO as a supplementary marker.

The Relationship between the Timing of Gestational Diabetes Screening and HbA1c Level and Neonatal Outcome / 대한진단검사의학회지

BACKGROUND: The aim of this study was to observe clinical outcomes of the mother and her infant who were possibly exposed to high blood glucose at least 2-3 months in the early and midterm pregnancy by checking gestational weeks (GW) and the first HbA1c level at initial diagnosis of gestational diabetes (GDM). METHODS: A total of 107 GDM patients and their newborns were subject of this study. GDM patients were newly diagnosed at the Holy Family Hospital of Catholic University from January 2003 until December 2007 and continuously managed in the diabetes center. Patients medical records were retrospectively reviewed to evaluate GW and HbA1c level at the time of diagnosis, and clinical outcomes of mother and newborn baby. RESULTS: The proportion of subjects who had been diagnosed of having GDM according to GW was 7.5%, in less than 24th week of pregnancy; 55.1% in the 24-28th week; 28.0% in the 29-32nd week; and 9.4% 33rd week or more. There were 39 out of 107 subjects (36.4%) with HbA1c levels > or =6.5% and 26 out of 39 subjects (24.3%) with HbA1c levels > or =7.0%. In clinical outcomes of newborn by HbA1c levels, the frequency of delivery of large for gestational age (LGA) infant was higher in mothers diagnosed with GDM after 29th week of pregnancy or with HbA1c levels 7.0% or more (P<0.001). CONCLUSIONS: If the screening test for gestational DM was delayed, HbA1c level and the risk for LGA seemed to be higher, so it may be necessary to screen GDM no later than 24th week of pregnancy.

Organization of Maximum Surgical Blood Order Schedule (MSBOS) according to the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) / 대한수혈학회지

BACKGROUND: The aim of this study is to organize the maximum surgical blood order schedule (MSBOS) of red blood cells (RBCs) for elective surgeries according to the International Classification of Diseases, Ninth Revision, Clinical Modification guidelines (ICD-9-CM) and we compared the results with the previously reported MSBOSs. METHODS: From 1 March to 31 August 2007, the data of the transfused RBCs for elective surgeries in our hospital were analyzed. The MSBOS was organized as the average number of units of transfused RBCs for the type of surgery, according to the ICD-9-CM. The results were compared with the MSBOSs that were previously reportedfrom 1982 to 2004 in Korea. RESULTS: A total of 121 types of 3,375 surgeries were performed. Type & screen for 91 types (81.3%), 1 unit for 20 types (13.8%), 2 units for 7 types (3.8%), 3 units for 1 type (0.4%) and 4 units for 2 types (1.8%) were recommended. There was a minimal difference between these results and the range for the previously reported MSBOSs. CONCLUSION: It seems that the MSBOS showed minimal change since 2004. We organized the MSBOS according to the guidelines of the ICD-9-CM. Standardization of the surgery name should be considered to achieve more useful utilization of MSBOS.

Distribution of Antigenic Aberration in the Bone Marrow of Acute Leukemia in Complete Remission / 대한진단검사의학회지

BACKGROUND: The aberrant, leukemia-associated antigen expression patterns allow us to discriminate leukemic blasts from normal precursor cells. Our major goal was to determine a guideline for the detection of minimal residual disease using CD20+/CD34+ and myeloid Ag+/CD19+ combination in the bone marrow of acute leukemia in complete remission (CR) after chemotherapy. METHODS: Bone marrow samples from 117 patients with acute leukemia in complete remission after chemotherapy and from 22 healthy controls were immunophenotyped by triple staining and measured by flow cytometry. RESULTS: The CD20+/CD34+ cells in the large lymphocyte gate (R1) ranged from 0% to 3.24% (0.8+/-0.82%, P=0.000) in CD20+/CD34+ B-lineage ALL CR (N=31), from 0.03% to 4.2% (0.7+/-0.83%, P=0.000) in CD20-/CD34- B-lineage ALL CR (N=66), from 0.1% to 0.96% (0.45+/-0.32%, P=0.016) in T-ALL CR (N=10), and from 0.02% to 0.48% (0.18+/-0.15%, P=0.776) in AML CR (N=10). The CD13,33+/CD19+ cells in R1 gate ranged from 0% to 2.69% (0.37+/-0.48%, P<0.001) in CD13,33+/CD19+ B-lineage ALL CR (N=31), from 0% to 1.8% (0.31+/-0.28%, P<0.001) in CD13,33-/CD19+B-lineage ALL CR (N=65), from 0.02% to 0.64% (0.29+/-0.22%, P=0.071) in T-ALL CR (N=9), and from 0% to 0.17% (0.07+/-0.09%, P=0.341) in AML CR (N=3). CONCLUSIONS: Using an immunophenotypic method for the detection of early relapse or minimal residual disease of B-lineage ALL bone marrow in CR after chemotherapy, different cutoff values should be applied according to antigen combination and gating. When the proportion of aberrant antigen combination was less than 5% in large lymphocyte gate, the results should be interpreted with caution.

Clinical Efficacy of HPV DNA Chip Test in the Era of HPV Vaccination: 1,211 Cases, A Single Institution Study / 대한진단검사의학회지

BACKGROUND: Human papillomavirus (HPV) prophylactic vaccines, bivalent types for HPV-16/18 with 70% prophylactic expectation, have been developed based on the genotypes found prevalent in the western countries, but little is known for those in Korea. Using a DNA chip test, we evaluated the clinical efficacy of HPV genotype based on cervical abnormalities. METHODS: As the initial diagnostic tests, HPV DNA chip tests and Papanicolaou smear (PAP) were used for 1,211 subjects. Cervical colposcopy directed biopsies were performed for 626 among the 1,211 subjects within one month. RESULTS: The most frequently found genotypes in all HPV-positive specimens (n=445) were HPV-16 (22.0%), 58 (13.9%), 52 (11.0%), 51 (9.0%), 56 (8.5%), and 18 (7.2%). HPV prevalence was significantly higher in specimens where PAP and biopsy results were closer to malignancy. The HPV genotype distribution of the histologically confirmed cervical high-grade squamous intraepithelial lesions (HSIL) or carcinoma cases showed HPV-16, 58, 52, 18, and 33, in descending order. The HPV DNA chip sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the detection of cervical HSIL or carcinoma were 76.9%, 70.1%, 72.1%, and 75.8%, respectively, Of these, the sensitivity and NPV were higher than those of PAP. PPV and NPV of HPV-16 were 90.5% and 60.7%, respectively, being the highest among the genotypes. CONCLUSIONS: We confirmed that HPV-16 genotype was also very important for the diagnosis of HSIL and cervical carcinoma in Korea. However, contrary to the findings in the western countries, the prevalence of HPV-58 was higher than that of HPV-18. Moreover, as the other HPV genotype reports were rare in Korea, further studies are required with the HPV DNA chip test before the nationwide adoption of the vaccines.

A case of neonatal amoebiasis with after-birth vomiting and bloody stool / 소아과

E. histolytica has a simple life cycle with two stages: an infective cyst and an invasive trophozoite. It lives on humans as its host. Its infection occurs through the ingestion of the cyst form, and the disease begins when the trophozoite, converted at the small intestine, adheres to colonic epithelial cells with a latent period of two days to four months. In some instances, amoebic abscess formations can occur at the liver, lung, brain, or spleen via the lymphoid system. Rare cases of amoebiasis in neonates have been reported, much less any intrauterine infections in the world that may have occurred during the gestation period. We've recently experienced a case of neonatal amoebiasis that entailed after-birth vomiting and bloody stool. The infant seemed pre-infected with E. histolytica before birth.

Operational Experience of a Quality Assurance System for HPV DNA Chip Tests / 임상검사와정도관리

BACKGROUND: A new HPV DNA chip test for the infection of 22 HPV genotypes was recently developed in Korea. This test using PCR and hybridization is inherently vulnerable to contamination, and to subjective qualitative test judgment. Hence, it warrants rigorous quality assurance measures. The authors would like to share operational experiences of the guidelines developed at Catholic University Holy Family Hospital. METHODS: Our quality assurance system of HPV DNA chip test comprised external quality controls, inter-laboratory proficiency tests, and internal quality controls. For the external quality controls, we analyzed the results of four years of participation in the quality assurance program by the Korean Laboratory Medicine Quality Assurance Association. The inter-laboratory proficiency tests with BioMedLab were done by single blind tests using patients' samples showing negative, single and multiple infections. The internal quality control dealt with methods to prevent contamination, and with reproduction tests. RESULTS: The results from the external quality control revealed consistency with HPV-16 in 7 trials during 4 years. The inter-laboratory proficiency tests showed a 82% consistency rate, 10 cases of inconsistency showing positive or negative mismatches, and 8 cases of genotypic mismatches. The 10 mismatches were due to the weak laser power of the scanner used in BioMedLab. The genotypic contamination rate found in the internal quality control was 3.3%, and the contamination by HPV-35 with low incidence rate was often observed. The contamination was not easily eliminated by re-tests from hybridization, but 80% of it was removed when re-tested with the remaining samples. The fluorescence intensity was not reproducible nor provide quantitative or semi-quantitative information. CONCLUSIONS: For quality assurance regarding HPV DNA chip tests, we suggest the following be implemented: technical quality control to rule out the false-negative and false-positive during PCR and hybridization; scanner quality control to prevent reading errors; and inter-laboratory proficiency tests.

Proportions of Cells Expressing CD38-/CD34+, CD38+/CD34+, CD19+/CD34+, or CD13,33+/CD34+ in the Regenerating Bone Marrows During Complete Remission of Acute Leukemia or After Bone Marrow Transplantation / 대한진단검사의학회지

BACKGROUND: The hemopoietic stem cells increase in number during the regeneration after chemotherapy or bone marrow transplantation (BMT). Although the proportion of hemopoietic stem cells and their differentiation have been studied by immunophenotyping using the flow cytometry, no substantial research efforts have been directed toward the regenerating marrow. We attempted to discover the proportions of undifferentiated stem cells, committed stem cells, B cell precursors, and myeloid precursors in the regenerating bone marrows during complete remission (CR) and after engraftment of BMT. METHODS: Bone marrow samples from 82 patients with acute leukemia in CR and from 25 patients after BMT engraftment, along with 22 control samples, were used to find the numbers of CD38-/CD34+, CD38+/CD34+, CD19+/CD34+, and CD13,33+/CD34+ cells in the large lymphocyte gate by flow cytometry. We cross-analyzed our results in terms of groups: CR, BMT, and initial diagnosis groups. We performed significance tests on age, relapse, chromosomal abnormalities, clinical outcomes, and initial immunophenotypes of the leukemic cells. RESULTS: The proportions of CD38-/CD34+, CD38+/CD34+, CD19+/CD34+, and CD13,33+/CD34+ cells are more highly distributed in acute B-lymphoblastic leukemia than the normal group and also in the CR than the BMT group. CD19+/CD34+ cells were increased in the relapse group and CD38+/ CD34+, CD19+/CD34+, and CD13,33+/CD34+ cells were increased in the group with chromosomal abnormality. The results were irrelevant to the initial immunophenotype of the leukemic blasts. CONCLUSIONS: The increases of the markers spanned too widely to apply one specific cutoff value to analyze them. They seemed to be the results of normal regeneration, irrelevant to relapse or initial immunophenotype of leukemic blasts.

A Case of IgA kappa Light Chain Deposition Disease and Combined Adult Fanconi Syndrome with Auer rod-like Intracytoplasmic Inclusions in Plasma Cells and Proximal Renal Tubular Cells / 대한진단검사의학회지

We report a case of IgA kappa light chain deposition disease and combined adult Fanconi syndrome with Auer rod-like intracytoplasmic inclusions in plasma cells and proximal renal tubular cells in a 54-yr-old female. Cytochemical stainings revealed a strong acid phosphatase activity of the inclusions and weak periodic acid-Schiff positivity, whereas the reactions for peroxidase and alpha-naphthyl acetate esterase were negative. An immunostaining verified IgA-kappa inside the plasma cells. Kidney biopsy revealed Bence Jones cast nephropathy with kappa light chain positivity, and Congo red staining was negative. Electron microscopy showed needle-shaped crystals located in tubular epithelial cells.

A Case of Clear-Cell Bowen's Disease Which May Have Developed Following Cryotherapy / 대한피부과학회지

Bowen's disease is a kind of squamous cell carcinoma in situ. In some cases, a pagetoid growth pattern can be observed with cytologically atypical clear cells arranged singly or in nests. The differential diagnosis of clear-cell Bowen's disease includes primarily Paget's disease and malignant melanoma in situ. A 66-year-old man presented with intermittently painful, solitary, well-demarcated, annular, erythematous, scaly plaque on the left fourth finger for 7 months. This had been treated by cryotherapy 8 months earlier, following the diagnosis of Bowen's disease. The skin biopsy specimen revealed nests of atypical clear cells in upper layer of the epidermis in addition to typical features of Bowen's disease. We, herein, report a rare case of clear-cell Bowen's disease, which is thought to have developed after cryotherapy.

A Case of Melanocytic Nevus Combined with a Vellus Hair Cyst / 대한피부과학회지

The combination of melanocytic nevus with other tumors of epidermal or adnexal origin has been described since the first case reported by Andresen and Enos in 1952. Most of reported cases are melanocytic nevi combined with common epidermoid cysts. The combination of nevus and cyst could possibly be misdiagnosed clinically as a malignant melanoma upon initial examination. A 58-year-old woman presented with an asymptomatic, solitary, skin-colored, erythematous 1.0 cm-diameter pedunculated nodule on the occipital scalp which had been present childhood. The histopathologic specimen of the nodule showed a solitary cyst surrounded by intradermal melanocytic nevus. The cyst was located in the dermis lined by squamous epithelium, which was ruptured and inflamed. It contained degenerated keratin materials and multiple transversely and obliquely cut vellus hairs. We herein report a rare case of melanocytic nevus combined with a vellus hair cyst.

A Case of Fibrosarcomatous Dermatofibrosarcoma Protuberans / 대한피부과학회지

Dermatofibrosarcoma protuberans(DFSP) is an unusual, locally aggressive, cutaneous neoplasm of low grade malignancy. Several histologic variants have been described. Among them, fibrosarcomatous dermatofibrosarcoma protuberans is associated with a higher rate of local recurrence and distal metastases as well as a shorter latency period of time until recurrence than does ordinary DFSP. As a result, it needs more intense treatments and more vigorous follow up. A 37-year-old man presented with an asymptomatic erythematous hard nodule and several reddish papules on the chest for 2 years. Histopathologic examination revealed fibrosarcomatous dermatofibrosarcoma protuberans. We, herein, report an unusual case of fibrosarcomatous dermatofibrosarcoma protuberans.