Automated cell counts on CSF samples: A multicenter performance evaluation of the GloCyte system.

OBJECTIVES: Automated cell counters have replaced manual enumeration of cells in blood and most body fluids. However, due to the unreliability of automated methods at very low cell counts, most laboratories continue to perform labor-intensive manual counts on many or all cerebrospinal fluid (CSF) samples. This multicenter clinical trial investigated if the GloCyte System (Advanced Instruments, Norwood, MA), a recently FDA-approved automated cell counter, which concentrates and enumerates red blood cells (RBCs) and total nucleated cells (TNCs), is sufficiently accurate and precise at very low cell counts to replace all manual CSF counts. METHODS: The GloCyte System concentrates CSF and stains RBCs with fluorochrome-labeled antibodies and TNCs with nucleic acid dyes. RBCs and TNCs are then counted by digital image analysis. Residual adult and pediatric CSF samples obtained for clinical analysis at five different medical centers were used for the study. Cell counts were performed by the manual hemocytometer method and with the GloCyte System following the same protocol at all sites. The limits of the blank, detection, and quantitation, as well as precision and accuracy of the GloCyte, were determined. RESULTS: The GloCyte detected as few as 1 TNC/µL and 1 RBC/µL, and reliably counted as low as 3 TNCs/µL and 2 RBCs/µL. The total coefficient of variation was less than 20%. Comparison with cell counts obtained with a hemocytometer showed good correlation (>97%) between the GloCyte and the hemocytometer, including at very low cell counts. CONCLUSIONS: The GloCyte instrument is a precise, accurate, and stable system to obtain red cell and nucleated cell counts in CSF samples. It allows for the automated enumeration of even very low cell numbers, which is crucial for CSF analysis. These results suggest that GloCyte is an acceptable alternative to the manual method for all CSF samples, including those with normal cell counts.

Implementation and analysis of a pilot in-hospital newborn screening program for glucose-6-phosphate dehydrogenase deficiency in the United States.

OBJECTIVE: The purpose of this study was to analyze a targeted screening program for glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDdef) and clinical outcomes of G6PD-deficient vs G6PD normal newborns. STUDY DESIGN: Retrospective chart review for 1578 male newborns was performed. The study group was those screened for G6PDdef. Comparisons between G6PD-deficient and normal infants were made with χ (2)-test and unpaired t-test. RESULT: A total of 1095 male newborns were screened, 11.1% had G6PDdef. 97.8% of screen results were reported by 48 h. Total bilirubin (TB) levels in deficient infants were significantly higher than in normal infants throughout birth hospitalization and they were more likely to receive phototherapy. Nineteen screened newborns were rehospitalized for hyperbilirubinemia, 47% had G6PDdef. CONCLUSION: In-hospital newborn screening for G6PDdef with rapid turnaround time is possible. G6PDdef is a risk factor for hyperbilirubinemia in American newborns. US centers with large at-risk populations can identify newborns at risk for severe hyperbilirubinemia with similar screening.

Reporting BRCA test results to primary care physicians.

PURPOSE: The main purpose of this study was to determine if comprehension of the cancer risk information presented in a hypothetical report for BRCA1/2 gene analyses was influenced by the format in which the information was presented. A secondary objective was to determine physician characteristics that might influence comprehension of the report. METHODS: A survey was conducted in which a case vignette describing a young woman at high risk for carrying a BRCA mutation was presented. Survey participants, all primary care practitioners, were asked to interpret a laboratory report that provided the patient's BRCA1/2 test result and accompanying data about the cumulative risk and incidence rates of breast cancer for BRCA1/2 mutation carriers and the general population. These data were presented in the report in either a tabular or a graphic format. The main outcome was measured by the responses to four questions that addressed the probabilistic cancer risk information. Physician predictor variables included medical specialty, practice setting, years in practice, continuing medical education in genetics, and knowledge of cumulative risk. RESULTS: Knowledge of cumulative risk was the only physician variable that influenced comprehension of the cancer risk information (OR = 31.9; P < 0.001). After adjusting for this variable, the graphic format tended to perform better than the tabular format in conveying breast cancer risk information (OR = 3.1; P = 0.102). CONCLUSIONS: Many physicians may be unprepared to interpret genetic risk information, due to lack of understanding of basic epidemiologic terms used to express the risk of disease.

Routine cultures of bone marrow and peripheral stem cell harvests: clinical impact, cost analysis, and review.

The American Association of Blood Banks requires routine culture of hematopoietic progenitor cells prior to bone marrow transplantation. We sought to evaluate the cost of that requirement and the incidence and clinical significance of positive cultures. We performed a retrospective analysis of transplant recipients at our institution. Of the 605 patients for whom 1,934 consecutive cultures of harvests were done between December 1992 and February 1996, 11 had positive cultures. Six patients received a culture-positive harvest with no adverse effects. The total cost of cultures was $35,660 (U.S. $). In North America and worldwide in 1995, routine culture of harvests would have prevented 7.9 and 18.9 cases of bacteremia, respectively, at a cost of $95,000 per bacteremia prevented. We conclude that routine culture of hematopoietic progenitor cells yields low rates of positivity and that infusion of contaminated harvests rarely results in clinically adverse outcomes.

A simplified method of CD34+ cell determination for peripheral blood progenitor cell transplantation and correlation with clinical engraftment.

Enumeration of CD34+ cells by flow cytometry is the recognized standard for quantitating progenitor cells for peripheral blood progenitor cell (PBPC) transplantation. Although many clinical studies have confirmed that the time to neutrophil and platelet engraftment is inversely proportional to the number of CD34+ cells infused, the minimum number of CD34+ cells necessary to acheive rapid engraftment has not been satisfactorily determined. The lack of a standardized method for quantitation of CD34+ cells by flow cytometry (FCM) is often cited as the reason for this ambiguity. This report describes an FCM method for CD34+ cell determination that is simple, highly reproducible, comparatively inexpensive, and validated by excellent correlation with clinical engraftment. Pheresis samples are stained and fixed within 4 hours of collection. Two hundred fifty thousand events are acquired as list mode data using a forward scatter threshold. The discrete CD34+ population is enumerated using a CD34-phycoerythrin FL2 vs. side scatter plot and Paint-A-Gate Pro software. The method was validated by excellent statistical correlation with clinical engraftment. Using this method, we determined the number of CD34+ progenitor cells necessary to achieve rapid engraftment to be 2 x 10(6)/kg.

The use of amplified variable number of tandem repeats (VNTR) in the detection of chimerism following bone marrow transplantation. A comparison with restriction fragment length polymorphism (RFLP) by Southern blotting.

Chimerism analysis after allogeneic bone marrow transplantation (alloBMT) allows detection of early marrow engraftment, disease relapse, and graft rejection. Our objective was to do retrospective and prospective studies of chimerism analysis by restriction fragment length polymorphism (RFLP) by Southern blotting and variable number of tandem repeats (VNTR) by polymerase chain reaction (PCR) to compare and contrast the methods. The retrospective group comprised 46 samples from 26 patients previously analyzed by RFLP, while the prospective group contained 34 samples from 25 patients. Using four different VNTR primers (D1S80, D17S30, D1S111, and APO-B), the recipient and donor samples amplified by the PCR were screened for unique banding patterns. The VNTR primer with the unique banding pattern was used to detect chimerism in each sample. A total of 635 VNTR analyses were performed. Interpretation was blinded for previous RFLP results. A comparison between the VNTR and RFLP results and a cost analysis of the two procedures were done. A unique VNTR banding pattern was present in 49 of 51 patients (identical twins in one case). The VNTR analysis showed complete chimerism in 68 samples, mixed chimerism in 9, and recurrences in 2. This agreed with the RFLP results in 64 (80%) of 80 samples. Failure to detect 1% to 10% of recipient DNA accounted for 15 (VNTR, 8; RFLP, 7) discordances. Follow-up revealed all donor DNA in five cases, decreasing quantities of recipient DNA in two cases (six samples), and no additional studies available in four cases. In one case, VNTR detected a complete chimerism when the DNA was insufficient for RFLP analysis. The cost analysis revealed an approximately 50% savings with the use of VNTR; VNTR is a viable alternative to RFLP in the detection of chimerism after bone marrow transplantation and offers substantial cost savings, faster turnaround time, easier preparation of the DNA, smaller DNA requirements, and the elimination of radioisotopes and cumbersome restriction enzymes.

Laboratory methods for the detection of hemoglobinopathies in the community hospital.

In the 1990s, community hospital laboratories can expect to evaluate anemia in patients from increasingly diverse racial and ethnic backgrounds, which will require the implementation of increasingly sophisticated laboratory methods for the detection of hemoglobinopathies. This article reviews the standard methods of alkaline and acid hemoglobin identification, and the methods of hemoglobin quantitation and ancillary tests are presented. Finally, a protocol is proposed that will allow the community hospital laboratory physicians to diagnose the majority of clinically significant hemoglobinopathies efficiently and accurately.

Significance of increased proportion of CD10-positive cells in nonmalignant bone marrows of children.

An immunophenotypic pattern characterized by increased proportions of CD10, CD19, and HLA-DR+ cells is observed in the bone marrow of some children with nonmalignant hematologic diagnoses. The purpose of this study was to ascertain the clinical and pathologic significance of this immunophenotype. The morphologic and immunophenotypic results of bone marrow specimens from 23 children with nonmalignant hematologic conditions are presented. In 11 children, an increased percentage of immature B-cell precursors was observed, suggesting the presence of B-lineage malignancy. None of the children have developed malignant lymphoproliferative disease in as long as 4 years of follow-up, despite the demonstration of a clonal lymphocyte proliferation in one patient. Although the biologic significance of the bone marrow immunophenotype is not yet understood, it is important to recognize that this lymphocyte pattern occurs commonly in children with nonmalignant hematologic conditions, and may reflect an increase in normally occurring lymphoid precursor cells.

Combined utility of gene rearrangement analysis and flow cytometry in the diagnosis of lymphoproliferative disease in the bone marrow.

The diagnosis of lymphoma involving bone marrow is often complicated by the presence of nonspecific lymphoid aggregates. Morphologic criteria may not permit the distinction of benign from malignant lymphoid aggregates with certainty in all cases. We combined morphology with immunophenotypic and immunogenotypic analyses of aspirated marrow cells to develop more reliable criteria for the diagnosis of marrow involvement by lymphoma. The presence of morphologically recognizable lymphoma cells in the bone marrow aspirate was always confirmed by immunogenotypic analysis. The yield of either immunophenotypic or immunogenotypic analyses on morphologically negative marrows was very low. Focal paratrabecular involvement by lymphoma was not always confirmed by immunophenotypic or immunogenotypic analyses, probably due to sampling error and factors interfering with aspiration of the lymphoid aggregates. Thus, the immunologic and molecular studies supported, but did not substantially improve upon the morphologic criteria that are in common usage for distinguishing benign from malignant lymphoid aggregates in the bone marrow. Finally, evidence of B-lymphocyte clonality was obtained in four of five cases in which there were nonspecific lymphoid aggregates in the bone marrow in the absence of otherwise clinically definable malignancy.

Plasma cell malignancy in the acquired immune deficiency syndrome. Association with Epstein-Barr virus.

The development of high-grade, malignant B-cell lymphoma is a well-recognized complication of human immunodeficiency virus (HIV) infection. Plasma cell neoplasms, however, have been rarely encountered in HIV-infected people. This study presents the morphologic and immunologic features of an unusual plasma cell tumor occurring in a 31-year-old HIV-antibody-positive male. The malignancy was characterized by widespread dissemination and hypercalcemia at presentation and a clinically aggressive course. Immunoperoxidase staining of tumor tissue obtained from biopsy and at autopsy had positive results for IgM and lambda. In the patient's serum, only an IgG kappa paraprotein was detected, indicating that the tumor was nonsecretory. DNA analysis of autopsy-derived tumor tissues demonstrated clonal rearrangements of the immunoglobulin (Ig) heavy chain gene locus and rearrangements in both kappa and lambda light chain gene loci. Furthermore, DNA hybridization studies revealed the presence of Epstein-Barr virus (EBV) genomes in tumor tissue but not in nontumor tissue from this patient.

Hematologic and bone marrow abnormalities in pediatric patients with human immunodeficiency virus (HIV) infection.

This study presents the peripheral blood and bone marrow findings in eight children with perinatally acquired HIV infection, ranging in age from 5 months to 3 years. The indications for bone marrow examination were comparable to those for adults with HIV infection and included cytopenia(s), slenomegaly, failure to thrive, and suspected tuberculosis. Thrombocytopenia was the most common indication, and platelet-associated antibodies were elevated in all patients with thrombocytopenia. The peripheral blood morphology was remarkable for the presence of plasmacytosis and eosinophilia in those patients with lymphocytic interstitial pneumonia. Five patients had trephine biopsies, and marrow cellularity was normal with normal or increased megakaryocytes in all cases. Lymphoid aggregates, also described in adult patients with acquired immunodeficiency syndrome (AIDS), were present in three of five trephine biopsies. In contrast to the adult patients, myelodysplasia was not observed in the pediatric age group. None of the eight children had malignancies or opportunistic infections that were diagnosed by bone marrow examination.

Clonal B-cell proliferation in an infant with congenital HIV infection and immune thrombocytopenia.

An infant with congenital human immunodeficiency virus (HIV) infection had immune thrombocytopenic purpura (ITP) develop at four months of age. A bone marrow aspirate had normal results in morphologic characteristics and cellularity. Flow cytometry analysis of the marrow cells showed that the predominant cell in the "lymphocyte" cluster was of B-lineage and common acute lymphocytic leukemia antigen (CALLA) positive. Southern blot analysis of marrow DNA demonstrated gene rearrangements in both the immunoglobulin (Ig) heavy chain and kappa light chain loci, confirming the presence of a clonal B-cell lymphoid proliferation. At one year of age the patient is clinically well without evidence of malignant lympho-proliferative disease. This case exemplifies a limited clonal B-cell expansion in the bone marrow of a patient with HIV infection and a benign hematologic condition.

Diffuse large-cell lymphoma with monoclonal IgM kappa and cold agglutinin.

A patient with diffuse large-cell lymphoma associated with a serum monoclonal IgM kappa and a cold agglutinin is described. The cold agglutinin was the initial manifestation of disease and was apparent for at least two months before the diagnosis of lymphoma. The lymphoma cells had surface and cytoplasmic IgM kappa.

Immunophenotyping of leukaemia: an immunoperoxidase method using air-dried smears.

An immunoperoxidase (IP) method utilizing monoclonal antibodies to demonstrate the presence of the common acute lymphocytic leukaemia antigen (CALLA) on leukaemic cells using air-dried blood and bone marrow smears is described. Two readily available CALLA-specific monoclonal antibodies, BA-3 and J5, and an avidin-biotin immunoperoxidase methodology were utilized. Nineteen cases of acute leukaemia or blast crisis of chronic myelogenous leukaemia were studied. Immunofluorescent studies correlated closely with the results of the immunoperoxidase technique.