SummaryBackgroundSurgical repair of craniosynostosis in young children is associated with copious bleeding and often coagulopathy. Typically, a reactive transfusion strategy is used to treat coagulopathy whereby fresh frozen plasma (FFP) is given only after clinical manifestation of clotting abnormality. This prospective, randomized clinical trial was designed to test the hypothesis that prophylactic FFP during craniofacial surgery reduces blood loss and blood transfusion requirements compared to a reactive FFP transfusion strategy.MethodsEighty‐one patients less than 2 years of age requiring primary repair of craniosynostosis were randomized to receive FFP using either a prophylactic or reactive strategy. Laboratory values were measured at four standardized time points. The volume of blood products transfused, length of stay in the pediatric intensive care unit (PICU), hospital length of stay, and number of donor exposures were recorded for each patient.ResultsThe prophylactic FFP group received a significantly greater average volume of FFP compared to the reactive group (29.7 ml·kg−1 vs 16.1 ml·kg−1; P < 0.001), which was associated with improvement in coagulation values at multiple time points. However, there was no difference in blood transfusion requirements or blood loss between the two groups. The two transfusion strategies resulted in similar median donor exposures. There was no difference in PICU or hospital length of stay.ConclusionA reactive FFP transfusion strategy required less plasma transfusion and was associated with similar rates of blood loss and PRBC transfusion as prophylactic FFP despite improvement in coagulation values in the prophylactic FFP group.

OBJECTIVE:To assess the performance of the QuantiFERON-TB Gold in-tube (QFT-GIT) assay for tuberculosis (TB) screening using a convenience sample from among a population of healthcare provider (HCP) employees of a hospital.METHODS:For the individuals in our cohort, we reviewed occupational health records, including TB risk factors, and the results of QFT-GIT testing. We considered a QFT-GIT result of greater than 0.35 IU/mL to be positive; when we obtained a positive result from a specimen from a particular individual, we repeated testing on a fresh specimen from that individual.RESULTS:Of the 758 HCP employees whose specimens we screened, 439 had negative QFT-GIT results with negative TB risk factors and 268 had a negative QFT-GIT result but had positive TB risk factors. QFT-GIT results were positive in 47 subjects. Of the positive participants, 12 had a mean TB antigen value (antigen minus nil stimulated concentrations [Ag-Nil]) of 0.61 on initial testing and had a negative result on repeat testing, 22 had a TB Ag-Nil of 1.19 on initial testing and had a positive result on repeat testing (P = .01).CONCLUSIONS:The QFT-GIT assay is useful for screening HCPs. However, false-positive results occur, particularly in a borderline zone of less than 1 IU/ml. Re-evaluation by repeat testing of fresh specimens from the same individual should be considered in subjects whose specimens test within the low-level positive cutoff.

BACKGROUND:There is little systematically derived evidence-based guidance to inform plasma transfusion decisions. To address this issue, the AABB commissioned the development of clinical practice guidelines to help direct appropriate transfusion of plasma.STUDY DESIGN AND METHODS:A systematic review (SR) and meta-analysis of randomized and observational studies was performed to quantify known benefits and harms of plasma transfusion in common clinical scenarios (see accompanying article). A multidisciplinary guidelines panel then used the SR and the GRADE methodology to develop evidence-based plasma transfusion guidelines as well as identify areas for future investigation.RESULTS:Based on evidence ranging primarily from moderate to very low in quality, the panel developed the following guidelines: 1) The panel suggested that plasma be transfused to patients requiring massive transfusion. However, 2) the panel could not recommend for or against transfusion of plasma at a plasma : red blood cell ratio of 1:3 or more during massive transfusion, 3) nor could the panel recommend for or against transfusion of plasma to patients undergoing surgery in the absence of massive transfusion. 4) The panel suggested that plasma be transfused in patients with warfarin therapy-related intracranial hemorrhage, 5) but could not recommend for or against transfusion of plasma to reverse warfarin anticoagulation in patients without intracranial hemorrhage. 6) The panel suggested against plasma transfusion for other selected groups of patients.CONCLUSION:We have systematically developed evidence-based guidance to inform plasma transfusion decisions in common clinical scenarios. Data from additional randomized studies will be required to establish more comprehensive and definitive guidelines for plasma transfusion.

The BD Directigen™ EZ RSV (BD) assay and the Binax NOW® RSV (BN) assays are lateral flow immunochromatographic assays used in the rapid diagnosis of respiratory syncytial virus (RSV) infection. A prospective study was undertaken to compare the performance characteristics of the BD and BN assays using 99 fresh nasopharyngeal aspirate specimens from children. All specimens were cultured by tube cultures and R-mix shell vials. Culture-negative specimens that tested positive by 1 or both antigen tests were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) for the presence of RSV nucleic acids. Specimens positive by culture and/or RT-PCR were considered true positives. The sensitivity, specificity, positive predictive value, and negative predictive value for BD were 90%, 94%, 94%, and 90%, respectively, and those for BN were 90%, 100%, 100%, and 90% respectively. The overall agreement between the 2 assays was 93% (confidence interval, 86.3–96.6%). Three specimens negative by both assays were positive by viral culture. The rapid lateral flow immunochromatographic assays were easy to perform and easy to interpret, and the results are available in less than 30 min. The reduced turnaround time offered by these assays is particularly useful in the rapid detection of RSV infections in children from outpatient settings.

Earlier we reported a case of plasma cell-rich acute cellular rejection of a transplanted kidney, which was associated with a serum antibody to the red blood cell (RBC) Kidd antigen, Jk-b, without a history of recent transfusion. We presented data to suggest that the plasma cells in the kidney made antibodies to the Jk-b antigen, which is also present in the kidney and may, in turn, have contributed to the rejection process (1). Since that time, we have identified a second patient with a milder but similar clinical course and antibodies to Jk-a, the polymorphic allele Kidd antigen. The patient was a 19-yr-old female who had received a kidney transplant from her father (three of six HLA match) seven yr earlier after developing end-stage renal disease secondary to focal segmental glomerulosclerosis. Her transplant status had been stable with a serum creatinine of 1.2–1.5 mg/dL with her immunosuppression consisting of alternate day prednisone, mycophenolate mofetile, and cyclosporine. She had a history of nephrolithiasis in the transplanted kidney and had recently experienced several urinary tract infections. A rise in her serum creatinine to 2.5 mg/dL and a recent history of medication non-adherence, as reflected by undetectable cyclosporine levels, prompted the performance of a kidney biopsy. The needle biopsy of the renal allograft revealed chronic allograft nephropathy with acute cellular rejection. The interstitium was infiltrated by lymphocytes and plasma cells that occupied 60% of its area. The plasma cells comprised less than 5% of the infiltrating mononuclear inflammatory cells. The tubules were infiltrated by lymphocytes that ranged from four to 10 lymphocytes per tubular cross-section. The blood vessels were free of inflammation. There were no viral inclusions or cytopathic viral changes. Immunohistochemical studies for CMV, EBV, and BK virus were negative. An immunofluorescence study for C4d using indirect immunofluorescence technique on frozen tissue with a monoclonal antibody (AbD Serotec, dilution 1:100) revealed no labeling of interstitial capillaries. The acute cellular rejection was graded as IA by the Banff criteria. Polymerase chain reaction (PCR) on blood was negative for BK and CMV virus, and there were only 500 copies/mL of EBV virus. PCR on urine revealed only 10 900 copies/mL of BK virus. The patient was treated with high dose steroids for three days and a repeat biopsy in one wk showed decreased lymphocytic infiltrates in the interstitium with only rare plasma cells. There was resolution of the acute cellular rejection with disappearance of lymphocytes from the tubular epithelium. The patient was discharged in stable condition and has remained so with a serum creatinine of 1.6–2.0 mg/dL. Although the patient had received two units of RBCs in association with her transplant seven yr earlier, her antibody screen for antibodies to red cells had been negative six months following the transplant and she had received no additional transfusions or treatment with intravenous immunoglobulin. Nevertheless, at the time of the first biopsy, the patient was noted to have low titer IgG serum antibodies to the RBC Kidd antigen, Jka. The patient s phenotype is Jk-a negative, Jk-b positive. The kidney donor was her father and his RBC phenotype is Jk-a positive, Jk-b positive. An eluate of the kidney biopsy contained anti-Jk-a antibodies. We previously discussed the possible functional relationship between kidney rejection and antibodies to the RBC Kidd antigen (1). The Pediatr Transplantation 2008: 12: 918–919 2008 Wiley Periodicals, Inc.

Plasma cell-rich acute cellular rejection of a transplanted kidney is described in association with the identification of serum antibody to the red cell Kidd antigen, Jk-b, which is also found in the kidney. This antibody was formed without a history of a recent blood transfusion or exposure to intravenous immunoglobulin. The role this antibody could have in the rejection of the transplant is discussed.

To the Editor: Mou and associates (Oct. 14 issue)1 report that the use of fresh whole blood for pump priming in cardiopulmonary bypass surgery in infants had no advantage over the use of reconstitu...

The term multiplex PCR refers to simultaneous amplification of more than one target in a single PCR. This method has some advantages but presents the possibility of competition between multiple targets for a finite number of reagents, which may invalidate the assay. The Roche Cobas AMPLICOR multiplex PCR for Chlamydia trachomatis and Neisseria gonorrhoeae, a U.S. Food and Drug Administration-approved assay, can detect both C. trachomatis and N. gonorrhoeae from a single specimen. PCR amplification of C. trachomatis and N. gonorrhoeae proceeds in one tube with a shared enzyme and shared nucleotides but independent biotinylated primers. An optional internal control (IC) permits detection of amplification inhibition. The IC DNA has primer binding sequences identical to those of the C. trachomatis target. Detection is accomplished by using oligonucleotide probes that are unique for C. trachomatis, N. gonorrhoeae, and IC, respectively, with colorimetric quantification by spectophotometer. A negative assay result is valid if the IC optical density (OD) is ≥0.2, indicating successful amplification (Method Manual, Cobas Amplicor, Roche Diagnostics, 12/1999, Revision 3.0). We performed 580 multiplex PCRs on endocervical and urethral swab specimens, and 58 assays yielded positive results for C. trachomatis alone (OD ≥ 2.0), 13 assays yielded positive results for N. gonorrhoeae alone (OD ≥ 3.5), and 4 assays yielded positive results for both. Of the 58 assays positive for C. trachomatis, 13 (22.4%) had IC OD values that were <0.2 (mean OD, 0.059). The results of these 13 assays were correctly interpreted as C. trachomatis positive (mean C. trachomatis OD, 3.187). Because of the failure of the IC to amplify, the N. gonorrhoeae OD values for these 13 were invalid. The limiting reagent could be the primer, shared by C. trachomatis and IC. In the absence of IC amplification, it was not clear that there were a sufficient number of reagents, aside from the primer, to amplify N. gonorrhoeae. Of these 13 assays, 11 had OD values interpreted as negative for N. gonorrhoeae (mean N. gonorrhoeae OD, 0.047). Five of these specimens from the 11 assays were cultured and yielded negative results. Two additional specimens that turned out to be N. gonorrhoeae culture positive had equivocal N. gonorrhoeae OD values of 2.013 and 3.492, respectively, resolvable as positive by duplicate repeat testing. A Roche Molecular Systems study suggested that competitive inhibition occurs when the relative concentration of one target is extremely high and that the competition is for reagents other than the primer. This Roche paper discusses the optional use of the IC for increased sensitivity as well as retesting of existing specimens to eliminate nonspecific, labile polymerase inhibition, which we saw in five specimens not discussed here (1). However, we had a very high rate of competitive inhibition not correctable by repetition. Experimental dilution of the specimens did result in IC amplification, but also, in one case, converted a positive C. trachomatis assay result to negative. The Molecular Pathology Checklist of the College of American Pathologists states that “the laboratory should be able to distinguish a true negative result from a false negative result due to PCR failure.” ICs accomplish this. The competitive inhibition described could be resolved by repeat amplification with only the N. gonorrhoeae primer. For the two equivocal specimens, positive for C. trachomatis, the initial OD value cutoff for N. gonorrhoeae positivity could be lower than the stated 3.5; perhaps 2.0 could be used as the cutoff OD value for duplicate repeat testing of specimens that produce equivocal results. Competitive inhibition is not fully addressed in the Method Manual. Undetected false negatives, due to failure to include the so-called optional IC, are misleading.

Culture and serology, the traditional mainstays of diagnostic testing for childhood respiratory infections, often take too long to be of real value in clinical decision making. Rapid diagnostic tests for confirmation of influenza, respiratory syncytial virus infection, and group A streptococcal infection lead to a reduction in the inappropriate use of antibiotics and lower costs by reducing the use of ancillary tests and, in some cases, the length of hospital stays.

ABSTRACT Exploiting the high sensitivity of the chemiluminescence phenomenon, an accurate and sensitive point-of-care test, called the ZstatFlu-II test (ZymeTx, Inc., Oklahoma City, Okla.), was developed to detect influenza virus infections. The ZstatFlu-II test takes 20 min and requires approximately 2 min of “hands-on” time for operational steps. The ZstatFlu-II test does not distinguish between infections with influenza virus types A and B. ZstatFlu-II test results are printed on Polaroid High-Speed Detector Film, allowing test results to be archived. A prototype version of the ZstatFlu-II test was evaluated during the 2000-to-2001 flu season with 300 nasal aspirate specimens from children at a pediatric hospital. Compared to culture, the ZstatFlu-II test had 88% sensitivity and 92% specificity. The Directigen test had a sensitivity of 75% and a specificity of 93%. The sensitivity of the ZstatFlu-II test was significantly higher than that of the Directigen test ( P < 0.0574).

BACKGROUND:During summer enteroviral meningitis is a common cause of febrile illness in children, who are typically hospitalized for 2 to 3 days if bacterial infection is suspected. It has been hypothesized that a sensitive polymerase chain reaction (PCR) assay could quickly confirm the diagnosis and subsequently decrease hospitalization costs. However, to have maximum impact results should be available within 24 h. This necessitates daily assays on small numbers of samples. METHODS:We examined the clinical utility of a PCR assay during two summers, comparing length of stay and charges. Only during the second summer were results reported to clinicians. Case controls were patients with negative PCR assay results but uncomplicated, presumed viral infections. We determined the cost per case identified with and without pleocytosis as a screen for PCR testing. RESULTS:During the first summer 25% (5/20) of patients with positive PCR assay results remained hospitalized for >2 days. During the second summer 10.2% (6 of 59) of children with positive enteroviral PCR assay results but 37.9% (25 of 66) of case controls remained hospitalized for >2 days. The mean length of hospitalization was significantly (P < 0.05) shorter for patients with positive PCR test results than for case controls. The material cost was approximately $238 per case identified. CONCLUSIONS:PCR testing has clinical utility for diagnosis of enteroviral meningitis. Although the demands for daily testing make the test expensive, it appears to be cost-effective with savings related to shorter hospital stays.

A monoclonal antibody (MAb), designated 4H12, was selected for reactivity to a surface antigen on PYS-2 teratocarcinoma cells. 4H12 was the product of a fusion of lymphoid cells of a non-immunized pregnant C57BL/6 mouse to NS-1 myeloma cells. Initial studies utilizing immunohistochemistry revealed that MAb 4H12 bound to an antigen found on cells in the decidua basalis of 7-, 8- and 10-day pregnant mice. Antigen-positive cells of 11--19-day pregnant mice were also found predominantly in the decidua. A few antigen-positive cells were found in the labyrinth of the placenta and up against Reichert's membrane. Antigen-positive cells were morphologically and spatially distinct, oval to round with large periodic acid Schiff positive granules. Indirect immunofluorescent (IIF) labeling of decidual cultures showed antigen on the surface of cells that were small, oval to round and adherent. The antigen recognized by MAb 4H12 was removed from tissue sections with trypsin and protease and therefore is suggested to be a protein. We conclude that MAb 4H12 recognizes a surface antigen found on cells historically described as granulated metrial gland (GMG) cells. This MAb should greatly facilitate the further analysis of the life history and function of GMG cells during pregnancy.

Monoclonal antibody MA21 recognized a 44kDa plasma membrane protein on F9 teratocarcinoma cells, trophectoderm of mouse peri-implantation-stage blastocyst and ectoplacental cone cells of 5 day postcoitum implanted blastocyst (Vernon, Linnemeyer and Hamilton, 1989). We show here that this antigen is expressed by trophoblast cells of the maturing placenta. Immunohistochemical assays of early and mature placental tissue sections, indirect immunofluorescence labelling of placental cultures and blastocyst outgrowths in vitro, and immunoprecipitation of 35S-labelled NP-40 extracts of placental cultures indicate the presence of a plasma membrane-associated antigen with the same characteristics as MA21 antigen of peri-implantation embryos and F9 teratocarcinoma cells. In sections of placentae, antigen-positive cells are always situated in a thin layer between trophoblastic giant cells and maternal tissue. In cultures of postimplantation stage embryos, attached trophoblast cells express MA21 antigen initially, but following transformation to the giant cell state, antigen is no longer expressed. These results indicate the presence of a plasma membrane protein antigen associated with a distinct population of cells believed to be trophoblast. We believe that these cells are the foremost trophoblast cells opposing maternal decidua and that they may give rise to secondary trophoblastic giant cells.

A monoclonal antibody (MAb) “MA21”, derived from lymphoid tissue of a multiparous mouse and selected for binding to mouse teratocarcinoma cell line F9, recognizes a surface antigen that appears on peri-implantation-stage mouse blastocysts. In indirect immunofluorescence assays, MAb MA21 does not bind to I-cell-through morula-stage embryos, nor to early, 3.5-day post-coitum (p.c.) blastocysts. When 3.5-day p.c. blastocysts are maintained 17 h in vitro and then assayed, MAb MA21 binds to a limited number of trophectoderm cells that are centered at the embryonic pole. As culture time lengthens, the number of antigen-expressing trophectoderm cells increases, forming a cap that spreads from the embryonic pole into the abembryonic region. Embryos maintained 48 h in vitro bind MAb MA21 over as much as 100% of the trophectoderm surface. MAb MA21 does not bind to the inner cell mass. When mouse pregnancy uteri are assayed by the immunoperoxidase method, MAb MA21 binds to extra-embryonic ectoderm and trophectoderm of 5-day p.c. implanted blastocysts, but does not bind to 6-day p.c. blastocysts. MAb MA21 recognizes a component with an estimated mol. wt of 44,000 from NP-40 detergent extracts of F9 cells and peri-implantation-stage mouse blastocysts. The component appears to be firmly associated with the plasma membrane; it is resistant to removal by high salt or moderate concentrations of non-ionic detergent.

Evolutionarily conserved antigens are present on spermatozoa of several mammalian species. We tested sera from infertile men and women containing antisperm antibodies (ASAs) for their reactivity with FA-1, an antigen known to be present on murine and human spermatozoa. Fifty percent of male sera and 63% of female sera contained anti-FA-1 antibodies, as judged by enzyme linked immunosorbent assay (ELISA). Fourteen percent of male sera and 50% of female sera were also shown to possess ASAs reactive with living mouse spermatozoa, and murine in vitro fertilization was inhibited by human antibodies. These results suggest that the transfer of immunoglobulins from human sera to spermatozoa of other species may provide a model to study how ASAs effect sperm function.

A panel of sperm-reacted monoclonal auto-antibodies developed from spleen cells of vasectomized mice (BDF1) were characterized. Immuogenic antigens were mainly located on the acrosome, midpiece and principal piece. All of the monoclonals were IgM; three demonstrated a multispecific reaction with testis and/or epididymis antigen extracts by immunoblotting. Immunobead studies indicated that most of the antibodies were to surface molecules, a finding supported by the observation that five of the seven antibodies caused complement-mediated immobilization (although not sperm agglutination) of mouse sperm. In vitro fertilization was significantly impaired when antibodies (Vx5, 8 and 10) were added to the sperm prior to exposure to the eggs. Furthermore, passive immunization with Vx5 antibody reduced in vivo fertilization. Our findings indicate that vasectomy-generated antibodies can reduce sperm function in vitro and in vivo.

It has been postulated that an acquired immunodeficiency syndrome develops in neonates as the result of a maternally induced graft-vs-host disease (GVHD) that develops when sufficient numbers of maternal lymphocytes are transferred to the fetus across the placental barrier. The present study was done to determine whether major histocompatibility complex (MHC) antigens or non-MHC minor histocompatibility antigens (MiHA) were involved. Female C57BL/6 mice were bred to males of eight selected strains such that maternal-fetal disparity existed at MHC antigens and/or minor histocompatibility antigens. Offspring were tested for immune function at 6-7 weeks of age using a Jerne plaque assay to measure the humoral response to that T dependent antigen sheep red blood cells (SRBC). None of the offspring developed clinical signs of GVHD, but 3 of 124 mice tested made no immune response to SRBC. Immunodeficiency was associated with maternal-fetal disparity only at a small number of MiHA and not at the MHC. We postulate that immunodeficiency in this model is mediated by a subclinical maternally induced GVHD to paternally derived MiHA of the fetus.

Antisperm antibodies are implicated as one causative factor of infertility, but the target antigens have not been identified. Immune responses to sperm antigens are qualitatively variable even within a single mouse strain. We took advantage of this variability and immunized individual female mice to allogeneic sperm to reflect their natural exposure during mating. We determined the ability of the individual sera to inhibit in vitro fertilization and to bind to sperm antigens separated by electrophoresis. Compared to preimmune sera, four of five immune sera significantly inhibited in vitro fertilization. The serum from individual mice bound variable panels of sperm antigens. By comparing the panels, we identified two polypeptides with molecular weights of 40,000 and 44,000 that were bound by all sera. We propose that these molecules may be good candidates for further investigation of the immunoprophylaxis of pregnancy.

CBA/J female mice mated to DBA/2 male mice have a high level of fetal resorption. The rate of resorption can be influenced by the environment in which the animals are maintained.