Robyn Rodwell

Early diagnosis of neonatal sepsis using a hematologic scoring system

Hematologic findings and published complete blood cell count criteria were evaluated as screening tests for neonatal sepsis. From the data obtained, a hematologic scoring system was formulated that assigns a score of 1 for each of seven findings: abnormal total leukocyte count, abnormal total neutrophil (PMN) count, elevated immature PMN count, elevated immature to total PMN ratio, immature to mature PMN ratio greater than or equal to 0.3, platelet count less than or equal to 150,000/mm3, and pronounced degenerative changes in PMNs. There were 298 evaluations for sepsis (243 in the first 24 hours of life and 55 between days 2 and 30). Twenty-six of 27 (96%) infants with sepsis and all 23 infants with probable infection had scores greater than or equal to 3, compared with 35 of 248 (14%) noninfected infants. The likelihood of sepsis with score greater than or equal to 3 was 31%, and this value differed with both gestational and postnatal ages (34% vs 8% in preterm and term infants less than 24 hours of age, and 65% thereafter). The higher the score the greater was the likelihood of sepsis. With score less than or equal to 2 the likelihood that sepsis was absent was 99%. The hematologic scoring system should improve the diagnostic accuracy of the complete blood cell count as a screening test for sepsis and could simplify and standardize the interpretation of this global test.

Hematologic scoring system in early diagnosis of sepsis in neutropenic newborns.

The hematologic profiles of 1000 newborns were prospectively examined to identify infants with neutropenia (N = 170) according to the system of Manroe et al. (J Pediatr 1979;95:89-98) and to evaluate a hematologic scoring system (Rodwell et al. J Pediatr 1988;112:761-7) as a screening test for sepsis. Neutropenia was more commonly of noninfectious than infectious origin (83.5% vs. 16.5%; P < 0.001). On the initial test a positive screen (scores > or = 3) identified 26 of 28 infants with sepsis or probable infection (sensitivity 93%; specificity 82%; positive and negative predictive values 50 and 98%, respectively). Corresponding values for an elevated immature:total neutrophil ratio were 100, 75, 43 and 100%. Overall mortality with neutropenia was 15% and was higher with an infectious than a noninfectious etiology (39% vs. 11%, P < 0.001) despite early antibiotic therapy. The combination of a neutrophil count < or = 500/mm3 and scores > or = 3 or an elevated immature:total neutrophil ratio identified a poor prognostic group: 67% (8 of 12) and 70% (7 of 10) infants, respectively, with these findings died, 6 in the infected group. The hematologic scoring system or immature:total neutrophil ratio in combination with the degree of neutropenia provides valuable diagnostic and prognostic information which could be applied to identification of possible candidates for granulocyte transfusions or other experimental treatments.

FLT3-Ligand Treatment of Humanized Mice Results in the Generation of Large Numbers of CD141+ and CD1c+ Dendritic Cells In Vivo

We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141+ DC, CD1c+ DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c+ DC and CD141+ DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c+ DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141+ DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c+ DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8+ T cells, CD141+ DC are superior in their capacity to cross-present protein Ag to CD8+ T cells following activation with polyinosinic-polycytidylic acid. CD141+ DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141+ and CD1c+ DC and evaluate adjuvants and DC-targeting strategies in vivo.

Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: A platform for cancer immunotherapy

The fundamental role of dendritic cells (DC) in initiating and directing the primary immune response is well established. Furthermore, it is now accepted that DC may be useful in new vaccination strategies for preventing certain malignant and infectious diseases. As blood DC (BDC) physiology differs from that of the DC homologues generated in vitro from monocyte precursors, it is becoming more relevant to consider BDC for therapeutic interventions. Until recently, protocols for the isolation of BDC were laborious and inefficient; therefore, their use for investigative cancer immunotherapy is not widespread. In this study, we carefully documented BDC counts, yields and subsets during apheresis (Cobe Spectra), the initial and essential procedure in creating a BDC isolation platform for cancer immunotherapy. We established that an automated software package (Version 6.0 AutoPBPC) provides an operator-independent reliable source of mononuclear cells (MNC) for BDC preparation. Further, we observed that BDC might be recovered in high yields, often greater than 100% relative to the number of circulating BDC predicted by blood volume. An average of 66 million (range, 17-179) BDC per 10-l procedure were obtained, largely satisfying the needs for immunization. Higher yields were possible on total processed blood volumes of 15 l. BDC were not activated by the isolation procedure and, more importantly, both BDC subsets (CD11c(+)CD123(low) and CD11c(-)CD123(high)) were equally represented. Finally, we established that the apheresis product could be used for antibody-based BDC immunoselection and demonstrated that fully functional BDC can be obtained by this procedure.

Granulocyte colony stimulating factor treatment for alloimmune neonatal neutropenia.

Granulocyte colony stimulating factor (G-CSF) treatment was successfully used in three preterm infants with alloimmune neonatal neutropenia (AINN). Two infants had persistent neutropenia despite treatment with intravenous immunoglobulin and random donor granulocyte transfusions for presumed sepsis. Neutrophil counts returned to normal with G-CSF treatment; the response was least convincing in one infant with fulminant necrotising enterocolits. It is suggested that treatment with G-CSF be considered early for the treatment of infants with AINN.

Long-Term Interferon-Alpha 2A Does not Induce Sustained Hematologic Remission in Younger Patients with Essential Thrombocythemia

The ability of Interferon alpha (alpha-IFN) to alter the natural history of essential thrombocythemia (ET) and induce sustained hematologic remission would provide further impetus to consider this agent in younger patients with this disease and may influence the decision to commence treatment in asymptomatic patients. This study has failed to demonstrate any sustained hematologic remissions after cessation of long-term (2 years) alpha-IFN administration in a group of 34 female patients with a median age of 41 years (range 14-68) who were considered at intermediate to high risk of thrombotic complications. In the twenty-one patients completing two years of therapy, 13 (62%) had complete hematological responses (CHR; platelet count <400 x 10(9)/L), 7 (33%) partial hematological responses (PHR; platelet count 400-600 x 10(9)/L) and no thrombotic or hemorrhagic complications occurred. In all patients who discontinued alpha-IFN at 2 years, platelet counts rose above the normal range within 1-4 months and the majority required reinstitution of some form of therapy. The inability of long-term alpha-IFN to induce sustained, unmaintained hematologic remission argues strongly against any significant effect on the neoplastic clone at the doses used in this study. This study does, however, confirm the efficacy of long-term alpha-IFN in younger female patients with ET, a group not previously well represented in clinical trials of the agent.

Compartmentalization of allogeneic T-cell responses in the bone marrow and spleen of humanized NOD/SCID mice containing activated human resident myeloid dendritic cells.

OBJECTIVE Human allogeneic (allo)-T-cell responses within recipient lymphoid tissues and the degree to which they are altered in the presence of activated tissue-resident dendritic cells (DC) remain unknown. This study examined allo-T-cell recruitment and the early allo-T-cell responses that occur in the bone marrow (BM) and spleen (SP) of humanized (hu) nonobese diabetic (NOD)/severe combined immunodeficient (SCID) recipients containing activated human tissue-resident myeloid DC (MDC). MATERIALS AND METHODS Human naïve allo-T cells were transferred into polyinosinic:polycytidylic acid [poly(I:C)]-treated or untreated huNOD/SCID recipients containing human tissue-resident DC derived from transplanted CD34(+) cells. Activation of human tissue-resident MDC mediated by poly(I:C) treatment, recruitment, proliferation, and effector differentiation of allo-T cells in the BM and SP of huNOD/SCID recipients were analyzed in vivo by flow cytometry. RESULTS Poly(I:C) treatment induced transient activation of human MDC within a maximum of 8 hours, as evidenced in the BM by an increased proportion of MDC-expressing CD86 while in the SP by MDC expressing CD86 and producing interleukin-12. Poly(I:C)-pretreated huNOD/SCID recipients showed changes in the recruitment of allo-T cells in the BM and SP and developed different allo-T cell responses within the BM and SP compartments. In the BM, allo-T cells underwent multiple divisions and increased numbers of interferon-gamma(+) and tumor necrosis factor-alpha(+) effector cells, while the majority of splenic allo-T cells underwent a single division and had fewer effector allo-T cells. CONCLUSIONS Our experimental transplantation model demonstrates that early allo-T-cell responses are regulated by compartmentalization in the BM and secondary lymphoid tissues; events potentially occurring after allotransplantation in human recipients.

Screening for cryptantigen exposure and polyagglutination in neonates with suspected necrotizing enterocolitis

Red blood cell (RBC) cryptantigens are receptors comprised of galactose residues which are normally concealed by a coating of neuraminic (sialic) acid in the sub-terminal structure of the RBC membrane.‘ They are commonly referred to as the T (Thomsen-Freidenreich) cryptantigens after the early investigators.’ Exposure on the surface membrane of the RBC or activation of the cryptantigens, previously thought to be a rare occurrence, was found in 0.6% of all neonatal admissions in a recent study.’ The frequency of this phenomenon is highest (range 11 -34%) in neonates with necrotizing enterocolitis (NEC).2-4 In infants with NEC, bacterial enzymes are responsible for this phenomenon which involves the display on the RBC surface of the normally masked crypt antigen^.^ Studies in neonates with NEC demonstrate that cryptantigen exposure is associated in the majority of cases with Clostridial infections, a fulminant course, intestinal and a four-fold increase in the likelihood of surgical inter~ention.~ Exposure of the cryptantigens renders the RBC polyagglutinable, that is, agglutinable by most ABO compatible adult sera which contain IgM anti-T, but not by sera from umbilical cord or infants less than 6 months who lack the antibody.’ Consequently, in neonates with Tactivation, there is minimal risk of haemolysis. However, if IgM anti-T containing blood products are transfused, polyagglutination and potentially lethal intravascular haemolysis may ensue.’-5 The risk of haemolysis can be minimized by the use of washed RBC or platelets2 Recent studies show screening of infants with suspected NEC for cryptantigen exposure can reduce the frequency of transfusion induced intravascular haemolysis2f6 and the mortality associated with necrotizing enterocolitis.6 Thus, in infants with suspected NEC, cryptantigen exposure provides valuable presumptive information as to the causative organism, an early guide to antibiotic therapy and thus has diagnostic, prognostic and therapeutic implication^.^*^ In this issue, Marshall et a/. report a fatal case of NEC associated with cryptantigen exposure, polyagglutination and intravascular haem~lysis.~ Cryptantigen exposure was not detected by standard pre-transfusion serological testing and this is a feature of this disorder. This case serves as a timely warning of the importance of early recognition of this phenomenon, and the transfusion precautions necessary to avoid polyagglutination and potentially fatal intravascular haemolysis. The recommendations of Marshall and associates merit emphasis and should prompt all neonatal units to introduce screening programmes for cryptantigen exposure in infants with suspected NEC, and to develop management strategies and transfusion regimens for infants whose RBC exhibit this phenomenon. A ETI 0 LOGY AN D PATH OG EN ES I S

Safe mobilization of normal progenitors in advanced chronic myeloid leukemia with intensive chemotherapy and granulocyte-colony stimulating factor.

Twenty-one patients with advanced chronic myeloid leukemia (late chronic phase (n = 8), accelerated phase (n = 11) and blast crisis (n = 2)) were treated with idarubicin, cytarabine, and etoposide followed by G-CSF and subsequent collection of peripheral blood progenitor cells in the early recovery phase. Treatment was reasonably well tolerated with no deaths or intensive care admissions. Despite the advanced phase of disease and heavy pretreatment with cytotoxics and interferon-alfa, 11 of 21 patients (52%) achieved a cytogenetic response. Of the nine major cytogenetic responses (complete (n = 3) and partial (n = 6)), seven achieved adequate progenitor collections for consideration for autologous transplantation. The only predictor of response was disease duration (P = 0.02). With a median follow-up of 1171 days from treatment it appears unlikely that G-CSF contributed to disease progression. Survival post-IcE was predicted by disease stage (P = 0.0001). Intensive chemotherapy followed by G-CSF allowed adequate yields of predominantly Philadelphia chromosome negative progenitor cells to be obtained from one-third of patients with advanced CML.

Reference intervals for plasma sulfate and urinary sulfate excretion in pregnancy.

BackgroundSulfate is important for fetal growth and development. During pregnancy, the fetus relies on sulfate from the maternal circulation. We report reference intervals for maternal plasma sulfate levels and fractional excretion index (FEI) for sulfate in pregnancy, as well as sulfate levels in cord blood from term pregnancies.MethodsPlasma and urine were collected from 103 pregnant women of 10-20 weeks gestation and 106 pregnant women of 30-37 weeks gestation. Venous cord plasma was collected from 80 healthy term babies. Sulfate levels were measured by ion chromatography. Plasma and urinary creatinine levels were used to calculate FEI sulfate in pregnant women. Analyses provide reference intervals, and explored the relationship between maternal sulfate data with several prenatal factors.ResultsMedian maternal plasma sulfate levels were 452 μmol/L and 502 μmol/L at 10-20 and 30-37 weeks gestation, respectively, and inversely correlated with FEI sulfate median values of 0.15 and 0.11. Overall reference intervals were 305-710 and 335-701 μmol/L (2.5th; 97.5th percentile; for 10-20 and 30-37 weeks gestation, respectively) for maternal plasma sulfate, and 0.06-0.31 and 0.05-0.28 for maternal FEI sulfate. Term venous cord plasma sulfate median levels were significantly (p = 0.038) higher in female babies (375 μmol/L) when compared to male babies (342 μmol/L), with an overall reference interval of 175-603 μmol/L.ConclusionsWe provide the first reference intervals for maternal plasma sulfate levels and FEI sulfate, as well as cord plasma sulfate levels. These findings provide reference data for further studies of sulfate levels in both mother and child.