Hematologic Abnormalities in Severe Neonatal Necrotizing Enterocolitis: 25 Years Later

Necrotizing enterocolitis (NEC), the most common surgical emergency in newborns, remains a therapeutic challenge for clinicians. The hematological manifestations associated with NEC were first described 25 years ago. This review discusses current knowledge of the pathophysiology involved in disturbances in megakaryocytopoiesis, coagulation, leukopoiesis, and erythropoiesis that accompany the clinical entity NEC. The discussion includes current understanding of and potential strategies for treating the hematopoietic disturbances that occur secondary to NEC.

Related papers:

title: Hematologic abnormalities in severe neonatal necrotizing enterocolitis.
abstract: Serial determinations of the absolute granulocyte and platelet counts were performed in 40 infants with severe neonatal necrotizing enterocolitis. Fourteen of the 38 infants had absolute granulocyte counts less than 1,500 nm3, the mean absolute granulocyte count was significantly lower in the group of infants who died during the acute episode of NEC as compared to that of the infants who survived. Thrity-five of 40 infants had nadir platelet counts less than 150,000/nm3, clinical bleeding occurred in 12 of the thrombocytopenic infants. Fourteen thrombocytopenic infants were evaluated for disseminated intravascular coagulation by additional coagulation studies; six were noted to have laboratory evidence of DIC. We conclude that (1) a low absolute granulocyte count in severe NEC is associated with a poor prognosis and (2) thrombocytopenia is a significant problem in severe NEC and may occur with or without evidence of fulminant intravascular coagulation.

title: Hematologic abnormalities in acute necrotizing enterocolitis.
abstract: This presentation emphasizes the possible clinical significance of hematologic abnormalities in acute necrotizing enterocolitis and presents the author’s experience.

title: Thrombocytopenia in the high-risk infant.
abstract: One-hundred twenty-nine high-risk infants with thrombocytopenia and 238 control infants without thrombocytopenia were evaluated. Mothers of thrombocytopenic babies had similar history to those of nonthrombocytopenic babies, although fewer chronic narcotic abusers were found among mothers of thrombocytopenic babies. Thrombocytopenia was more common in babies less than 37 weeks' gestation and in sick babies compared to healthy babies. Sixty percent of infants had no recognizable cause of thrombocytopenia. Features associated with thrombocytopenia included umbilical line placement, respiratory assistance, hyperbilirubinemia, phototherapy, prematurity, respiratory distress syndrome, low Apgar score, sepsis, meconium aspiration, and necrotizing entercolitis. Thrombocytopenic babies had Apgar score, sepsis, meconium aspiration, and necrotizing entercolitis. Thrombocytopenic babies had more complications, more hemorrhage, and greater mortality than nonthrombocytopenic babies. Platelet size was increased in two babies with immune thrombocytopenia and in none of the others. This study shows that neonatal thrombocytopenia is often associated with high-risk factors and with increased hemorrhage, morbidity, and mortality. This relationship suggests an important prognostic value to platelet size was increased in two babies with immune thrombocytopenia and in none of the others. This study shows that neonatal thrombocytopenia is often associated with high-risk factors and with increased hemorrhage, morbidity, and mortality. This relationship suggests an important prognostic value to platelet counts, although the extent to which the thrombocytopenia contributed directly to morbidity and mortality is not clear.

title: Hypoxia causes ischemic bowel necrosis in rats: the role of platelet-activating factor (PAF-acether).
abstract: We have previously shown that injection of platelet-activating factor causes necrotizing enterocolitis in the rat and that platelet-activating factor is an endogenous mediator in lipopolysaccharide-induced bowel necrosis. Because hypoxia is a known predisposing factor for neonatal necrotizing enterocolitis, we investigated the effect of hypoxia on platelet-activating factor formation and intestinal necrosis. Young male Sprague-Dawley rats were made severely hypoxic by placing them in a 100% N2 chamber for 2 minutes; moderate hypoxia was accomplished using 10% O2 for 15 or 30 minutes. To evaluate the role of platelet-activating factor on intestinal perfusion and injury, two platelet-activating factor antagonists, SRI 63-441 and WEB 2086, were injected 10 minutes before the hypoxic exposure. We found that plasma platelet-activating factor levels were significantly elevated after 2 minutes of severe hypoxia (13.8 +/- 2.9 ng/mL vs. control 2.1 +/- 0.8 ng/mL) and after 30 minutes of moderate hypoxia (41.1 +/- 11.7 ng/mL). This increase in platelet-activating factor level was not caused by decreased degradation, because neither plasma nor intestinal platelet-activating factor acetylhydrolase was decreased in the hypoxic rats. (Intestinal acetylhydrolase activity was actually increased). Intestinal perfusion was markedly decreased at 30 minutes in hypoxic animals. In contrast, all platelet-activating factor antagonist-treated animals had normal intestinal perfusion. Histological examination of affected bowel from hypoxic animals showed early intestinal necrosis which was completely prevented by pretreatment with SRI 63-441 and WEB 2086. Because 30 minutes of hypoxia also resulted in metabolic acidosis, we further investigated if acidosis alone could induce platelet-activating factor release and bowel injury. We found that acidosis alone resulted in moderate increase of plasma platelet-activating factor but did not produce bowel injury. We conclude that platelet-activating factor plays a central role in mediating hypoxia-induced intestinal necrosis. Acidosis may enhance the effect of hypoxia on platelet-activating factor production.

title: Endotoxin and Hypoxia-Induced Intestinal Necrosis in Rats: The Role of Platelet Activating Factor
abstract: We have previously shown that intravascular platelet activating factor (PAF) causes ischemic bowel necrosis in rats morphologically similar to neonatal nec-rotizing enterocolitis (NEC). Because endotoxin (LPS) and hypoxia are risk factors for NEC, we studied their effect on PAF metabolism and the development of intestinal injury. Young mate Sprague-Dawley rats were anesthetized with pentobarbital and divided into six experimental groups: 1) control, 2) LPS alone (2 mg/kg), 3) hypoxia atone (5% O2), 4) LPS + hypoxia, 5) WEB 2086 (PAF antagonist) + LPS + hypoxia, and 6) SRI 63–441 (PAF antagonist) + LPS + hypoxia. Evaluations included blood pressure recording, superior mesenteric artery blood flow, arterial blood gas, white blood cell count, hematocrit, plasma PAF, plasma acetylhydrolase, plasma tumor necrosis factor, intestinal perfusion, and intestinal injury at 3 h. We found that LPS + hypoxia synergistically contributed to hypotension (mean blood pressure 27 ± 5.6% baseline versus 101 ± 3.9% control) metabolic acidosis (pH 7.05, base deficit 24 mEq/L), hemoconcentration, decreased superior mesenteric artery blood flow (2.2 ± 03 mL/min versus 5.8 ± 0.2 mL/min control), and intestinal injury. The morbidities resulting from LPS + hypoxia were partially or completely prevented by PAF antagonists. In addition, animals treated with LPS + hypoxia had neutro-penia, elevated plasma acetylhydrolase, and elevated plasma TNF. These results suggest that endogenous PAF may contribute to LPS + hypoxia-induced intestinal hy-poperfusion and necrosis.

title: The Role of Recombinant Platelet-Activating Factor Acetylhydrolase in a Neonatal Rat Model of Necrotizing Enterocolitis
abstract: Previous studies have shown that the endogenous inflammatory mediator platelet-activating factor (PAF) plays an important role in the pathophysiology of neonatal necrotizing enterocolitis (NEC). This study was designed to investigate the role of the PAF-degrading enzyme acetylhydrolase(PAF-AH) in a neonatal rat model of NEC. To study the absorption, localization, and activity of human recombinant PAF-AH (rPAF-AH), newborn rats were treated with enteral rPAF-AH, and plasma and intestines were sampled at 8 and 24 h for determination of PAF-AH enzyme activity and rPAF-AH concentration using a specific enzyme-linked immunoassay. To study the effect of rPAF-AH on neonatal NEC, rats were treated with rPAF-AH via the enteral route every 3 h, and then subjected to formula feeding and asphyxia per an established neonatal rat protocol for NEC. Pretreatment with enteral rPAF-AH significantly reduced the incidence of NEC compared with controls (6/26 versus 19/26,p < 0.001). We found that enteral rPAF-AH administration resulted in significant intestinal PAF-AH activity but no circulating PAF-AH activity despite immunohistochemical localization of the administered rPAF-AH to the intestinal epithelial cells. These findings suggest that rPAF-AH is functional and stable in the gut of neonatal rats. We conclude that enteral administration of rPAF-AH remains locally active and reduces the incidence of NEC in our experimental animal model.

title: The central role of PAF in necrotizing enterocolitis development.
abstract: We have addressed two critical questions concerning NEC development. 1) Why is the neonatal intestine particularly susceptible to necrosis? and 2) Does PAF play a critical role in NEC development? We have found that intestinal tissue of the newborn has the highest specific activity for the acetyltransferase of the de novo pathway. It is suggested that the high capacity of this tissue to synthesize PAF may contribute to the fact that the necrosis of the newborn is more prevalent in this tissue. We have previously reported that dexamethasone lowers the activity of acetyl-CoA:lyso-PAF acetyltransferase in liver and spleen. This hormone also cause an increase in plasma PAF-acetylhydrolase activity and an increased secretion of PAF-acetylhydrolase by various macrophages. It would, therefore, appear that the beneficial effects of glucocorticoids on the prevention of NEC may be due to both increased inactivation of PAF as caused by the increase in PAF-acetylhydrolase as well as a decrease in PAF synthesis. We are presently investigating the effect of glucocorticoids on acetyl-CoA: alkyl-lyso-sn-glycero-3-phosphate acetyltransferase. The reported studies in which NEC was prevented by intravenous infusion of recombinant PAF-acetylhydrolase provides further documentation as to the importance of PAF in the development of NEC. The specific activity of PAF-acetylhydrolase required for protection by dexamethasone was similar. This finding would be suggestive of the fact that the mechanisms by which dexamethasone causes a complete protection against NEC may be mediated by increasing the plasma activity. Other mechanisms have been proposed such as facilitating the maturation of the small bowel. As discussed, other factors such as hypoxia, endotoxins, TNF alpha, and enternal feeding have been suggested to be contributing agents of NEC development. Many of these factors and procedures are known to increase in PAF. We have suggested a mechanism to explain the increase in PAF formation as caused LPS, TNF alpha, and interleukins being the inhibition of the secretion of PAF-AH by macrophages. Our previous reports on the mechanisms involve in the prevention of NEC by glucocorticoids and the reported findings that human recombinant PAF-acetylhydrolase can prevent NEC provide further support for a central role for PAF in NEC development. Furthermore, the presence of a high PAF biosynthetic activity in the neonatal intestine affords an explanation as to why this tissue is highly susceptible to this disease.

title: Platelet-activating factor in infants at risk for necrotizing enterocolitis.
abstract: Platelet-activating factor (PAF) is a heterogeneous phospholipid that has been implicated as participating in a number of perinatal disease processes including necrotizing enterocolitis (NEC).



Baseline blood levels of PAF and related lipids (PAF-LL) were measured for 164 infants at risk for NEC from 3 neonatal intensive care units. Serial levels were obtained from the 11 infants in whom NEC developed.



The mean peak PAF-LL in the infants without NEC was 2.03 +/- 1.96 ng/mL. Infants with stage II (n = 6) and III (n = 5) NEC had elevated peak PAF-LL values (mean peak value 13.6 +/- 6.9 ng/mL). No PAF-LL measurements obtained from infants during stage II or III NEC were <2.03 ng/mL. Three infants had PAF-LL elevations before the development of any clinical or radiographic evidence of NEC. PAF-LL level increased as the severity of NEC increased and decreased with its resolution. Setting a PAF-LL level of 10.2 ng/mL as a cutoff for NEC had a positive predictive value of 100%.



PAF-LL determinations can complement clinical and radiographic studies to diagnose and follow the progression of NEC. PAF-LL may have a role in the evolution of NEC.

title: Presence of platelet-activating factor-acetylhydrolase in milk.
abstract: Human milk contains numerous factors such as immunoglobulins, lactoferrin, lysozyme, macrophages, etc., which serve an immunoprotective role. Platelet-activating factor (PAF) is one of the most proinflammatory agents thus far described. PAF is metabolized to the biologically inactive lysoPAF by the enzyme PAF-acetylhydrolase (PAF-AH). In the present study we have demonstrated that PAF-AH activity is present in human milk. The activity was associated with aqueous phase and was not stimulated by the addition of bile salts or Ca2+. The activity of PAF-AH in human milk was not affected by the addition of propranolol or NaCl. PAF, and 1-acyl-2-acetyl-glycerophosphocholine were the only substrates cleaved by the enzyme. Based on these properties it is concluded that the milk PAF-AH is not the lipoprotein or bile salt-stimulated lipase known to be present in milk. Inhibitor studies revealed that the enzyme in human milk was the plasma type PAF-AH. The activity of PAF-AH was stable at pH 4.0 at 37 degrees C and the activity varied in milk samples obtained from various species. The enzyme was secreted by milk macrophages. The presence of PAF-AH in human milk may explain, in part, the beneficial effects of breast feeding in the prevention of necrotizing enterocolitis by inactivating the potent proinflammatory autacoid, PAF.

title: Breast milk and neonatal necrotising enterocolitis
abstract: In a prospective multicentre study on 926 preterm infants formally assigned to their early diet, necrotising enterocolitis developed in 51 (5.5%). Mortality was 26% in stringently confirmed cases. In exclusively formula-fed babies confirmed disease was 6-10 times more common than in those fed breast milk alone and 3 times more common than in those who received formula plus breast milk. Pasteurised donor milk seemed to be as protective as raw maternal milk. Among babies born at more than 30 weeks' gestation confirmed necrotising enterocolitis was rare in those whose diet included breast milk; it was 20 times more common in those fed formula only. Other risk factors included very low gestational age, respiratory disease, umbilical artery catheterisation, and polycythaemia. In formula-fed but not breast-milk-fed infants, delayed enteral feeding was associated with a lower frequency of necrotising enterocolitis. With the fall in the use of breast milk in British neonatal units, exclusive formula feeding could account for an estimated 500 extra cases of necrotising enterocolitis each year. About 100 of these infants would die.

title: gp130 and the IL-6 family of cytokines: signaling mechanisms and thrombopoietic activities.
abstract: gp130 is a common signal transducing component of the functional receptor complexes for the interleukin (IL)-6 family of cytokines, ie, IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. These cytokines exhibit pleiotropic biological activities in, for instance, immune, hematopoietic, and neural systems, and function in a redundant manner owing to the shared usage of gp130. Thrombopoiesis is regulated by a variety of polypeptide factors including some from the IL-6 family. A polypeptide that has the most potent thrombopoietic activity among the thus far identified molecules is thrombopoietin (TPO). TPO exerts its biological functions through its cognate receptor, c-MPL, that interestingly is classified into the hematopoietic cytokine receptor family in which gp130 also belongs. Although each member of the IL-6 family as well as TPO signals through the distinct receptor complex, the underlying signaling mechanism is similar in each case: the first event after cytokine stimulation is dimerization of a respective set of receptor components, and activation of receptor-associated tyrosine kinases in the janus kinase (JAK) family then follows. Activation of JAK kinases is the pivotal step to initiate downstream cytoplasmic signaling cascades involving signal transducer and activator of transcription transcription factors and mitogen activated protein kinase. In this article, we focus on gp130-mediated cytokine signal transduction by comparing that mediated by c-MPL.

title: Effects of recombinant interleukin 11 on human megakaryocyte progenitor cells.
abstract: Utilizing a serum depleted, fibrin clot assay system, we investigated the proliferative effects of recombinant human IL-11 on both CD34 + DR + and CD34 + DR - human bone marrow subpopulations

title: Cytokine elevations in critically ill infants with sepsis and necrotizing enterocolitis.
abstract: We hypothesized that plasma levels of cytokines such as interleukin-6 and tumor necrosis factor (TNF) are elevated in critically ill infants with sepsis and necrotizing enterocolitis (NEC) and that the magnitude of their elevation is correlated with mortality rate. We measured plasma levels of interleukin-6 and TNF in 62 newborn infants with suspected sepsis or NEC. Eighteen infants had bacterial sepsis, 9 had bacterial sepsis plus NEC, and 15 had NEC but negative culture results. Twenty comparably ill infants with negative results on culture of systemic specimens served as study control subjects. Interleukin-6 levels were five- to tenfold higher in infants with bacterial sepsis plus NEC at the onset of disease than in infants with bacterial sepsis alone, in infants with NEC but negative culture results, and in control infants (p < 0.01). These differences persisted throughout the 48-hour study period. Interleukin-6 levels were also significantly higher in nonsurvivors than in survivors (p < 0.001). In contrast, plasma TNF values were not consistently increased in any of the groups. We conclude that plasma interleukin-6 is a more reliable indicator of bacterial sepsis and NEC than plasma TNF and may identify infants who might benefit from immunotherapeutic strategies.

title: Erythropoietin and the incidence of necrotizing enterocolitis in infants with very low birth weight.
abstract: The presence of erythropoietin (Epo) in human milk and the expression of Epo receptors on intestinal villous enterocytes of neonates suggest that Epo has a role in growth and development of the gastrointestinal tract. On this basis, the authors hypothesized that recombinant Epo (rEpo) given for prevention or treatment of the anemia of prematurity would protect against necrotizing enterocolitis (NEC).



A retrospective cohort study was conducted from a university neonatal intensive care unit of 483 very low birth weight (500 to 1,250 g) neonates born from July 1, 1993 to January 1, 1998.



A total of 260 neonates received rEpo, and 223 did not (control group). The rEpo and control groups were similar in gender distribution (52% v. 48% boys), gestational age (26.8+/-2.1 v. 27.6+/-2.9 weeks; mean +/- SD), birth weight (895+/-198 v. 911+/-208 g), 1 and 5 minute Apgar scores (4.2 and 6.1 v4.7 and 6.7), and incidence of severe intraventricular hemorrhage (8.9% v. 10.3%). The rEpo group had a lower incidence of NEC (12 of 260, 4.6% v. 24 of 223, 10.8%; P = .028, 95% confidence interval for difference: -0.108 to -0.015).



In very low birth weight infants, the incidence of NEC is lower in those who received rEpo.

title: Protective effects of interleukin-11 in a murine model of ischemic bowel necrosis.
abstract: The present study examined the effect of interleukin-11 (IL-11) in a murine model of bowel ischemia (BI). Prophylactic IL-11 administration in BI mice (induced by occluding the superior mesenteric artery for 90 min) was associated with significantly decreased morbidity and mortality. IL-11-treated mice demonstrated rapid recovery of intestinal mucosa as evidenced by an increase in mitotic activity and suppression of apoptosis in intestinal crypt cells as well as increased peripheral platelet and leukocyte counts primarily due to an increase in peripheral lymphocyte number in BI mice. In contrast to vehicle-treated mice, which uniformly developed thrombocytopenia after ischemic injury, no IL-11-treated BI mice developed thrombocytopenia during the experimental period. These data suggest a role for IL-11 in the treatment of gastrointestinal mucosal diseases due to a wide variety of causative injuries.

title: Intestinal cytokine gene expression in infants with acute necrotizing enterocolitis: interleukin-11 mRNA expression inversely correlates with extent of disease.
abstract: The authors have shown previously that surgical specimens from infants with acute necrotizing enterocolitis (NEC) show upregulation of inducible nitric oxide (NO) synthase (iNOS) and interferon-gamma mRNA. However, the contribution of other inflammatory cytokines such as interleukin-8 (IL-8), IL-11, and IL-12 has not been defined. Likewise, the role of GTP-cyclohydrolase, the rate-limiting enzyme in tetrahydrobiopterin synthesis, and thus NO production by iNOS is unclear. In this study, the authors sought to further define the pattern of cytokine expression seen in infants with acute NEC.



The authors measured intestinal cytokine mRNA expression by semiquantitative reverse transcriptase polymerase chain reaction in 21 infants with histologically confirmed NEC, 18 with other inflammatory conditions, and in 9 patients without intestinal inflammation. Guanosine triphosphate-cyclohydrolase (GTP-CH) activity was measured by specific enzyme assay. Univariate exact logistic regression analysis was performed to identify predictors of outcome.



IL-8 and IL-11 mRNA were upregulated in patients with acute NEC compared with those with other inflammatory conditions or those without disease; these levels returned to baseline at the time of stoma closure. Increased IL-11 mRNA decreased the likelihood of pan-necrosis (odds ratio, 0.93; P =.002). Increased IL-12 levels (but not IL-8) seemed to protect against pan-necrosis (odds ratio, 0.70; P =.06).



Local upregulation of IL-11 may represent an adaptive response designed to limit the extent of intestinal damage in NEC. Decreased IL-12 levels may contribute to the pathogenesis of NEC by allowing bacteria to escape host defenses.

title: Association between serum macrophage colony-stimulating factor levels and monocyte and thrombocyte counts in healthy, hypoxic, and septic term neonates.
abstract: Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that mainly stimulates the growth, differentiation, and proliferation of cells of the monocyte-macrophage lineage. There are only limited numbers of studies about M-CSF levels in neonates, but high levels of serum M-CSF have been reported in septic and some thrombocytopenic adult patients. In this study, we investigated the serum M-CSF levels in healthy, septic, and hypoxic term neonates on the first day of life and examined the relationship of serum M-CSF levels and circulating monocyte and thrombocyte counts in these newborn infants.



Three groups were defined in this prospective study: group 1, healthy neonates with no risk factors (n = 40); group 2, neonates who had severe hypoxia (n = 20); and group 3, neonates who fulfilled the criteria for early-onset sepsis (n = 18). Blood samples were collected for complete blood cell count and serum M-CSF levels by peripheral venipuncture from each infant in the first 24 hours after birth before any medical therapy.



The gestational ages and birth weights did not differ significantly between the groups. Serum M-CSF levels of the septic neonates were significantly higher than of both healthy and hypoxic neonates, but did not differ significantly between the healthy and hypoxic neonates. There was no significant correlation between serum M-CSF levels and circulating monocyte counts, but there was a significant inverse correlation between serum M-CSF levels and thrombocyte counts. When this correlation was analyzed according to groups, we determined that this inverse correlation between M-CSF levels and thrombocyte counts was especially significant in the septic neonate group, but not significant in the healthy and hypoxic neonate groups.



Serum M-CSF levels are significantly higher in neonates with sepsis. High serum M-CSF levels may have a possible role in the pathogenesis of thrombocytopenia in neonates with sepsis.

title: Age‐related changes in thrombopoietin in children: reference interval for serum thrombopoietin levels
abstract: We studied thrombopoietin (TPO, Mpl ligand) values using a sensitive ELISA in 254 serum samples obtained from disease‐free children and adult volunteers. TPO was detected in all samples, and its values ranged widely from 0.25 to 9.18 fmol/ml. When analysed by dividing the subjects into 11 age groups, the mean TPO levels from birth to 1 month of age were increased (3.73–5.92 fmol/ml). The highest values were found 2 d after birth; TPO levels then gradually decreased to adult levels (0.83 fmol/ml). The relationship between TPO values and platelet counts was not significant in all subjects (r = 0.27) or in children alone (r = 0.12). In children > 1 month of age a 95% reference interval for serum TPO values was determined from 0.58 to 3.27 fmol/ml. A significant correlation was found between TPO values in serum and plasma; serum TPO values = −0.257 + 4.039 × plasma TPO values (r = 0.951, P < 0.001, n = 22). This study is the first to report age‐dependent changes in blood TPO levels throughout child development. Serum TPO values were significantly high up to 1 month of age and were correlated with plasma TPO levels.

title: Endogenous Thrombopoietin Levels and Effect of Recombinant Human Thrombopoietin on Megakaryocyte Precursors in Term and Preterm Babies
abstract: Thrombocytopenia (platelets <150 × 109/L) is common in the early newborn period. Recent evidence suggests this is due mainly to impaired fetal megakaryocytopoiesis/platelet production. The cause remains unknown. Thrombopoietin (Tpo) is now recognized as the major regulator of platelet production in adults. However, nothing is known about Tpo levels, or the role of Tpo in megakaryocytopoiesis/platelet production, in the fetus/newborn. To answer these questions we: 1) measured plasma Tpo levels by ELISA at birth in healthy term (n = 17) and preterm (gestational age, 24-34 wk) babies (n = 16), and in thrombocytopenic preterm babies(n = 13); and 2) assessed the in vitro Tpo dose response characteristics of circulating megakaryocyte precursor cells (MKp) from all three groups. The median Tpo levels were similar in term babies 145 pg/mL (range 52-237 pg/mL) compared with preterm babies 132 pg/mL (32-318 pg/mL). In the thrombocytopenic preterm babies the median Tpo level of 185(46-264) was not significantly higher than in healthy babies, despite the fact that their median platelet counts were significantly lower-82 × 109/L (range 21-135), compared with the healthy preterm babies 252× 109/L (152-320)- p < 0.0001. Tpo levels in the thrombocytopenic preterm babies were also much lower than levels measured in three thrombocytopenic children (905, 2138, and 2700 pg/mL). MKp from all three groups showed dose-dependent proliferation in response to Tpo(p < 0.01 at 100 ng/mL Tpo). Increases in MKp were greater in healthy and thrombocytopenic preterm babies when compared with term babies: 48.2-, 24.6-, and 9.8-fold, respectively (p < 0.05 for both comparisons). These results strongly suggest that: 1) Tpo is a major regulator of megakaryocytopoiesis/platelet production in the fetus/newborn,2) impaired fetal Tpo production may be a factor in early thrombocytopenia in preterm babies, and 3) recombinant human Tpo is likely to be effective in the treatment of early neonatal thrombocytopenia.

title: Plasma thrombopoietin concentrations in thrombocytopenic and non‐thrombocytopenic patients in a neonatal intensive care unit
abstract: Thrombocytopenia is a frequent occurrence in the neonatal intensive care unit (NICU), but the role of thrombopoietin (Tpo) in the pathophysiology is unknown. We obtained serial plasma Tpo concentrations in 20 thrombocytopenic neonates in our NICU, and performed bone marrow studies in 15. The initial Tpo levels ranged from undetectable (<41 pg/ml) to 1112 pg/ml and did not correlate with gestational age or platelet count. Neonates with decreased marrow megakaryocytes did not have plasma Tpo levels as high as those reported in adults, particularly in small for gestational age infants (Tpo < 300 pg/ml). In 14/15 neonates followed until resolution, the Tpo concentration decreased as the platelet count increased.

title: Circulating thrombopoietin levels in neonates with infection
abstract: Thrombocytopenia is a commonly encountered hematologic complication in neonates with sepsis. Thrombopoietin (TPO) is the principal physiologic regulator of megakariocytopoiesis and platelet production. This study was carried out to determine whether variations in circulating TPO levels would occur in infected neonates and/or if they would correlate with platelet counts. In a prospective study of 36 sick neonates (gestational age 24–42 wk) admitted to a regional Neonatal Intensive Care Unit (NICU), blood was collected for TPO measurements and platelet counts on admission to the NICU, if infection was inferred, and at recovery before discharge. An additional group of 15 apparently healthy neonates was also studied (median postnatal age at the time of blood sampling for TPO assessment: 4 d, range 1–10) as control. TPO was measured on plasma samples using a commercially available enzyme‐immunosorbent assay (ELISA). On admission, the majority (21/36) of the sick neonates had non‐infectious diseases, 2 had early onset sepsis, and 13 had infection (defined as the presence of clinical signs of sepsis, abnormal leukocyte counts or C‐reactive protein values, and positive results on local cultures, but negative blood culture results). During the hospital stay, 5 neonates developed sepsis (positive blood culture) and 6 had infection (as previously defined) or necrotizing enterocolitis (NEC). The median TPO level (1704pg/ml, range 51–3912) was higher during sepsis (either early or late) than during infection (included NEC) (198pg/ml, range 21–2504), or non‐infectious disease (659 pg/ml, range 0–2533), while platelet counts (median value 37,000 cells/μl, range 15,000‐486,000) were lower than during either infection (included NEC) (median value 238,000 cells/μl, range 49,000‐655,000) or noninfectious disease (median value 110,000 cells/μl, range 45,000‐549,000). When infants had recovered from these illnesses, TPO concentrations markedly dropped (median value 59 pg/ml, range 0–825). These values were similar to those found in the control neonates (median TPO level 85 pg/ml, range 43–620). In infected neonates (sepsis plus infection), TPO levels inversely correlated with platelet counts (r= ‐0.634, p= 0.001) as did those of infants with non‐infectious disease (r= ‐0.574, p= 0.006), while there was no significant correlation between TPO levels and platelet counts in the samples obtained after recovery or in the control infants. We conclude that infected neonates have high circulating TPO levels in the face of low platelet counts. Whether larger TPO concentrations following exogenous administration of recombinant TPO would restore the number of circulating platelets warrants further investigation.

title: Necrotizing enterocolitis: laboratory indicators of surgical disease.
abstract: The timely distinction between infants with necrotizing enterocolitis (NEC) who need surgery and those who are likely to recover with medical management is important, but it may be difficult clinically. Because pneumoperitoneum is not always present, additional markers of bowel gangrene are needed. Among 73 babies managed for NEC over the study period, 49 (67%) met the study criteria of Bell's stage > 1, and their records were reviewed to determine the usefulness of common laboratory tests in predicting outcome. The patients were divided into three groups based on management. Group 1 (7 patients) required surgery at the time of initial presentation because of pneumoperitoneum. The remaining 42 patients were initially managed medically, 19 of whom (group 2) recovered successfully; the other 23 (group 3) required surgery. The combination of certain laboratory tests, ie, white blood cell count (WBC), immature:total neutrophil ratio (I:T), platelet count (PLT), and base excess (BE), was of significance in distinguishing between infants who would need surgery and those who would recover with medical therapy (group 3 v group 2) 4 to 12 hours or 12 to 24 hours after the diagnosis of NEC was established. A scoring scale was developed, with a point for each of the following: WBC < 9,000/mm3, I:T > .5, PLT < 200,000/mm3, and BE < or = -2. A score of > or = 3 during 4 to 12 hours after diagnosis of NEC strongly predicted the presence of surgical disease (positive predictive value, 100%; negative predictive value, 76%; specificity, 100%; sensitivity, 64%).(ABSTRACT TRUNCATED AT 250 WORDS)

title: Risk factors and severity indices in necrotizing enterocolitis
abstract: In retrospective analysis we set up a chart review of 61 consecutively newborn infants with proven diagnosis of ants were admitted to the intensive care unit of the Department of Pediatric Surgery from 1985 to 1991. Birth weights varied from 790 to 4680 g (mean value 2408 ± 853), gestational ages from 27 to 43 weeks (mean value 36 ± 4.0); 33 patients received conservative treatment and 28 patients were operated on. An analysis of operated (group 1) and conservatively (group 2) treated patients revealed no statistically significant difference in birth weight, gestational age, peripartal complicanecrotizing enterocolitis. The 26 female and 35 male inftions or type of feeding. Yet a few parameters measured at admission remained as severity indices. Operated babies had significantly higher counts of immature granulocytes and an elevation in the I:T‐ratio, as well as lower counts of total granulocytes, lymphocytes, platelets and lower weights of the placenta. However, when taking into account the gestational age, the results showed that these parameters became insignificant in the low birthweight group below 34 weeks, and were of higher validity in the gestational age group over 35 weeks. In the latter group, C‐reactive protein also proved to be a good parameter with statistical significance respecting the severity of necrotizing enterocolitis.

title: The clinical significance of thrombocytopenia in neonates with necrotizing enterocolitis.
abstract: The aim of this study was to evaluate the clinical significance of low platelet count in the surgical management of neonates with necrotizing enterocolitis (NEC).



The clinical course of 58 consecutive neonates with advanced NEC (Bell's stages II or III) treated in the authors' hospital between 1995 and 1998 was reviewed. NEC in neonates who required operation was classified as isolated, multifocal, or pan-intestinal. Severe thrombocytopenia was defined as platelet count less than 100 x 10(9)/L. Rapid fall in platelet count was defined as fall greater than 150 x 10(9)/L within 24 hours to a level less than 100 x 10(9)/L.



Median birth weight was 1,564 g (range, 550 to 4,270) and gestational age was 31 weeks (range 23 to 41). Twenty-two neonates (38%) were below 1,000 g. Age at the onset of the disease was 13 days (range, 1 to 62). NEC was treated medically in 7 neonates (12%). Indications for operation included pneumoperitoneum in 23 neonates (45%), clinical deterioration in 19 (37%), and intestinal obstruction in 9 (18%). The nadir platelet count (lowest level during the course of disease) was lower in patients with stage III disease than in patients with stage II disease (P <.05). The greater the extent of the disease, the lower the platelet count (P =.012). The nadir platelet count was lower in infants who died than in survivors (P <.05). None of the patients with platelet count greater than 100 x 10(9)/L died. In predicting intestinal gangrene, severe thrombocytopenia has a sensitivity of 69%, specificity of 60%, and positive predictive value of 89%; rapid fall in platelet count has a sensitivity of 32%, specificity of 89%, and positive predictive value of 92%.



(1) A platelet count less than 100 x 10(9)/L or a rapid fall in platelet count represent poor prognostic factors. (2) Monitoring the platelet count during the course of NEC is useful; however, it cannot be used in isolation to predict the extent of the disease or survival rate.

title: Velocardiofacial Syndrome Patients with a Heterozygous Chromosome 22q11 Deletion Have Giant Platelets
abstract: Patients with a microdeletion on chromosome 22q11 demonstrate the clinical picture of the velocardiofacial syndrome. We report on three members of the same family with this microdeletion and velocardiofacial syndrome, all having an increase in platelet size and a mild decrease in platelet number. Their platelet function, however, tested by aggregation and by adherence to collagen in a whole blood perfusion system, was normal. We retrospectively studied the files of 35 other patients with 22q11 deletion and also found that their platelets had an increased size compared with cardiac controls. Moreover, their platelet size correlated negatively with platelet number. Knowing that patients with 22q11 deletion are obligate carriers for a heterozygous glycoprotein Ibβ deletion, these patients can be considered to be heterozygous Bernard-Soulier patients. In addition, a significant increase in platelet size may be a positive predictor for the clinical diagnosis of the velocardiofacial syndrome.

title: Modified bleeding time in the infant
abstract: The present authors developed a modified template bleeding time for use in the newborn infant. The sensitivity of the bleeding time to the presence of antibiotics, indomethacin, generalized illness, and thrombocytopenia was tested in 242 infants. Both indomethacin and thrombocytopenia similarly prolonged the BT, and the latter could be corrected by raising the platelet count.

title: Micromethod for bleeding time in the newborn.
abstract: A new method of measuring bleeding time using an Autolet device is described. Normal ranges of bleeding time and volume of blood used in the test have been defined in a population including preterm babies. Abnormal values have been shown in newborns with a variety of problems, and an abnormality of bleeding time has been found to precede intraventricular haemorrhage.

title: Platelet function in the newborn.
abstract: Whole blood flow cytometry, an important new method for the assessment of platelet function, is particularly advantageous for neonatal studies because only minuscule volumes (approximately 2 microL) of blood are required. By this method, we have demonstrated that neonatal platelets are less reactive than adult platelets to physiological agonists in whole blood, as determined by the activation-induced increase in the platelet surface expression of P-selectin and the glycoprotein (GP) IIb-IIIa complex and by the activation-induced decrease in the platelet surface expression of the GPIb-IX complex. Our data suggest that the mechanism of neonatal platelet hyporeactivity is, at least in part, a relative defect in a common signal transduction pathway. We have further demonstrated that the platelets of very low birth weight (VLBW) preterm neonates are maximally hyporeactive on days 3 to 4 of life but return to almost the adult range by days 10 to 14. Given that intraventricular hemorrhage (IVH) is also maximal on days 3 to 4, these defects may contribute to the propensity of VLBW preterm neonates to IVH.

title: Effects of Melatonin Treatment in Septic Newborns
abstract: Free radicals have been implicated in the pathogenesis of neonatal sepsis and its complications. This study was conducted to determine the changes in the clinical status and the serum levels of lipid peroxidation products [malondialdehyde (MDA) and 4-hydroxylalkenals (4-HDA)] in 10 septic newborns treated with the antioxidant melatonin given within the first 12 h after diagnosis. Ten other septic newborns in a comparable state were used as “septic” controls, while 10 healthy newborns served as normal controls. A total of 20 mg melatonin was administered orally in two doses of 10 mg each, with a 1-h interval. One blood sample was collected before melatonin administration and two additional blood samples (at 1 and 4 h) were collected after melatonin administration to assess serum levels of lipid peroxidation products. Serum MDA + 4-HDA concentrations in newborns with sepsis were significantly higher than those in healthy infants without sepsis; in contrast, in septic newborns treated with melatonin there was a significant reduction (p < 0.05) of MDA + 4-HDA to the levels in the normal controls at both 1 and 4 h (p < 0.05). Melatonin also improved the clinical outcome of the septic newborns as judged by measurement of sepsis-related serum parameters after 24 and 48 h. Three of 10 septic children who were not treated with melatonin died within 72 h after diagnosis of sepsis; none of the 10 septic newborns treated with melatonin died. To our knowledge, this is the first study where melatonin was given to human newborns.

title: Modulation of hemostatic mechanisms in bacterial infectious diseases
abstract: Life-threatening complications from bacterial infections are a major and growing clinical problem, aggravated by the emergence and spread of antibiotic resistance in bacterial pathogens and by an increase in the number of immunocompromised patients.[1-3][1] During bacterial infection, the host

title: Multisystem organ failure and capillary leak syndrome in severe necrotizing enterocolitis of very low birth weight infants.
abstract: Classification systems for necrotizing enterocolitis (NEC) in preterm infants have been developed to define severity grades relevant for treatment and prognosis. Multisystem organ failure (MSOF) and capillary leak syndrome (CLS) also have prognostic value in these patients. The aim of this retrospective study was to investigate the incidence and predictive value of MSOF and CLS according to the classification criteria.



The records of 1,022 very low birth weight infants admitted from 1982 to 1996 were reviewed for diagnosis of NEC stage IIA or higher (classification of Walsh and Kliegman). Among those patients (n = 50) the incidence of MSOF and CLS was determined, separately for surgical or conservative treatment.



Twelve patients were assigned to stage II, 22 to stage IIIa, and 16 to stage IIIb; 31 infants underwent operation. Mortality rate was not influenced by the grade. In eight patients only gastrointestinal symptoms were found, whereas in 23 patients, up to three organ systems and in 19 patients, four or more organ systems were affected. Mortality depended on the number of involved organ systems. CLS occurred postoperatively in 10 of the 31 infants; eight of them died.



The prognostic values of MSOF and CLS are higher than that of classification criteria in NEC of VLBW infants.

title: Indications of Coagulation and/or Fibrinolytic System Activation in Healthy and Sick Very-Low-Birth-Weight Neonates
abstract: A combined hemostatic defect consisting of a reduction in certain procoagulants, anticoagulants (antithrombin III-ATIII-, protein C-PC-) and components of the fibrinolytic system (plasminogen-Plg-) was demonstrated in very-low-birth-weight infants (VLBW <1,500 g) with gestational age 26–32 weeks. Sixteen of them were healthy, 28 were suffering from RDS and 24 from septicemia. The hemostatic defect was more profound in the RDS group, nevertheless increased TAT (thrombin + ATIII complex) and/or PAP values (plasmin + a2-antiplasmin complex) was a more frequent finding in the septicemic group of infants (91.8 vs. 17.9%). Moderate-to-severe thrombocytopenia was detected in a higher percentage in the septicemic (70.8%) than in the RDS group (50%), and increased D-dimers were demonstrated in 34.8 and 28.6% of the infants, respectively. Elevated TAT or PAP values were not always associated with gross coagulation abnormalities, and advanced disseminated intravascular coagulation (DIC) was only documented in 16.7% of the septicemic and 7.1% of the RDS infants. None of the VLBW neonates presented with clinical evidence of thrombosis, although hemorrhagic manifestations were apparent in 34.8 and 14.3% of the neonates with septicemia or RDS, respectively, mainly due to DIC or severe thrombocytopenia. In conclusion, increased TAT and/or PAP values are good indicators of the in vivo activation of the hemostatic system, but still their impact on sick neonates morbidity and mortality remains unknown.

title: Resistance to activated protein C in newborns with necrotizing enterocolitis
abstract: Sir: Case control studies have failed to identify any consistent risk factors for the development of necrotizing enterocolitis (NEC) in preterm infants, but thrombosis of large mesenteric vessels is frequent in such infants . Recently the resistance to activated protein C, a hereditary defect of factor V, was identi®ed to be the most common risk factor for deep vein thrombosis in the Caucasian population with a carrier frequency of about 5% . In children with thromboembolism, APC resistance has also been reported to be common [6]. As thrombosis of large mesenteric vessels is a frequent ®nding in infants with NEC, we raised the question whether APC resistance is a risk factor for the development of neonatal NEC. All patients with NEC 3 II according to Bell et al.  (n  39) treated in our intensive care unit between January 1990 and December 1996 were included in the study. DNA of the patients was prepared from blood samples stored on ®lter paper as part of the neonatal screening programme. Filter paper was cut into small pieces and DNA extracted with a commercially available kit (Qiamp tissue kit, QIAGEN, Hilden, Germany). A 266 base-pair fragment of the factor V gene was ampli®ed by the polymerase chain reaction (PCR) and digested with the restriction enzyme Mnl 1 as described elsewhere [3]. For each patient with NEC, a control patient matched by birth weight was also studied. DNA was prepared from whole blood after informed consent had been given by the parents. APC resistance was tested as described above. The study was approved by the local committee on research in human subjects of the Medical University of LuÈ beck. Statistical analysis were performed with chi-square and Mann-Whitney rank sum test. DNA extraction and PCR analysis were successful in 85% (n  33) of the infants with NEC (including all children who died, n  5) and in all corresponding controls. There were no dierences between patients and controls in terms of birth weight, gestational age, gender, number of children requiring ventilatory support and prenatal glucocorticoid therapy (Table 1). In both groups one infant was heterozygote for APC resistance. The APC positive NEC patient was a female born at 25 weeks of gestation. She developed NEC on day 17 and required resection of 15 cm of her ileum. During the operation thrombosis of a branch of the mesenteric vein was noted. No other thromboembolic complications were reported and the infant left the hospital without severe disability. Out of 32 APC negative NEC patients, 26 received surgical treatment and thrombosis of large mesenteric vessels was noticed in 2 of them. Pathogenesis of NEC is complex and not yet fully understood. Large studies have failed to de®ne any consistent risk factors in premature infants, except for prematurity itself. Ischaemic necrosis is one of the hallmarks in the pathology of NEC and animal studies in a dog model suggest that NEC is associated with intravascular coagulopathy . Thrombosis of mesenteric vessels is a frequent ®nding at autopsy of infants who died because of NEC [5]. In a recent study by Ballance et al. , 5 out of 19 patients had thrombi within the superior mesenteric artery or large mesenteric artery branches on autopsy. Thromboembolic intestinal infarction probably represents a distinct pathological subgroup of NEC patients. The resistance to activated protein C, which is caused by a point mutation in the factor V gene is by far the most common genetic risk factor for deep vein thrombosis in adults . It has been shown in children that APC resistance is a risk factor for venous and arterial thrombosis. Furthermore, 47% of APC-resistant children and neonates with arterial or venous thrombosis had additional exogenous risk factors (e.g. central lines, sepsis) . Since exogenous risk factors for thrombosis are quite common in very preterm infants, hereditary thrombophilia might turn the scale towards thrombotic events in mesenteric vessels, initiating or aggravating NEC. We were able to study a cohort of children with NEC covering a period of 7 years. Despite the limitations imposed by the retrospective study design, our data clearly indicate that APC resistance is not an independent risk factor for the development of NEC. We found one APC-resistant child each in case and control group. The heterozygote frequency inferred from this is 3% and is within the expected range of heterozygote frequency in the Caucasian population . Nevertheless the fact that the only child with NEC and APC resistance had thrombosis of a mesenteric vein suggests that APC resistance might be an additional risk factor for thromboembolic events, even in extremely low birth weight infants.

title: Development of the human coagulation system in the healthy premature infant.
abstract: This study was designed to determine the postnatal development of the human coagulation system in the healthy premature infant. Consecutive mothers of healthy premature infants born at either St Joseph's Hospital or McMaster University Medical Centre in Hamilton were asked for consent. One hundred thirty-seven premature infants (30 to 36 weeks of gestational age) entered the study. The premature infants did not have any major health problems and did not require ventilation or supplemental oxygen. Demographic information and a 20-mL blood sample were obtained in the postnatal period on days 1, 5, 30, 90, and 180. Between 40 and 96 premature infants were studied on each day for each of the following tests: prothrombin time, activated partial thromboplastin time, thrombin clotting time, plasminogen; 13 factor assays [fibrinogen, II, V, VII, VIII, IX, X, XI, XII, XIII, high-mol-wt kininogen (HMWK), prekallikrein (PK), von Willebrand factor (vWF)] and eight inhibitors [antithrombin III (AT-III), heparin cofactor II, alpha 2-antiplasmin, alpha 2-macroglobulin, alpha 1-antitrypsin, C1 esterase inhibitor, protein C (PC), and protein S (PS)]. A combination of biologic and immunologic assays were used. Between 30 to 36 weeks there was a minimal effect of gestational age for levels of AT-III, PC, and factors II and X only; therefore, the entire data set was used to generate reference ranges for these components of the coagulation system for premature infants. Next, the results for the premature infants were compared with those of a previously published study in 118 fullterm infants and with those for adults. An effect of gestational age was shown for plasminogen, fibrinogen, factors II, V, VIII, IX, XI, XII, HMWK, and all eight inhibitors. In general, the postnatal maturation towards adult levels was accelerated in premature infants as compared with the fullterm infants. By 6 months of age, most components of the coagulation system in premature infants had achieved near adult values.

title: Neonatal sepsis: a challenge in hemostaseology.
abstract: Several therapeutic approaches to sepsis and disseminated intravascular coagulation (DIC) have shown promising results in animal models. Large controlled trials in humans, however, have failed to show a clearly beneficial effect of a single drug or substance on outcome and survival so that treatment remains uncertain. As one researcher stated: ". . . sepsis is a classical example of a disease greater than the sum of its parts; it is a complex process in which intervention in one area might have only a modest effect on the final outcome." We believe that the complex pathophysiological setting of septic shock will undoubtedly require a multifaceted approach. Consequently, we attempt to arrest DIC and restore adequate tissue perfusion by intervention with heparin, AT and if possible protein C (PC) in the earliest stage of the disease, with the aim of blocking ongoing microthrombus formation and to support fibrinolysis. Growing understanding of the basic underlying mechanisms teaches us how to successfully stabilise the individual decompensated sub-systems like coagulation in septic patients. We should learn to accept these steps to reach the goal of a better outcome in terms of survival in this devastating illness.

title: Efficacy and safety of recombinant human activated protein C for severe sepsis.
abstract: Drotrecogin alfa (activated), or recombinant human activated protein C, has antithrombotic, antiinflammatory, and profibrinolytic properties. In a previous study, drotrecogin alfa activated produced dose-dependent reductions in the levels of markers of coagulation and inflammation in patients with severe sepsis. In this phase 3 trial, we assessed whether treatment with drotrecogin alfa activated reduced the rate of death from any cause among patients with severe sepsis.



We conducted a randomized, double-blind, placebo-controlled, multicenter trial. Patients with systemic inflammation and organ failure due to acute infection were enrolled and assigned to receive an intravenous infusion of either placebo or drotrecogin alfa activated (24 microg per kilogram of body weight per hour) for a total duration of 96 hours. The prospectively defined primary end point was death from any cause and was assessed 28 days after the start of the infusion. Patients were monitored for adverse events; changes in vital signs, laboratory variables, and the results of microbiologic cultures; and the development of neutralizing antibodies against activated protein C.



A total of 1690 randomized patients were treated (840 in the placebo group and 850 in the drotrecogin alfa activated group). The mortality rate was 30.8 percent in the placebo group and 24.7 percent in the drotrecogin alfa activated group. On the basis of the prospectively defined primary analysis, treatment with drotrecogin alfa activated was associated with a reduction in the relative risk of death of 19.4 percent (95 percent confidence interval, 6.6 to 30.5) and an absolute reduction in the risk of death of 6.1 percent (P=0.005). The incidence of serious bleeding was higher in the drotrecogin alfa activated group than in the placebo group (3.5 percent vs. 2.0 percent, P=0.06).



Treatment with drotrecogin alfa activated significantly reduces mortality in patients with severe sepsis and may be associated with an increased risk of bleeding.

title: Replacement therapy with protein C concentrate in infants and adolescents with meningococcal sepsis and purpura fulminans.
abstract: We report the effects of substitution with a virus-inactivated protein C (PC) concentrate in disseminated intravascular coagulation (DIC) in infants and children with meningococcal sepsis associated with purpura fulminans. It was a prospective open-label study. Eight pediatric and adolescent patients age 0.2 to 18.25 years with DIC associated with severe acquired PC deficiency (range 0.02 to 0.48 IU/mL; median, 0.22 IU/mL) in meningococcal septic shock and purpura fulminans were studied. Replacement therapy was initiated with a virus-inactivated PC concentrate with an initial intravenous bolus of 80 to 120 IU/kg followed by 50 IU/kg up to six times per day as an adjunctive therapeutic regimen to otherwise optimal intensive care treatment. After initial PC administration, plasma PC levels rose to normal ranges and were maintained under PC replacement therapy. Improving or even normalizing global hemostatic parameters were assessed in all patients. Markedly elevated plasminogen activator inhibitor type 1 (PAI-1) levels prior to treatment, reflecting a reduced fibrinolytic potential, decreased rapidly under PC substitution. Concomitantly improving signs of purpura fulminans reflected by decreasing size of skin lesions, demonstrated a restoring microcirculation. Six of the eight patients survived. One patient required limb amputation; two patients died because of multiorgan failure. Both presented with a severely low plasma PC activity of 0.02 IU/mL on admission to the hospital. No adverse effects were observed with the PC concentrate administration. It can be concluded that the administration of PC concentrate had a marked benefit on the deranged coagulation status of patients with purpura fulminans and meningococcal septicemia. Normalization or even partial correction of hemostasis as well as improvement of microcirculation accompanied by improving signs of purpura fulminans were demonstrated in all patients.

title: Severe sepsis--a new treatment with both anticoagulant and antiinflammatory properties.
abstract: In the last half of the 20th century, the use of antibiotics for the treatment of bacterial infections transformed the practice of medicine, resulting in sharp reductions in morbidity and mortality...

title: High dose of intravenous antithrombin III without heparin in the treatment of disseminated intravascular coagulation and organ failure in four children
abstract: In several animal experiments, high doses of antithrombin III concentrates have shown beneficial effects on mortality and reversal of coagulation abnormalities which had resulted from disseminated intravascular coagulation. Other experiments have suggested that antithrombin III infusion without heparin is effective in the treatment of organ failure. We clinically treated children suffering disseminated intravascular coagulation only with antithrombin concentrate. Four patients suffering disseminated intravascular coagulation with organ failure were selected. We started antithrombin III concentrate infusion as soon as the diagnosis was established. The dosage of antithrombin III was 120–250 units/kg/day for 2 or 3 days. Heparin was not used. All 4 patients recovered completely and quickly without any complications within 14 days. We suggest that the high‐dose antithrombin III infusion without heparin is an effective and safe therapy for disseminated intravascular coagulation with organ failure.

title: A placebo-controlled randomized trial of antithrombin therapy in neonatal respiratory distress syndrome.
abstract: Neonatal respiratory distress syndrome (RDS) is associated with decreased plasma activity of antithrombin (AT) and increased formation of thrombin. We tested whether AT reduces thrombin formation, improves gas exchange, and decreases the duration of mechanical ventilation and supplemental oxygen. One hundred twenty-two infants were randomized to pasteurized AT concentrate or to placebo. Two ml/kg (equivalent to 100 IU AT/kg) were followed by 1 ml/kg (50 IU/kg) every 6 h for 48 h. Outcome measures included plasma AT activity, thrombin-AT (TAT) complex, prothrombin fragment (F1+2), the ratio of arterial to alveolar oxygen pressure [(a/A)PO2], and the ventilator efficiency index (VEI). In the AT group (n = 61), mean (SD) birth weight was 1,198 (301) g, mean (SD) gestational age (GA) was 28.3 (2.0) wk, 54% were male. In the placebo group (n = 61), mean (SD) birth weight was 1,201 (315) g, mean (SD) GA was 28.8 (2. 3) wk, 51% were male. In treated infants, AT activity was raised to means of 1.69 and 2.25 U/ml at 24 and 48 h, respectively. Corresponding means in control infants were 0.37 and 0.44 U/ml (p < 0.0001). F1+2, but not TAT, was significantly reduced by AT (p = 0. 004). VEI and (a/A)PO2 were similar in both groups throughout the first week of life. Median days receiving mechanical ventilation were 7.1 (AT) versus 4.8 (placebo), p = 0.0014. Median days receiving supplemental oxygen were 7.9 (AT) versus 5.5 (placebo), p < 0.0001. There were seven (11.5%) deaths in the AT group and three (4.9%) deaths in the placebo group. We conclude that treatment with AT cannot be recommended in premature infants with RDS.

title: Human granulocyte colony-stimulating factor may improve outcome attributable to neonatal sepsis complicated by neutropenia.
abstract: To determine whether adjunctive therapy with recombinant human granulocyte colony-stimulating factor (rhG-CSF) could reverse sepsis-associated neonatal neutropenia and improve neonatal survival compared with conventional therapy in a phase I/II-type trial.



An intravenous infusion of rhG-CSF (10 microg/kg/d x 3 d) was administered to 14 septic neutropenic neonates. Neutrophilic responses and outcome of these neonates were compared with 11 concurrently treated, retrospectively selected, case-matched control septic patients identified by using a search of medical records coded for sepsis with neutropenia (>/=24 hours).



Seven neonates with early-onset sepsis with neutropenia at birth and seven neonates with late-onset sepsis plus neutropenia (all with necrotizing enterocolitis) were entered in the rhG-CSF treatment group. Results were compared with a conventional therapy control group (five early onset, six late onset). No significant differences existed in the birth weight, gestational age, use of antibiotic therapy, magnitude of respiratory support, severity of metabolic acidosis, use of vasopressors, or other supportive therapy between the two groups. In the rhG-CSF-treated group and in the conventionally treated control group, the absolute neutrophil count (ANC) (mean +/- SEM) was 585 +/- 138 and 438 +/- 152, respectively. The ANC increased to more than baseline in the rhG-CSF-treated group by 10-fold versus 2-fold at 24 hours, 18-fold versus 4-fold at 48 hours, 24-fold versus 5-fold at 72 hours (significant by one-way analysis of variance in the rhG-CSF group only), and 29-fold versus 16-fold at 7 to 10 days when compared with the conventional therapy group. There were no nonresponders in the rhG-CSF group by 24 hours after the first dose of study drug. Monocyte cell counts also increased significantly in both groups by 7 days after entry into this protocol but remained within normal range for age. No clinically significant effect on lymphocytes, erythrocytes, or platelet counts was noted. Thirteen patients in the rhG-CSF-treated group (92%; 13 out of 14) and five in the conventionally treated group (55%; 5 out of 11) survived to 28 days after the onset of the signs of sepsis. No adverse effects were noted in the rhG-CSF-treated group.



rhG-CSF can increase the neutrophil count in critically ill septic neutropenic neonates. This finding suggests that rhG-CSF may be effective in a therapeutically useful time frame to treat septic neonates with neonatal neutropenia attributable to bone marrow suppression or neutrophil consumption. Future randomized trials are needed to validate the beneficial effects of rhG-CSF and to determine whether any significant side effects of therapy exist.

title: Administration of recombinant granulocyte colony-stimulating factor to neonates with septicemia: A meta-analysis.
abstract: A meta-analysis was used to determine whether administering recombinant granulocyte colony-stimulating factor (rG-CSF) to neonates with bacterial septicemia reduces mortality. Five studies were identified, involving 73 rG-CSF recipients and 82 control subjects. Mortality was lower among the rG-CSF recipients (odds ratio, 0.17; CI, 0.03-0.70; P <.05). However, when the non-randomized studies were excluded, the P value was.13. For the subgroups "<2000 g" or "neutropenia," the P value was <.02. Thus the routine use of rG-CSF cannot be recommended for all neonates with sepsis.

title: A randomized trial of granulocyte-macrophage colony-stimulating factor in neonates with sepsis and neutropenia.
abstract: To determine whether adjunctive therapy with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) could reverse sepsis-associated neonatal neutropenia and improve neonatal survival and to assess its safety compared with conventional therapy in a control group.



This prospective, randomized, controlled trial was performed in 60 infants with neutropenia and clinical signs of sepsis. A subcutaneous injection of rhGM-CSF (5 microgram/kg/day) was administered to 30 of the patients for 7 consecutive days. Hematologic parameters (absolute neutrophil, eosinophil, monocyte, lymphocyte counts, and platelet number) and outcome were compared with 30 conventionally treated (control) patients.



Twenty-five patients from the GM-CSF-treated group and 24 from the conventionally treated group had early-onset sepsis (</=3 days' postnatal age), and the other 11 patients had late-onset sepsis (>3 days' postnatal age). There was no difference between groups in terms of birth weight; gestational age; gender; maturity; maternal age; and incidence of prolonged rupture of membranes, maternal hypertension, or severity of sepsis. All neonates tolerated GM-CSF well with no adverse reactions. The absolute neutrophil count on day 7 was significantly increased in the GM-CSF-treated group compared with the conventionally treated group: 8088 +/- 2822/mm(3) versus 2757 +/- 823/mm(3). The mean platelet count was significantly higher on days 14 in the GM-CSF-treated group compared with conventionally treated group: 266 867 +/- 55 102/mm(3) versus 229 200 +/- 52 317/mm(3). Hematologic parameters were otherwise similar between groups before treatment and on day 28. Twenty-seven neonates in the rh-GMCSF group and 21 in the control group survived to hospital discharge. The mortality rate in the rhGM-CSF group (10%) was significantly lower than in the conventionally treated group (30%).



Treatment with rhGM-CSF is associated with an increase in absolute neutrophil, eosinophil, monocyte, lymphocyte, and platelet counts and decreased mortality in critically ill septic neutropenic neonates. These results suggest that rhGM-CSF may be effective in the treatment of neonatal sepsis with neutropenia, and further randomized trials are needed to confirm its beneficial effects.

title: Variations in transfusion practice in neonatal intensive care.
abstract: To compare the transfusion practices between two neonatal intensive care units (NICUs) to assess the impact of local practice styles on the timing, number, and total volume of packed red cell transfusions in very low birth weight infants. To derive multivariate models to describe practice and to identify potential areas for improvement in the future.



We reviewed phlebotomy losses and transfusion rates between two NICUs (A and B) for 270 consecutive admissions of birth weight < 1500 g. We stratified for birth weight and for illness severity by the Score for Neonatal Acute Physiology (SNAP). Measures of short-term outcome were compared. We derived multivariate models to describe and compare the practices in the two NICUs.



Patients in NICU A had smaller phlebotomy losses than those in NICU B. A lower percentage of the patients in NICU A (65% vs 87%) received transfusions, but they tended to receive a greater total volume per kg per patient (67 mL/kg vs 54.8 mL/kg). Transfusion timing differed between the NICUs; in NICU A only approximately one-half of their transfusions occurred in the first 2 weeks, whereas in NICU B almost 70% of the transfusions were given in this time period. Multivariate models showed that phlebotomy losses were significantly related to lower gestational age (GA) and higher SNAP. Hospitalization in NICU B resulted in 10.7 cc of additional losses relative to NICU A for a comparable GA and illness severity score. The volume of blood transfused per kilogram of body weight was a function of GA, SNAP, and hospital. Care practices in NICU A added an additional 19 cc of transfused volume in the first 14 days of life, and an additional 26 cc thereafter when adjusted for GA and SNAP. These differences in phlebotomy and transfusion were not associated with differences in the days of oxygen therapy or mechanical ventilation, the oxygen requirement at 28 days, the incidence of chronic lung disease, or the rate of growth by day 28.



We identified significant differences in phlebotomy and transfusion practices between two NICUs. We found no differences in short-term outcome, suggesting that the additional use of blood in one of the NICUs was discretionary rather than necessary. Our multivariate models can be used to characterize and quantify transfusion and phlebotomy practices. By predicting which patients are likely to require multiple transfusions, clinicians can target patients for erythropoietin therapy and identify those patients for whom donor exposure can be reduced by a unit of blood for multiple use. The models may help in monitoring changes in practice as they occur.

title: T cryptantigen activation is associated with advanced necrotizing enterocolitis.
abstract: Thomsen-Friedenreich cryptantigen activation (TCA) exposes neonates with necrotizing enterocolitis NEC to the risk of hemolysis after transfusion of blood products. The authors aimed to determine the prevalence of TCA in neonates with NEC and to correlate TCA with severity of disease and outcome.



One hundred four neonates with NEC were tested for TCA on admission. Patients with TCA requiring transfusion were given packed red cells, low-titer anti-T fresh frozen plasma, and washed platelets to avoid hemolysis.



Twenty-three infants had TCA, and 96% of these had stage III disease. The incidence of TCA was significantly higher in infants with stage III disease compared with those with stage II (30% v 4%; P <.01). A total of 91% of infants with TCA required laparotomy compared with 81% of those with no activation. At laparotomy, widespread disease was more common in the TCA group (71% v 55%). TCA did not significantly increase mortality rate (TCA, 39% v no TCA, 28%); this may reflect the transfusion policy of our unit.



Twenty-two percent of neonates with NEC referred to our unit had TCA. There is an association between TCA and advanced NEC. Screening of neonates with advanced NEC for TCA is advised to identify those at risk of hematologic complications.

title: Modulation of the production of erythropoietin by cytokines: in vitro studies and their clinical implications.
abstract: The etiology of the anemia of chronic disorders is complex. Factors which clearly contribute to the suppression of erythropoiesis are (a) reduced iron availability and (b) stimulation of the synthesis of immunomodulatory peptides such as IL-1, TNF-alpha and IFN-gamma, which inhibit the proliferation of erythrocytic progenitors in the bone marrow. The question as to whether lack of EPO is of general importance in the pathogenesis of the anemia of chronic inflammatory and malignant diseases is still a subject of controversy. The present in vitro studies show that IL-1 and TNF-alpha, but not IFN-gamma significantly lower the pO2-dependent formation of EPO in HepG2 cultures. In addition, clinical examples are given of anomalously low or high EPO levels in association with diseases involving the immune system. It is proposed that monokines and related immunomodulatory peptides could play a role in the control of the production of EPO.

title: Monokines inhibiting erythropoietin production in human hepatoma cultures and in isolated perfused rat kidneys.
abstract: The blood level of erythropoietin (Epo) is often anomalously low in anemic patients with inflammatory or malignant diseases. Therefore, we studied effects of pure recombinant immunomodulatory peptides on Epo formation in cultures of the human hepatoma cell line, HepG2. Interleukin (IL)-1 beta, IL-1 alpha, and tumor necrosis factor alpha lowered Epo production with half-maximal inhibition at 2, 5, and 20 U/ml, respectively. IL-6, transforming growth factor beta 2 and interferon gamma did not inhibit. Furthermore, IL-1 beta (10 U/ml) proved to block Epo formation in isolated serum-free perfused rat kidneys. Proposedly, monokines play a role in the pathogenesis of Epo deficiency in various diseases.

title: Why Is Erythropoietin Present in Human Milk? Studies of Erythropoietin Receptors on Enterocytes of Human and Rat Neonates
abstract: Erythropoietin receptors (Epo-R) are expressed on cells in the small bowel of human fetuses, but their function has not been defined. We hypothesized that intestinal Epo-R are present postnatally, and that recombinant erythropoietin (rEpo) would increase enterocyte migration and decrease cytokine-induced apoptosis. We used reverse transcriptase-polymerase chain reaction and immunohistochemistry to evaluate the presence of Epo-R mRNA and protein in rat intestinal epithelial cells (IEC-6), and in postnatal human and rat bowel. The effect of rEpo on rates of cell migration and proliferation were established in IEC-6 cells by using cell counting and incorporation of bromodeoxyuridine. To determine whether rEpo affects response to injury, cells were pretreated with rEpo, then were damaged with 25 or 50 ng/mL tumor necrosis factor-α plus 2.5 µg/mL cycloheximide. Cell death was determined by colorimetric bioassay. We found that Epo-R mRNA and protein were expressed by IEC-6 cells and by enterocytes of postnatal rat and human small bowel. Cells that had been exposed to 0.05 or 5.00 U/mL rEpo migrated faster than did the controls (p < 0.05), but no difference was noted in cell proliferation. Treatment of IEC-6 cells with rEpo before or at the time of injury resulted in a lower percentage of cell death, and this effect was neutralized by anti-Epo antibody. We conclude that Epo-R is expressed in enterocytes postnatally in rats and humans. Recombinant Epo increases the rate of migration of IEC-6 cells and decreases cytokine-induced apoptosis. These studies suggest that Epo within human milk has actions on neonate's intestinal function.

title: Human Milk as a Potential Enteral Source of Erythropoietin
abstract: In addition to its content of traditional nutrients, milk is a rich source of hormones and peptides, which survive digestion in the neonatal gastrointestinal tract secondary to lower proteolytic activity and increased protein permeability. Previous studies have shown accelerated erythropoiesis or elevated serum erythropoietin (Epo) levels in neonatal (suckling) animals after maternal phlebotomy or maternal hypoxia exposure. We sought to determine whether significant quantities of Epo are present in human milk and whether Epo remains intact under physiologic digestion conditions. Immunoreactive Epo concentrations were determined in 409 human milk samples obtained from mothers of term and premature infants. Samples collected between birth and postpartum d 134 were divided into 11 postpartum day groups. Mean milk-borne Epo concentrations were within the normal range for plasma Epo concentrations and rose with postpartum day (F10,398 = 5.82, p < 0.0001). No differences were observed between milk collected from mothers of premature versus term infants. Estimated weekly human milk-borne Epo intakes approximated the lower range of published parenteral therapeutic doses. In simulated digestion at physiologic pH levels of 3.2, 5.8, and 7.4, milk-borne Epo resisted degradation at 1 and 2 h, compared with baseline. Therefore, we conclude that human milk contains considerable amounts of Epo which resist degradation after exposure to gastric juices at physiologic pH levels. These results support continued investigation into the fate and developmental roles of Epo in human milk.

title: Human milk feeding of the micropremie.
abstract: There is increasing evidence that mother's milk is an appropriate feeding even for LBW and VLBW infants. During early neonatal life, supplements in the form of human milk fortifiers or single nutrients may be necessary to maintain an adequate biochemical status especially for sodium, phosphorus, and protein. The ideal amount and balance of supplemental nutrients to add to mother's milk for small premature infants remain unknown. From research to date, it is clear that growth responses to fortified mother's milk fed in early life may not be the most important outcomes in relation to long-term growth and development. Infants who receive human milk in early life may be at reduced risk for developing infections and allergy as well as osteopenia and growth failure. Further studies are needed to provide a better understanding of the role of human milk as the sole source of nutrition in premature infants, especially the micropremie.

title: Necrotizing enterocolitis and hematopoietic cytokines.
abstract: The names of the hematopoietic cytokines are misleading because in addition to their effects on bone marrow and bone marrow-derived cells, they have many diverse effects, including effects on the gastrointestinal tract. These effects may be directly mediated by interaction with specific receptors on gastrointestinal epithelial cells, or they may result from their effects on circulating or bowel wall leukocytes and the cytokines these cells produce. As might be expected of factors largely defined by their effects on inflammatory cells, the hematopoietic cytokines are intimately involved in the processes of bowel injury. Further investigations are needed to define the role of hematopoietic cytokines in the human neonate's balance between local gastrointestinal host defense and bowel wall injury. This could lead to effective strategies for the treatment and prevention of NEC.

title: A decreased incidence of necrotizing enterocolitis after prenatal glucocorticoid therapy.
abstract: In a large multicentered, collaborative randomized and blinded trial utilizing antenatal corticosteroids, the goals included determining the effectiveness of these agents in accelerating lung maturation, as well as monitoring any short-term or long-term adverse effects of this treatment on the parturient, fetus, and/or infant. More than 100 specific items, pertaining to diagnoses, complications, and outcomes were recorded for the 696 mothers enrolled in the study and their 745 infants. A significantly decreased incidence of necrotizing enterocolitis (P = .002) was found in the infants treated with steroids. The possibility of accelerated intestinal maturation induced by antenatal maternal steroid therapy exists. This treatment regimen is particularly attractive as adverse aspects of steroid therapy at the dosage utilized have not been demonstrated.

title: Prenatal and postnatal corticosteroid therapy to prevent neonatal necrotizing enterocolitis: a controlled trial.
abstract: To determine whether prenatal corticosteroid therapy would reduce the incidence of neonatal necrotizing enterocolitis (NEC), we assigned a total of 466 women admitted in premature labor either to receive placebo (group A, n = 256), if delivery was expected to occur within 24 hours of admission, or to receive betamethasone (group B, n = 210) if delivery was expected to take place more than 24 hours after admission. All women were free of severe medical complications or drug therapy; cases of intrauterine growth retardation or premature rupture of the membranes were excluded. Their newborn infants, excluding malformed, congenitally infected, and growth-retarded infants, were enrolled in the study unless they had died before the age of 10 postnatal days. Babies born to group A mothers (n = 248) were further assigned to a treatment group (group A1, n = 130) receiving dexamethasone, 2 mg/kg/day by intravenous injection during the first 7 days of life, or to a control group (group A2, n = 118) receiving 10% dextrose solution placebo. Group B infants (prenatal betamethasone, n = 205) received neither treatment nor placebo. The incidence of NEC in group A1 was 6.9% (9/130), and in group A2 it was 14.4% (17/118) (p less than 0.05). In group B the incidence was 3.4% (7/205); this was much lower than in group A2 (p less than 0.01) and lower than in group A combined (10.4%) (p less than 0.01). There was no death from NEC and no surgical intervention among group B patients. The mortality rate for group A1 (11%) was lower than for group A2 (56%) (p less than 0.02). There were fewer indications for surgical intervention for NEC in group A1 than in group A2. Histologic studies confirmed bowel ischemia in all specimens analyzed. These data support the hypothesis that the incidence of NEC is significantly reduced after prenatal steroid treatment. Although postnatal therapy with steroids does not decrease the incidence as effectively as prenatal therapy, it improves clinical outcome of NEC.

title: Hormonal regulation of platelet-activating factor-acetyltransferase activity in rat tissues.
abstract: Platelet-activating factor (PAF) plays a very important role in a number of physiological or pathological conditions. Recently, we and others have suggested that the PAF concentration in various tissues may be regulated by the enzyme that inactivates PAF, PAF acetylhydrolase (PAF-AH). Furthermore, it has been shown that the plasma isozyme of PAF-AH is regulated by various steroid hormone. Lyso-PAF:acetyl-CoA acetyltransferase (PAF-AT) is a key enzyme in PAF biosynthesis by the remodeling pathway. In the present study, the hormonal effects on the tissue levels of PAF-AT activity were investigated in various organs of the rat. The microsomal fraction was prepared from the spleen, lung, uterus, and liver of female rats which had been pretreated with dexamethasone, 17 alpha-ethynylestradiol, medroxyprogesterone-acetate, or vehicle and the PAF-AT activity was assayed. The rats treated with dexamethasone demonstrated significantly lower PAF-AT specific activity in the liver (P < 0.001) and spleen (P < 0.01), compared to the control group. A dose-dependent decrease of PAF-AT specific activity was demonstrated following dexamethasone treatment. Medroxyprogesterone injection also caused a significant decrease of PAF-AT activity in the liver (P < 0.01). No change of PAF-AT activity in any tissue was observed in the 17 alpha-ethynylestradiol-injected animals. Plasma PAF-AH activity was increased significantly by the administration of dexamethasone (P < 0.001) or medroxyprogesterone (P < 0.001), and decreased by 17 alpha-ethynylestradiol (P < 0.001), as previously described. The observation that dexamethasone or medroxyprogesterone treatment decreases the activity of PAF-AT and increases the plasma PAF-AH activity provides a mechanism for the tight control of PAF in tissues.