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JOURNAL CLUB

• Chairperson: Dr.Yashodha.H.T

• Presenter: Dr.Mohan.T.Shenoy

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Introduction Hematologic Profile of Sepsis in Neonates: Neutrophil CD64 as a Diagnostic Marker

• Vineet Bhandaria, Chao Wangb, Christine Rinderb and Henry Rinderb

• a Department of Pediatrics, Division of Perinatal Medicine, and

• b Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut

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Pediatrics 2008;121;129-134

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• Global problem and is a significant contributor to morbidity and death.

• Most studies focus on infants of very low birth weight, given their immature immune system and the added contribution of a variety of risk factors.

• It is important to realize that even late-preterm infants have a compromised immune system and are susceptible to infections in the NICU

• Early recognition and diagnosis of neonatal sepsis are difficult because of the variable and nonspecific clinical presentation of this condition.

SEPSIS IN NEONATES

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NEED FOR THE STUDY It is difficult to identify neonates with sepsis before receipt of the blood culture results.

Because there is a possibility of sepsis even in the presence of negative blood culture results (for example, if the neonate had been exposed to antibiotics in utero), clinicians may opt to continue antibiotic treatment on the basis of the clinical profile

The readily achievable complete blood count and leukocyte differential assays have relatively poor specificity for diagnosing sepsis.

The associated band count and leftward shift of myeloid immaturity measurements may improve diagnostic yield, but their subjective measurement is problematic.

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Acute phase reactants (for example, procalcitonin and C-reactive protein) have also been studied as markers of neonatal sepsis.

Among the various cytokines, most studies have confirmed the utility of Interleukin-6 as an early marker of neonatal sepsis. The cutoff values for interleukin-6 to diagnose sepsis have ranged from 18 to 31 pg/mL.

Interleukin-8, in contrast, has not lived up to its initial promise as a diagnostic marker for neonatal sepsis. It is also important to note that, although manual immunoassays can yield results in 1.5 to 4 hours, readily available automated processes for measurement of cytokine levels are necessary if cytokine values are to be used to facilitate clinical decisions to initiate or to discontinue antibiotic therapy

Unfortunately, acute-phase reactants have similar diagnostic properties, no single marker has been found to have a significant advantage over the others

Therefore, the need persists for improved diagnostic indicators of neonatal sepsis

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It is extremely important to make an early diagnosis of sepsis, because prompt institution of antimicrobial therapy improves outcomes.

Isolation of bacteria from a central body fluid (usually blood )is the standard and most-specific method to diagnose neonatal sepsis.

The readily achievable complete blood count and leukocyte differential assays have relatively poor specificity for diagnosing sepsis.

The associated and count and leftward shift of myeloid immaturity measurements may improve diagnostic yield, but their subjective measurement is problematic.

Therefore, the need persists for improved diagnostic indicators of neonatal sepsis.

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Recently, attention has been directed to the leukocyte cell surface antigens as diagnostic markers of sepsis.

CD64 (Cluster of Differentiation 64) is a type of integral membrane glycoprotein known as an Fc receptor that binds monomeric IgG-type antibodies with high affinity.

It is more commonly known as Fc-gamma receptor 1 (FcγRI).

CD64 is expressed at low concentration on the surface of nonactivated neutrophils

CD64 surface upregulation is induced by granulocyte colony-stimulating factor (G-CSF) and interferon-γ (INF-γ) within 4–6 hours of stimulation

quantitatively upregulated during infection and sepsis,under the influence of inflammatory cytokines;

this increase in surface density occurs in a graded manner dependent on the intensity of the cytokine stimulus

CD64 expression is stable for 24 hours.

Neutrophil surface CD64

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However, each laboratory would need to establish its own cut off values

Technological advances in flow

cytometry

Rapidly quantitate, with precision, and, importantly for neonates, with minimal blood volumes,

Could be performed at any laboratory with flow cytometry facilities and does not need other special equipment or expertise.

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Authors hypothesized that neutrophil surface CD64 expression would be a sensitive index for the detection of neonatal sepsis.

This study was supported by a grant from Trillium Diagnostics.

The goal in this prospective study

A. Enroll consecutive infants undergoing sepsis evaluation from Yale-New Haven Children’s Hospital newborn special care unit.

B. Measure the neutrophil CD64 index in blood as an adjunct to our previously validated hematologic scoring system for detecting neonatal sepsis

STUDY DETAILS

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OBJECTIVE The goal was to determine the utility of neutrophil CD64 as a diagnostic marker for sepsis in neonates.

METHODS

A prospective study that enrolled consecutive infants with suspected sepsis performed in the Yale-New Haven Children’s Hospital newborn special care unit.

Consecutive patients (May 2005 to July 2006) undergoing sepsis evaluation (as deemed necessary by the attending neonatologist) were enrolled.

The length of antibiotic therapy varied on the basis of the severity of the illness and the discretion of the attending neonatologist.

Complete blood count with differential, blood culture, and CD64 index measurement were performed, and neutrophil CD64 indices were correlated with the diagnoses of confirmed and suspected sepsis

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2 or more of the following clinical features were used to identify patients forsepsis evaluations:

(1) Respiratory compromise,

a) Tachypnea (RR- 60 breaths per minute),b) Increased apnea (cessation of respiration for 20 seconds 2 per

hour),

c) Severe apnea (any single episode requiring positive pressure

ventilation), d) Increased ventilatory support (with no other obvious cause eg,

pneumothorax),

e) Desaturation (pulse oximetry readings of 85%);

(2) Cardiovascular compromise

a) Bradycardia (heart rate 100 beats per minute),b) Pallor, c) Decreased perfusion (capillary refill of 3 seconds or cold extremities),

d) Hypotension;

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(3) Metabolic changes,

a) Hypothermia (rectal temperature of < 36°C), b) Hyperthermia (rectal temperature of >38°C),c) Feeding intolerance (increased gastric residuals of >30% of food

volume in 2 feedings within 24 hours),

d) Glucose instability (blood glucose level of <45 mg/dL or >125 mg/dL),

e) Metabolic acidosis (pH < 7.25);

(4) Neurological changes

lethargy, hypotonia, or decreased activity.

These clinical features were validated in an earlier study to be strongly suggestive of infection.

As part of the evaluation, blood was drawn for a complete blood count, including manual differential,blood culture, and CD64 index.

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The following previously validated hematologic criteria were used as indicators for sepsis:

A. absolute neutrophil count (ANC) < 7500 or >14,500 cells per mm3,

B. absolute band count of 1500 cells per mm3, C. immature/total neutrophil ratio of 0.16, and D. platelet count of 150 000 cells per mm3.

All blood cultures were collected by using standard sterile techniques, according to protocol.

The Bactec microbial detection system (Becton-Dickinson, Sparks, MD) was used to detect positive blood cultures.

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A.Confirmed sepsis

Infants whose culture results were positive

B. Suspected sepsis

Those with a positive sepsis score (2 of the hematological criteria enumerated above) among the infants with negative culture results

C. No sepsis : Remaining infants were categorized.

SEPSIS STRATIFICATION

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The first commercially available assay for PMN CD64, developed by TrilliumDiagnostics, LLC is a fluorescence based, no wash flow cytometric assay,

namely the Leuko64.

The assay kit contains

A. Cocktail of monoclonal antibodies including

I. to CD64 and II. to CD163,

B. Red cell lysis buffer,

C. Fluorescence quantitation beads, and

D. Software program for automated analysis of the flow cytometric data that reports PMN CD64 as a CD64 index.

CD64 Index

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Briefly, 50 microL of whole blood or phosphate-buffered saline–diluted whole blood was incubated for 10 minutes at room temperature with a saturating amount of fluorescein isothiocyanate-conjugated anti-CD64 monoclonal antibody or isotype control (Leuko64 kit; TrilliumDiagnostics, Scarborough, ME), followed by ammonium chloride-based red cell lysis.

Samples were washed once and resuspended in 0.5 mL of phosphate-buffered saline with 0.1% bovine serum albumin.

The PMN CD64 index is designed so that

Normal inactivated PMNs yield values of < 1.00 and Documented infection or sepsis typically show values > 1.50.

Using clinical flow cytometers, assay can be completed within 30 minutes

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There were 10 confirmed sepsis episodes with positive culture results:

• 4 involved coagulase-negative staphylococcal species, • 2 Escherichia coli, and • 1 each group B streptococci, Klebsiella oxytoca, Klebsiella pneumoniae, and Enterobacter sp.

For these culture-positive sepsis episodes, the CD64 index had the highest area under the curve (0.852) of all hematologic variables, with a sensitivity of 80% and a specificity of 79%, with a cutoff value of 4.02.

RESULTS

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There were 293 episodes of sepsis evaluations for 163 infants.

Infants with sepsis episodes (confirmed or suspected; n = 40) were of greater gestational age (34.7 0.9 weeks), compared with those (n= 123) with no sepsis (32.6 0.5 weeks), but had similar birth weights (2325 200 vs 1969 94 g) and Apgar scores at 1 and 5 minutes.

There was no difference in the duration of hospitalization for the 2 groups.

RESULTS

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Sepsis episodes had higher neutrophil CD64 indices (5.61 vs 2.63)

As expected, hematologic profiles of sepsis episodes (n =128) were characterized by higher white blood cell counts,absolute neutrophil counts, absolute band counts, and immature/total neutrophil ratios but lower platelet counts.

RESULTS

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In the present study, CD64 index was found to have the highest AUC, compared with commonly used hematologic parameters (including band count and

immature/ total neutrophil ratio), for diagnosing confirmed neonatal sepsis

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SUMMARY OF INFLAMMATORY MARKERS in HEMATOLOGICAL

PROFILE

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In the present study, we found the CD64 index to have the highest AUC, compared with commonly used hematologic parameters for diagnosing confirmed neonatal sepsis.

This finding alone justifies additional prospective studies of CD64 in neonatal sepsis evaluations.

Moreover, CD64 showed surprising utility in all sepsis episodes, even given the a prior use of other hematologic indices to establish that definition.

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CONCLUSION

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In fact, the combination of the CD64 index and the ANC criterion, without any other hematologic indices, demonstrated 95% sensitivity for all sepsis episodes and a 93% negative predictive value ( for predicting which episodes did not fit septic criteria )

These findings strongly suggest that the objective CD64 index could replace the band count (and its derived immature/total neutrophil ratio) as a hematologic indicator of sepsis

Moreover, the high negative predictive value of the CD64/ANC combination suggests that these 2 values could be efficacious in guiding decisions to withhold antibiotic therapy.

Future studies are warranted for direct comparison of the CD64 index with the band count and immature/total neutrophil ratio, to determine whether the former may supplant these subjective criteria in conjunction with the ANC.

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The quantitation of CD64 is rapid (60 minutes) with the use of flow cytometry and uses minimal blood volume.

In fact, for the present study, no extra blood was obtained from the neonates to perform this test; the samples sent for the complete blood count proved adequate.

ROLE OF CD 64 TESTING

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MERITS

CD64 index was elevated significantly during neonatal sepsis episodes and was the most-diagnostic measure of confirmed sepsis;

In conjunction with the ANC, the CD64 index showed promise for replacing the subjectively measured band count and immature/total neutrophil ratio.

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DEMERITS

Only available in specialized research labs

Before this test can be accepted, however, critical issues such as availability and cost must be evaluated carefully in routine clinical settings.

CD64 expression has not been studied extensively in ill preterm neonate

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In accord with other reports, author’s findings suggest

A. CD64 index be incorporated into sepsis scoring systems for neonates and evaluated prospectively for its

incremental (or replacement) diagnostic value.

B. Future studies are warranted for direct comparison of CD64 index with the band count and immature/total neutrophil ratio, to determine whether the former

may supplant these subjective criteria in conjunction with the ANC

SUGGESTIONS

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Statistical Analyses

Comparisons between the sepsis and no-sepsis groups were performed by using Student’s t test or 2 analysis, as appropriate. All analyses were performed by usingGraphPad Prism 3.02 (GraphPad Software, San Diego,CA).

Receiver operating characteristic curves were generated and analyzed for the area under the curve (AUC); pairwise comparisons were performed by using MedCalcfor Windows 9.2.0.2 (MedCalc Software, Mariakerke, Belgium).

A P value of .05 was considered statistically significant.

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