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 Table of Contents  
Year : 2012  |  Volume : 37  |  Issue : 4  |  Page : 193-199

CD64 ( diagnostic accuracy compared with the hematologic scoring system and CRP in neonatal sepsis)

1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Pediatric, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Date of Submission16-Feb-2012
Date of Acceptance28-Mar-2012
Date of Web Publication21-Jun-2014

Correspondence Address:
Hosneia K. Akl
Department of Clinical Pathology, Faculty of Medicine, Zagazig University, P.O. Box 44519, Zagazig
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Source of Support: None, Conflict of Interest: None

DOI: 10.7123/01.EJH.0000418698.37863.30

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Despite the advances in prenatal and neonatal care, there is still a high incidence of neonatal sepsis and new accurate and rapid tests are required that facilitate the identification of this condition in order to initiate an early effective treatment for a successful outcome.


To evaluate the diagnostic performance of CD64 expression in relation to the hematologic scoring system (HSS) and C-reactive protein (CRP) either individually or in combination in neonatal sepsis.

Participants and methods

This study included 36 neonates, 13 with culture proven sepsis (group I), 13 with clinically suspected sepsis and culture-negative results (group II), and 10 age-matched and sex-matched healthy neonates (control group). All neonates were examined thoroughly. Complete blood counts were performed, from which HSS was formulated and CRP was measured. Bacterial blood cultures (urine and cerebrospinal fluid when indicated) were carried out only for patients. Neutrophil surface expression of CD64 was assessed by flow cytometry.


Neutrophil CD64 expression was significantly higher in neonates with proven sepsis than the other groups (P=0.0001). A significant positive correlation was found between CD64 expression and HSS (r=0.6581, P<0.001) and CRP (r=0.7531, P<0.001). In terms of the validity of CD64 expression compared with HSS and CRP, the accuracy was 86.1% with a specificity and a positive predictive value (PPV) of 82.6 and 75%, respectively; HSS and CRP sensitivities were 92.3 and 100%, respectively, but with lower specificities (73.9 and 52.2%, respectively) and PPVs (66.7 and 54.2%, respectively). Combinations of CD64 with the two parameters showed that the best was that of CD64 and CRP as sensitivity reached 100% with a constant specificity and slightly increased PPV and accuracy (76.5 and 88.9%, respectively).


CD64 expression is significantly related to definite sepsis and correlated to HSS and CRP. It is a reliable diagnostic test to differentiate infected from noninfected neonates.

Keywords: CD64, C-reactive protein, flow cytometry, hematologic scoring system, neonatal sepsis

How to cite this article:
Mohamed HE, Akl HK, El Banna EA. CD64 ( diagnostic accuracy compared with the hematologic scoring system and CRP in neonatal sepsis). Egypt J Haematol 2012;37:193-9

How to cite this URL:
Mohamed HE, Akl HK, El Banna EA. CD64 ( diagnostic accuracy compared with the hematologic scoring system and CRP in neonatal sepsis). Egypt J Haematol [serial online] 2012 [cited 2022 Aug 19];37:193-9. Available from: http://www.ehj.eg.net/text.asp?2012/37/4/193/134964

  Introduction Top

Sepsis is a clinical syndrome characterized by the presence of both infection and a systemic inflammatory response 1. It is a pathogen-initiated cytokine-mediated process in which immune, inflammatory, and coagulation homeostasis is disturbed 2.

This process is defined by an initial phase (systemic inflammatory response syndrome), a phase of proinflammatory and anti-inflammatory cytokine production, and a phase of a compensatory anti-inflammatory response 3.

The evolution of disease and clinical syndromes is dependent on a complex and delicate balance between the proinflammatory and anti-inflammatory factors 4. Neonatal sepsis is one of the most common causes of neonatal morbidity and mortality despite the extensive research and development in its understanding and management 5.

The accurate diagnosis of sepsis is particularly challenging in neonates as blood culture and clinical manifestations are less informative 1.

The isolation of the organism by a culture remains the gold standard for a definitive diagnosis; however, the confirmation or exclusion of positive culture requires days and more importantly its sensitivity is frequently low because of a concomitant antibiotic therapy 4.

Inflammatory markers for example acute-phase reactants, cytokines, and chemokines have been evaluated in the diagnosis of sepsis, most of which are useful but may be nonspecifically elevated in inflammatory states such as surgery or trauma 6.

In addition, in neonates, the clinical signs of sepsis are poor and nonspecific particularly in preterm infants, in whom the onset of sepsis may be acute and the clinical course can deteriorate rapidly 4.

Therefore, early diagnosis of this life-threatening disease is a mandatory prerequisite for a timely treatment 7.

CD64 (FCγRI) is a neutrophil surface marker with a high affinity for the FC portion of the immunoglobulin G to which attention has been directed recently because of its potential utility in the diagnosis of sepsis 8,9.

It is usually expressed at a low concentration on the surface of nonactivated neutrophils and markedly upregulated by binding of bacterial membrane components and antigenic stimulation by microbial products 10,11.

The aim of the study is to evaluate the diagnostic performance of CD64 and compare it with different conventional tests such as hematologic scoring system (HSS) and C-reactive protein (CRP) in neonatal sepsis.

  Participants and methods Top

This study was carried out at the pediatric and clinical pathology departments of our University Hospital, and included 36 neonates in the following groups:

Group I: proven-sepsis: This group included 13 neonates (seven males and six females) with clinical evidence of infection and the isolation of microorganisms from blood, cerebrospinal fluid (CSF), and/or urine 4.

Group II: suspected-sepsis: This group included 13 neonates (eight males and five females); they had two or more of symptoms or signs of infection with negative blood, CSF, and urine cultures, and no evidence of localized infection such as pneumonia or necrotizing enterocolitis.

Group III: control group: This group included 10 (six males and four females), healthy full-term neonates.

All neonates were subjected (after obtaining the consent of their parents) to the following.


Including prenatal, recent neonatal, and nursery history if the infant had been incubated.

Risk factors for sepsis

Maternal risk factors

Prolonged rupture of membranes (more than 24 h), chorioamnionitis, maternal pyrexia, uterine tenderness, and foul-smelling amniotic fluid 12.

Neonatal risk factors

Prenatal asphyxia, low birth weight, prematurity, invasive procedures, and presence of open congenital anomalies.


General and systemic examination including neonatal reflexes CNS, chest and heart, abdominal and genitalia with special attention to manifestations of sepsis: (fever, hypothermia, apnea, tachycardia, flaring, grunting, poor feeding, abdominal distension, cyanosis, hepatomegaly, and jaundice). Anthropometric measurements of weight, length, and BMI (kg/m2) for all the individuals of each group were carried out.

Laboratory investigations

Complete blood count: 2 ml of venous blood was transferred into a clean glass vial containing 50 µl EDTA for the determination of complete blood count including hemoglobin concentration, red blood cell counts, hematocrit value, platelet count, and total leukocyte count (TLC) and differential leukocytic count (DLC) by Sysmex SF-3000 (TOA Medical Electronics Co., Ltd, Kobe, Japan).

Peripheral blood smears were drawn on clean glass slides and stained with Leishman stain as described by Dacie et al. 13. A DLC was performed to obtain a total neutrophil count (TNC), immature neutrophil count (INC) (including band count), and mature neutrophil count (MNC). Band forms were identified when the width of the nucleus at any constriction was not less than one-third the width at its widest portion 14. Using these values, immature to total neutrophil (I/T) and immature to mature neutrophil (I/M) ratios were calculated. Therefore, the data can be collected for the HSS, which be calculated using the six hematologic values (TLC, TNC, INC, I/T ratio, I/M ratio, and platelet count); sepsis was confirmed when HSS was greater than or equal to 3 [Table 1] 15.
Table 1: Hematologic scoring system

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Blood culture on the BacT/Alert 3D culture system (BioMerieux Inc.): Up to 4 ml venous blood was drawn using standard recommended antisepsis procedures from each patient. All blood cultures were processed using the BacT/Alert 3D system, automated continuously monitoring blood culture instrument (BioMerieux Inc.). Bacterial growth was monitored every 10 min daily for 5 days. Bottles flagged as positive by the BacT/Alert instrument were subcultured on blood, MacConkey, Chocolate, and Sabouraud dextrose agar media, along with CSF or urine samples, and incubated aerobically. Also, culture on GN and NS media (BioMerieux Inc.) was performed and incubated anaerobically.

All colonies that appeared on any culture were examined macroscopically and different microbial pathogens were identified by Gram stain, and finally by API20 strip biochemical identification strips (BioMerieux Inc.) and the available biochemical reactions. Negative cultures were incubated until completion of the rest of the period.

Newborn infants with positive blood cultures were considered to have proven sepsis, whereas the others were still considered to be ‘clinically suspected’ of having an infection.

C-reactive protein estimation 16

Serum was collected and stored at +2 to +8°C until use. BN ProSpec was used in CRP estimation; the results were evaluated by comparison with a standard of known concentration (CardioPhase*; Dade Behring, Newark, New Jersey, USA). The cut-off value of greater than 0.5 mg/dl was considered evidence of infection.

Flow cytometric analysis of CD64 expression

On EDTA anticoagulated blood by the whole-blood lysis technique using a FACscan flow cytometer (Becton Dickinson). Samples were stained with a phycoerythrin-labeled anti-CD64 monoclonal antibody (CD64-phycoerythrin) from Immunotech (Merseille, France). Negative isotype-matched controls were used to determine the nonspecific binding. At least 10.000 cells/sample were acquired and analyzed. The cut-off point of positivity (5.3%) was calculated from the mean values of the percent of CD64-expressing cells in the control samples (cut-off=mean±2 SD).

Statistical analysis

Statistical analysis was carried out using the SPSS version 10.0. Data are represented as median, range, and percentage. The χ2 test and the Kruskal–Wallis test were used when appropriate. Diagnostic performance and accuracy were calculated. P<0.05 was considered to be statistically significant in all tests.

  Results Top

[Table 2] summarizes the demographic data of the neonates studied; there was no statistical difference between the three groups studied.
Table 2: Demographic data of the studied groups

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[Table 3] shows the statistical differences among the three groups studied in TLC, TNC, INC, I/T ratio, I/M ratio, and platelet count. Also, there was a significant decrease in TLC, TNC, I/T, and I/M ratios and an increase in the platelet count in group II compared with group I (P<0.05).
Table 3: Blood indices in the studied groups

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The organisms isolated from the cultures were mostly Gram-positive organisms (69.2%), among them Staphylococcus aureus 4 (30.7%), Staphylococcus coagulase negative 3 (23.1%), Streptococcus pneumoniae 1 (7.7%), and enterococci 1 (7.7%). The Gram-negative organisms (30.8%) Klebseilla 3 (23.1%) and Escherichia coli 1 (7.7%) as shown in [Table 4].
Table 4: Isolated organisms in the proven-sepsis group

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[Table 5] shows highly significant differences between the three groups studied in terms of CD64 expression, HSS, and CRP. [Figure 1] includes dot-plot histograms showing the pattern of positive expression of CD64 that was observed in the control group (3.1%), the group with suspected sepsis (4.5%), and the group with documented sepsis (25.7%).
Table 5: CD64, HSS, and CRP in the studied groups

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Figure 1: Dot-plot histograms showing the pattern of positive expression of CD64 that was observed in (a) the control group (3.1%), (b) the group with suspected sepsis (4.5%), and (c) the group with documented sepsis (25.7%).

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A significant positive correlation between CD64 expression and both HSS and CRP among the three groups studied (r=0.6581, P<0.001 and r=0.7531, P<0.001, respectively) is shown in [Table 6].
Table 6: Correlation between CD64 expression and other parameters

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The performance of each test is shown in [Table 7], CD64 expression was the most specific and accurate (82.6 and 86.1%, respectively) and the CRP test was the most sensitive test (100%).
Table 7: Validity of the laboratory parameters used in the detection of sepsis

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[Table 8] shows the validity of different combinations of the three laboratory parameters. When CRP was added to any combination, sensitivity and negative predictive value (NPV) reached 100%, whereas specificity was the same as that of CD64 alone (82.6%).
Table 8: Validity of the laboratory parameters used in the detection of sepsis

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  Discussion Top

Neonatal systemic infection is the most important cause of neonatal mortality and morbidity, and consequently, early identification of sepsis is still a critical issue in the neonatal area. An accurate inflammatory marker with high diagnostic sensitivity, specificity, and NPV for neonatal sepsis would be a valuable tool for therapeutic decision-making and avoidance of unnecessary use of antibiotics 17,18.

In the present study, the most common organism isolated from the sepsis-confirmed group was Gram-positive organisms (69.2%): staphylococci 53.8%, Streptococcus pneumoniae 7.7%, and enterococci 7.7%. Gram-negative bacilli (30.8%) were Klebseilla 23.1% and E. coli 7.7%. These results were nearby and online with that of other reports 19–21 who reported the current shift from Gram-negative to Gram-positive bacteria in causing blood stream infections.

The aim of this study was to evaluate CD64 expression as a diagnostic measure and to compare it with the HSS and CRP.

The neonates studied were divided into three groups: proven sepsis, suspected sepsis, and healthy controls.

CRP is an acute-phase reactant found in the blood that is produced by hepatocytes in the setting of an infection or tissue injury 22. Although not specific for neonatal sepsis, CRP has high sensitivity and NPV as reported by Anwar and Mustafa 23.

Ahmed et al. 24 reported that the sensitivity, specificity, NPV, and positive predictive value (PPV) of CRP were 85.7, 95, 82.7, and 95.9%, respectively. Santana et al. 25 reported that the sensitivity and specificity of CRP were 80 and 92%, respectively, whereas Garland and Bowman 26 have documented 86% NPV of CRP. In the present study, the sensitivity and the NPV of CRP were 100%, whereas a low specificity and PPV were reported, 52.2 and 54.2%, respectively, yielding an accuracy of 69.4%. These high sensitivity and NPV in most of the studies indicate that the likelihood of sepsis being present in the absence of CRP positivity is very low.

The discrepancy in the validity of CRP in different studies may be because of variations in the diagnostic criteria, the time of onset of infection (early or late), and the different methods of assay 24.

Ng et al. 27 have reported that serial measurements at 24 and 48 h after the onset of illness improve the sensitivity considerably (82 and 84%, respectively). CRP increases at around 24 h after the onset of infection 28. The specificity and PPV of CRP can be considered as a ‘specific’ but ‘late’ marker of neonatal infection 27–29.

Using a cut-off of greater than or equal to 3, the score had a high sensitivity of 96%, but a disappointingly low PPV of 31% 30. This scoring system was not widely adopted because of its unfavorable diagnostic values, the complexity of the scoring method, and the fact that some of the tests were labor intensive and required a highly trained technician to produce an accurate result. Low platelet counts and morphological changes in neutrophils were often severe, but late signs of infection 30–32. It also has several advantages as it is applicable to all infants, including those who have received antibiotic therapy before evaluation; it simplifies the interpretation of the hematologic score (the higher the score, the greater the certainty that sepsis is present) 30.

Rodwell et al. 30 evaluated the role of hematologic findings as a screening test for neonatal sepsis; in that study, 298 infants were evaluated for sepsis and 27 had culture-proven sepsis. Twenty-six (96%) of those 27 infants had greater than or equal to 3 hematologic criteria present at the time of the sepsis episode. It was concluded that the presence of greater than or equal to 3 hematologic scores was 96% sensitive and 78% specific. These criteria had a PPV of 31% and a NPV of 99%. Modified Rodwell criteria have been used in other studies to differentiate infants with and without sepsis 33–35.

In this study, for the HSS, the sensitivity, specificity, NPV, and PPV were 92.3, 73.9, 66.7, and 94.4, respectively, with an accuracy of 80.5%.

However, the wide variability in the diagnostic accuracy of leukocyte indices in neonatal sepsis, especially the band count and its derived I/T, must be kept in mind 36. Also, it must be kept in mind that white blood cell counts and absolute neutrophil count may be affected by many factors besides infection including age, blood sampling method, mode of delivery, maternal hypertension, and sex 37.

CD64 is normally expressed in very low concentrations by unstimulated neutrophils, whereas it is considerably upregulated with the onset of bacterial invasion within an hour of acute inflammation 38,39.

Bhandari et al. 40 reported that the CD64 index had the highest area under the curve of all hematological variables, with a higher sensitivity and specificity for blood culture-positive cases. Davis and Bigelow 41, Nuutila et al. 42, and Rudensky et al. 43 reported that neutrophil CD64 expression is an improved diagnostic indicator of infection/sepsis. The results of our study are in agreement with this as we found significantly elevated levels of CD64 in septic neonates when compared with healthy controls. These findings are also in agreement with other studies 10, 44, 45 that have reported the same results in neonates with early-onset sepsis. Another study reported similar results in very low-birth-weight neonates with late-onset neonatal sepsis 46.

Layseca-Espinosa et al. 44 found that the enhanced expression of CD64 was a highly specific indicator of neonatal infection (96.8%), although its diagnostic sensitivity was low (25.8%) and NPV was intermediate (57.4%) and mentioned that the neonates with septicemia were older than the controls, which might have an influence on CD64 expression. Ng and coworkers reported that the measurement of neutrophil CD64 expression by quantitative flow cytometric analysis had a very sensitive and moderately specific diagnostic value (96 and 90%, respectively) with an NPV of 97% 10, 45, 46.

This study showed that CD64 had high sensitivity and NPV (92.3 and 95%, respectively), with considerable specificity (82.6%).

CD64 has already been identified as a high-affinity Fc-γ receptor of immunoglobulin G antibody in the process of phagocytosis and intracellular killing of opsonized microbes 47,48. The pathogens that require opsonization are encapsulated bacteria 49; thus, CD64 expression is not related to viral infection.

In the current study, three infants from the suspected sepsis group who had a severe clinical course but had negative blood culture were subsequently found to have both increased CD64 expression and elevated serum CRP concentrations. All received a full course of antibiotics, with subsequent improvement. Therefore, the clinical and biochemical evidences suggested that these infants were most likely to be infected rather than false-negative cases. Inclusion of these cases in the proven sepsis group would improve the specificity and PPV of CD64 to 95 and 93.8%, respectively.

Although CRP showed 100% sensitivity and NPV its specificity was very low, whereas CD64 showed respectable sensitivity and specificity. HSS-reported sensitivity was also respectable but its subjective measurement is problematic compared with CD64, which is objective 40.

Recent investigations have focused on the combination of markers ensuring greater diagnostic accuracy. Ng et al. 10 found that the use of CD64 in combination with other diagnostic markers such as CRP improved the sensitivity and NPV to 97 and 98%, respectively, for early-onset neonatal infection. Our data indicate that the addition of CD64 to CRP can marginally enhance the sensitivity and NPV from 92.3 to 100%, whereas the specificity remains equal to that of CD64 when assayed alone. The same results were obtained when the three diagnostic markers were combined together.

The advantages of using CD64 as a diagnostic marker are as follows: (a) the flow cytometric analysis can be performed with minimal blood volume (50 µl of whole blood), (b) the result is rapidly available within (<60 min), (c) the measurement is ‘quantitative’ and thus enables comparison of results among different centers, (d) unlike cytokines, which are usually assayed in batches 50, the measurement of cell surface antigens is performed on an ad-hoc basis, (e) the persistent expression of CD64 for at least 24 h gives the marker a wide diagnostic window, and (f) the very favorable diagnostic utilities render CD64 one of the best infection markers for the identification of early-onset and late-onset neonatal sepsis 10.

Although diagnostic utilities such as sensitivity, specificity, PPV, and NPV eventually determine the accuracy and usefulness of a clinical test, a high sensitivity and NPV would be the most important in neonatal infection because all genuinely infected newborns should be identified and treated. In addition, acceptable specificity is considered to be greater than 80% so that the unnecessary use of antibiotics can be minimized 48.

In conclusion, the results of this study suggest that CD64 is a reliable diagnostic test to differentiate infected from noninfected neonates.[50]

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  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]


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