Context.—The number of band forms and immature neutrophils increases during acute bacterial infection. However, the determination of band counts and other neutrophil morphologic changes, such as the presence of toxic granulation, toxic vacuolization, and Dohle bodies in the cytoplasm, is labor intensive and time consuming, as it requires manual examination by an experienced medical technologist.

Objective.—To investigate the value of the neutrophil volume distribution width (NDW), generated by VCS technology of the Coulter LH 750 hematology analyzer, as an additional predictor of acute infection.

Design.—Total white blood cell count, percentage of neutrophils, and NDW data from 70 patients with positive blood cultures for bacteria and from 35 age-matched control subjects were retrospectively analyzed.

Results.—A significant increase in the NDW was observed in the bacteremic patients compared with the controls (24.7 ± 4.5 vs 19.0 ± 1.5; P < .001). Such increase was observed even in patients with white blood cell counts less than 11 000/μL (23.0 ± 5.6 vs 19.0 ± 1.5; P < .001) or with percentages of neutrophils less than 85% (24.0 ± 4.9 vs 19.0 ± 1.5; P < .001). The more dramatic increases were seen in patients with leukocytosis (25.7 ± 3.2, P < .001) or with neutrophilia (25.9 ± 3.4, P < .001). Using an NDW cutoff of 23, a 100% specificity and a 69% sensitivity were achieved.

Conclusions.—As a quantitative parameter, the NDW has potential for use as an additional indicator for diagnosing acute infection.

The complete blood cell count with differential is among the most commonly ordered tests for the diagnosis of acute infection. When performing a manual differential count on a peripheral blood smear, the medical technologist traditionally classifies cells using a series of visual criteria, such as size of the cell, density of the nuclear chromatin, presence of the nucleolus, nucleus-cytoplasm ratio, and presence of cytoplasmic granules. Automated hematology analyzers are now able to provide accurate and precise differential counts in a fast and cost-effective manner. Among these, the Coulter LH 750 (Beckman Coulter, Inc, Fullerton, Calif) uses a unique process of simultaneously collecting data from more than 8000 leukocytes, including direct current impedance to measure cell volume for accurate size of all cell types, radio frequency opacity to characterize conductivity for internal composition of each cell, and a laser beam to measure light scatter for cytoplasmic granularity and nuclear structure.1,2 The Coulter LH 750 measures not only the mean channels of cell volume, conductivity, and light scatter but also the SD of each of these parameters. This so-called VCS technology is analogous to microscopic evaluation of a peripheral blood smear but uses the most modern technology to refine the output.

It has been previously demonstrated that VCS parameters can detect morphologic changes in immature and reactive neutrophils.3,4 Evaluation of the clinical utility of these findings showed that the mean channel of neutrophil light scatter and the mean neutrophil volume could be indicators of acute infection. The mean neutrophil volume had a better sensitivity than the white blood cell (WBC) count or the percentage of neutrophils and was elevated even in patients with infection with otherwise unremarkable hematologic profiles, suggesting its use as a new and promising indicator for the diagnosis of infection.3,4 

During acute bacterial infection, there is a left shift of the circulating granulocytes, with the presence of band forms and other immature neutrophils. We expect the entire cell population not only to increase in volume or size but also to become less homogeneous, with increased variability of cell sizes and shapes. Therefore, we hypothesize that the SD of neutrophil volume (or the neutrophil volume distribution width [NDW]), quantitatively measured by the Coulter LH 750 with VCS technology, could be used as an indicator of acute infection.

Patient Selection

We retrospectively analyzed peripheral blood samples from 70 patients with positive blood cultures for bacteria. All samples were drawn within 2 days of the blood culture collection, and patients whose blood cultures yielded bacteria that were likely to be contaminants, such as coagulase-negative staphylococci, were excluded from the study. The patients' medical records were reviewed for clinical correlation and for exclusion of any concomitant hematologic diseases, such as leukemia or myelodysplasia, that would affect the leukocyte morphologic structure. Thirty-five control subjects were selected from among patients whose complete blood cell count and differential data were within normal limits (WBC count range, 4000–11 000/μL; and percentage of neutrophils, <85%) and who had no signs of infection or hematologic disease.

Data Collection

Data collected included total WBC count, percentage of neutrophils, and the NDW. The NDW was calculated as the SD of neutrophil volume, measured by the direct current impedance of each cell passing through the aperture. Therefore, the NDW reflects the neutrophil size variability.1,2 Manual differential count was not performed in the present study. The study protocol was approved by the institutional review board.

Statistical Analysis

All analyses were performed using SPSS version 12.0 software (SPSS Inc, Chicago, Ill). Results were expressed as mean ± SD. Comparisons between means were performed by analysis of variance. Comparison between 2 means was performed by Student t test. P < .05 was considered significant.

Clinical Data

We retrospectively analyzed data from 70 patients with positive bacterial blood cultures, including 43 gram-positive and 27 gram-negative cases. The most common bacteria cultured were Staphylococcus aureus (n = 20), followed by Escherichia coli (n = 8), Bacillus (n = 6), and Proteus (n = 5); α-hemolytic streptococcus, Enterococcus faecalis, group B streptococcus, Klebsiella pneumoniae, and Streptococcus pneumoniae (n = 3 each); Clostridium perfrigens and Enterobacter cloacae (n = 2 each); and β-hemolytic group G streptococcus, Diphtheroid, Enterococcus, group A streptococcus, group C streptococcus, group G streptococcus, gram-negative cocci—unidentified, gram-negative rods— unidentified, Morganella morganii, Streptococcus viridans, Salmonella, and Serratia (n = 1 each). The mean patient age was 52 years, and the male-female ratio was 1.2:1. The WBC count ranged from 1700/μL to 39 200/μL (mean, 12 700/μL). The percentage of neutrophils ranged from 29% to 97% (mean, 76%). All patients had clinical indications of acute infection. Thirty-five control subjects with normal complete blood cell counts with differential (WBC count range, 4100–10 900/μL) were age matched (mean age, 51 years), with a mean WBC count and a mean percentage of neutrophils of 6930/μL and 61%, respectively.

NDW in Acute Infection

We initially investigated the changes in the overall NDW, which reflects the neutrophil size variability. As seen in the Figure, a significant increase in the NDW was observed in the bacteremic patients compared with the controls (24.7 ± 4.5 vs 19.0 ± 1.5; P < .001). We then stratified patients based on WBC count (11 000/μL cutoff) and on percentage of neutrophils (85% cutoff). Such increase in the NDW was observed even in patients with a WBC count less than 11 000/μL (n = 32; 23.0 ± 5.6 vs 19.0 ± 1.5; P < .001) or with a percentage of neutrophils less than 85% (n = 47; 24.0 ± 4.9 vs 19.0 ± 1.5; P < .001). The more dramatic increases in the NDW were seen in patients with leukocytosis (n = 38; 25.7 ± 3.2, P < .001) or with neutrophilia (n = 23; 25.9 ± 3.4, P < .001), indicating that such numerical changes correlate with the release of a heterogeneous population of immature cells from the bone marrow.

Neutrophil volume distribution width (NDW) in acute infection. A significant increase in the NDW was observed in the bacteremic patients (n = 70) compared with control subjects (n = 35) (24.7 ± 4.5 vs 19.0 ± 1.5; P < .001)

Neutrophil volume distribution width (NDW) in acute infection. A significant increase in the NDW was observed in the bacteremic patients (n = 70) compared with control subjects (n = 35) (24.7 ± 4.5 vs 19.0 ± 1.5; P < .001)

Close modal

Evaluation of Sensitivity and Specificity in Predicting Acute Infection

We then calculated the sensitivity and specificity of the NDW for predicting infection at designated cutoffs (Table). When we selected an NDW cutoff of 22 or higher, we achieved a sensitivity of 79% and a specificity of 94%. These results were slightly better than the sensitivity and specificity of the mean neutrophil volume using a cutoff of 150 or higher, which were 70% and 91%, respectively.3 In addition, this was significantly higher than the 55% sensitivity achieved using 11 000/μL as the WBC count cutoff and the 33% sensitivity achieved using 85% as the percentage of neutrophils cutoff.3 We achieved 100% specificity and 69% sensitivity using an NDW cutoff of 23 (Table). Therefore, the NDW may be another reliable indicator for predicting acute bacterial infection.

Neutrophil Volume Distribution Width (NDW) in Predicting Acute Bacterial Infection

Neutrophil Volume Distribution Width (NDW) in Predicting Acute Bacterial Infection
Neutrophil Volume Distribution Width (NDW) in Predicting Acute Bacterial Infection

The correct and timely diagnosis of severe acute infectious processes, such as septicemia, is critical for proper patient management. Laboratory tests most often ordered in this scenario are blood culture, complete blood cell count with differential, and manual differential count. For many years, WBC count, percentage of neutrophils or absolute neutrophil count, increased banded neutrophils, and immature-total neutrophil ratio have been used to predict acute infection.5–9 The diagnostic value of the band count as an indicator of acute infection, however, is the subject of ongoing debate.8–14 In addition, the determination of band counts and other neutrophil morphologic changes, such as the presence of toxic granulation, toxic vacuolization, and Dohle bodies in the cytoplasm, is labor intensive and time consuming, as it requires manual examination by an experienced medical technologist.8,9,11–13 Furthermore, the results are subjective because they depend on human interpretation, and only a few hundred cells can be analyzed for any given sample. Although the blood culture is considered the gold standard for diagnosing septicemia, major pitfalls include low sensitivity,6 the need for proper collection techniques to avoid contamination, and the delay of at least a few days before results are available. Therefore, there is a need for a more objective and faster way to predict acute infection.

It was previously demonstrated that the morphologic changes of reactive neutrophils can be quantitatively determined by VCS parameters of the Coulter LH 750.3,4 Compared with controls, the neutrophils in septic patients had significant increases in the mean neutrophil volume.4 In addition, the mean channel of neutrophil light scatter, reflecting cytoplasmic granularity and nuclear structure, was significantly decreased in septic patients compared with controls.3 

In this study, we investigated the value of the neutrophil volume SD (the NDW), generated by VCS technology of the Coulter LH 750 hematology analyzer, as an additional predictor of acute infection. We demonstrated that, compared with controls, the neutrophil population of patients with infection is less homogeneous, with a wider variation of individual cell sizes, as shown by the increased NDW. Furthermore, change in the NDW has clinical value in the diagnosis of acute bacterial infection, as it was associated with positive blood cultures, higher WBC counts, and higher percentages of neutrophils and was present even in patients who did not have leukocytosis or neutrophilia. Because a significant proportion of patients with acute infection have unremarkable WBC count profiles, the ability of the NDW to predict infection even in these patients highlights at least 1 important possible clinical application of this new parameter. On the opposite end of the spectrum, the value of the NDW as a discriminator in the differential diagnosis between leukemoid reaction due to infection and chronic myelogenous leukemia is under investigation. Our preliminary results appear to indicate a more significant increase in the NDW of patients with chronic myelogenous leukemia compared with that in patients with infection and in controls (D.S.X., unpublished data, 2005).

The use of VCS parameters could have an important effect on the practice of laboratory medicine because their clinical application offers several advantages. These parameters are generated during automated differential analysis without additional specimen requirements. They are quantitative, more objective, and more accurate than manual differential counts because more than 8000 leukocytes are simultaneously evaluated.1 In addition, the VCS parameters have shown better diagnostic performance than WBC count and percentage of neutrophils, which are traditionally used as indicators of infection. For example, using an NDW cutoff of 23, a 100% specificity and a 69% sensitivity were achieved (Table), compared with sensitivities of 55% achieved using 11 000/μL as the WBC count cutoff and 33% achieved using 85% as the percentage of neutrophils cutoff.3 The results obtained in this retrospective proof-of-concept pilot study are promising, considering that the clinical value of the WBC count, the percentage of neutrophils, and other traditional tests such as the band count has been the subject of much controversy in the medical literature.8–14 To further elucidate this matter, we are collecting data for a prospective study comparing the sensitivity and specificity of the neutrophil VCS parameters, including the NDW, with those of the band count and the C-reactive protein levels.

In conclusion, we believe that the NDW may be a sensitive and reliable VCS parameter for use in the diagnosis of acute infection. Its clinical value should be investigated in the differential diagnosis of several other conditions associated with neutrophilic leukocytosis, such as tissue infarction or ischemia (hypoxia), exercise or epinephrine medication, use of glucocorticoids, chronic inflammation, tumors, and myeloproliferative disorders. Use of the NDW is analogous to the approach used in the differential diagnosis of anemias, in which the mean corpuscular volume and the red blood cell distribution width can discriminate among the primary anemia subtypes and direct further diagnostic workup.

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The authors have no relevant financial interest in the products or companies described in this article.

Author notes

Reprints: Dongsheng Xu, MD, PhD, Department of Pathology and Laboratory Medicine, Boston Medical Center, H3600, 88 E Newton St, Boston, MA 02118 ([email protected])