Hematopathology / NEUTROPHIL VCS PARAMETERS

Quantitative Determination of Neutrophil VCS Parameters by the Coulter Automated Hematology Analyzer New and Reliable Indicators for Acute Bacterial Infection
Fernando Chaves, MD, Bethany Tierno, MD, and Dongsheng Xu, MD, PhD
Key Words: Neutrophil; VCS parameters; Automated hematology analyzer; Bacterial infection
DOI: 10.1309/LLF75W0FWQQ8TCC5

Abstract
We studied the clinical usefulness of the VCS parameters (mean channels of cell volume, conductivity, and light scatter) in reactive neutrophils for predicting acute bacterial infection, which are obtained by the Coulter LH 750 hematology analyzer (Beckman Coulter, Fullerton, CA) during automated differential counts. Peripheral blood samples from 69 patients with positive blood cultures for bacteria and 35 control subjects were studied. We observed a significant increase in the mean channel of neutrophil volume (MNV) from septic patients compared with control subjects (156 13.5 vs 143 4.8; P < .001). The mean channel of neutrophil light scatter was decreased significantly in patients (140 10.1 vs 146 7.3; P = .002). An elevation of the MNV was associated with a higher WBC count and percentage of neutrophils and was present even in patients who did not have leukocytosis or neutrophilia. With a cutoff of 150 for the MNV, a specificity of 91% and sensitivity of 70% were achieved. As a quantitative, objective, and more sensitive parameter, we believe that the MNV has a potential to be an additional indicator for acute bacterial infection.

The correct and timely diagnosis of severe acute infectious processes, such as septicemia, is extremely important for proper patient management. However, given the overlapping clinical signs and symptoms with those of other acute critical medical conditions, achieving a high level of certainty while diagnosing septicemia can be a difficult challenge in which the laboratory has a crucial role providing invaluable information. The tests most often ordered in this scenario are the CBC count with differential, blood culture, and often a manual differential count. Although an automated hematology analyzer can readily obtain the CBC count with differential, the expected leukocytosis with neutrophilia might not always be identified. In fact, these numbers fluctuate widely during the course of a septic episode, depending on a balance between the release of neutrophils and their precursors from the bone marrow in response to cytokine stimulation and, later, sequestration of these cells in lung capillaries due to the increased rigidity of cellular membranes seen when they are activated.1,2 The blood culture is considered the gold standard for diagnosing septicemia. Major pitfalls, however, include low sensitivity,3 the need for proper collection techniques to avoid contamination, and the delay of at least a few days before results are available, often leading clinicians to initiate empirical antibiotic therapy that later must be discontinued. Review of peripheral blood smears can also yield important diagnostic information through the identification of the morphologic changes characteristically seen in reactive neutrophils during infection.3-6 Such changes include the presence of toxic granulation, toxic vacuolization, and Dhle bodies in the cytoplasm. Younger forms (left shift), such as bands and metamyelocytes, also can be identified. This approach,
American Society for Clinical Pathology

440 440

Am J Clin Pathol 2005;124:440-444

DOI: 10.1309/LLF75W0FWQQ8TCC5

Hematopathology / ORIGINAL ARTICLE

however, is labor-intensive and time-consuming because it requires manual examination by an experienced medical technologist. Furthermore, the results are subjective because they depend on human interpretation, and only a few hundred cells can be analyzed for any given sample. The VCS technology of the Coulter LH 750 hematology analyzer (Beckman Coulter, Fullerton, CA) can obtain directly data from more than 8,000 WBCs using direct current impedance to measure cell volume (V) for accurate size of all cell types, radio frequency opacity to characterize conductivity (C) for internal composition of each cell, and a laser beam to measure light scatter (S) for cytoplasmic granularity and nuclear structure.7,8 These data then are used to identify each cell as a neutrophil, lymphocyte, monocyte, eosinophil, or basophil, generating an automated differential count. However, the use of the VCS technology to evaluate morphologic changes within the same cell population, such as the previously described neutrophil changes during acute bacterial infection, has never been well studied. Band forms and other immature granulocytes (metamyelocytes, myelocytes), as well as reactive segmented neutrophils, tend to be larger and have lower nuclear complexity than their normal resting counterparts. Therefore, we proposed that the morphologic changes seen in the left-shifted and reactive segmented neutrophils could be analyzed quantitatively by using the Coulter LH 750 with VCS technology. Furthermore, we evaluated the clinical usefulness of these morphologic parameters as possible indicators of an acute infectious process.

and were optically and electronically measured by the Coulter LH 750. Such parameters reflect the mean channels of neutrophil volume (MNV), neutrophil conductivity (MNC), and neutrophil light scatter (MNS).7,8 Manual differential count was not performed. Statistical Analyses All analyses were performed using SPSS software, version 12.0 (SPSS, Chicago, IL). Results were expressed as the mean SD. Comparisons between means were performed by analysis of variance. Comparison between 2 means was performed by using the Student t test. A P value less than .05 was considered significant.

Results
Clinical Data We retrospectively analyzed data from 69 patients with a positive bacterial blood culture including 43 gram-positive and 26 gram-negative cases. The most common bacteria cultured were Staphylococcus aureus (n = 20), followed by Escherichia coli (n = 7) Table 1. The mean patient age was 52 years, and the male/female ratio was 1.1:1. The WBC count ranged from 1,700/L (1.7 109/L) to 39,200/L (39.2 109/L; mean, 12,700/L [12.7 109/L]). The percentage of neutrophils ranged from 29% (0.29) to 97% (0.97; mean, 76% [0.76]). All patients had clinical indications of acute infection. Thirty-five

Materials and Methods

Case Selection In this retrospective case-control study, we studied peripheral blood samples from 69 patients with positive blood cultures for bacteria. All samples were obtained within 2 days of the blood culture collection, and cases in which blood cultures yielded bacteria likely to be contaminants, such as coagulase-negative staphylococci, were excluded from the study. The patients medical records also were reviewed for clinical correlation. Thirty-five control samples were selected randomly from among cases with CBC count and differential data within normal limits (WBC count, <11,000/L [<11.0 109/L]; percentage of neutrophils, <85% [0.85]) and without signs of infection. Data Collection Data included total WBC counts, percentage of neutrophils, and the VCS parameters of neutrophils, which were generated by each individual cell passing through the aperture
American Society for Clinical Pathology

Table 1 Blood Culture Data for 69 Cases

No. (%) of Cases

Staphylococcus aureus Escherichia coli Bacillus species Proteus species -Hemolytic Streptococcus Enterococcus faecalis Group B Streptococcus Klebsiella pneumoniae Streptococcus pneumoniae Clostridium perfringens Enterobacter cloacae -Hemolytic group G Streptococcus Diphtheria Enterococcus species Streptococcus Group A Group C Group G Gram-negative cocci, unidentified Gram-negative rods, unidentified Morganella morganii Streptococcus viridans Salmonella species Serratia species

20 (29) 7 (10) 6 (9) 5 (7) 3 (4) 3 (4) 3 (4) 3 (4) 3 (4) 2 (3) 2 (3) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1)

Am J Clin Pathol 2005;124:440-444

441
DOI: 10.1309/LLF75W0FWQQ8TCC5

441 441

Chaves et al / NEUTROPHIL VCS PARAMETERS

control subjects with normal CBC counts with differential were age-matched (mean age, 51 years); the means for WBC count and percentage of neutrophils were 6,930/L (6.9 109/L) and 61% (0.61), respectively. Mean Channels of VCS Parameters Because neutrophils fulfill the major role in the immune response to acute bacterial infections, we focused mainly on the VCS parameter changes in reactive neutrophils. As shown in Table 2, we observed a significant increase in the MNV for bacteremic cases compared with control samples. On the other hand, the MNS was decreased significantly in patient samples, indicating left-shift transition with increased bands and other immature granulocytes that classically have hypolobated, less complex nuclear morphologic features. We did not observe any difference in the MNC between the 2 groups. These results indicate that the VCS parameters could quantitatively identify the morphologic changes seen in reactive neutrophils of patients with acute bacterial infection. Correlation of VCS Parameters With WBC Count We then studied whether changes in VCS parameters correlated with patients WBC counts Table 3. We initially subdivided the patients into 3 groups based on WBC count: group 1 (n = 31), WBC count, 1,700/L to 11,000/L (1.7-11.0 109/L; mean, 6,600/L [6.6 109/L]); group 2 (n = 18), WBC count, greater than 11,000/L but less than 15,000/L (>11.0 to <15.0 109/L; mean, 12,700/L [12.7 109/L]); and group 3 (n = 20), WBC count, 15,000/L to 39,200/L (15.0-39.2 109/L; mean,

22,300/L [22.3 109/L]). We observed a significant increase of the MNV in all 3 groups vs the control group, including group 1, indicating that the MNV may be a more sensitive and reliable indicator of acute bacterial infection than the classically used WBC count. The MNV increased progressively in groups 2 and 3. However, we observed a significant decrease in the MNS in groups 1 and 3 but not in group 2. This might be because the left-shifted changes (decreased MNS) in this group were compensated by increased toxic cytoplasmic granularity (increased MNS). There was no difference in MNC between control samples and any patient group (Table 3). Correlation of VCS Parameters With Percentage of Neutrophils We studied whether changes in VCS parameters correlated with the percentage of neutrophils Table 4. Patient samples were stratified based on the percentage of neutrophils into 2 groups: 1 (n = 46), neutrophils, less than 85% (<0.85); and 2 (n = 23), neutrophils, 85% or more (85%). We observed significant MNV increases in both groups compared with control samples. The increase in the MNV was more pronounced in patients with a higher percentage of neutrophils. However, a significant MNS decrease was noted only in group 2, suggesting a better correlation of the MNS with a higher percentage of neutrophils. Because the bone marrow stimulatory mechanisms that produce neutrophilia also lead to a release of immature granulocytes (left shift), this finding strengthens our supposition that the decreased MNS in infected patients is due to the lower nuclear complexity seen in such cells. There was no statistical difference in the MNC between patient and control samples (Table 4). Evaluation of Sensitivity and Specificity in Predicting Acute Infection The sensitivity and specificity of the MNV and MNS, as well as the sensitivity of the WBC count and percentage of neutrophils, for predicting infection were analyzed at designated cutoff points Table 5. The specificity of the WBC count and percentage of neutrophils could not be analyzed because normal values were inclusion criteria for control samples. When we selected a cutoff value of MNV equal to or

Table 2 Parameters in Acute Bacterial Infection

Patient Samples (n = 69) MNV MNC MNS 156 13.5 141 3.9 140 10.1 Control Samples (n = 35) 143 4.8 142 2.6 146 7 .3 P .001 .233 .002

MNC, mean channel of neutrophil conductivity; MNS, mean channel of neutrophil light scatter; MNV, mean channel of neutrophil volume.

Table 3 Correlation of VCS Parameters With WBC Count*

Patient Groups Control Group (n = 35) MNV MNC MNS 143 4.8 142 2.6 146 7 .3 1 (n = 31) 152 13.5 141 4.40 139 12.7 2 (n = 18) 157 15.8 142 3.10 143 6.90 3 (n = 20) 161 9.1 141 3.8 139 7 .2 All Groups .0001 .392 .015 Control vs 1 .015 .445 .021 Control vs 2 .0001 .999 .801 P Control vs 3 .0001 .868 .045 1 vs 2 .339 .506 .391 1 vs 3 .027 .949 1 2 vs 3 .757 .858 .521

MNC, mean channel of neutrophil conductivity; MNS, mean channel of neutrophil light scatter; MNV, mean channel of neutrophil volume; VCS parameters, mean channels of cell volume, conductivity, and light scatter. * For a definition of the groups, see the text.

442 442

Am J Clin Pathol 2005;124:440-444

DOI: 10.1309/LLF75W0FWQQ8TCC5

American Society for Clinical Pathology

Hematopathology / ORIGINAL ARTICLE

Table 4 Correlation of VCS Parameters With Percentage of Neutrophils

Patient Groups Control Group (n = 35) MNV MNC MNS 143 4.8 142 2.6 146 7 .3 1 (<85%) (n = 46) 154 9.8 142 3.6 143 9.0 2 (85%) (n = 23) 161 13.7 141 4.50 136 10.4 All Groups .0001 .131 .0001 Control vs 1 .0001 .942 .287 P Control vs 2 .0001 .14 .001 1 vs 2 .057 .194 .003

MNC, mean channel of neutrophil conductivity; MNS, mean channel of neutrophil light scatter; MNV, mean channel of neutrophil volume.

Table 5 MNV in Predicting Acute Bacterial Infection*

Parameter MNV MNS WBC count ( 103/L) Neutrophil (%) Cutoff Points 145 150 152 141 11 85 Sensitivity (%) 83 (57) 70 (48) 61 (42) 46 (32) 55 (38) 33 (23) Specificity (%) 54 (19) 91 (32) 97 (34) 80 (28)

MNS, mean channel of neutrophil light scatter; MNV, mean channel of neutrophil volume. * Numbers in parentheses are the numerators for determining sensitivity and specificity; 69 cases were used to determine sensitivity and 35, specificity. The specificity for the WBC count and percentage of neutrophils could not be analyzed because normal values were inclusion criteria for control samples.

greater than 150 (Table 5), we achieved a sensitivity of 70% and specificity of 91%. This sensitivity was significantly higher than the 55% achieved using 11,000/L (11.0 109/L) as the cutoff for the WBC count and the 33% achieved using 85% (0.85) as the cutoff for percentage of neutrophils. Thus, the MNV could be an even more sensitive indicator for acute bacterial infection. Although the sensitivity improved to 83% using 145 as the MNV cutoff, the specificity decreased to 54%. When we selected a cutoff value of the MNS equal to or less than 141, the sensitivity and specificity were 46% and 80%, respectively (Table 5). Thus, the MNV was the single most predictable indicator of acute bacterial infection.

Discussion
For many years, the WBC count, absolute neutrophil count, percentage of neutrophils, and, especially, increases in bands and other immature neutrophils have been used to predict bacterial infection.3-6,9 Morphologic changes in neutrophils, such as toxic granulation, toxic vacuolization, and Dhle bodies, also provided important diagnostic information. However, assessing such information by manual examination is costly and labor-intensive. Other problems of the 100- or 200cell manual differential count include imprecision, variation
American Society for Clinical Pathology

owing to the heterogeneous distribution of leukocytes on the peripheral blood smear, and interobserver variation in cell identification.8,10-12 Therefore, the diagnostic value of the manual differential count as an indicator of infection remains the subject of ongoing debate.6,8-15 The use of automated hematology analyzers has revolutionized the way we practice laboratory medicine.8 This technology is capable of rapidly examining very large numbers of cells to provide comprehensive hematology profiles.7 Usual parameters obtained include WBC count with differential, RBC counts, and hemoglobin, hematocrit, and reticulocyte values. It also provides measures of cell variability, such as the red cell distribution width, and cell size, such as the mean corpuscular volume, that together give valuable information for the differential diagnosis of anemias. In addition, with the VCS technology of the Coulter LH 750,7 more than 8,000 WBCs can be evaluated automatically using much less time for differential analysis and classification compared with 100or 200-cell differential counts performed by most medical technologists. In this proof-of-concept study, we demonstrated that the morphologic changes seen in left-shifted, reactive neutrophils during an episode of acute bacterial infection could be measured quantitatively using the Coulter LH 750. We showed that the MNV was elevated significantly and the MNS was decreased significantly in samples from septic patients. An elevation of the MNV was associated with positive blood cultures, higher WBC counts, and higher percentages of neutrophils and was present even in samples from patients who did not have leukocytosis or neutrophilia. This suggests that the MNV could be a more sensitive and reliable indicator of acute bacterial infection. In addition, we observed that neutrophil size variability also was increased significantly in samples from septic patients compared with those in control samples (data not shown). Clinical application of VCS parameters offers several advantages. These are the parameters generated during differential analysis without additional specimen requirements. They are quantitative, more objective, and more accurate than manual differential counts because more than 8,000 WBCs are evaluated automatically.7
Am J Clin Pathol 2005;124:440-444
443
DOI: 10.1309/LLF75W0FWQQ8TCC5

443 443

Chaves et al / NEUTROPHIL VCS PARAMETERS

In this pilot study, statistically significant differences of the MNV and MNS were identified between samples from bacteremic patients and control subjects. The clinical usefulness of these findings was evaluated by comparing the sensitivity of these parameters with that of 2 other laboratory tests commonly used in the diagnosis of infection: the WBC count and the percentage of neutrophils. Of these 4 laboratory values, the MNV had the highest sensitivity for predicting infection (Table 5). Furthermore, the MNV was elevated significantly even in samples from patients with normal WBC counts and percentages of neutrophils. This highlights the clinical importance of these findings because a significant proportion of infected individuals are in this category, as represented by the study population in which 31 (45%) and 46 (67%) of the 69 patients had unremarkable WBC counts and percentages of neutrophils, respectively. If the diagnosis of infection had been based on the WBC count or the percentage of neutrophils, the correct diagnosis easily could have been overlooked. Therefore, we believe that the MNV has the potential to be used as an additional indicator of acute bacterial infection, particularly when the other hematologic parameters do not correlate well with the patients clinical signs and symptoms. Large prospective cohort studies are needed to further validate the clinical usefulness of the VCS parameters in other causes of neutrophilic leukocytosis. These causes may include the increased release from bone marrow stores owing to tissue infarction or ischemia (hypoxia); decreased margination owing to exercise or epinephrine medication; decreased extravasation into tissue due to use of glucocorticoids; and increased numbers of bone marrow precursors owing to chronic inflammation, tumors, or myeloproliferative disorders. Several other clinical applications of the VCS technology remain to be studied. With the vast amount of data that could be obtained relating to the morphologic features of neutrophils, lymphocytes, monocytes, and eosinophils separately, the prospects are promising. Valuable possible clinical applications could include, for example, a fast, emergency departmentbased, differential diagnosis of upper respiratory tract infections, because viral pathogens are known to change the morphologic features of lymphocytes. Correlation of manual differential counts and serum C-reactive protein levels9 with the VCS parameters of neutrophils, as well as their value as predictors of acute infection, is under investigation.
From the Department of Pathology and Laboratory Medicine, Boston Medical Center, Boston, MA. Address reprint requests to Dr Xu: Dept of Pathology and Laboratory Medicine, H3600, Boston Medical Center, 88 E Newton St, Boston, MA 02118.

References
1. Worthen GS, Schwab B III, Elson EL, et al. Mechanics of stimulated neutrophils: cell stiffening induces retention in capillaries. Science. 1989;245:183-186. 2. Linderkamp O, Ruef P, Brenner B, et al. Passive deformability of mature, immature, and active neutrophils in healthy and septicemic neonates. Pediatr Res. 1998;44:946-950. 3. Procop GW, Hartman JS, Sedor F. Laboratory tests in evaluation of acute febrile illness in pediatric emergency room patients. Am J Clin Pathol. 1997;107:114-121. 4. Wenz B, Gennis P, Gaehler S, et al. The clinical utility of the leukocyte differential in emergency medicine. Am J Clin Pathol. 1986;86:298-303. 5. Mathy KA, Koepke JA. The clinical usefulness of segmented vs stab neutrophil criteria for differential leukocyte counts. Am J Clin Pathol. 1974;61:947-958. 6. Wile MJ, et al. Manual differential cell counts help predict bacterial infection: a multivariate analysis. Am J Clin Pathol. 2001;115:644-649. 7. Richardson-Jones A. An automated hematology instrument for comprehensive WBC, RBC, and platelet analysis. Am Clin Lab. 1990;9:18-22. 8. Krause JR. Automated differentials in the hematology laboratory. Am J Clin Pathol. 1990;93(4 suppl 1):S11-S16. 9. Manucha V, Rusia U, Sikka M, et al. Utility of haematological parameters and C-reactive protein in the detection of neonatal sepsis. J Paediatr Child Health. 2002;38:459-464. 10. Gulati GL, Hyun BH, Ashton JK. Advances of the past decade in automated hematology. Am J Clin Pathol. 1992;98(4 suppl 1):S11-S16. 11. Ardron MJ, Westengard JC, Dutcher TF. Band neutrophil counts are unnecessary for the diagnosis of infection in patients with normal total leukocyte counts. Am J Clin Pathol. 1994;102:646-649. 12. Pierre RV. Peripheral blood film review: the demise of the eyecount leukocyte differential. Clin Lab Med. 2002;22:279297. 13. Seebach JD, Morant R, Ruegg R, et al. The diagnostic value of the neutrophil left shift in predicting inflammatory and infectious disease. Am J Clin Pathol. 1997;107:582-591. 14. Pierre RV. Left shift and inflammation: a never-ending story [letter]. Am J Clin Pathol. 1998;109:114-115. 15. Swaim WR. Laboratory and clinical evaluation of white blood cell differential counts: comparison of the Coulter VCS, Technicon H-1, and 800-cell manual method. Am J Clin Pathol. 1991;95:381-388.

444 444

Am J Clin Pathol 2005;124:440-444

DOI: 10.1309/LLF75W0FWQQ8TCC5

American Society for Clinical Pathology