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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2015  |  Volume : 58  |  Issue : 4  |  Page : 459-463
Volume, conductivity, and scatter parameters as diagnostic aid to bacterial sepsis: A tertiary care experience


1 Department of Haematology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India
3 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India

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Date of Web Publication4-Nov-2015
 

   Abstract 

b>Introduction and Materials and Methods: Early diagnosis of sepsis is extremely important to reduce high mortality and morbidity. In this study, clinical usefulness of the volume, conductivity and scatter parameters (mean channels of cell volume, conductivity, and light scatter) in neutrophils was analyzed for predicting acute bacterial infection, which are obtained by the Coulter LH 750 Hematology Analyzer (Beckman Coulter, Fullerton, CA, USA) during automated differential counts. Results: Peripheral blood samples from 162 patients with positive blood cultures for bacteria and 40 healthy controls 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). Discussion and Conclusion: An elevation of the MNV was associated with a higher white blood cell count and percentage of neutrophils and was present even in patients who did not have leukocytosis or neutrophilia. With a cut-off of 149 for the MNV, a specificity of 91.4% and sensitivity of 88.7% were achieved. As a quantitative, objective, and more sensitive parameter, we propose that the MNV has a potential to be an additional indicator for acute bacterial infection.

Keywords: Neutrophilia, leukocytosis, sepsis, volume, conductivity and scatter parameters

How to cite this article:
Purohit AH, Kumar P, Sharma S, Kapil A, Gupta A, Mukhopadhyay AK. Volume, conductivity, and scatter parameters as diagnostic aid to bacterial sepsis: A tertiary care experience. Indian J Pathol Microbiol 2015;58:459-63

How to cite this URL:
Purohit AH, Kumar P, Sharma S, Kapil A, Gupta A, Mukhopadhyay AK. Volume, conductivity, and scatter parameters as diagnostic aid to bacterial sepsis: A tertiary care experience. Indian J Pathol Microbiol [serial online] 2015 [cited 2019 Dec 7];58:459-63. Available from: http://www.ijpmonline.org/text.asp?2015/58/4/459/168853



   Introduction Top


Bloodstream infection is a major cause of morbidity and mortality.[1] Sepsis is the most common cause of death in hospitalized patients worldwide, especially in a developing country like India.[2] Early diagnosis of bacteremia is extremely important to reduce high mortality.[1] Blood culture remains the gold standard for diagnosing bacteremia or sepsis.[3] Although viral and fungal agents are also implicated in sepsis, bacteria are still the most important implicated agent.[4] The complete blood cell count and peripheral blood smear examination along with blood culture have been the cornerstone of diagnosing sepsis for many years. Increased total leukocyte count (TLC), immature/total neutrophil ratio and neutrophilia are common findings in sepsis.[5],[6] Moreover, characteristic morphological changes regarding the size of the cell, the density of the nucleus, number of nuclear lobes, along with the presence of toxic granules, vacuolization and occasional Döhle bodies are evident in sepsis.[7],[8] However, this approach is not only labor-intensive and time-consuming, but also requires the technical expertise of a hematologist to identify correctly these changes.[9] It is subjective and is not based on totally objective criteria. Further, the decision depends on observation of 100 or 200 cells in the peripheral smear. It also has a limited scope as only a few cells could be examined under microscopic examination. Blood culture too is not infallible because of its low sensitivity and easy amenability to contamination.[3] Moreover, a positive blood culture report takes at least 48 h, which delays the initiation of early antibiotic therapy.

With the introduction of newer automated analyzers for determining hemogram, technology has a new role to play. The volume, conductivity and scatter (VCS) technology of the Coulter LH 750 hematology analyzer (Beckman Coulter, Fullerton, CA, USA) can obtain data from more than 8000 white blood cells (WBCs) in a few seconds, using 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. The data from VCS technology can thus be a comparable reflection of cell morphology.[9],[10],[11]

Volume, conductivity and scatter parameters, therefore, could be a promising tool for the early diagnosis of sepsis. Although there have been previous studies from the west which support the same, but their different study population limits its relevance in India.[10],[11],[12] Therefore, in the present study we have tried to investigate the VCS parameters if they have any role in diagnosing bacteremia in suspected population. We also tried to find out a cut-off value of mean channel of neutrophil volume (MNV) that could be an aid as supportive evidence in diagnosing bacteremia or sepsis.


   Materials and Methods Top


This observational descriptive study was conducted in the department of Laboratory Medicine at a tertiary care center between October 2011 and October 2012 for a duration of 12 months. During the study period 162 confirmed bacterial blood culture positive cases were taken as "cases" and 40 voluntary healthy donors from the blood bank of the hospital were taken as 'control'. Informed consent was taken from all study participants. We excluded the cases in which blood cultures yielded bacteria likely to be contaminants.

All blood samples from clinically suspected sepsis cases received at our laboratory for hemogram and blood culture were screened. Hemogram samples were run on LH 750, Beckman Coulter, USA according to prescribed protocol.[13] For blood culture, a single sample was collected from a peripheral vein or artery under aseptic conditions. Blood cultures were done in a brain heart infusion biphasic medium. Three milliliter of blood was inoculated into the brain heart infusion broth and incubated at 37°C. Subcultures were done on sheep blood agar and MacConkey agar at the earliest visual detection of turbidity on days 1, 4, and 7 if the bottles did not show turbidity. The isolate was identified by their respective characteristic appearance on media and confirmed by the pattern of biochemical reactions using the standard method.[14] Blood culture positive cases were identified. Cases wherein blood culture did not grow any bacteria were excluded from the further study. VCS parameters of these cases and controls including MNV, mean channel of neutrophil conductivity (MNC) and mean channel of neutrophil scatter (MNS) were retrospectively studied. Data was compiled and entered into Microsoft Excel and was statistically analyzed.

Statistical analysis

All statistical analyses were performed using Software STATA version 11.2 (Statacorp data analysis & statistical software). Results were expressed as the mean ± standard deviation (SD) and median with range. Comparisons between means were performed by using the Student's t-test.


   Results Top


There were 162 confirmed blood culture positive cases during the study period. Complete blood counts and VCS parameters of the cases and the controls were recorded. Out of the cases, gram-positive bacteria were identified in 72 cases and gram-negative bacteria in 90 cases. The commonest bacteria isolated was Staphylococcus aureus (n = 52), followed by Acinetobacter and  Escherichia More Details coli (n = 28 each) [Table 1].
Table 1: Blood culture data of 162 blood culture positive cases

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Baseline characteristics of the cases and the control were as shown in [Table 2]. The age and sex difference between two groups were not statistically significant. However, the difference in mean total leucocyte count and differential neutrophil was statistically significant (P = 0.0001).
Table 2: Patient and control baseline characteristics

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Volume, conductivity and scatter parameters of study and control group were analyzed [Table 3]. It was observed that MNV and MNS were higher in patients compared to controls. The difference was statistically significant (P = 0.0001). The difference in MNC values between cases and controls was not statistically significant. These changes suggest that VCS parameters can be a true reflection of morphological changes of cells.
Table 3: Comparison of VCS parameters between patient and control group (overall)

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Correlation between VCS parameters and increased TLC: The case group was found to have higher TLC (median TLC was 9.8 × 109/L, range 3.9-21.1 × 109/L)than the control group (median TLC 7.5 × 109/L, range 4 × 109/L-12.9 × 109/L) and the difference was statistically significant (P = 0.0001).

The case group was further divided into two groups with respect to TLC-group A (n = 114) (TLC <11.0 × 109/L) and Group B (n = 48) (TLC ≥11.0 × 109/L). The difference in MNV values between Group A and Group B (161.68 ± 7.89 vs. 164.16 ± 10.48) was not statistically significant (P = 0.67) [Table 4]. However, when compared with controls, both the groups were seen to have higher MNV values, and the differences were statistically significant (P = 0.0001). On analyzing groups for MNS values, both the groups had lower MNS values compared with controls. However, the difference was statistically significant for group A only (P = 0.0001).
Table 4: Correlation of VCS parameters with WBC count

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For analyzing the changes in VCS parameters with percent neutrophils, we further divided the cases into two groups with group 1 comprising neutrophil count <75% and group 2 with ≥75% neutrophil [Table 5]. The changes in MNV, MNS, and MNC of both groups when compared with controls showed statistically significant difference. However, the difference between two groups was not statistically significant.
Table 5: Correlation of VCS parameters with percentage of neutrophils

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Determination of cut-off point for MNV to predict culture positivity: It was observed that taking the cut-off of MNV as 149 gave a sensitivity of 91.4% and specificity of 88.7% with area under the curve (AUC) of 0.96. To compare, the cut-off of WBC count of 9.0 × 109/L gave a sensitivity of 66.7% and specificity of 66.7% with AUC of 0.74 [Table 6]. Receiver-operating characteristic curve analysis also showed that the mean neutrophilic volume had larger area under curve when compared with total leucocyte count. [Figure 1]
Table 6: MNV and TLC as predictor of sepsis

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Figure 1: Receiver-operating characteristic curve analysis showing that mean neutrophilic volume had larger area under curve when compared with total leucocyte count

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On analyzing MNV values with respect to Gram-positive and Gram-negative cases no difference was observed (P = 0.8260).


   Discussion Top


For many years, the WBC count, absolute neutrophil count, percentage of neutrophils, and especially increases in bands and immature neutrophils have been used to predict bacterial infection.[7],[8]

Although morphologic changes in neutrophils or the changes in plasma/nuclear ratio in lymphocytes provides important diagnostic information, but such assessment by manual examinations is costly, labor-intensive and subjective and is based on limited number of cells observed in peripheral smear. The advent of automated hematology analyzers changed the laboratory practice with this technique being capable of rapidly assessing large numbers of cells (more than 8000 in few seconds) to provide comprehensive hematology profiles completely removing subjective factors from the data.[15]

In the present study with the VCS technology, we demonstrated that the morphologic changes seen in left shifted neutrophils during acute bacterial infection proven by positive blood culture analysis could be measured quantitatively objectively on large number of cells without additional specimen requirements.

Clinical application of VCS parameters offers several advantages. These are the parameters generated objectively during differential analysis without additional specimen requirements. They are quantitative, more objective, and more reproducible than manual differential counts because more than 8000 WBCs are evaluated automatically.[11]

The present study, which included 162 septic patients with positive blood cultures and 40 healthy controls, evaluated VCS parameters of neutrophils in the early diagnosis of sepsis and sought to establish the cut-off levels, sensitivity and specificity for useful parameter.

The mean channel of neutrophil volume and MNS were found to be elevated significantly in culture-positive patients. Higher MNV values indicate the reactive increase in the size of neutrophils in the cases, whereas higher MNS values indicate a shift to the left with increased bands and immature granulocytes that are hypolobated and relatively simple nuclear characteristics. MNV was increased irrespective of the WBC count or percentage neutrophil.

The difference in MNV values between Group A and Group B (groups based on TLC) was not statistically significant (P = 0.67) [Table 4]. However, when compared with controls, both the groups were seen to have higher MNV values and the differences were statistically significant (P = 0.0001). Furthermore, we observed that neutrophil size variability suggested by SD was increased significantly in samples from culture positive compared with those in control samples. However, no significant changes were observed between the two groups indicating an increase in neutrophil size was independent of increase in patients TLC.

The changes in MNV, MNS, and MNC of two groups (those with neutrophil count <75% and other with ≥75% neutrophil) when compared with controls showed statistically significant difference. However, the difference between two groups was not statistically significant indicating that the morphological characteristics of neutrophils have altered in bacterial sepsis in terms of size, granularity, and nucleus irrespective of the degree of neutrophilia.

This has a special significance in view of the fact that only a few culture positive cases had TLC of more than 11.0 × 109/L or neutrophil count ≥75%. And these cases could have been missed by the counts. We have taken a cut-off of 149 for calculating sensitivity and specificity so that a reasonably high value of sensitivity and specificity (sensitivity of 91.4% and specificity of 88.7% respectively) could be obtained. These findings suggest that the MNV could be a more sensitive yet independent reliable indicator of acute bacterial infection.

In the past, various studies have demonstrated VCS parameters as reliable markers of sepsis. Chaves et al.[10] studied VCS parameters in 69 culture positive cases and concluded that cut-off of MNV as 150 gives a sensitivity of 70% and specificity of 91%, respectively. MNV of control was 143 ± 4.8. In another study done by Celik et al.[12] in 76 culture positive neonates, cut-off of 148.4 ± 11 gave a sensitivity 79% and specificity of 82% with AUC 85%. They also studied interleukin-6 (IL-6) and C-reactive protein (CRP) as a surrogate marker in the prediction of sepsis. It must be noted that study population is different in terms of demography and thus cut-off cannot be applicable to all.

Mardi et al. studied the diagnostic significance of MNV and mean monocytic volume (MMV) compared with IL-6, CRP and WBC count for predicting sepsis and observed a significant increase in MNV and MMV in the sepsis group compared with limited infections and controls. Further, they found that MNV at a cut-off of 150 had a comparable sensitivity and specificity was the most predictive VCS parameter.[15]

One needs to establish the research population data by a more robust prospective cohort study. Our present study could be limited by the fact that we could not follow-up the patients in the due treatment course. However, we have initiated this effort in our laboratory to follow-up the patients VCS parameters after the present encouraging results.


   Conclusion Top


Volume, conductivity and scatter parameters are important diagnostic tools, which can serve as an adjunct to the clinical diagnosis of bacterial sepsis and can help in the early institution of empirical antibiotic therapy.

Financial support and sponsorship

This study was funded by Intramural Grant, All India Institute of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Wenzel RP, Edmond MB. The impact of hospital-acquired bloodstream infections. Emerg Infect Dis 2001;7:174-7.  Back to cited text no. 1
    
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Todi S. Sepsis: New horizons. Indian J Crit Care Med 2010;14:1-2.  Back to cited text no. 2
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Ntusi N, Aubin L, Oliver S, Whitelaw A, Mendelson M. Guideline for the optimal use of blood cultures. S Afr Med J 2010;100:839-43.  Back to cited text no. 3
    
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Kaufman D, Fairchild KD. Clinical microbiology of bacterial and fungal sepsis in very-low-birth-weight infants. Clin Microbiol Rev 2004;17:638-80.  Back to cited text no. 4
    
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Wile MJ, Homer LD, Gaehler S, Phillips S, Millan J. Manual differential cell counts help predict bacterial infection. A multivariate analysis. Am J Clin Pathol 2001;115:644-9.  Back to cited text no. 5
    
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Osrin D, Vergnano S, Costello A. Serious bacterial infections in newborn infants in developing countries. Curr Opin Infect Dis 2004;17:217-24.  Back to cited text no. 6
    
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Wenz B, Gennis P, Canova C, Burns ER. The clinical utility of the leukocyte differential in emergency medicine. Am J Clin Pathol 1986;86:298-303.  Back to cited text no. 7
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Mathy KA, Koepke JA. The clinical usefulness of segmented vs. stab neutrophil criteria for differential leukocyte counts. Am J Clin Pathol 1974;61:947-58.  Back to cited text no. 8
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Krause JR. Automated differentials in the hematology laboratory. Am J Clin Pathol 1990;93 4 Suppl 1:S11-6.  Back to cited text no. 9
    
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Chaves F, Tierno B, Xu D. Quantitative determination of neutrophil VCS parameters by the Coulter automated hematology analyzer: new and reliable indicators for acute bacterial infection. Am J Clin Pathol 2005;124:440-4.  Back to cited text no. 10
    
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Richardson-Jones A. An automated hematology instrument for comprehensive WBC, RBC, and platelet analysis. Am Clin Lab 1990;9:18-22.  Back to cited text no. 11
    
12.
Celik IH, Demirel G, Sukhachev D, Erdeve O, Dilmen U. Neutrophil volume, conductivity and scatter parameters with effective modeling of molecular activity statistical program gives better results in neonatal sepsis. Int J Lab Hematol 2013;35:82-7.  Back to cited text no. 12
    
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Collee JG, Marr W. Culture of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th ed. New York: Churchill Livingstone; 1996. p. 113-29.  Back to cited text no. 14
    
15.
Mardi D, Fwity B, Lobmann R, Ambrosch A. Mean cell volume of neutrophils and monocytes compared with C-reactive protein, interleukin-6 and white blood cell count for prediction of sepsis and nonsystemic bacterial infections. Int J Lab Hematol 2010;32:410-8.  Back to cited text no. 15
    

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Correspondence Address:
Ashok K Mukhopadhyay
Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.168853

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    Figures

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    Tables

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

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