The software autocalculated leukocyte roundness value (rv), which showed no difference between sepsis and control patients, with an overall roundness value of 2.2 ± 1.0 rv for septic patients, and, a roundness value of 2.2 ± 1.2 rv, P = 0.8 with an AUC = 0.51 (0.35–0.66) for noninfected controls (Table 2).
Magnetic properties represented by SD
We measured the median levitation height, which for sepsis patients was 293.1 ± 61.4 px and 288.8 ± 23.7 px for controls, P = 0.8, with an AUC = 0.52 (0.35–0.69), which proved insignificant (Fig. 3E). The cells’ levitation height varied more within a sample in sepsis patients compared with a noninfected control, likely due to alterations of density after granular content release, and also change in magnetic properties due to intracellular ROS generation. We next compared the SDs of the levitation heights of the samples, which was 71.5 ± 24.8 px for sepsis patients and 47.4 ± 16.0 px for controls, P = 0.0003, with an AUC = 0.80 (0.67; 0.93) (Table 2).
Correlation with complete blood cell and differential laboratory testing
To understand the relationship between leukocyte parameters quantified by magnetic levitation and parameters from complete blood cell testing, we assessed the correlation between these findings in the patients with infection (noninfected controls did not have these parameters available). There was incomplete correlation between mean cell area and neutrophil percentage (r = 0.32), lymphocyte percentage (r = −0.48), band percentage (r = 0.13), and white blood cell (WBC) count (r = 0.19) on the complete blood count (cbc) with differential. This was similar for the correlation between length and neutrophil percentage (r = 0.43), lymphocyte percentage (r = −0.54), band percentage (r = 0.07), and WBC count (r = 0.18), as well as width and neutrophil percentage (r = 0.38), lymphocyte percentage (r = −0.54), and band percentage (r = 0.07) and WBC count (r = 0.25). Further studies are needed to determine if magnetic levitation is either superior or provides new information compared with the cbc with differential.
Previous reports suggest that during sepsis, circulating pathogen and host chemotactic factors mobilize and activate leukocytes, promote increase in neutrophil size, and stimulate cell degranulation (14); however, these are not routinely assessed parameters. Other methods, such as Automated Hematology Analysis (AHA), have been introduced to measure morphology, such as size and granularity of cells, using VCS (volume, conductivity, and scatter) (15). Our findings are consistent with other studies using VCS to measure neutrophils from controls and septic patients, which all show septic patients had larger white cells than those of patients with localized infections and noninfected controls (15–17). Flow cytometry has also been used to determine the size of the cells and shows similar results for septic patients, patients with localized infection, and noninfected controls (18, 19). Furthermore, recent reports have shown a positive correlation between the size of activated neutrophil granulocytes and size increase in infected samples (20, 21).
During activation, previous observations suggest leukocyte plasma membranes have a shape that includes “pointed spikes” due to increased exocytosis and release of certain granular content (3). At the resolution used in our approach, we were unable to detect these differences, which is not unexpected given the resolution of the 20 × 0.50 objective of about 0.6 μm. The neutrophil granulocyte has several stages of activation, each of which has specific morphological parameters, so even though they increase in size, the “spikiness” might not parallel that pattern (21). Thus, although using our system, the roundness parameter was not found to be useful in distinguishing infected patients from noninfected.
When activated by bacterial by products or host anaphylatoxins, circulating neutrophils generate reactive oxygen species (ROS), and exocytose granular content (22). The highly paramagnetic ROS increases the magnetic signature of activated neutrophils, positioning the cells closer to the bottom magnet. Conversely, degranulation promotes a significant decrease in cell density process, which would position the neutrophils closer to the middistance between the two magnets. These concurrent yet opposing mechanisms explain the observed wide distribution range in the levitation height of neutrophils from septic patients compared with noninfected controls. Further investigation is required to better understand the dynamics of these conflicting mechanisms and their implications for sepsis screening and detection using magnetic levitation height.
Furthermore, we show promising diagnostic accuracy in a convenience sample, especially for the size parameters (area, length, and width) ranging from 0.89 to 0.92 AUC. For example, calculating the diagnostic operating characteristics with a cutoff of 23.3 px for width yielded a sensitivity of 91% and a specificity of 74% with a positive likelihood ratio of 3.5. If we alter the cutoff to 24.6 px the sensitivity decreases to 82%, but the specificity increases to 92%, with a positive likelihood ratio of 10.6. In comparison to AHA technology using VCS parameter, the sensitivity was 83% and specificity 78% (16). The challenge of AHA is that it requires an expensive piece of equipment, the UniCel DxH 800 Coulter system and highly trained technicians. As previously described, other technologies such as flow cytometry have proven useful (18, 19). However, these are more complex and time-consuming procedures. Furthermore, published diagnostic accuracies show variable results, most of which are less discriminatory compared with our preliminary findings (23, 24).
For this study, we measure all the leukocytes in a field from whole blood as we intend for the technology to ultimately be implemented at the bedside as a quick, inexpensive, and easy test. Even though the leukocytes mainly consist of neutrophils and lymphocytes, 60% and 30%, respectively, for a healthy individual (25), an alternative approach would be to include only subpopulations such as neutrophil granulocytes in our analyses, which will be applied in future studies.
There are a number of limitations to this investigation. First, as this was an initial investigation into the methodology, our sample size was small. Second, we enrolled a convenience sample population, which is prone to selection bias. For future studies, we will need to test the technology in an undifferentiated population to assess validity of the test. Third, we compared patients to noninfected control presumed to not be acutely ill; this may be different in clinical application to acutely ill patients when we are trying to decide on whether infection is the source of the illness. Fourth, we did not compare the leukocyte measurements to commonly used laboratory parameters such as a complete blood count with differential to determine if the new method is superior to existing technologies. Fifth, we performed an offline analysis. Although the bedside application is theoretically identical and will ultimately be automatic, this will need to be verified. Sixth, we do not know how comorbidities will impact our test. Finally, this was an observational trial, and future investigations on how the results of testing inform practice are needed to improve patient-oriented outcomes.
In this pilot investigation, we describe a novel methodology using magnetic levitation to separate leukocytes from circulating RBCs and assess leukocyte parameters. The technique demonstrated a promising diagnostic accuracy for identifying patients with an infection regarding size parameters (area, length, and width) especially. Infected patients had leukocytes that were larger in size and had a pattern consistent with more variable density and magnetic properties. Secondary analysis suggests that these parameters may also be associated with sepsis severity. This technique, which is potentially inexpensive, portable, and implementable at the bedside, shows promise as a novel screening and diagnostic test for infection.
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Keywords:© 2019 by the Shock Society
Leukocytes; magnetic fields; magnets; point-of-care systems; sepsis; systemic inflammatory response syndrome