Supplementary Material- Calculating the Delta Neutrophil Index (DNI)

Delta Neutrophil Index (DNI) as a novel diagnostic and prognostic marker

of infection: a systematic review and meta-analysis

Jae Hyon Park1*, Hyeong Ju Byeon 1*, Geum Hwa Lee2, Jong Wook Lee3,4, Andreas

Kronbichler5, Michael Eisenhut6, and Jae Il Shin2

1. Yonsei University College of Medicine, Seoul, Korea 2. Department of Pediatrics, Yonsei University College of Medicine, Seoul, Korea 3. Jincheon Sungmo Hospital, Jincheon, Korea 4. Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea 5. Department of Internal Medicine IV (Nephrology and Hypertension), Medical University Innsbruck, Innsbruck, Austria 6. Department of Pediatrics, Luton & Dunstable University Hospital NHS Foundation Trust, Luton, United Kingdom

Note: All reference numbers refer to the reference list in the main manuscript text.

*These authors contributed equally to this work An automated hematology analyzer in general automatically performs “cell-counts”. Several basic parameters such as haemoglobin, RBC, platelet count, red cell indices, RDW and WBC with 3 part differential can be retrieved using an automated hematology analyzer. Herein, we used a high volume hematology analyzer ADVIA 120 (Siemens Healthcare Diagnostics, Bayer, USA) to show an example of how to calculate the DNI value (%).

DNI percentage can be calculated using the following equation.

Delta Neutrophil Index (DNI) Equation:

DNI (%) = (% neutrophil + % eosinophil) - % polymorphonuclear (PMN) cells

*The name “delta” here () signifies the “difference” between these different measures.

In a high volume hematology analyzer such as ADVIA 120 (Siemens Healthcare Diagnostics, Bayer, USA), there exist (1) the peroxidase channel and (2) the basophil lobularity channel shown as the following:

Peroxidase Channel Basophil Lobularity Channel

X-axis: peroxidase activity; X-axis: lobularity/nuclear density; Y-axis: cell volume; Y-axis: cell size Purple: Neutrophils Yellow: Basophils Yellow: Eosinophils Purple: Polymorphonuclear cells Green: Monocytes Blue: Mononuclear cells (including lymophocytes) Blue: Lymphocytes and Basophils White (left-side): Blast cells Turquoise: Largely Unstained Cells (LUC)

In the peroxidase channel, cells are stained by peroxidase reagent and analyzed for size and peroxidase stain intensity. The peroxidase channel stains cells with peroxidase granules and as can be seen on the right side of the x-axis in the peroxidase channel: eosinophils are strongly stained and neutrophils receive medium staining; monocytes are weakly stained; lymphocytes and basophils are not stained. Generally, specific lysis reagents are used to separate basophils from all other white blood cells and thus by subtracting basophils from the lymphocyte and basophil cluster, the lymphocytes are calculated by the peroxidase channel. Since it is difficult to count basophil cells using the peroxidase channel, basophil lobularity channel is separately used.

The % of neutrophils and % of eosinophils calculated by the peroxidase channel can be seen in the routine WBC differential window (left) where in the example, the % of neutrophil is 85.3% and the % of eosinophil is 2.2%. (Additional note: notice that the WBC count in the example is 8240 cells/L which is within the normal range of 4500 to 11000 cells/L. Thus, in this example, the total WBC count itself is unable to predict infectious status. Only with additional information such as neutrophil differential % > 75% can infection be speculated without the calculation of DNI)

In the basophil lobularity channel, surfactant and phthalic acid are added to lyse the RBC and strip away the cytoplasmic membrane from all leukocytes except basophils. Only basophils with retained cytoplasm remain and are identified. A high-angle detector is then used to analyze the shape and structure (lobularity and density) of the remaining nuclei. More importantly, in the basophil lobularity channel, we can also observe the number of polymorphonuclear cells colored purple and mononuclear cells colored blue.

The % of polymorphonuclear cell is calculated by the basophil lobularity channel can be seen in the additional routine parameter window, where the % polymorphonuclear cell (PMN) is estimated to be 61.6% in the example.

Using the following equation:

DNI = (% neutrophil + % eosinophil) - % polymorphonuclear (PMN) cells

DNI = (85.3% + 2.2%) – 61.6% = 25.9% = (% of band neutrophil, promyelocytes, myelocytes, and metamyelocytes)

Generally, in a healthy individual, the sum of neutrophil and eosinophil is almost equivalent to the number of PMN cells. However, with infection, due to the increase in the number of band neutrophils, meta-myelocytes, promyelocytes and myelocytes, all of which are one nuclear cells, the % of PMN cells recorded by the basophil lobularity channel decreases. Thus, the difference between the summed percentages of neutrophil and eosinophil recorded by the peroxidase channel and the percentage of PMN cells increases. In summary, with infection, the DNI parameter increases markedly.