2.4. WBC histograms and DLC scatterplots

The WBC histograms and the differential leucocyte count scatterplots were analysed for five different infections and compared with healthy control group. In a WBC histogram, there are 2 discriminators, the first being lower discriminator (LD) towards 35fL and upper discriminator (UD) towards 450fL and the cells falling between these two discriminators are the WBCs. There are two troughs, valley discriminators, T1 (78‐114fL) and T2 (less than 150fL). The area between LD and T1 has lymphocytes (F1‐ 35 to 90fL); between T1 and T2 has monocytes, basophils and eosinophils along with immature granulocytes—blasts, promyelocytes, myelocytes and metamyelocytes (F2‐ 90 to 160fL) and area between T2 and UD has neutrophils (F3‐ 160 to 450fL) [F stands for fraction; Figure 1A]. 13 , 14 The DLC scatterplots in healthy controls is explained in Figures 2A, ​,3A3A and ​and3B3B.

WBC Histogram: A: Healthy control: LD and UD discriminators at 35fL and 450fL; first imaginary discriminator T1 at 78‐114fL and second imaginary discriminator T2 at 150fL. F1 peak (between LD and T1) consists of lymphocytes, F2 (between T1 and T2) represents monocytes, eosinophils, basophils and immature granulocytes and F3 (between T1 and LD) represents mature neutrophils. B: Malaria: Presence of pre‐threshold peak at LD (35fL) representing parasitized un‐lysed RBCs that enters WBC chamber is highly characteristic (arrow). There is a shift to right of T1 and obliteration of T2. C: Dengue: There is shift to right of T1 and T2 with widening of F1 peak. D: Typhoid: The shift to right of T1 is mild. The WBC histogram is almost comparable to that seen in healthy control. Similar histograms were noted in leptospirosis and rickettsia

WBC differential count scatterplot: A: Healthy control scatterplot with separate windows for lymphocytes (blue), monocytes (green), neutrophils (purple) and eosinophils (orange). Note the windows where blasts, variant lymphocytes, immature neutrophils and un‐lysed RBCs can fall. B: Malaria: Lymphocytes plots extend into variant lymphocyte window and monocyte plots extend into blast window. Note the obliteration of clear demarcation between the windows of lymphocytes, neutrophils and monocytes. C: Dengue: Lymphocytes plots are seen extending into variant lymphocyte window and monocyte plots into blast window. Leucopenia was noted in this patient. D: Typhoid fever: The WBC differential count scatterplot was comparable to that seen in healthy control. Similar scatterplots were noted in leptospirosis and rickettsia

Nucleated RBC plot: A (2D plot), B (3D plot): Healthy control: Axial light loss is plotted on x‐axis and low‐angle light scatter on y‐axis. Measurement of AL2 separates the leucocytes from nucleated red blood cells. Nucleated RBC plot can be used in discrimination of leucocytes (blue) from presence of NRBC (red), platelet (green) clumps, giant platelets and parasitized un‐lysed RBCs. C,D: Malaria: The white arrow (C) and red arrow (D) shows tight green cluster representing parasitized un‐lysed RBCs. E,F: In dengue fever, leptospirosis, typhoid fever and rickettsia, nucleated RBC plot was comparable to that seen in healthy control