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Automation in hematology Part (2)

Automation in hematology Part (2) 


- Graphical description of numerical data of different cell populations in a cell counter. 

- x-axis => cell size 

- y-axis => no: of cells 

- Gives information on: 

▪Average size 

▪Distribution of size 

Discrimination thresholds 

- Moving or fixed discriminator differentiate the distribution curve for the volume 

- WBC Discriminator 

▪The automated counter sets an LD fluctuating between 30-60 fl & a UD fixed at 300 fl. 

▪WBC is calculated from particle counts > than this LD 

- RBC Discriminator 

▪Has two flexible discriminators LD (25-75 fl) & UD (200-250 fl). 
▪RBC is calculated from particle counts between this LD & UD.
- Platelet Discriminator
▪Three discriminators – LD (2-6 fl), UD (12-30 fl) & a fixed discriminator (8-12 fl) 

Evaluation of RBC Count & MCV – RBC Histogram 

▪The normal RBC distribution curve is a Gaussian bell-shaped curve. 
▪The analyzer counts RBCs as those which range from 36-360 fl. 
▪MCV is a perpendicular line from the peak of the curve to the base.
▪The peak of the curve should fall within the normal MCV range of 80-100 fl.
▪There are 2 flexible discriminators – LD (25-75 fl) & UD (200-250 fl)

Abnormalities of RBC Histogram 

▪A Left shift of the curve in microcytosis. 

▪A right shift of the curve in macrocytosis.
▪The bimodal peak of the curve in the dimorphic population of cells.

Estimation of Hematocrit, MCH & MCHC 

▪Hematocrit (%) = Mean cell volume (fl)/ Red cell count (106/μl) 

▪Mean cell hemoglobin (pg)= Hemoglobin (g/L)/ Red cell count (106/μl) 

▪Mean cell hemoglobin concentration (g/dL) =  Hemoglobin (g/L)/ Hematocrit (%) 

Estimation of RDW 

▪RDW is expressed as a coefficient of variation of RBC size distribution. 
▪RDW- CV is a better indicator of anisocytosis than RDW-SD. 

▪RBC distribution curve ll get wider as RBC vary more in size. 

▪Normal Range – RDW-CV – 11.0-15.0% RDW-SD – 40.0 - 55.0 fL






- Iron Deficiency

- HbH Disease

- S/Beta Thalassemia

- HbAC


- Severe anemia of chronic disease.

- Early Iron Deficiency

- Early B12/ Folate deficiency


- Sickle/C disease

- B12/ Folate deficiency

- Immune hemolytic anemia

- Cold Agglutinins



- Thalassemia Trait

- Anemia of chronic disorders

- Hereditary spherocytosis

-Sickle cell trait

- Normal

- Myelodysplasia

- Aplastic anemia

Estimation of WBC Count

Hematology analyzers can generate a: 

- 3-part differential count – lymphocytes, monocytes & granulocytes based on the principle of electrical impedance 


- 5-part differential count - lymphocytes, monocytes, neutrophils, eosinophils & basophils. 

based on different principles  

▪light scatter 
▪electrical impedance 

▪electrical conductivity
▪peroxidase staining 

Estimation of WBC Count – WBC Histogram 

- Cells greater than 35 fl are counted as WBCs in the WBC/Hb chamber. 

- Cells with volume 35-90 fl à Lymphocytes. 

- Cells with volume 90-160 fl à Mononuclear cells. 

- Cells with volume 160-450 fl à Neutrophils. 

Abnormalities of WBC Histogram 

- Peak to the left of lymphocyte peak – nucleated cells. 

- Peak between lymphocytes & monocytes – blast cells, eosinophilia, basophilia, plasma cells & atypical lymphocytes. 

- Peak between monocytes & neutrophils – left shift 

Platelet Histogram 

- PLT size: 8-12 fL 

- PLT detection: between 2 and 30 fL 

- Fixed discriminator at 12 fL 

Estimation of Platelet Count 

- Platelets are counted by the electrical impedance method in the RBC aperture. 

- Particles more than 2 fl and less than 20 fl are classified as platelets by the analyzer. 

- MPV is a measurement of the mean size of platelets found in blood Normal MPV is 7-10 fl. 

- Increased MPV ( > 10 fl) d/t destruction of platelets in circulation. 

- Decreased MPV ( < 7 fl) d/t decreased production of platelets in circulation. 

- Plateletocrit (PCT) is a volume of circulating platelets in a unit volume of blood. Normal PCT is 0.19-0.36%. 

- PCT ↑ in thrombocytosis and ↓ thrombocytopenia. 

- PDW is a measure of platelet size variation. Standard PDW ranges from 9 to 14 fL 

- ↑ PDW is observed in megaloblastic anemia, CML & after chemotherapy.


- Flags are signals that happen when an abnormal result is detected by the automated blood analyzer. 

- Flags are showed by certain ‘asteriks’ on the report. 

- They minimize the False +ve & False –ve results by mandating the results of blood smear examination. 

RBC Flags

RL Flag 

- Seen when LD greater than preset height by 10 % 

- Shown by RBC Count, HCT, MCV, MCH, and MCHC 

- This occurs when there is platelet aggregation or RBC fragments.
RU Flag 
- Seen when UD greater than preset height by 10%.
- Shown by RBC Count,HCT, MCV,MCH,MCHC
- Occurs when there are Cold agglutinins 


- Shown by RDW-SD.
- Seen in Post Blood transfusion and Treated Fe deficiency anemia. 

WBC Flags 

WL -flag 

- Generated when the curve deviates from the baseline on the LD. 

- Various causes for this are 

▪ Platelet aggregates (clotted sample, EDTA incompatibility) 

▪ Lyse-resistant RBCs. 

▪ Erythroblasts. 

▪ Cryoagglutinates. 

▪ Giant platelets. 

WU Flag 

- Generated when there is a deviation of the curve on the UD or if it does not end at the baseline.
- Caused by hyperleukocytosis. 
T1 & T2 Flags 

- The peak between T1-T2: The middle cell count - Eosinophils, Monocytes, Blasts, promyelocytes, myelocytes, and metamyelocytes. 

- The peak between LD-T1: Lymphocytes 

- The peak between T2-UD: Neutrophils. 

- T1 & T2 flags appear when is not possible to differentiate between lymphocytes, middle cells & neutrophils which happens in the presence of abnormal/higher leucocyte counts as in Chronic Myeloid Leukemia. 
F1, F2, F3 Flags 

- Sometimes, the cell Fractions may be mixed. 

- F1 & F2 or F2 or F3 combine into each other over large areas. 

- F1 (small cell inaccurate) flag: Acute Lymphocytic Leukemia 

- F2 (middle cell inexact) flag: eosinophil, Acute Myeloid Leukemia, monocytosis, etc 

- F3 (large cell inaccurate) flag: height of T2 greater than the limit of 50%. 

Platelet Flags 

PL Flag 

- This happens when the LD> the preset height by 10% 

- Shown by Platelet count, MPV, P-LCR
- Occurs due to noise. 

PU Flag 
- This happens when the UD> the preset height by > 40%. 

- This occurs in Hemolytic anemias with fragmented cells and Large platelets.