Automation in hematology Part (2)
Histograms
- 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
RDW
|
|
MCV LOW |
MCV NORMAL |
MCV HIGH |
|
RDW HIGH |
- Iron Deficiency - HbH Disease - S/Beta Thalassemia - HbAC - MAHA - Severe anemia of chronic disease. |
- Early Iron Deficiency - Early B12/ Folate deficiency - SCA - Sickle/C disease |
- B12/ Folate deficiency - Immune hemolytic anemia - Cold Agglutinins -Alcoholism |
|
RDW NORMAL |
- 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
Or
- 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.
Flagging
- 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
MP-flag
- 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.
 Histograms - Graphical description of numerical data of different cell populations in a cell counter.…){kind=link}
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