histogram made easy

1. INTERPRETATION OF HISTOGRAMS

2. PRINCIPLE OF CELL COUNTER
Electronic impedence
• Wallace coulter (1956)
• Passage of cell displaces the volume of diluent and this increases
resistance
• Impulse are generated due to difference in potential at two
electrodes which is read at oscilloscope
• Number of impulse indicate number of cells and height indicates
volume of cells

3. Optical light scatter
• A diluent suspension flows through a aperture so that the cells
pass in a single file in front of light source
• Light is scattered by the cells passing through the light beam
• The amount of light scatter is detected by phomultiplier and
photodiodes
• Electrical impulse are generated for counts

4. • Peroxidase based cell counter
• Fluorscence based cell counters
• Immunological based cell counters

5. HISTOGRAMS
• These are the graphical representation of neumerical data of
different cell population on cell counter
• Y axis represents the number of cells and X axis represents the
cell size

6. • Platelets have
volume b/w 8-12 fl
and counted b/w 2
to 25 fl.
• RBC have volume
80-100 fl and are
counted b/w 25 to
250 fl.

7. Normal RBC histogram
• Normal RBC distribution curve is Gaussian bell shaped curve
• Peak of curve should fall within the normal MCV range of 80-100 fl
• MCV is perpendicular line from peak of the curve to base
• There are two flexible discriminators LD (25-75 fl) and UD (200-
250fl)

8. The distribution should always starts and ends on base line and
should be located between the two discriminators

9. RED CELL FLAGS
RL flag
• When lower discriminator exceeds the preset height by 10 %
• RBC count, HCT, MCV, MCH and MCHC show RL flag.

11. Possible causes of RL flag
• Giant platelets
• Microerythrocytes
• Fragmented RBCs
• Platelet clumps
• In case of fragmented RBC and extreme microerythrocytosis the
there is no clear separation in volume between platelets and
erythrocytes. Due to high numbers of RBC the platelet result
might be false high and should be checked with alternative
methods.

12. RU flag
• Flag is seen when UD exceeds the preset height by
greater than 5 %.

14. Possible causes of RU flag
• Cold agglutination
• RBC aggluatination
• Rouleax formation
• RBC agglutination might cause a low incorrect RBC count
and effect also the parameter Hct, MCV, MCH and MCHC. In
case of cold agglutinates warm the sample up to 37°C.
(MCHC should trop back to normal value if the problem is
solved)

15. Multiple peaks (MP)
• RDW SD shows the MP flag

16. Possible causes
• Iron defiecieny anemia in recovery
• Post transfusion
• Extreme leucocytosis

17. Thrombocyte histogram
• The histogram curve should lay within the lower and upper platelet discriminator
(PL & PU) and start and end on the base line.
• PLT counted between 2 fl and 30 fl.
1 flexible Discriminator PL 2 to 6 fl.
1 flexible Discriminator PU 12-30 fl.
1 fixed Discriminator at 12 fl

18. Parameters of platelet histogram
• MPV ( 8 - 12 fl)
• P-LCR - ratio of large platelets Reference range 15 - 35 %
• PDW – Platelet distribution width curve (9-14 fl)

19. PL flag
• When lower discriminator exceeds preset height by
10%
• Platelet count, P-LCR and MPV will show PL flag

20. Possible causes
• High blank value
• Cell fragments
• High numbers of bacteria
• Contaminated reagent
• Platelet aggregation

21. • In case of high background numbers (blank), check reagent for contamination
(bacteria). Check expiry date.
• In order the background check is within range, the patient sample should be
checked – platelet results might be incorrect high due to cell fragments or
bacteria's.
• In some cases platelet aggregates might cause the problem. In this case the
histogram curve would also show an abnormal distribution at the upper
discriminator. Platelet aggregation might cause low incorrect platelet results.

22. PU flag
• This occurs when UD exceeds the preset height by more than
40%

23. Possible causes
• PLT clumps
EDTA-incombatibility
Clotted sample
• Giant Platelets
• Microerythrocytes
• Fragmentocytes or dysplastic RBC
In case of platelet aggregation, the PLT count is incorrect low.
Check EDTA incombatibility –e.g. re-collect the sample and use citrate as anticoagulance to
avoid clocking caused by EDTA.
In case of extreme microerythrocytes or fragmented RBC the PLT count might be incorrect
high. PLT results should be confirmed with alternative methods

24. Multiple peaks (MP)

25. WBC histogram

26. • Lower discriminator in this fluctuates between 30 -60 fl
• Upper discrminator is fixed at 300 fl
• The number of cells between LD and UD is WBC count

27. • WBC histogram consists of two troughs, valley discriminators,
T1 (78-114 fl) and T2 (<150 fl)
• Peak between LD and T1 represents small cells i.e. lymphocytes
• Peak between T1 and T2 includes eosinophils, monocytes,
blasts, promyelocytes, myelocytes and metamyelocytes
• Peak after T2 represents neutrophils

29. WL flag
• Deviation of base line at lower discriminator

30. Possible causes
• Lyse resistant RBC
• PLT Clumps
EDTA-incombatibility
coagulated sample
• Erythroblasts (NRBC)
• Cold agglutinates

31. AG flag
• Abnormal curve in front of lower discriminator
Large platelet clumps (> 30fl) are
detected in the area before the lower
WBC discriminator. Due to their
enhanced size they may not affect the
PLT histogram curve. If “AG” mark will
be generated, the sample should be
checked for platelet clumps (e.g.
microscopic slide review).

32. WU flag
• Deviation on upper discriminator curve
Possible cause:
• extreme leukocytosis
• rare: WBC aggregation
The histogram curve does not match the
basis line at upper discriminator due to
high numbers of large particles (WBC
aggregation) or if the linearity of the white
blood cell count exceeds the limit. (WBC>
100 x 10³/µl)
Pre-dilution (e.g. 1:5) of the sample might
help to obtain correct results.

33. • T1 and T2 flags when discrimination between various cell
population cannot be done due to presence of abnormal
leucocytes

34. • F1, F2, F3 flags

39. RBC - 05.18

HB - 15.02 
HCT - 45.08

MCV - 88.07

RDW - 16.03

RBC - 03.19 
HB - 06.40 
HCT - 20.05 
MCV - 64.04 
RDW - 18.00 

46. • THANKS