- The document discusses histograms generated by cell counters that graphically represent cell population data. It focuses on histograms for red blood cells (RBCs), white blood cells (WBCs), and platelets. - Key parameters for each type of histogram are defined, such as how cells are counted and measured, normal ranges, and flags that indicate potential issues. Common causes for shifts or abnormalities in the histograms are also outlined. - The information provides guidance on interpreting cell counter histograms and histograms to help evaluate a patient's blood cell counts and detect possible blood disorders or interferences.

1. Histogram

2. TWO SIDES OF INSTRUMENTS
(THREE PART INSTRUMENTS)
RBC SIDE (Dilu-
1:50,000)
WBC SIDE
(Dilu-1:500)
>30 fl size2-30 fl size
RBCPLATELET
35-90 fl 90-160 fl 160-450 fl
Lymphocytes Mononuclear cells
Monocytes
Eosinophils
Basophils
Granulocytes
Neutrophils

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

6. Normal RBC histogram
• 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)

7. • If RBCs are larger than normal  shift to right
• If RBCs are smaller than normal  shift to left

9. • Although the size ranges for RBC histograms are between 24 fL and
360 fL, the instrument counts only those cells with volume sizes
between 36 fL and 360 fL as red cells.
• Those cells counted in the 24 fL to 36 fL range are rejected and not
included in the RBC count.
• They are enumerated and displayed in the histogram area between
the 24 fL and 36 fL

11. RDW

13. RDW- SD

16. RDW SD
• The RDW-SD is the arithmetic width of the distribution curve
measured at the 20% frequency curve.
• The normal RDW-SD is 39 to 47 fL

17. RDW CV
• ratio of 1 standard deviation (SD) to the MCV.
• Since it is a ratio, changes in either the SD or MCV will influence the
results.
• Microcytosis will tend to elevate the RDW-CV simply by decreasing
the denominator (MCV) of the ratio.
• As a result, the RDW is probably a more sensitive parameter in
microcytic than in macrocytic red cell disorders.
• Conversely, macrocytosis, by increasing the denominator, may offset
the change in the SD and reduce the RDW-CV.

20. IDA and beta thalassemia trait
• although their histograms are similar, the degree of anisocytosis, as
measured by the RDW, differentiates them.
• Iron deficiency anemia is characterized by elevated RDW, reflecting
the heterogeneity in the acquired erythrocyte populations.
• In thal trait however, the RDW is usually within range due to the
homogeneity of the inherited population of erythrocytes.

21. RED CELL FLAGS
• RL flag
• When lower discriminator exceeds the preset height by 10 %
• RBC count, HCT, MCV, MCH and MCHC show RL flag.
• RU flag
• Flag is seen when UD exceeds the preset height by greater than 5 %.

22. • Normally, the space below 36 fL remains clear, but in certain conditions the
histogram may begin above the baseline or show a high takeoff on the far
left of the curve which generally indicates the presence of small particles.
• red cell fragments,
• microspherocytes,
• nucleated RBCs,
• nonlyzed RBCs,
• elliptocytosis,
• macrothrombocytes,
• platelet clumps,
• bacteria, parasitic organisms
• interfering substances such as cryoglobulin, cold agglutinin, and macroglobinemia

24. Erythrocyte histogram Flagging
• Possible causes:
• Cold agglutination
• RBC agglutination
• Rouleux formation

25. • Incase of cold agglutinates warm the sample up to 37°C (room
temperature)
• (MCHC should drop back to normal value if the problem issolved)

27. multiple peak of RBC histogram
• MP
• Possible causes:
• Iron deficiency in recovery
(therapy)
• Dimorphic picture
• Multiple RBC transfusions
• Extreme leukocytosis (> 600 x
10³/µl)

29. Shift to left
• In IDA and beta thalassemia (thal) trait, the
red cell distribution curves are shifted to
the left, and the percentage of microcytosis
is increased

31. WB histogram

34. • 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

38. In the WBC histogram the dotted curve before the lymphocyte
peak indicates nucleated RBC. Nucleated RBCs (normoblasts) will
be counted as WBC in the haemanalyser

39. there are two peaks. The second peak in the RBC distribution (with higher
corpuscular volume) curve indicates reticulocytes. Presence of significant
number of nucleated RBCs and reticulocytes are highly suggestive of
haemolytic anaemia.

40. Detection of malarial parasites
• A spurious increase in the mixed
cell population can be an
indicator of the presence of
malaria parasites in the red blood
cells .
• This occurs because the parasite
infected RBCs cannot be lysed by
the stromatolyser solution and
will enter the WBC counting
block.
• In a study, using Sysmex XS-800i
analyser, a spurious increase in
the mixed cell population was
moderately sensitive and highly
specific in diagnosing malaria

41. Flag “ WL “, curve does not start at the base
line
Possible causes:
• PLT Clumps
EDTA-Incombatibility
Coagulated Sample
• high osmotic resistance (Erythrocytes not lysed)
• Erythroblasts
• Cold agglutinins

42. Flag WU
• curve does not end at the base line.

43. Platelet histogram

44. • Platelet histogram starts and ends on base line

45. • Platelet are counted between 2 t 20 fL
• Particles interfering at 2 fL
• EDTA
• Dust
• Particles interfering at 20 fL
• RBC and WBC fragments

46. • Platelet discriminator
• Lower discriminator (LD)
• 2-6fL
• Upper discriminator (UD)
• 12 – 30 fL
• Fixed discriminator at 12 fL

49. Mean platelet volume
(MPV )
• calculated measure of platelet volume expressed in femtoliter (fL).
• Normal range of MPV is reported to be between 7.2 and 11.7 fL.
• MPV is inversely related to platelet counts. When marrow depression is the
cause of thrombocytopenia, a rising trend in MPV heralds platelet recovery,
and platelet transfusions may be put on hold
• increased MPV
• hypoproduction of platelets, immature platelets are activated and increase in size by
pseudopod formation
• increased MPV can be used as a marker of production rate and platelet activation.
• High MPV with ongoing thrombocytopenia represents peripheral destruction.
• Low MPV indicates underproduction/bone marrow suppression.

50. • MPV is calculated by the formula,
• MPV (fL) = ([PCT (%)/platelet count (×10^9/l)]) × 10^5 .

52. Plateletcrit
• volume of platelets expressed as a percentage of total blood volume
• The normal range for PCT is 0.22%–0.24%.
• PCT parallels the platelet count.
• PCT = platelet count × MPV/10,000.

53. Platelet larger cell ratio
• indicator of circulating larger platelets (>12 fL), expressed as percentage.
• The normal percentage range is 15%–35%.
• It has also been used to monitor platelet activity.
• Platelet larger cell ratio (P-LCR)
• inversely related to platelet count
• directly related to PDW and MPV.
• decreased in patients with thrombocytosis
• increased in thrombocytopenia.
• Further, studies have shown that P-LCR was significantly decreased in
reactive thrombocytosis compared to neoplastic thrombocytosis. A greater
increase in P-LCR is seen in destructive thrombocytopenia than those with
hypoproliferative thrombocytopenia

54. Immature platelet fraction
• IPF indicates the percentage of immature platelets containing higher
concentration of RNA released into the circulation
• normal 1.1% and 6.1% in healthy individuals
• An increased IPF is seen as production of platelets increases and low
IPF indicate suppressed thrombopoiesis

55. Platelet distribution width
• measures variability in platelet size, changes with platelet activation,
and reflects the heterogeneity in platelet morphology.
• indicator of platelet anisocytosis.
• PDW is increased in the presence of platelet anisocytosis.
• The PDW reported varies markedly, with reference intervals ranging
from 8.3% to 56.6%.
• There is a direct relation between MPV and PDW, that is, a high PDW
is associated with a high MPV

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

59. • 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

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

63. • shows a significant number of cells with size more than 10 fL.
• Immune Thrombocytopenic Purpura (ITP) is a condition where
there is accelerated platelet destruction by autoantibodies,
with a compensatory increase in platelet production, hence
circulating platelets in patients with ITP are younger and have
larger size

64. Mechanism of thrombocytosis in iron
deficiency anaemia:
• In iron deficiency anaemia
• microcytes with size less than 30 fL will be counted as platelets. Even a small
percentage of RBCs when falsely counted as platelets can significantly affect
the platelet count as RBC count is in millions and platelet count is in lacs
• EPO increases platelet counts by stimulating of thrombopoesis

65. Interference caused by fragments