WBC Composite Histogram

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INTERPRETATION OF HISTOGRAM

Histograms are graphic representation of cell frequencies verses size.Histogram provide information about erythrocytes ,leukocytes and platelet frequency and distribution as well as presence of subpopulation.Shift in one direction or another can be of diagnostic importance.

Produced from thousands/millions of signals generated by the cells passing through detector where they are differentiated by:

Their sizeFrequency of occurrence in the population

3-part differential usually cont Granulocytes or large cellsLymphocytes or small cellsMonocytes(mononuclear cells) or (middle cells)

5-part classify cells toNeutrophilsEosinophilsBasophilsLymphocytes Monocytes

A sixth category designated “large unstained cells” include cells larger than normal and lack the peroxidase activity this include

◦Atypical lymphocytes◦Various other abnormal cells.

Other counters identifies 7 categories including◦Large immature cells(composed of blasts and

immature granulocytes)◦Atypical lymphocytes(including blast cells).

Hematology analyzer provide mathematical results obtained by electrical and light signals generated when blood cells pass through sensing zone of the machine.Two method- 1- electrical impedance counting2- light scatter method.

Cell counting

Coulter PrincipleDilutionVacuum and pressureElectrical impedanceReagent systems

Sensing Zone

Red Blood Cell

The Coulter PrincipleThe Coulter Principle

A red cell passes through RBC aperture

Oscilloscope

Sensing Zone

Neutrophil

Oscilloscope

The Coulter PrincipleThe Coulter Principle

A white cell passes through WBC aperture

Before adding lysing reagent After Cell diameter in μm fl

Neutrophils 10 - 15 120 - 250Basophils 9 - 14 70 - 130 Eosinophils 11 - 16 80 - 140 Monocytes 12 - 20 60 - 120 Lymphocytes 7 – 12 30 - 80

Discriminations thresholds

Platelet- with a volume of 8-12 fl are counted from 2-30 fl.RBC- with volume of 80-100 fl is detected from 30 -250 fl.WBC- RBC are lysed by lytic reagent .the different WBC discriminator set at different levels between the ranges of 30-450 fl.

Normal Histogram

Three parts differential white blood cells:

30 to 125µ3 : lymphocytes

125 to 160µ3 : monocyte

160 to 450 µ3 : granulocytes

WBC Composite Histogram

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Coulter WBC Histogram

Lymphs

30 – 90 fL

Monos

90 -160 fL

Neuts

160 - 450 fL

RBC HISTOGRAM

NORMALNORMAL

RBC HISTOGRAM

RBC FRAGMENTS, MICROCYTIC RBCs, Giant PLTRBC FRAGMENTS, MICROCYTIC RBCs, Giant PLTDI RBCsDI RBCs

MACROCYTIC, TARGET CELLS, DI RBCMACROCYTIC, TARGET CELLS, DI RBCCOLD AGGLUTININCOLD AGGLUTININ

Post TransfusionPost Transfusion

PLT HISTOGRAMS

NORMALNORMAL

PLT HISTOGRAMS

Giant Platelets

Small Platelets

WBC HISTOGRAMS

ImmNE2 Eosinophilia

ImmNE1 & ImmNE2

Blasts

Lymphocytosis

ImmNE1 = band formsImmNE2 = immature neutrophils : metamyelocytes, myelocytes, promyelocytes

WBC

WBC Adults 4-10 x 103/μl Childs till 12 x 103/μl Newborns till 15 x 103/μl

Lymph. Adults -25-40 % Childs, Newborns- till 70 % MXD - Adults 3-13 % Neutro. - Adults 50-70 %

Red Blood Cell Count

RBC Men 4.6-6.2 x 106/μl Women 4.2-5.4 x 106/μl

HGB Men 14-18 g/dl Women 12-16 g/dl HCT Men 43-49 % Women 36-46 %

MCV- 85-95 fl MCH -27-33 pg

MCHC- 32-36 g/dl RDW-SD 37-46 fl (Width in 20% of the Peak

hight)

RDW-CV 11-16 % (calc. width of the 68 % Peak hight)

PLATELET

PLT 150-400 x 103/μl x 109/l PDW 9-14 fl (Width in 20% of the Peak hight) MPV 8-12 fl P-LCR 15-35 %

Anemia is not yet apparent MCV still is in the normal range Peripheral Smear shows mild Anisocytosis BUTRDW is increased (Earliest Indicator) Histogram is Unimodal but is wider Increased RDW combined with normal RBC values (MCV , Hb , Hct ) distinguishes Iron deficiency from Normal Subjects 10

Anemia is present, MCV is very low, and the smear is very abnormal RDW is abnormally high; Histogram remains abnormal. The diagnosis is easily made at this point, but earlier identification would improve management

The red cell count is increasing, MCV is not yet normal, and Two populations of red cells are seen-preexisting microcytes, and newly formed normocytes. The two populations are distinguished easily on the red cell histogram but not so easily on the peripheral blood smear.

EARLY FOLATE DEFICIENCY-• The MCV is still normal RBC count and Hb slightly reduced but• RDW is clearly increased , even before apparent anemia.

SEVERE FOLATE DEFICIENCY –• RBC Count is low.• MCV is high.•RDW is increased

Normocytic recovery a small peak of cells in the normal range • RDW is higher than untreated megaloblastic anemia due to two cell population contributing to the heterogeneity.

Microcytic recovery Two Cell population is clearly seen in this histogram – old macrocytes and newly produced microcytes . Concomitant iron deficiency has been unmasked. RDW is markedly increased..

Case -12 yr old boy with purpura, marked pallor, fever •Pancytopenia •MCV 100.5, RDW 15.9% •RBC histogram skewed to right •WBC histogram: lymphocyte peak, faint dome of neutrophils •PLT histogram- abn shape,descending slope not touching baseline •BM Bx confirmed AA

Case -

WBC LYM% MXD% NEUT%

+ 23.8 x 109/L 8.1% 7.9% 84.0%

Case -WBC 7.9 x 109/L LYM% + 64.7% MXD% 15.8% NEUT% – 19.5%

Case

WBC 7.7 x 109/L LYM% F1 * 13.2% MXD% F2 * 37.7% NEUT% 49.1%

Case WBC 4.3 x 109/L LYM% 18,3% MXD% + 62,2% NEUT% 19.5%

Case -

Case

WBC 2.3 x 109/L LYM% 39.7%MXD% 32.2% NEUT% 28.1%

Case -6

RBC 4.48 x1012/LHGB 8.8g/dlHCT 29.3% MCV 65.4fl MCH 19.6pg MCHC 30.0g/dlRDW-CV 18.2%

Case- RBC

HGB HCT MCV MCH MCHC RDW

1.64 x1012/L 6.2g/dl 18.2% 110.0fl 37.8pg 34.1g/dl 15.2%

Case

RBC HGB HCT MCV MCH MCHC RDW

4.15 x1012/L 14.0g/dl 40.8% 98.3fl 33.7pg 34.3g/dl 22.7%

Anisocytosis

Case

RBCHGBHCTMCVMCHMCHCRDW

3.62 x1012/L11.1g/dl31.9%88.1fl30.7pg34.8g/dl+ 25.5%

Poikilocytosis

Case PLTPDWMPVP-LCR

71 x109/LPUDWDW

Giant platelet

Although the wide distribution on the PLT histogram suggests the appearance of large platelets, the distribution curve intersects the discrimination line at a high point

Case WBC

LYM% MXD% NEUT %

PLT PDW MPV P-LCR

6.0 x109/L 27.5% 7.9% 64.4%

86 x109/L 18.6fl 12.8fl 43.7%

Platelet Aggregation The smear clearly shows that platelets are aggregating. The WBC histogram shows a peak in the ghost area ( ) , PLT histogram shows a wide distribution. Although these large particles usually affect the leucocyte counts, the leukocytes distribution of case 1 is well separated from the ghost area on the WBC histogram, probably without any effect of small particles in the ghost area. There is no WL Alarm given .

Case

RBC HGB HCT MCV MCH MCHC RDW

2.23 x1012/L 14.4g/dl 24.9% 111.7fl 64.6pg 57.8g/dl 25.4fl

Cold Agglutinins

Incubation 30 min

RBC HGB HCT MCV MCH MCHC RDW

4.35 x1012/L 14.5g/dl 43.5% 100.0fl 33.3pg 33.3g/dl 14.7fl

Because in this case erythrocytes have passed through the detector as clusters of several cells, the RBC, HCT,MCH, MCV, MCHC and RDW values are abnormal. The RBC histogram shows a second peak.After the clusters have been dissolved by incubation, all erythrocytes aredetected as single cells. Therefore the second peak on the RBC histogram doesnot appear and the RBC, HCT, MCV, MCH, MCHC and RDW values are

Case

WBC LYM% MXD% NEUT %

49.4 x109/L -.--- -.--- -.---

Insufficient Lysing of Erythrocytes

The histogram show On the WBC histogram the distribution curve intersects the WBC lower discrimination line at an abnormally high point.

This is frequently seen with blood samples taken from hepatic disease patients or newborns. These problems are solved by diluting the sample or replacing plasma with cellpack. The smear photo shows large platelets and acantocytes, suggesting hepatic diseases

RL: Abnormal height at lower discriminator of RBC Histogram (LD)

RU: Abnormal height at upper discriminator of RBC Histogram (UD)

MP: Multiple peaks: Distinguish ?? of two RBC Populations DW:The distribution (RDW) can not be detected because the Histogram does not cross the 20 % limit twice

WL: Abnormal height at lower discriminator of WBC Histogram (LD) WU: Abnormal height at upper discriminator of WBC Histogram (UD) T1: Valley 1 not found T2: Valley 2 not found F1, F2, F3: Abnormal height at the points T1 or T2; adjacent fractions are marked

PL: Abnormal height at lower discriminator of PLT Histogram (LD)

PU: Abnormal height at upper discriminator of PLT Histogram (UD)

MP: Multiple Peaks found

DW:The distribution (PDW) can not be detected because the Histogram does not cross the 20 % limit twice

Mark “ RL “, abnormal height at lower discriminator Possible causes: • Giant Platelets • Micro-Erythrocytes • Platelet Clumps

Mark “ RU “, abnormal height at the upper discriminator Possible causes: Cold Agglutinins (check MCHC > 40 g/dl) Erythroblasts / Normoblasts

MP “, multiple peaks found Possible causes: Iron deficiency in therapy Infection or Tumor Anemia (visceral iron deficiency) Transfusions

“DW “, abnormal histogram distribution

Distribution curve does not cross 20% level twice. The overall height of the curve is always 100 %. The width is calculated on the 20 % height of the curve. Hint for extreme Aniso- or. Poikilocytosis

Thrombocyte-Histogram

MPV (mean PLT volume) Ref range: 8 - 12 fl P-LCR (ratio of large platelets) Ref range: 15 - 35 % Increase could be a sign for: • PLT Clumps • Giant PLT • Microerythrocytes

PDW, (platelet distribution width at 20 % of peak height Ref range: 9 - 14 fl

Increase could be a sign for: PLT Clumps Microerythrocytes Fragments

Mark “ PL “, abnormal height at lower discriminatorPossible cause: High blank value Cell fragments

Mark “ PU “, abnormal height at upper discriminator Possible Cause : • PLT Clumps EDTA-Incombatibility Clotted sample • Giant Platelets • Microerythrocytes

Mark “ MP “, Multi Peaks found

Possible Cause: Platelet transfusion

Mark “ DW “, Distribution With The distribution can not be detected because the Histogram does not cross the 20 % limit twice. • This curve in only an example but could also show another course. • The overall height of the curve is always 100 %. The width is calculated on the 20 % height of the curve.

Leukocyte-Histogram Flag “ WL “, Curve does not begin at the basis line

Possible causes : • PLT Clumps EDTA-Incombatibility coagulated Sample • high osmotic resistant (Erythrocytes not lysed) • Erythroblasts • cold agglutinate

RBC Histogram

ABN / INDICATOR PROBABLE CAUSE COMMENTLeft of curve does not touch baseline

Schistocytes and extremely small red cells

Review smear CBC and Platelet histogram

Bimodal peak Transfused cells, therapeutic response

Review Smear

Right portion of curve extended

Red cell autoagglutination

Review CBC & Smear

Left shift of curve Microcytes Review smear & CBC

Right shift of curve Macrocytes Review smear & CBC

WBC Histogram

ABN / INDICATOR PROBABLE CAUSE COMMENTTrail extending downward at extreme left, or lymph peak not starting at baseline

NRBC, Plt clumping, unlysed RBC, cryoproteins, parasites

Review smear and correct WBC for NRBC

Peak to the left of lymph peak or widening of lymph peak towards left

NRBC Review smear & correct WBC for NRBC

Widening of lymph peak to right

Atypical lymphs, blasts, plasma cells, hairy cells, eosinophilia, basophilia

Review smear

Wider mono peak Monocytosis, plasma cells, eosinophilia, basophilia, blasts

Review smear

WBC Histogram

ABN / INDICATOR PROBABLE CAUSE COMMENT

WBC histogram (lymph peak) does not start at baseline

Giant platelets, NRBC, Plt clumping

Review smear, correct WBC for NRBC

Elevation of left portion of granulocyte

Left Shift Review smear

Elevation of right portion of granulocyte peak

Neutrophilia Review smear

Platelet Histogram

ABN / INDICATOR PROBABLE CAUSE COMMENTPeak or spike at left end of histogram (2-8 fl)

Cytoplasmic fragments

Review smear

Spike towards right end of histogram

Schistocytes, microcytes, giant platelets

Review smear + CBC

( MCV & RDW)

( MPV & PDW)

Bimodal peak Cytoplasmic fragments

Review smear

R1- RBC precursors, Giant or clumped platelets, cryoglobulins.

R2- Blast, basophilia, eosinophilia, monocytosis,plasma cells and abnormal size lymphocytes.R3-eosinophilia and immature granulocytes.R4-absolute granulocytosis.

CONCLUSION Histogram in conjunction with absolute

counts give valuable information about the abnormality of the sample & the need for follow up peripheral blood examination.Histogram should be used as quality check but not diagnostic for any pathological condition.The manual blood film remains the definitive tool for complete haematological analysis.

Take home messages

Shapes of histograms identified pathology before the blood smear could be examined.Newer parameter like RDW and PDW have added new dimension to understand blood cells and classify there abnormality.The manual blood film remains the definitive tool for complete haematological analysis.

Histograms Interpretation

LYMPH% 31,2 %MXD% 6,8 %NEUT% 62,0 %LYMPH# 1,8 x103/µlMXD# 0,4 x103/µlNEUT# 3,6 x103/µl

250

RBC

RDW-SD 40,0 fl

40

PLT

PDW 13,1 flMPV 10,4 flP-LCR 28,1 %

WBC

300THANYOU

SPEAKER- DR NARMADA PRASAD TIWARI

Known interfering substance

RBCs• High WBCs esp if RBCs is low

RBCs• Agglutinated RBCs RBCs

Hb Turbidity of the blood sample Hb• Elevated WBCs• Elevated lipids• Fetal bloods

Known interfering substance

RBCs• High WBCs esp if RBCs is low

RBCs• Agglutinated RBCs RBCs

Hb Turbidity of the blood sample Hb• Elevated WBCs• Elevated lipids• Fetal bloods

MCVRed cell agglutination number of large platelets

HTRed cell agglutination

RDWAgglutination of RBCsNutritional deficiencyBlood transfusion

MCVRed cell agglutination number of large platelets

HTRed cell agglutination

RDWAgglutination of RBCsNutritional deficiencyBlood transfusion

WBCs interferring subs.Normoblasts WBCs

Unlysed RBCs WBCs

MM WBCs (ppt protein)

Hemolysis WBCs (red cell stroma)

Leukemia WBCs ( cell fragility)

In CLL small lymph not counted

Cryoglobulin all parameters of blood

WBCs interferring subs.Normoblasts WBCs

Unlysed RBCs WBCs

MM WBCs (ppt protein)

Hemolysis WBCs (red cell stroma)

Leukemia WBCs ( cell fragility)

In CLL small lymph not counted

Cryoglobulin all parameters of blood

PlateletsRBCs fragments plat (microcytes)

WBCs fragments plat (microcytes)

Chemotherapy plat ( plat. fragments)

Hemolysis Plat (red cell strom)

ACD blood plat (plat. Aggregation)

RBCs inclusion plat. (Malaria, H.j bodies)

Plat. agglutination plat

PlateletsRBCs fragments plat (microcytes)

WBCs fragments plat (microcytes)

Chemotherapy plat ( plat. fragments)

Hemolysis Plat (red cell strom)

ACD blood plat (plat. Aggregation)

RBCs inclusion plat. (Malaria, H.j bodies)

Plat. agglutination plat

LymphocytesNucleated RBCs lymphParasites lymphResistent RBCs lymph

Monocytes in large lymphocytes, atypical lymph,

blasts and basophils

Granulocytes in eosinophilia, blasts, promyelo, myelo,

metameyl and plasma cell

LymphocytesNucleated RBCs lymphParasites lymphResistent RBCs lymph

Monocytes in large lymphocytes, atypical lymph,

blasts and basophils

Granulocytes in eosinophilia, blasts, promyelo, myelo,

metameyl and plasma cell