slide review rules 3 rd basic haematopathology course tmh mumbai

Slide Review Rules

3rd Basic Haematopathology CourseTMH

Mumbai

Healthy Man

CBC DIFF

Retic

Results – 43 yrs Male

• Hb – 14.3• Hct – 48• RCC – 5.7• MCV – 84• MCH – 31• MCHC – 34• RDW – 14• Plt – 234• MPV – 6.9

Results – 43 yrs Male

• Hb – 12.3• Hct – 38• RCC – 6.3• MCV – 60• MCH – 26• MCHC – 32• RDW – 14• Plt – 84• MPV – 11.8

Results – 43 yrs Male

• Hb – 7.3• Hct – 23• RCC – 4.0• MCV – 58• MCH – 24• MCHC – 28• RDW – 22• Plt – 434 • MPV – 8.9

Results – 43 yrs Male

• Hb – 14.3• Hct – 48• RCC – 5.7• MCV – 84• MCH – 31• MCHC – 34• RDW – 14• Plt – 234• MPV – 6.9

• Hb – 7.3• Hct – 23• RCC – 4.0• MCV – 58• MCH – 24• MCHC – 28• RDW – 22• Plt – 434 • MPV – 8.9

• Hb – 12.3• Hct – 38• RCC – 6.3• MCV – 60• MCH – 26• MCHC – 32• RDW – 14• Plt – 84• MPV – 11.8

Results – 43 yrs Male

• Hb – 14.3• Hct – 48• RCC – 5.7• MCV – 84• MCH – 31• MCHC – 34• RDW – 14• Plt – 234• MPV – 6.9

• Hb – 7.3• Hct – 23• RCC – 4.0• MCV – 58• MCH – 24• MCHC – 28• RDW – 22• Plt – 434 • MPV – 8.9

• Hb – 12.3• Hct – 38• RCC – 6.3• MCV – 60• MCH – 26• MCHC – 32• RDW – 14• Plt – 84• MPV – 11.8

Normal

Results – 43 yrs Male

• Hb – 14.3• Hct – 48• RCC – 5.7• MCV – 84• MCH – 31• MCHC – 34• RDW – 14• Plt – 234• MPV – 6.9

• Hb – 7.3• Hct – 23• RCC – 4.0• MCV – 58• MCH – 24• MCHC – 28• RDW – 22• Plt – 434 • MPV – 8.9

• Hb – 12.3• Hct – 38• RCC – 6.3• MCV – 60• MCH – 26• MCHC – 32• RDW – 14• Plt – 84• MPV – 11.8

NormalMicrocyticHypochromicModrate ThrombocytopeniaGiant Platelets

Results – 43 yrs Male

• Hb – 14.3• Hct – 48• RCC – 5.7• MCV – 84• MCH – 31• MCHC – 34• RDW – 14• Plt – 234• MPV – 6.9

• Hb – 7.3• Hct – 23• RCC – 4.0• MCV – 58• MCH – 24• MCHC – 28• RDW – 22• Plt – 434 • MPV – 8.9

• Hb – 12.3• Hct – 38• RCC – 6.3• MCV – 60• MCH – 26• MCHC – 32• RDW – 14• Plt – 84• MPV – 11.8

MicrocyticHypochromicModrate ThrombocytopeniaGiant Platelets

MicrocyticHypochromicAnisocytosis

Normal

Definitive Flags Description

Anemia Low RBC or Low HGB

Eosinophilia High EO (percentage

Erythrocytosis High RBC

Hypochromia (1+, 2+, 3+) Low MCHC

Leukocytosis High WBC

Lymphocytosis# High LY#

Neutropenia Low NE (percentage)

Thrombocytopenia Low PLT

Thrombocytosis High PLT

Macrocytosis (1+, 2+, 3+) High MCV

Microcytosis (1+, 2+, 3+ Low MCV

Hypochromia Low MCH

Results – 43 yrs Male

• Hb – 14.3• Hct – 48• RCC – 5.7• MCV – 84• MCH – 31• MCHC – 34• RDW – 14• Plt – 234• MPV – 6.9

• Hb – 7.3• Hct – 23• RCC – 4.0• MCV – 58• MCH – 24• MCHC – 28• RDW – 22• Plt – 434 • MPV – 8.9

• Hb – 12.3• Hct – 38• RCC – 6.3• MCV – 60• MCH – 26• MCHC – 32• RDW – 14• Plt – 84• MPV – 11.8

MicrocyticHypochromicModrate ThrombocytopeniaGiant Platelets

MicrocyticHypochromicAnisocytosis

Normal

Reference Range1-2 years of age

WBC 5000-16000/cummRBC 3.8-5.2Hemoglobin 11-15gm%PCV 30-40%MCV 72-90flMCH 25-29pgMCHC 32-36%Platelet count 1,50,000-5,50,000/

cummABSOLUTE DIFFERENTIAL COUNT #

NEUTROPHIL 2.0-7.0

LYMPHOCYTE 3.5-11.0EOSINOPHIL 0.2-1.0MONOCYTE 0.2-1.0

BASOPHIL 0-0.2

2-6 years of ageWBC 5000-15000/cummRBC 4.0-5.2 million/cummHemoglobin 11-14gm%PCV 35-40%MCV 75-90flMCH 24-30pgMCHC 31-37%Platelet count 1,50,000-4,50,000/

cummABSOLUTE DIFFERENTIAL COUNT #

NEUTROPHIL 2.0-8.0LYMPHOCYTE 6.0-9.0EOSINOPHIL 0.2-0.6

BASOPHIL 0-0.2

MONOCYTE 0.2-1.0

6-12 years of ageWBC 5000-13000/cummRBC 4.0-5.2million/cummHemoglobin 12.0-16.0gm%PCV 35-45%MCV 77-95flMCH 25-33pgMCHC 31-37%Platelet count 1,50,000-4,50,000/cumm

ABSOLUTE DIFFERENTIAL COUNT #NEUTROPHIL 2.0-8.0LYMPHOCYTE 1.0-5.0EOSINOPHIL 0.1-1.0BASOPHIL 0.0-0.2

MONOCYTE 0.2-1.0

Adult RangesMale Female

WBC 4000-12000/cumm 4000-12000/cumm

RBC 4.4-5.9 millions/cumm 3.8-5.2 millions/cummHB 13.0-17.0gm/dl 11.0-15.0gm/dlPCV 40-50% 35-46%MCV 80-100fl 80-100flMCH 26-34pg 26-34pgMCHC 31.5-36.3% 31.4-35.8%RDW 11.5-14.5% 11.5-14.5%

PLATELET 150000-450000/cumm 150000-450000/cummESR ( mm/ 1 Hour)Age 17- 50 4-10 mm 6- 12

Age 51-60 6- 12 9-19

> 60 6-14 10-20

ABSOLUTE DIFFERENTIAL COUNT #

NEUTROPHIL 2.0-7.0LYMPHOCYTE 1.0-4.0MONOCYTE 0.2-1.0EOSINOPHIL 0.2-0.6BASOPHIL 0-0.2

OLD LAB CRITERIA FOR MAKING SLIDE Parameter Lower limit

Male Upper limit Male

Lower limit Female

Upper limit Female

WBC 2000 20000 2000 20000 RBC NO NO NO NO HGB 7 20 7 18 PCV 20 55 20 56 MCV 70 100 70 100 MCH NO NO NO NO MCHC NO NO NO NO RDW NO NO NO NO PLT COUNT 50000 10L 50000 10L Absolute Eosinophil count

2 2

Modified New lab Validation Criteria

Parameter Rules Comments

WBC <4.0 OR >20.0 SLIDE REVIEWPLT <50,000(irrespective of MPV) SLIDE REVIEW

50,000 - 100,000 NO SLIDE REVIEW MPV >11.5Hb < 7g/dl or > 20g/dl SLIDE REVIEWMCHC >35.8 SLIDE REVIEWRDW >22 SLIDE REVIEWMCV & RDW <70 & >22 MCV >105 SLIDE REVIEWDifferentialNE % >80 NO SLIDE REVIEWLY % >50 (Adults) >60 (Child) SLIDE REVIEWMO % >12 SLIDE REVIEWEOS % > 20 NO SLIDE REVIEWBA % > 2 SLIDE REVIEWNRBC any value SLIDE REVIEWRETICS % > 4% & & SLIDE REVIEWRETIC # > 0.1 #RETICS > 10% SLIDE & SMEAR

REVIEW

Suspect flags : Imm 1 & 2, Variant lymphVerify diffAtypical lymphBlast flags - NE Blasts - LY Blasts - MO BlastsNRBCLow event

Slide Review Rule – Revision based on Truth table

Christian Medical CollegeVellore

India

  ISLH LAB RULES

MODIFIED NEW RULES

True positive 35.0% 27.5% 18.7%

False Positive 2.8% 2.1% 1.8%

False Negative 1.1% 0.9% 0.6%

True Negative 61.1% 69.4% 78.9%

Definitive Flags Description

Anemia Low RBC or Low HGB

Eosinophilia High EO (percentage

Erythrocytosis High RBC

Hypochromia (1+, 2+, 3+) Low MCHC

Leukocytosis High WBC

Lymphocytosis# High LY#

Neutropenia Low NE (percentage)

Thrombocytopenia Low PLT

Thrombocytosis High PLT

Macrocytosis (1+, 2+, 3+) High MCV

Microcytosis (1+, 2+, 3+ Low MCV

Hypochromia Low MCH

Results – 43 yrs Male

• Hb – 14.3• Hct – 48• RCC – 5.7• MCV – 84• MCH – 31• MCHC – 34• RDW – 14• Plt – 234• MPV – 6.9

• Hb – 7.3• Hct – 23• RCC – 4.0• MCV – 58• MCH – 24• MCHC – 28• RDW – 22• Plt – 434 • MPV – 8.9

• Hb – 12.3• Hct – 38• RCC – 6.3• MCV – 60• MCH – 26• MCHC – 32• RDW – 14• Plt – 84• MPV – 11.8

MicrocyticHypochromicModrate ThrombocytopeniaGiant Platelets

MicrocyticHypochromicAnisocytosis

Normal

Parameter Rules Comments

PLT < 50,000 (irrespective of MPV) 50,000- 100,000 MPV >11.5

Slide ReviewNO Slide Review

HGB <7g/dl Slide Review

MCHC >35.8 Slide Review

MCV <70 and/or MCH <26 Slide Review

MCV <70 and/or MCH <26 RDW > 22 No Slide Review

Lymph% >50 (adults) ; >60(child) Slide Review

NRBC Any value Slide Review

Retic% & Retic # > 10% & > 0.1 Slide Review

All Suspect and System Flags Slide review for all the following

Current Validation Criteria (currently in use after consensus)

  ISLH LAB RULES

MODIFIED NEW RULES

CURRENT MODIFIED

True positive 35.0% 27.5% 18.7% 14.5%

False Positive 2.8% 2.1% 1.8% 0.9%

False Negative 1.1% 0.9% 0.6% 0.8%

True Negative 61.1% 69.4% 78.9% 83.8%

Flag Sensitivity

  ISLH LAB RULES

MODIFIED NEW RULES

CURRENT MODIFIED

True positive 35.0% 27.5% 18.7% 14.5%

False Positive 2.8% 2.1% 1.8% 0.9%

False Negative 1.1% 0.9% 0.6% 0.8%

True Negative 61.1% 69.4% 78.9% 83.8%

KAPPA VALUE 0.916 0.927 0.925 0.934

Parameter Rules Comments

PLT < 50,000 (irrespective of MPV) 50,000- 100,000 MPV >11.5

Slide ReviewNO Slide Review

HGB <7g/dl Slide Review

MCHC >35.8 Slide Review

MCV <70 and/or MCH <26 Slide Review

MCV <70 and/or MCH <26 RDW > 22 No Slide Review

Lymph% >50 (adults) ; >60(child) Slide Review

NRBC Any value Slide Review

Retic% & Retic # > 10% & > 0.1 Slide Review

All Suspect and System Flags Slide review for all the following

Current Validation Criteria (currently in use after consensus)

Suspect/System/Defenetive Flags

Suspect Flagging

NE Blasts

Imm NE 2

Imm NE 1

LY Blasts

Variant LY

MO Blasts

NRBCs

Depending on the Mean and SD of each parameter in the V, C and S, the flagging is programmed.

Suspect message Description

Abnormal Hemoglobin Pattern characteristic of specimen with abnormal hemoglobin clearing observed during retic analysis

Cellular Inter Pattern consistent with NRBC detection during a CBC only cycle. This Cellular Interference Suspect msg is not associated with a Review (R) flag

Dimorphic Reds Evidence of the presence of at least two populations of red cells

Giant Platelets Patterns characteristic of specimen containing Giant Platelets

Imm Grans Pattern characteristic of specimen containing: a) metamyelocytes and myelocytes and/or promyelocytes, or b) myelocytes and/or promyelocytes without metamyelocytes.

Left Shift Pattern is characteristic of specimen containing metamyelocytes, but without myelocytes, promyelocytes, or blasts

LY Blast Blasts in the Lymphocyte region of the dataplot

MO Blast Blasts in the Monocyte region of the dataplot

NE Blast Blasts in the Neutrophil region of the dataplot

NRBC CBC & Diff pattern is characteristic of specimen with NRBCs.This Suspect msg applies when the NRBC analysis is disabled but a CD/CDR cycle is run.

RBC Frag/Microcytes The specimen may contain red cell fragments &/ some microcytic red cells

Red Cell Agglut Red cells may be clumped or display rouleaux on peripheral smear

Sickled Cells Pattern characteristic of specimen containing irreversibly sickled cells

Variant LY Pattern characteristic of specimen with variant lymphs, including mature lymph such as those observed viral infections, as well as immature&/ abn lymph

System Message Description

Abn Diff Pattern Undefined abnormal Diff pattern observed during Diff analysis.

Cellular Inter Poor separation between a WBC population and interference. WBC correction was performed as a best estimation, and the WBC was flagged with R

MCV Inter: PLT Interference with MCV, RBC, and RDW and RDW-SD due to PLT.

MCV Inter: WBC Low confidence in MCV due to interference from high WBC level

MO-NE Overlap Population labeled as neutrophils appeared in the monocyte region during Diff analysis

NE-EO Overlap Neutrophil and eosinophil populations were shifted or overlapped during Diff analysis

Platelet Clumps Pattern is characteristic of specimen containing platelet clumps.

RBC-PLT Overlap Interference with larger platelets; may occur with the Giant Platelet Suspect message.

RET-RBC Overlap The retics could not be clearly separated from the mature red cells

Excessive Debris The number of debris events too high compared to white events during Diff analysis

Excessive Low DC Events

Too many low DC events during NRBC analysis

Flow Cell Clog: D Hardware detected a flow cell was clogged during Diff analysis

Healthy Man

CBC DIFF

Retic

Normal RPD/ Positional Parameters

VOLUME CONDUCTIVITY SDATTER

MEAN SD MEAN SD MEAN SD

NEUTROPH 138.05 – 151.31

19.55 – 23.22

148.2 – 153.02

5.71 – 8.15

143.61 – 154.03

11.33 – 13.39

LYMPHOCY 75.67 – 82.23

13.72 – 18.82

115.63 – 119.99

12.24 – 16.92

71.68 – 78.88

18.4 – 20.68

MONOCYTE 154.04 – 168.32

17.23 – 20.09

124.83 – 128.17

4.66 – 5.66

90.25 – 95.73

10.67 – 13.13

Referred for High Neutrophil count

Referred for High Neutrophil count

VOLUME CONDUCTIVITY SDATTER

MEAN SD MEAN SD MEAN SD

NEUTROPH 138.05 – 151.31

19.55 – 23.22

148.2 – 153.02

5.71 – 8.15

143.61 – 154.03

11.33 – 13.39

High Neutrophil count

High Neutrophil count

VOLUME CONDUCTIVITY SDATTER

MEAN SD MEAN SD MEAN SD

NEUTROPH 138.05 – 151.31

19.55 – 23.22

148.2 – 153.02

5.71 – 8.15

143.61 – 154.03

11.33 – 13.39

Haemopoiesis

Production of RBCs, WBCs & Platelets

Bone Marrow• Is the Factory that produces and Designs blood

corpuscles from precursor cells• This production is at a regular baseline level to

maintain normal levels replacing the dead corpuscles that completes their life span– Red cells (90-120 days), Platelets (7-10 days), Neutrophils

(few min to not more than one day)

• When the need arises the production is increased.

• The Design is to Fit the function. It utilizes structural elements of the marrow environment

Red blood cells

• FunctionCarries Haemoglobin

- Tranport blood gases- Maintain blood pH

Proper structure, size and shape - Flexibility to get to every tissue and cell. - Biconcave shape allows collapse

NeutrophilFunction1st line of defence against infectionGood Phagocytic cell • ability to move well • pseudopodia forming

phagosomes• Lots of cytoplasm to

– accomodate phagocytosed– pack with cytotoxic lysosomal

granules

Eosinophils• Function• Designed like Neutrophil but

unable to have pseudopodia, phagocytosis or phagosomes due to large granules

• David vs Goliath• Kills large organisms even more

than 10 times its size – by spitting the granules instead of phagocytosing→ increasing toxic oxygen radicles – frustrated phagocytosis

• Anti-allergic – destroys histamin

Basophils/Mast cells

• Function• Causes allergy, -

contains large granules that contain Histamin and heparin

Monocytes/Macrophage/Reticulum cell

• Function• Phagocytosis• Lot of cytoplasm

and all capabilities for killing organisms

Lymphocyte• Function• Antibodies and

Cytokines production• Active protein

sysnthesis• Therefore the cell has

more nuclei and very little cytoplasm

• Not to store but to secrete

Platelets (Thrombocytes)• Function• Provides a lot of necessary

procoagulant surface for clotting in Haemostasis

• Obtained from megakaryocyte which has the same surface - but 1,000 platelets from 1 megakaryocyte provides 10,000 times the surface than one megakaryocyte

Erythropoiesis & Myelopoiesis

Megakaryocyte

Marrow Environment

Marrow Environment to Peripheral Blood

Marrow Environment to Peripheral Blood

Time line • Maturation time from Proerythroblast to Reticulocyte

takes 5 days• Maturation from Myeloblast to Neutrophil takes 7 days• At times of need the days are shortened.• Shortens to 1 day in case of Neutrophils

– Toxic changes – immature granules– Dohle bodies – retains the color of immature cytoplasm– Macro

Infection

Macrocytic Anaemia

Macrocytic anaemia without Macrocytes

VOLUME

MEAN SD

NEUTRO 138.05 – 151.31

19.55 – 23.22

LYMPHO 75.67 – 82.23

13.72 – 18.82

MONO 154.04 – 168.32

17.23 – 20.09

43 year old Male – Pre employment

43 year old Male – Pre employment

VOLUME

MEAN SD

NEUTRO 138.05 – 151.31

19.55 – 23.22

LYMPHO 75.67 – 82.23

13.72 – 18.82

MONO 154.04 – 168.32

17.23 – 20.09

43 year old Male – Pre employment

VOLUME

MEAN SD

NEUTRO 138.05 – 151.31

19.55 – 23.22

LYMPHO 75.67 – 82.23

13.72 – 18.82

MONO 154.04 – 168.32

17.23 – 20.09

CLL

70 year old male – thrombocytopenia (78,000)

VOLUME CONDUCTIVITY SDATTER

MEAN SD MEAN SD MEAN SD

NEUTRO 138.05 – 151.31

19.55 – 23.22

148.2 – 153.02

5.71 – 8.15

143.61 – 154.03

11.33 – 13.39

Abnormal Neutrophils

Morphology of the neutrophil with pseudo Pelger Huet anomaly

VOLUME CONDUCTIVITY SDATTER

MEAN SD MEAN SD MEAN SD

NEUTRO 138.05 – 151.31

19.55 – 23.22

148.2 – 153.02

5.71 – 8.15

143.61 – 154.03

11.33 – 13.39

MDS – RAEB

ROC ANALYSIS AND CUT OFFS FOR NEUTROPHIL PARAMETERS FOR MDS

NEUTROPHIL PARAMETERS

sensitvity specificity Cut off AUC P value

NEU MEAN VOL 35.9 97.4 <137.9 0.582 0.2

NEU MN VOL SD 69.2 84.2 >22.5 0.718 0.0008

NEU MEAN COND 48.7 97.4 <147.7 0.532 0.67

NEU MN COND SD 74.4 92.1 >7.04 0.886 0.0001

NEU MEAN SCATTER

61.4 94.7 <142 0.719 0.0006

NEU MN SC SD 87.2 84.2 >11.6 0.893 0.0001

MDS on Cell counter• The mean cell scatter (MNES) was significantly lower.

• Distribution of Cell volume (DNEV), Conductivity (DNEC) as well as Scatter (DNES) of neutrophils in the MDS group were significantly higher.

• Thus the cell counter identifies Neutrophil hypogranularity which could be obvious on smears depending on the stain used.

• In addition it identifies Neutrophil Anisocytosis (Variation in Cell size – DNEV), Neutrophil Anisogranularity (Variation in Granularity – DNES) and also Variation in cytoplasmic/nuclear structure among Neutrophils (DNEC). These features are not obvious on smears.

VOLUME CONDUCTIVITY SDATTER

MEAN SD MEAN SD MEAN SD

NEUTRO 138.05 – 151.31

19.55 – 23.22

148.2 – 153.02

5.71 – 8.15

143.61 – 154.03

11.33 – 13.39

MDS – RAEB

Platelet Histogram

Normal distribution with normal platelets

Case - 1

Normal distribution

1023340818

Case - 5

Normal distribution

1023340102

Case - 4

Normal distribution with platelet anisocystosis but with no haitus

Case - 3

Normal distribution with only fewer platelets of intermediate sizeCurve still fitted in ascending mode

Case - 61023340058

No curve fit

S-10;n-20;L-60;vL-10; 2.0

So very steep due to lesser small plts but peak continues after 10 at same amplitude Even after 10 - like there are two populationVery Large population is significant – Creating a major hiatus

1016140199Case -1

S 10;N10;L60;VL20; 2.5 – same scenario like previous case

1016140179

Case -4

Platelet on smear 0.5 – mostly small pltsPeak before 10fl. Major Hiatus created by clumps and very large platelets - so curve not fitted – but has not significantly impacted platelet count since they are few

1016140503

Case -5

Platelets on smear 0.8 – same scenario as previous case

1016140406

Case -7

S0;N0;L80;VL20; 0.5 MCV70.7

VL + small RBCsLesser platelets with more counts

1016140671

Case -11

20 year old pregnant female with fever for 1 month

WBC CBC histogram showed the presence of population below 35fl.

VCS

VCS

Malaria diagnosis ok

III

III IVNormal RPD

I

Parameter Rules Comments

PLT < 50,000 (irrespective of MPV) 50,000- 100,000 MPV >11.5

Slide ReviewNO Slide Review

HGB <7g/dl Slide Review

MCHC >35.8 Slide Review

MCV <70 and/or MCH <26 Slide Review

MCV <70 and/or MCH <26 RDW > 22 No Slide Review

Lymph% >50 (adults) ; >60(child) Slide Review

NRBC Any value Slide Review

Retic% & Retic # > 10% & > 0.1 Slide Review

All Suspect and System Flags Slide review for all the following

Newer Flags based on Ref Range on CPD

Lymphocyte MicrocytosisNeutrophil MacrocytosisLymphocyte anisocytosis

Current Validation Criteria (currently in use after consensus)

Thank you

Any questions?