slide review rules 3 rd basic haematopathology course tmh mumbai
TRANSCRIPT
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?