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Slide Review Rules 3 rd Basic Haematopathology Course TMH Mumbai.

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Presentation on theme: "Slide Review Rules 3 rd Basic Haematopathology Course TMH Mumbai."— Presentation transcript:

1 Slide Review Rules 3 rd Basic Haematopathology Course TMH Mumbai

2 Healthy Man CBC DIFF Retic

3 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

4 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

5 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

6 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

7 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

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 Microcytic Hypochromic Modrate Thrombocytopenia Giant Platelets

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 Microcytic Hypochromic Modrate Thrombocytopenia Giant Platelets Microcytic Hypochromic Anisocytosis Normal

10 Definitive FlagsDescription AnemiaLow RBC or Low HGB EosinophiliaHigh EO (percentage ErythrocytosisHigh RBC Hypochromia (1+, 2+, 3+)Low MCHC LeukocytosisHigh WBC Lymphocytosis#High LY# NeutropeniaLow NE (percentage) ThrombocytopeniaLow PLT ThrombocytosisHigh PLT Macrocytosis (1+, 2+, 3+)High MCV Microcytosis (1+, 2+, 3+Low MCV HypochromiaLow MCH

11 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 Microcytic Hypochromic Modrate Thrombocytopenia Giant Platelets Microcytic Hypochromic Anisocytosis Normal

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20 Reference Range 1-2 years of age WBC5000-16000/cumm RBC3.8-5.2 Hemoglobin11-15gm% PCV30-40% MCV72-90fl MCH25-29pg MCHC32-36% Platelet count1,50,000- 5,50,000/cumm ABSOLUTE DIFFERENTIAL COUNT # NEUTROPHIL 2.0-7.0 LYMPHOCYTE3.5-11.0 EOSINOPHIL0.2-1.0 MONOCYTE 0.2-1.0 BASOPHIL 0-0.2 2-6 years of age WBC5000-15000/cumm RBC4.0-5.2 million/cumm Hemoglobin11-14gm% PCV35-40% MCV75-90fl MCH24-30pg MCHC31-37% Platelet count1,50,000- 4,50,000/cumm ABSOLUTE DIFFERENTIAL COUNT # NEUTROPHIL2.0-8.0 LYMPHOCYTE6.0-9.0 EOSINOPHIL 0.2-0.6 BASOPHIL 0-0.2 MONOCYTE 0.2-1.0 6-12 years of age WBC5000-13000/cumm RBC4.0-5.2million/cumm Hemoglobin12.0-16.0gm% PCV35-45% MCV77-95fl MCH25-33pg MCHC31-37% Platelet count1,50,000-4,50,000/cumm ABSOLUTE DIFFERENTIAL COUNT # NEUTROPHIL2.0-8.0 LYMPHOCYTE1.0-5.0 EOSINOPHIL0.1-1.0 BASOPHIL 0.0-0.2 MONOCYTE0.2-1.0 Adult Ranges MaleFemale WBC 4000-12000/cumm RBC 4.4-5.9 millions/cumm3.8-5.2 millions/cumm HB 13.0-17.0gm/dl11.0-15.0gm/dl PCV 40-50%35-46% MCV 80-100fl MCH 26-34pg MCHC 31.5-36.3%31.4-35.8% RDW 11.5-14.5% PLATELET 150000-450000/cumm ESR ( 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 2.0-7.0 LYMPHOCYTE 1.0-4.0 MONOCYTE 0.2-1.0 EOSINOPHIL 0.2-0.6 BASOPHIL 0-0.2

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23 Modified New lab Validation Criteria Parameter Rules Comments WBC 20.0 SLIDE REVIEW PLT <50,000(irrespective of MPV) SLIDE REVIEW 50,000 - 100,000 NO SLIDE REVIEW MPV >11.5 Hb 20g/dl SLIDE REVIEW MCHC >35.8 SLIDE REVIEW RDW >22 SLIDE REVIEW MCV & RDW 22 MCV >105 SLIDE REVIEW Differential NE % >80 NO SLIDE REVIEW LY % >50 (Adults) >60 (Child) SLIDE REVIEW MO % >12 SLIDE REVIEW EOS % > 20 NO SLIDE REVIEW BA % > 2 SLIDE REVIEW NRBC any value SLIDE REVIEW RETICS % > 4% & & SLIDE REVIEW RETIC # > 0.1 # RETICS > 10% SLIDE & SMEAR REVIEW Suspect flags : Imm 1 & 2, Variant lymph Verify diff Atypical lymph Blast flags - NE Blasts - LY Blasts - MO Blasts NRBC Low event

24 Slide Review Rule – Revision based on Truth table Christian Medical College Vellore India

25 ISLHLAB RULES MODIFIED NEW RULES True positive35.0%27.5%18.7% False Positive2.8%2.1%1.8% False Negative1.1%0.9%0.6% True Negative61.1%69.4%78.9%

26 Definitive FlagsDescription AnemiaLow RBC or Low HGB EosinophiliaHigh EO (percentage ErythrocytosisHigh RBC Hypochromia (1+, 2+, 3+)Low MCHC LeukocytosisHigh WBC Lymphocytosis#High LY# NeutropeniaLow NE (percentage) ThrombocytopeniaLow PLT ThrombocytosisHigh PLT Macrocytosis (1+, 2+, 3+)High MCV Microcytosis (1+, 2+, 3+Low MCV HypochromiaLow MCH

27 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 Microcytic Hypochromic Modrate Thrombocytopenia Giant Platelets Microcytic Hypochromic Anisocytosis Normal

28 ParameterRulesComments PLT< 50,000 (irrespective of MPV) 50,000- 100,000 MPV >11.5 Slide Review NO Slide Review HGB<7g/dlSlide Review MCHC>35.8Slide Review MCV <70 and/or MCH <26 Slide Review MCV <70 and/or MCH <26 RDW > 22No Slide Review Lymph%>50 (adults) ; >60(child)Slide Review NRBCAny valueSlide Review Retic% & Retic #> 10% & > 0.1Slide Review All Suspect and System FlagsSlide review for all the following Current Validation Criteria (currently in use after consensus)

29 ISLHLAB RULES MODIFIED NEW RULES CURRENT MODIFIED True positive35.0%27.5%18.7%14.5% False Positive2.8%2.1%1.8%0.9% False Negative1.1%0.9%0.6%0.8% True Negative61.1%69.4%78.9%83.8%

30 Flag Sensitivity

31 ISLHLAB RULES MODIFIED NEW RULES CURRENT MODIFIED True positive35.0%27.5%18.7%14.5% False Positive2.8%2.1%1.8%0.9% False Negative1.1%0.9%0.6%0.8% True Negative61.1%69.4%78.9%83.8% KAPPA VALUE0.9160.9270.9250.934

32 ParameterRulesComments PLT< 50,000 (irrespective of MPV) 50,000- 100,000 MPV >11.5 Slide Review NO Slide Review HGB<7g/dlSlide Review MCHC>35.8Slide Review MCV <70 and/or MCH <26 Slide Review MCV <70 and/or MCH <26 RDW > 22No Slide Review Lymph%>50 (adults) ; >60(child)Slide Review NRBCAny valueSlide Review Retic% & Retic #> 10% & > 0.1Slide Review All Suspect and System FlagsSlide review for all the following Current Validation Criteria (currently in use after consensus)

33 Suspect/System/Defenetive Flags

34 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.

35 Suspect messageDescription Abnormal HemoglobinPattern characteristic of specimen with abnormal hemoglobin clearing observed during retic analysis Cellular InterPattern consistent with NRBC detection during a CBC only cycle. This Cellular Interference Suspect msg is not associated with a Review (R) flag Dimorphic RedsEvidence of the presence of at least two populations of red cells Giant PlateletsPatterns characteristic of specimen containing Giant Platelets Imm GransPattern characteristic of specimen containing: a) metamyelocytes and myelocytes and/or promyelocytes, or b) myelocytes and/or promyelocytes without metamyelocytes. Left ShiftPattern is characteristic of specimen containing metamyelocytes, but without myelocytes, promyelocytes, or blasts LY BlastBlasts in the Lymphocyte region of the dataplot MO BlastBlasts in the Monocyte region of the dataplot NE BlastBlasts in the Neutrophil region of the dataplot NRBCCBC & 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/MicrocytesThe specimen may contain red cell fragments &/ some microcytic red cells Red Cell AgglutRed cells may be clumped or display rouleaux on peripheral smear Sickled CellsPattern characteristic of specimen containing irreversibly sickled cells Variant LYPattern characteristic of specimen with variant lymphs, including mature lymph such as those observed viral infections, as well as immature&/ abn lymph

36 System MessageDescription Abn Diff PatternUndefined abnormal Diff pattern observed during Diff analysis. Cellular InterPoor separation between a WBC population and interference. WBC correction was performed as a best estimation, and the WBC was flagged with R MCV Inter: PLTInterference with MCV, RBC, and RDW and RDW-SD due to PLT. MCV Inter: WBCLow confidence in MCV due to interference from high WBC level MO-NE OverlapPopulation labeled as neutrophils appeared in the monocyte region during Diff analysis NE-EO OverlapNeutrophil and eosinophil populations were shifted or overlapped during Diff analysis Platelet ClumpsPattern is characteristic of specimen containing platelet clumps. RBC-PLT OverlapInterference with larger platelets; may occur with the Giant Platelet Suspect message. RET-RBC OverlapThe retics could not be clearly separated from the mature red cells Excessive DebrisThe 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: DHardware detected a flow cell was clogged during Diff analysis

37 Healthy Man CBC DIFF Retic

38 Normal RPD/ Positional Parameters VOLUMECONDUCTIVITYSDATTER MEANSDMEANSDMEANSD NEUTROPH138.05 – 151.31 19.55 – 23.22 148.2 – 153.02 5.71 – 8.15 143.61 – 154.03 11.33 – 13.39 LYMPHOCY75.67 – 82.23 13.72 – 18.82 115.63 – 119.99 12.24 – 16.92 71.68 – 78.88 18.4 – 20.68 MONOCYTE154.04 – 168.32 17.23 – 20.09 124.83 – 128.17 4.66 – 5.66 90.25 – 95.73 10.67 – 13.13

39 Referred for High Neutrophil count

40 VOLUMECONDUCTIVITYSDATTER MEANSDMEANSDMEANSD NEUTROPH138.05 – 151.31 19.55 – 23.22 148.2 – 153.02 5.71 – 8.15 143.61 – 154.03 11.33 – 13.39

41 High Neutrophil count

42 VOLUMECONDUCTIVITYSDATTER MEANSDMEANSDMEANSD NEUTROPH138.05 – 151.31 19.55 – 23.22 148.2 – 153.02 5.71 – 8.15 143.61 – 154.03 11.33 – 13.39

43 Haemopoiesis Production of RBCs, WBCs & Platelets

44 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

45 Red blood cells Function Carries Haemoglobin - Tranport blood gases - Maintain blood pH Proper structure, size and shape - Flexibility to get to every tissue and cell. - Biconcave shape allows collapse

46 Neutrophil Function 1 st line of defence against infection Good Phagocytic cell ability to move well pseudopodia forming phagosomes Lots of cytoplasm to – accomodate phagocytosed – pack with cytotoxic lysosomal granules

47 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

48 Basophils/Mast cells Function Causes allergy, - contains large granules that contain Histamin and heparin

49 Monocytes/Macrophage/Reticulum cell Function Phagocytosis Lot of cytoplasm and all capabilities for killing organisms

50 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

51 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

52 Erythropoiesis & Myelopoiesis

53 Megakaryocyte

54 Marrow Environment

55 Marrow Environment to Peripheral Blood

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57 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

58 Infection

59 Macrocytic Anaemia

60 Macrocytic anaemia without MacrocytesVOLUMEMEANSD 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

61 43 year old Male – Pre employment

62 VOLUMEMEANSD 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

63 43 year old Male – Pre employment

64 VOLUMEMEANSD 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

65 70 year old male – thrombocytopenia (78,000)

66 VOLUMECONDUCTIVITYSDATTERMEANSDMEANSDMEANSD 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

67 Morphology of the neutrophil with pseudo Pelger Huet anomaly

68 VOLUMECONDUCTIVITYSDATTERMEANSDMEANSDMEANSD 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

69 ROC ANALYSIS AND CUT OFFS FOR NEUTROPHIL PARAMETERS FOR MDS NEUTROPHIL PARAMETERS sensitvityspecificityCut offAUCP value NEU MEAN VOL 35.997.4<137.90.5820.2 NEU MN VOL SD 69.284.2>22.50.7180.0008 NEU MEAN COND 48.797.4<147.70.5320.67 NEU MN COND SD 74.492.1>7.040.8860.0001 NEU MEAN SCATTER 61.494.7<1420.7190.0006 NEU MN SC SD 87.284.2>11.60.8930.0001

70 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.

71 VOLUMECONDUCTIVITYSDATTERMEANSDMEANSDMEANSD 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

72 Platelet Histogram

73 Normal distribution with normal platelets Case - 1

74 Normal distribution 1023340818 Case - 5

75 Normal distribution 1023340102 Case - 4

76 Normal distribution with platelet anisocystosis but with no haitus Case - 3

77 Normal distribution with only fewer platelets of intermediate size Curve still fitted in ascending mode Case - 6 1023340058

78 No curve fit

79 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 population Very Large population is significant – Creating a major hiatus 1016140199 Case -1

80 S 10;N10;L60;VL20; 2.5 – same scenario like previous case 1016140179 Case -4

81 Platelet on smear 0.5 – mostly small plts Peak 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

82 Platelets on smear 0.8 – same scenario as previous case 1016140406 Case -7

83 S0;N0;L80;VL20; 0.5 MCV70.7 VL + small RBCs Lesser platelets with more counts 1016140671 Case -11

84  20 year old pregnant female with fever for 1 month

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

86 VCSVCS VCSVCS Malaria diagnosis ok I II III IV Normal RPD I

87 ParameterRulesComments PLT< 50,000 (irrespective of MPV) 50,000- 100,000 MPV >11.5 Slide Review NO Slide Review HGB<7g/dlSlide Review MCHC>35.8Slide Review MCV <70 and/or MCH <26 Slide Review MCV <70 and/or MCH <26 RDW > 22No Slide Review Lymph%>50 (adults) ; >60(child)Slide Review NRBCAny valueSlide Review Retic% & Retic #> 10% & > 0.1Slide Review All Suspect and System FlagsSlide review for all the following Newer Flags based on Ref Range on CPD Lymphocyte Microcytosis Neutrophil Macrocytosis Lymphocyte anisocytosis Current Validation Criteria (currently in use after consensus)

88 Thank you Any questions?


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