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Pankhi Dutta MD DM (haematopath) Consultant haematopathologist SevenHills Hospital, Mumbai.

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Presentation on theme: "Pankhi Dutta MD DM (haematopath) Consultant haematopathologist SevenHills Hospital, Mumbai."— Presentation transcript:

1 Pankhi Dutta MD DM (haematopath) Consultant haematopathologist SevenHills Hospital, Mumbai.

2 Introduction  Automated cell counter-backbone of the haemat lab  Wallace Coulter in 1956 – impedance method  Various technologies today  More accurate, more precise reports at a faster rate  Basic CBC + newer parameters  Inherent technological limitations

3 Basic parameters – 3 part counter  Haemoglobin  RBC, WBC, PLT count  Red cell indices  RDW  3-part differential  Histograms NO. 4 DATUM:9/10/9515:11 MODE:VOLLBLUT WBC5,8x 10 3 /µl RBC4,84 x10 6 /µl HGB13,7 g/dl HCT42,0% MCV86,8fl MCH28,3pg MCHC32,6g/dl PLT257x10 3 /µl LYMPH%31,2% MXD%6,8% NEUT%62,0% LYMPH#1,8x10 3 /µl MXD#0,4x10 3 /µl NEUT#3,6x10 3 /µl 250 RBC RDW-SD40,0fl 40 PLT PDW 13,1fl MPV 10,4fl P-LCR 28,1% WBC 300

4 3-part differential analyser Two chambers  Hb + WBCs  RBCs + PLTs Vacuum Blood cell DC supply Registor (constant current) Internal electrodeExternal electrode Aperture Transducer chamber Blood cell suspension

5 V07063-part Diff technology Haemoglobin molecule RBC Ammonium salts   Fe 2+   RBC    Fe Lysis of RBC Fe 2+ Haemoglobin estimation

6 V part Diff technology Haemoglobin molecule RBC   Fe 2+   RBC    Fe Change of conformity

7 V07063-part Diff technology Haemoglobin molecule Methemoglobin-complex  Stable coloumetric complex – directly proportional to Hb  Absorbance of solution is measured against standard    Fe 3+   Fe 2+   O2O2 3. Oxidation Fe 2+

8 DC detection method Particle counting  DC - direct current - impedance principle - volumetric measurement  WBC count and 3-part differential  RBC count  PLT count V07063-part Diff technology

9 DC Detection Method V07063-part Diff technology external electrode internal electrode aperture vacuum U = R x I Impulse

10 Impedance Principle External Electrode Internal Electrode Aperture V = R x C V = Voltage C = Current R = Resistance

11 Impedance Principle V = R x C V = Voltage C = Current R = Resistance External Electrode Internal Electrode Aperture

12 Problems- recirculation and coincidence A B C pulse A pulse B pulse C aperture cells

13 V07063-part Diff technology Samples are passing through the centre of the aperture with sheath flow solution for RBC & PLT Hydrodynamic Focusing  Recirculation and coincidence are prevented  Enhanced linearity & accuracy

14 V07063-part Diff technology time pulse height From pulse to histogram: pulse diagram DC Detection Method

15 V07063-part Diff technology Histogram Cumulative Distribution Curve cells DC Detection Method

16 NO. 4 DATUM:9/10/9515:11 MODE:VOLLBLUT WBC5,8x 10 3 /µl RBC4,84 x10 6 /µl HGB13,7 g/dl HCT42,0% MCV86,8fl MCH28,3pg MCHC32,6g/dl PLT257x10 3 /µl LYMPH%31,2% MXD%6,8% NEUT%62,0% LYMPH#1,8x10 3 /µl MXD#0,4x10 3 /µl NEUT#3,6x10 3 /µl 250 RBC RDW-SD40,0fl 40 PLT PDW 13,1fl MPV 10,4fl P-LCR 28,1% WBC 300

17 V part Diff technology fl fl Erythrocyte (RBC) Histogram  RBC detection: between 25 and 250 fL  Distribution curves are separated by flexible discriminators: RL & RU RL RU RBC PLT

18 V07063-part Diff technology fl fl  The histogram curve should start and end at the base line within the discriminators RL RU RBC PLT Erythrocyte (RBC) Histogram

19 V07063-part Diff technology fl fl  In case of abnormal histogram curves the flag messages: RL; RU or MP are generated and results must be checked  RL : Abnormal height at lower discriminator  RU : Abnormal height at upper discriminator  MP : (Multi Peak) RBC Anisocytosis RL RU RBC PLT Example: RL flag message 100% 20% Abnormal Erythrocyte (RBC) Histogram

20 V07063-part Diff technology 2-6 fl12-30 fl fixed at 12 fl PL PU PLT RBC 100% 20%  PLT detection: between 2 and 30 fL  Fixed discriminator at 12 fL Platelet (Plt) Histogram

21 V07063-part Diff technology 2-6 fl12-30 fl PL PU PLT RBC 100% 20%  In case of abnormal histogram curves the flag messages: PL; PU or MP are generated and results must be checked  PL : Abnormal height at Lower discriminator  PU : Abnormal height at Upper discriminator  MP : (Multi Peak) Platelet Anisocytosis Example: abnormal PLT curve PU message Abnormal Platelet (Plt) Histogram

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29 Lysing reaction to the WBCs Structure of WBS Mitochondria Nucleus Nucleolus Cell membrane Ribosome Cytoplasm Lysing reaction on the WBC Leukocyte (WBC) Histogram

30 Before lysing reaction Neutrophile Basophile Eosinophile Monocyte Lymphocyte Cell size in µm Lysing reaction and WBC After lysing reaction Lymphocyte Monocyten Basophile Eosinophile Neutrophile Cell volume in fl Lymphocyte Monocyte Basophile Eosinophile Neutrophile Leukocyte (WBC) Histogram

31 V07063-part Diff technology 2-6 fl12-30 fl fixed at 12 fl WL WU 100% 20%  WBC detection: between 30 and 300 fL  Leukocytes are separated in 3 parts: lymphocytes, mixed cells (mono, eo, baso) and neutrophils by discriminators: T1, T2 T1 T2 Leukocyte (WBC) Histogram

32 V07063-part Diff technology ~30 fl -300 fl WL WU 100% 20%  The histogram curve should start within the lower and upper discriminator at the base line  Abnormal curves are flagged with WL, WU, T1, T2, F1, F2  results must be checked T1 T2 Example: abnormal WBC curve WL message in case of Lyse resistant RBC Abnormal Leukocyte (WBC) Histogram

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39 QUESTIONS?

40 5-part differential counters Various technologies :-  Fluorescence flowcytometry  Volume Conductivity Scatter  Peroxidase staining

41 VOLUME MEASUREMENT VCS utilises the Coulter Principle of counting and sizing to measure the volume of the cell by using Direct Current (DC) across the two electrode in a flow cell. Beckman Coulter

42 CONDUCTIVITY MEASUREMENT Cell exposed to RF, the RF energy penetrates into cell and reveal information about its size and internal structure.

43 SCATTER MEASUREMENT As cells are pass in single stream (flow cell) they are struck by laser strike which gets scattered. The light scatter at angles between 10 and 70 deg is used by VCS instruments. The scattered light gives information about cell surface and granularity

44 3D Data Analysis Lymphs Monos Basos NRBCs Eos Neuts

45 ADVIA TECHNOLOGY WBC and Differential  Peroxidase Channel Stain Cells With Peroxidase :Eosinophils- Strong Staining :Neutrophils- Medium Staining :Monocytes- Weak Staining :Lymphocytes and Basophils- No Staining :Large Unstained Cells (LUC) No staining  Also Measure Cell Size Using Low Angle ScatterPlot 2D  Scattergram To Give 4 Part Differential Eosinophils Neutrophils Monocytes LUC Lymphocytes + Basophils Perox Activity Volume

46 The ADVIA WBC differential is calculated from a 3 step process. Cells are stained by peroxidase reagent and analyzed for size and peroxidase stain intensity. Cell specific lysis reagents are used to separate basophils from all other white cells. Basos are subtracted from the lymph/baso cluster in the perox channel to calculate the lymphs. ADVIA TECHNOLOGY

47 Sysmex X-class analyzers- Fluorescence flow cytometry

48 Fluorescence flow cytometry- (light scatter and fluorescent dyes)

49 49 Differential- FSc vs SSc (baso channel) SFL vs SSc (diff channel)

50 ACAS / Centroids SSC SFL Ghost Neut + Ba Mono Lymph Eo 1. The first centroids are provided: The starting position of centroids has been determined from thousands of samples. These values are stored in the instrument and are used as the starting position for cluster analysis.

51 ACAS / Mahalanobis Distance 2. Cluster analysis of scattergram If a cell is detected, distances between this signal and the given centroids are calculated (Mahalanobis distance). This distance reveals to which given cell population the signal belongs. SSC SFL Ghost Neut + Ba Mono Lymph Eo 1. calculated centroids Mahalanobis- Distance

52 Differential fluorescent staining- Immature granulocytes(Sysmex)

53 Diff scattergram: IG positive vs IG negative IG MASTER

54 Reticulocyte parameters (RET channel)  Separate channel  Polymethine dye stains N.A. in WBCs, nRBCs, retics & platelets.  Size vs fluorescence  Retic count

55 Retic channel  Reticulocyte count  Reticulocyte fractions (LFR, MRF, HRF)  Immature reticulocyte fraction  Ret-He (reticulocyte Hb content)  Platelet –O (fluorescent platelets)  Fragmented red cells (FRC) An example of efficient multitasking!!

56 New haematological parameter can predict iron deficiency where classical serum tests fail Reticulocyte Haemoglobin Equivalent--- Ret-He CHr- Siemens (FDA approved)

57 Ret-He  Ret-He - Hb content equivalent of reticulocytes  Units of “pg”(normal range- 28 – 35 pg)  Monitors state of iron supply during the course of erythropoiesis, provides information on availability of functional iron.  Can classify hypochromic anemia, classical vs functional ID, helps to select optimal therapy & to monitor response to EPO and iron treatment.

58 Spurious platelet counts- Siegenthaler & Spertini, NEJM May 2006  48 yr old M with severe burns  Automated CBC- Hct- 37%, MCV-91fl, RDW- 15.8%, WBC-7,400/ul, PLT count /ul  PS – microspherocytes and spherocytes  Manual platelet count- 85,000/ul

59 Overcoming the problems with impedance counting  Manual method- haemocytometer with phase contrast (Time consuming, laborious, operator dependency is more)  Flowcytometric method using RBC/PLT ratio (anti CD41, anti CD61) Am J Clin Path 2001 (Expensive, requires a flowcytometer and experience with FCM)

60 Optical (fluorescent) platelets (good correlation with reference methods) Sysmex fluorescent PLT-O results are unmatched by ordinary platelet technologies of other analyzers

61 Fluorescent PLT (platelet- O) Microcytic RBC Giant PLT

62 PLT abn. Distribution giant thrombocytes

63 IPF – Immature Platelet Fraction Immature PLT are identified by its increase in fluorescence (more RNA), FSC is also higher. IPF

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67 Peripheral smear examination still required!!

68 Thrombocytopenia

69 Platelet clump(EDTA induced)

70 Platelet count after collection in citrate !!

71 Criteria for smear review  Smear review- increases manual work, reduces TAT & productivity  Need to reduce smear review rate without risk of missing anything significant  Different labs – different criteria  Criteria depend upon patient population, type of analyser in use, etc.

72 Consensus rules for smear review  International consensus group for hematology review – ISLH, 2002 ( Dr. Berend Houwen)  Laid down rules for action following automated CBC including smear review  Rules tested in 15 labs (13,298 samples)  Data analysed, rules refined, 43 rules laid down  Guidelines for individual laboratories

73 Review : Criteria for automated CBC & WBC diff analysis

74 Examples of some consensus rules (Lab Haematol, 2005) Rule noParameterPrimaryAnd/orAction 1neonate1 st sampleSlide review 10MCV 105flSpecimen <24hrs old Slide review 15RDW>221 st timeSlide review 16No WBC diff/incomplete Manual diff & slide review 7Platelet st timeSlide review

75 flaggednormal Analyser Routine technician ? Abnormal RBCs ? Atypical mononuclear WBCs Common RBC abnormality Granulocyte left shift Atyipcal (variant) lymphocytes normoblasts Sr. technician ? Blasts ? Organisms Myelocytes Plasma cells Dohle bodies Targets Auer rods Physician Diagnostic cells Report Examiner Differentiation Hierarchial blood film evaluation

76 QUIZ-

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81 Fragments ?

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83 Eosinophilia

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85 Iron Deficiency Anemia

86 Photo of slide

87 Lymphocytosis

88 Summary  Automated cell counters-backbone of the diagnostic laboratory  Fast, accurate, precise  Impedance and various other technologies  All have various limitations  Accurate information on the technologies helpful to recognize problematic areas  Maintenance, calibration, QC procedures  Slides still need to be reviewed (criteria) Finally………

89 IT IS THE MAN BEHIND THE MACHINE WHO MATTERS MOST!! THANK YOU!!


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