Automated cell counters: principle and types

Automated cell counters

1. BY: SIVARANJINI. N GUIDE : DR. YOGITA.V. KARELIYA AUTOMATED CELL COUNTERS: PRINCIPLE AND TYPES

2. The complete blood count (CBC) including DLC are the backbone of any laboratory evaluation and provide valuable information to diagnose anemia, acute and chronic illnesses, white cell disorders, leukemias and platelet disorders.

3. Haematologic evaluation nearly always requires- Enumeration of the formed elements of the blood Determination of the morphology of each type. Manual methods are - time consuming, - tedious - imprecise.

4. Wallace H. Coulter an electrical engineer and businessman while working with the US Navy in the late 1940s developed and first applied the Coulter Principle

5. TYPES OF CELL-COUNTERS THREE PART FIVE PART SEVEN-PART Differentiate cells into three categories Granulocytes Lymphocytes Monocytes/mixed cells Differentiate cells into the five basic Leukocyte types 1.Neutrophils 2.Eosinophils 3.Basophils 4.Lymphocytes 5.Monocytes In addition are able to distinguish   1.Nucleated RBCs, Abnormal and atypical cells and immature cells

6. ADVANTAGES Speed with efficient handling of large number of samples. Accuracy and precision in quantitative blood tests. Ability to perform multiple tests on a single platform. Significant reduction of labor requirements. Invaluable for accurate determination of red cell indices .

7. DISADVANTAGES Flagging of a laboratory test result demands labour intensive manual examination of a blood smear Comments on red cell morphology cannot be generated Platelet Clumps are counted as single ,so low count. Erroneously increased or decreased results due to interfering factors Expensive with high running costs

8. Principle Electrical impedance Optical light scatter Fluorescence Light absorption Electrical conductivity

9. ELECTRICAL IMPEDANCE

10. COULTER PRINCIPLE The poorly conductive blood cells are suspended in a conductive diluent (liquid). The diluent is passed through an electric field created between two electrodes. The liquid passes through a small aperture (hole). The passage of each particle through the aperture momentarily increases the impedance (resistance) of the electrical path between the electrodes. The increase in impedance creates a pulse that can be measured.

11. The number of pulses = blood cell count The amplitude (height) of the pulse = Volume of cell Depending on this, frequency distribution curves and size distribution histograms are plotted. Threshold limit based on size of RBCs, WBCs and platelets are established to separate individual cells. Applied in 3 part differential count

12. RBC SIDE DILUTION (1:50,000) >30fl size RBC 2-30fl size PLATELETS

13. WBC SIDE DILUTION (1:500) 35-90 fl lymphocytes 90-60fl Monocytes, eosinophils, basophils 160-450fl Neutrophils LYSING REAGENT

14. Discrimination threshold Moving or fixed discriminator separates the distribution curves for the volume. E.g., Platelets: 8-12 fl, (range is 2-30 fl) RBCs: 80-100 fl (range is 40-250) WBC: 30-300 fl (after RBC lysis) subdivided into, Lymphocytes: <50 fl Neutrophils: 100-250 fl Intermediate forms (eosinophils, basophils, monocytes) : 50-100 fl (T1-T2)

15. The Size Distribution Curve should always start on the base line and fall between the lower and the upper discriminator

16. OPTICAL SCATTER

17. FORWARD ANGLE LIGHT SCATTER Illuminating beam that has been bent to a small angle from direction of the original beam .It measures size or volume of cells

18. SIDE SCATTER The illuminating beam that is scattered by particle to an angle of 90* from the illuminating beam. This depends on cell's surface texture and internal structure as well as to its size and shape and granularity. It is sometimes referred to as a granularity signal or an orthogonal light scatter signal

19. The patterns of scatter are measured at various angle. Scattered light provides information about cell structure, shape, and reflectivity. These characteristics can be used to differentiate the various types of blood cells and to produce scatter plots(scattergram) with a five-part differential

20. OTHER METHODS Peroxidase based counter : Used to count neutrophils. Lymphocytes are not stained. Fluorescence based : Reticulocyte and platelet count. Immature platelet are detected best. Immunological based: Accurate platelet count using CD41 and CD61 antibodies.

21. ERRORS 1. Recirculation error Cells that re circulate through the edge of an electrical field produce an aberrant impulse, which is smaller than cell passing through the aperture.  2. Coincidence error  Cells that pass through the aperture simultaneously, or almost so, are counted and sized as a single large cell called coincidence. 3 . Non central flow error Cell pass through the aperture off centre produce aberrant impulses and appear larger than their actual size

22. HYDRODYNAMIC FOCUSING AND LAMINAR FLOW OVERCOMES THESE ERRORS

23. Principles of Measurement Direct Measurement: RBC –Impedance, hydrodynamic focusing WBC -Impedance, hydrodynamic focusing Platelets –Impedance (2-20 fl), hydrodynamic focusing Hb–mod. Cyanmethemoglobin(525 nm) MCV –mean RBC volume (histogram) Indirect Measurement: Hct, MCHC, and MCH (calculations) RDW and MPV (CV of respective histograms) WBC differential VCS –volume, conductivity, scatter; employs differential shrinkage Reticulocyte Supravital stain Fluorescent detection

24. Parameters provided by automated hematology analyzers Hemoglobin ( Hb ) Hematocrit ( HCT ) RBC count ( RBC # ) Platelet count ( PLT # ) WBC count ( WBC # ) WBC differential ( WBC % ) MCH (Hb/RBC #) MCV (HCT/RBC #) MCHC (Hb/HCT) RDW (RBC volume) MPV (Plt TV/PLT) Histograms (RBC, WBC & PLT)

25. Hemoglobin estimation Blood mixed with RBC lysing agent Cyan-meth Hb method Sulph -meth Hb method

26. Red Cell Indices

27. Hematocrit calculation The volume of the red cells as compared to the volume of the whole blood sample. The volume of each red cell is measured as it is counted and a mean cell volume is derived. i.e. (MCV) multiplied by the red cell count (RBC(. Hematocrits are reported in L/L or the traditional %.

28. Correlating Hemoglobin and Hematocrit Values The hemoglobin times three roughly equals the hematocrit in most patients. Example : 14.8 x 3 = 44 (patient's hematocrit result is 45 L/L) 11.0 x 3 = 33 (patient's hematocrit result is 32 L/L) The exception to this rule is in patients with hypochromic red cells. These patients will have hematocrits that are more than three times the hemoglobin

29. MCV The counter provides us with MCV which is derived from the histogram (sum of pulse height / sum of pulse) MCH is Mean Corpuscular Hemoglobin weight in picograms. This is the average weight of the hemoglobin in picograms in a red cell. MCH =hemoglobin in pg/L / red cell count in millions/L MCHC is Mean Corpuscular Hemoglobin Content. This indicates the average weight of hemoglobin as compared to the cell size. MCHC = (Hemoglobin in g/dL / HCT) x 100

30. X axis: Volume of cells Y axis: No. of cells LD: 25-75 fLUD: 200-250 fL MEDIAN = MCV

31. RDW : The RDW (red cell distribution width) is a measurement of the width of the bases of the RBC histogram. MPV : The MPV is a measure of the average volume of platelets in a sample and is analogous to the erythrocytic MCV. Pct : (plateletcrit) analogues to HCT for RBCs

32. Known interfering substances RBCs High WBC count esp. with low RBC count Agglutinated RBCs Hemoglobin Increased turbidity of sample: Leukocytosis, Lipemic sample, Fetal blood. MCV RBC aggregation Large platelets

33. Hematocrit : RBC agglutination RDW Nutritional deficiencies Blood transfusion RBC agglutination

34. Interference with WBC counts High TC Normoblasts Lyse- resistant RBCs In certain leukemias increased cell fragility can give rise to low counts Increased Lymphocytes Nucleated RBCs Lyse resistant RBCs

35. Increased monocytes Increased large lymphocytes Blast cells Increased neutrophils Blast cells Eosinophilia

36. Interference with platelet counts High count RBC fragments Microcytic RBCs WBC fragments Low count Platelet fragmentation i.e.. in chemotherapy Platelet agglutination

37. RBC histogram Main RBC population RBC doublets

38. Platelets have a size between 8 and 12 fl and are counted between 2 and 30 fl. Erythrocytes have a size of 80-100 fl and are counted between 25 and 250 fl.

39. RBC Normal Histogram RBC histogram starts at 50fl and ends at 150fl

40. Discriminant functions in thalassemia

41. RU-Flag Normoblasts Cold agglutinins: disappears on incubating.

42. RL- Flag When LD exceeds preset height by more than 10 % Large platelets Fragmented RBCs Platelet aggregation

43. MP-Flag (multiple peaks) Blood transfusion Dimorphic anaemia Treated IDA

44. Platelet histogram

45. The histogram curve should lay within the lower and upper platelet discriminator (PL & PU) and start and end on the base line. PLT counted between 2 fl and 30 fl. 1 flexible Discriminator PL 2 to 6 fl. 1 flexible Discriminator PU 12-30 fl.  1 fixed Discriminator at 12 fl

46. PL-Flag When LD exceeds preset ht. by 10%. Cell Fragments Contamination Bacteria Noise

47. PU-Flag When UD exceeds preset ht. by > 40%. Clotted Blood Fragmented RBCs Pseudo-thrombocytosis Large platelets

48. WBC HISTOGRAM

49. WBC Normal Histogram The WBC graph shows a normal distribution of Lymphocytes, Monocytes and Granulocytes. Lympho Mono Granulo

50. WL-Flag Curve does not start at base line Causes : Platelet aggregation Lyse resistant RBCs cryoaggutinates nRBCs. Giant platelets

51. WU- Flag Curve does not end at baseline Immature WBCs Hyperleucocytosis

52. T1 Flag When discrimination b/w lymphocytes & middle cells is not possible.----T1 is not found Abnormal leukocytes in CML

53. T2 Flag When discrimination b/w neutrophils & middle cells is not possible.----T2 is not found. Higher WBC count in CML.

54. F flags No other flags are present But valleys are far from base line F1(small cell inaccurate):Height of T1 exceeds limit of 40% ALL F2(medium cell inaccurate):Heights of T1 & T2 exceeds limit of 40% & 50% Respectively AML, Eosinophilia, monocytosis F3(Large cell data inaccurate): Height of T2 exceeds limit of 50%

55. FIVE PART AND SEVEN PART COUNTERS

56. VOLUME =SIZE CONDUCTIVITY = INTERNAL COMPOSITION SCATTER= CELL SHAPE/ SURFACE

57. VOLUME VCS utilizes the Coulter Principle of (DC) Impedance to physically measure the volume that the entire cell displaces in an isotonic diluent. This method accurately sizes all cell types regardless of their orientation in the light path.

58. CONDUCTIVITY Alternating current in the radio frequency (RF) range short circuits the bipolar lipid layer of a cell's membrane allowing the energy to penetrate the cell. This powerful probe is used to collect information about cell size and internal structure, including chemical composition and nuclear volume.

59. SCATTER When a cell is struck by the coherent light of a LASER beam, the scattered light spreads out in all directions. Using a proprietary new detector, median angle light scatter (MALS) signals, are collected to obtain information about cellular granularity, nuclear lobularity and cell surface structure

60. BASOPHILIA

61. NEWER PARAMETERS Cellular Hb Concentration Mean(CHCM): Uses Light scatter technology.  True estimate of hypochromia in IDA. Hb Distribution Width: Degree of variation in red cell hemoglobinization. Range-1.82 to 2.64.   Nucleated Red Cells:   nRBCs identified, Separated & corrected count obtained. WBCs have high fluorescence & forward scatter)  P-LCR(Platelet Large Cell Ratio): % of platelets with a volume >12fl. Due to platelet aggregates, microerythrocytes, giant platelets.   Reticulated Platelets /Immature Platelet Fraction(IPF): Newly produced platelets that have remains of RNA in their cytoplasm. Reflects rate of thrombopoiesis

62. RETICULOCYTES

63. RETICULOCYTE INDICES Various dyes & flurochromes bind with RNA. RNA content- 3 Maturation stages; LFR,MFR & HFR  Immature reticulocyte Fraction(IRF): Sum of MFR & HFR. Early and sensitive index for erythropoeisis.   Reticulocyte Hb Equivalent(RET-He ): Hb content of freshly prepared RBCs. Real time information on Fe supply to erythropoiesis. Early detection of Fe deficiency. Differentiate IDA & ACD. Monitoring of erythropoietin & Fe therapy.  

64. Technical operations

65. Before starting sample run…. Two essential procedures : Calibration Quality control.

66. Accuracy: Refers to closeness to the true value. It implies freedom from error. Precision: Refers to reproducibility of test. It implies freedom from variation.

67. *Calibration* (setting accuracy) Calibration -is done to standardize the instrument for accuracy. Calibrator -Certified reference material (CRM) used to calibrate a measurement on an analyzer. Calibration factors -If any deviation from calibration references is observed necessary calibration correction factors are applied to set the accuracy of the instrument

68. When to Calibrate You should calibrate your instrument : At installation. When a different or new control material is used. When results have changed.

69. Calibration should be done at least once per year as per NABL guidelines Automated hematology analyzers should be calibrated using calibrators provided by manufacturers Controls lack accuracy so not recommended as calibrators If calibrators are not available then controls must be assigned values reliably by reference method and can be used as calibrators

70. Pre calibration check Total maintenance of the instrument Reagents(replenish or replace) Calibrator(check for expiry) Calibration procedure Done by company personal. Precision check Carryover check Calibration in both (open and closed modes) Post calibration validation Run calibrator as samples Run 3 level controls

71. PRECISION CHECK (N=10) Sample Requirements - For reproducibility studies, ensure the patient for the sample that is being tested: Is receiving no medication Has normal hematologic parameters, with a WBC count of 5.0 ± 1.0. Has normal erythrocyte, leukocyte, and platelet morphology and, if you want to check the Diff parameters, with Diff values Neutrophils 40 to 72% Lymphocytes 17 to 45% Monocytes 4 to 12% Eosinophils 0 to 10% Basophils 0 to 1% Ensure you have enough normal whole blood from a single donor for 11 cycles.

72. Quality Control

73. QUALITY CONTROL QA is the sum of all those activities in which the laboratory is engaged to ensure that information generated by laboratory is correct. QA is not restricted to the development and retention of quality control charts but rather includes all aspects of laboratory activities that affects the results produced, from the choice of methods, to the education of personnel, to the handling of specimens and reporting results.

74. PURPOSE OF QC Assure proper functionality of instrumentation Means of assuring accuracy of unknowns Monitoring the integrity of the calibration When controls begin to show evidence of unusual trends When controls exceed the manufacturer’s defined acceptable limits

75. QA in hematology lab is intended to ensure the reliability of the lab tests . The objective is to achieve precision and accuracy

76. 3 components of QA programme : 1 ) Internal Quality Control ( IQC ) 2 ) External Quality Control ( EQC ) 3 ) Standardization

77. CONTROLS Prepared in house or obtained commercially. 10 consecutive values of control recorded and Mean & SD calculated. 3 concentration (high, normal, low) of control to be analyzed. The results are plotted on a graph to create Levey-Jennings Chart .

78. Retained sample testing Retained sample is yesterdays patient sample having normal value for the measurable parameters and preserved at 4’c The same preserved sample is run on next day after the commercial control and values are compared with the yesterdays value. The values should fall within the acceptable limit for the observed parameters. i.e. for WBC+/- 0.75 ,Hb+/-0.4 and platelet +/-30. Then the same sample can be used through out the day as a control for monitoring the performance of the analyser.

79. Continuous detection and rectification of the Analytical Process. Reagent-Equipment-Personnel-Specimen Ensure the degree of both precision & accuracy of your results Assure the quality and clinical applicability of your laboratory reports Generate objective evidence of your analytical performance. Impact of Internal Quality Control

80. 2 ) External Quality Control ( EQC) {Proficiency Testing} Is the objective evaluation by an outside agency of the performance by a number of laboratories on material which is supplied specially for the purpose is usually organized on a national or regional basis By comparing results from different laboratories it is possible to establish between-laboratory and between-method performances. Use: Quality assessment of laboratory. Identifying and recommending best methods for various tests.

81. INTER-LAB QUALITY CONTROL Regional level external quality control. We send same sample after running it in our lab for testing into another lab. Results are compared. If they are in acceptable limit, then it confirms accuracy and precision of lab. If results are out of acceptable range, then we must take corrective actions like calibration.

82. EQAS External quality assurance services National level external quality control Reference sample are sent by AIMS to numerous laboratories of the country. Mean and SD is calculated from the results. Proficiency of the labs is evaluated from whether their results are within the range of mean +/- 2SD or not.

83. 3 ) Standardization Refers to both materials and methods A material standard or reference preparation is used to calibrate analytic instruments and to assign a quantitative value to calibrators A reference method is an exactly defined technique which provides sufficiently accurate and precise data for it to be used to assess the validity of other methods International reference preparations are not freely available for routine use but are intended to act as standards for assigning values to commercial ( or lab produced ) ‘secondary standard’ or calibrators

84. Thank you