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- 1. AUTOMATED RETICULOCYTE ANALYSIS Fery Soedewo Dept.of Clinical Pathology Airlangga University Medical School/ Sutomo General Hospital 2011 7/18/2011 1
- 2. Reticulocyte - The last immature erythrocyte stage - Spend 2-3 days in marrow and 1 day in circulation → mature erythrocyte - Contain remnant cytoplasmic RNA and organelles (mitochondria & ribosomes) - Retics = non-nucleated erythrocyte that contains ≥ 2 particles of blue-stained granulofilamentous material after NMB- staining 7/18/2011 2
- 3. Fig. 1. Reticulocyte ( New Methylene Blue staining) 7/18/2011 3
- 4. - The amount of reticulocytes in circulation reflects erythropoietic activity . - Retic.maturation started from extrusion of orthochromatic normoblast nucleus , to complete loss of ribosomes and RNA ,is thought to take 4 days (only the last day occurs in the circulation ) 7/18/2011 4
- 5. Fig. 2 . Erythropoiesis stages 7/18/2011 5
- 6. 1930 : Heilmeyer classify 4 groups of reticulocytes : Grup 0 - normoblast Grup I - Reticulum as a clumped precipitate (0.1%) Grup II - Reticulum as a form of wreath ( 7% ) Grup III - show an opened wreath ( 32% ) Grup IV - only shown a few granules of the reticulum ( 61% ) 7/18/2011 7/18/2011 6
- 7. Fig.3. Stages of Red Cell Maturation 7/18/2011 7
- 8. Fig. 4 . Heilmeyer’s reticulocytes maturation stages 7/18/2011 8
- 9. Retic.count Reporting Methods 1. Percentage : % Retics/ 1000 red cells ( N: 1±0.5%) 2. Corrected Retics - correction made for anemic patients - increased retic.count : → increased of retics in circulation → decreased of red cells in circulation - observed retic.count corrected to normal Hct / PCV (0.45) 7/18/2011 9
- 10. Corrected Retic.count = patient’s PCV observed retics(%) x ------------------ 0.45 7/18/2011 10
- 11. 3. Absolute Retic.Count 4. Retic.Production/Maturation Index : - peripheral retic.number is combination of the rate of release of retics from marrow and the degree of immaturity of freshly released retics . 7/18/2011 11
- 12. In ↑ erythrop’s stimulation : 1. younger retics (shift cells) released into circulation ( = basophilic macroretics) 2. shortened retic.maturation time in marrow and longer maturation time in circulation 7/18/2011 12
- 13. Fig. 5. Reticulocyte’s Maturation time 7/18/2011 13 13
- 14. Reticulation Production/Maturation Index = Pt’s PCV observed retic.count (%) x ----------- 0.45 ------------------------------------------------- maturation time in circulation 7/18/2011 14
- 15. Younger Retics (Shift Cells) - Reticulocyte = red cell containing ≥ 2 stained intra erythrocytic particles - Circ.Retics are not distinguishable from mature red cells morphologically in Wright-stained smears - Young retics (shift cells) have the greatest quantity of RNA 7/18/2011 15
- 16. Young retics (Shift cells) Young retics have a bluish cast after fixation and staining with Wright’ stain, larger than mature red cells , called Polychromatophilic macrocytes ( N: < 5% of total Retics) Polychr.macrocytes = a good indication of EPO-mediated increase in erythropoiesis 7/18/2011 16
- 17. Young Retics (Shift Cells) Hb level of 10.5 g/dl (Hct 31%) is the critical threshold for increased Polychrm.macrocytes 7/18/2011 17
- 18. Assessment of Manual Retic.Count : - High interlab’s CV (25-48%) due to : 1. interobserver variation in retic’s definition 2. the number of red cells evaluated 3. the types of blood film stained 4. the use of standard area-reduction device (Miller disc) 7/18/2011 18
- 19. Table 1. The number of counted erythrocytes and reticulocytes’ precision Retic.count(%) CV 2% CV 5% 1 27,700 4,500 3 11,100 1,350 5 7,750 1,100 10 5,000 900 20 4,000 650 30 3,500 550 40 3,000 500 7/18/2011 19 19
- 20. Manual reticulocytes count : - imprecise - inaccurate - labour intensive → ↓ cannot yield a quantitative measurement . 7/18/2011 20 20
- 21. Clinical interests ↓ only on ↑ retics (in hemorrhage, hemolysis, hematinic therapy’s response) ↓ imprecision/inaccuracy was tolerable 7/18/2011 21 21
- 22. Modern medicine → increase of reticulocyte’s clinical utilities → need more precise & accurate retic.counts 7/18/2011 22 22
- 23. Reticulocyte’s Clinical Utilities 1. For hematological diagnosis : - Classify anemic patients - Assess bone marrow’s function - Aplastic crisis - Myelodysplastic Syndrome (MDS) - Hemorrhages or hemolysis 7/18/2011 23
- 24. 2. Treatment Monitoring : - In EPO therapy - As an indicator of Marrow’s regeneration after chemotherapy or BMT - Timing the Stem Cell harvest 7/18/2011 24
- 25. Automated reticulocytes count is more accurate ( Manual : CV › 25% → 5-7%) Blood cell counters permit precise measurements of RNA content and cellular indices (Volume, Hb-concentration and Hb- content) 7/18/2011 25 25
- 26. CV from manually Reticulocyte count & Analyzers : CV CV Manual Automatic Reticulocyte 1% 47.3% 6.4% Reticulocyte 9% 27.2% 5.8% 26 7/18/2011 26
- 27. Hematology’s Automation - 2 basic principles operation of Hematology Analyzers : 1. Electronic Impedance / low-voltage DC resistance (Coulter, 1950s) 2. Optical Scatter 7/18/2011 27
- 28. Reticulocyte’s Automation Earlier there were only 2 automatic methods for counting reticulocyte : - Computer-controlled automated microscope for blood smear analysis (early ’80s) - Flow cytometric methods 28 7/18/2011 28
- 29. Automatic microscope → analogue with manual light-microscope, and scanned automatically NMB-stained blood films using a pattern recognition devices . → a good equipment for its better reproducibility , the analysis results is as good as the manual methods . Unfortunately it is not so popular . 29 7/18/2011 29
- 30. Automatic retic. Count method using acridine-orange fluorescence (1952) → the dye binds to retic’s ribosomal-RNA → fluorescent in UV-light . → the intensity of fluorescence is proportional to the RNA present → ~ maturity of the reticulocytes . There is satisfactory agreement between manual and automated methods in categorizing the 4 Heilmeyer maturation groups 30 7/18/2011 30
- 31. Fluorescence staining combined with flow-cytometry → led to the new automated systems for reticulocyte counting . Several different dyes have been used for flow cytometric retics count . 31 7/18/2011 31
- 32. Flow cytometric Retic.counting - Many fluorochromes have been used , i.e : - Acridine orange - Auramine O - Di-methyloxacarbocyanide - Ethidine bromide - Pyronin -Y - Thioflavine-T - Thiazole orange most require 30 minutes incubation → semi- automated ? 7/18/2011 32 32
- 33. - Ethidium bromide at relatively high pH require only few minutes to enter the cells - Auramine O requires only a few seconds 7/18/2011 33 33
- 34. Cell Information - FCM Side scatter Information on internal structure Dichroic mirror Fluorescence Information on amount of Laser RNA and DNA Beam Forward scatter Information on cell size 7/18/2011 34 34
- 35. Table 2. Evolution on parameters’ content of CBC No Parameters Year 1 Hb, Hct, RBC, WBC 1950s 2 MCV, MCH, MCHC 3 PLT,RDW,PDW,MPV,Pct, P-LCR,RDW-SD 4 LUC,CHCM,HDW,LI,MPXI 1960s 5 LYM,MONO,GRAN (%,#) 1970s 6 NEUT,EOS,BASO (%,#) 1980s 7 FLAGGING 8 RETICULOCYTE COUNT 1990s 7/18/2011 35 35
- 36. Current Options on Reticulocyte Counting : Method Dye Technique Usage CV-% Microscopy, Supravital NMB/BCB Manual ± 70% 25 Flow Cytometry Thiazole O Fluorescence < 1% 15-20 Flow Cytometry NMB, Optical scatter ± 10% 5-10 spered Flow Cytometry Oxazine 750 Optical scatter ± 4% 5-10 Flow Cytometry Auramine O Fluorescence ± 5% 5-10 7/18/2011 36
- 37. Retic.count methods on Hematology instruments Coulter Sysmex Abbott Bayer LH-750 XE-2100 CD-4000 Advia 2120 Supravital staining Supravital staining CD4K530 stain Supravital staining (NMB) ; Volume, (Auramin O) Multiangle scatter (Oxazine 750) Conductivity, Fluorescent and Fluorescent Low-angle (2-30) Optical Scatter detection detection and High-angle (5- (VCS technology) 150, optical scatter and absorbance 7/18/2011 37
- 38. The improved precision for Flow Cytometric methods arises from : 1. Removal of inter-observer variation 2. The larger number of cells counted (10000 – 30000 RBC events , compare to 1000 for the visual method) 3. The fluorescence measured is proportional to the amount of RNA present in the cell 7/18/2011 38 38
- 39. How the methods inform the maturation of Retics ? - The analyzers divide the retic area on the scattergram by 2 vertical discriminators → producing 3 populations : - Low Fluorescence Ratio (LFR) – the most mature forms - Middle Fluorescence Ratio (MFR) - High Fluorescence Ratio (HFR) – the least mature forms 7/18/2011 39 39
- 40. Fig. 6. Frequency’s curve of red cell maturation 7/18/2011 40
- 41. Reticulocyte’s Automation Fluorescence-based (Thiazole Orange, Auramine O, fluorescence’dyes) Absorbance-based (Oxazine O, New Methylene Blue, nucleic acid dyes) Interacting with RNA Immature Retics high fluorescence Thiazole Orange overestimated because of DNA / RNA content of another cells/components 41 7/18/2011 41
- 42. Table 3. Reticulocyte’s parameters in various hematology analyzers Methods Parameters CD 4000 Fluorescen (Auramine O) 3 Retic(%,#), IRF (Abbott) GEN-S Absorbans (New 4 Retic(%,#), (Coulter) Methylene Blue) IRF,RMI SE-9000 Fluorescen (Auramine O) 5 Retic(%,#), (Sysmex) HFR,MFR,LFR Advia 120 Absorbans (Oxazine 750) Retic(%,#),CHr, (Bayer) 8 MCVr, RDWr, HDWr,CHCMr, CHDWr 7/18/2011 42 42
- 43. Fig. 7. Reticulocyte Analysis High angle detector (5o - 15o) 670nm Laser Diode Absorbance RNA Content Oxazine 750 RNA Stain Low angle detector (2o-3o) 43 7/18/2011 43
- 44. Fig.8. Reticulocyte Technology Reticulocytes are stained with a nucleic High Angle (5-15 degrees) acid dye - Oxazine 750 Scatter and Absorption are measured using laser channel RBC and Reticulocyte indices are measured simultaneously Oxazine 750 Absorption 8 44 7/18/2011 44
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- 46. Fig. 9. Reticulocyte Cytogram and Relationship to Maturity High Angle (5-15 degrees) Oxazine 750 Absorbance 46 7/18/2011 46
- 47. Fig. 10. CBC results 7/18/2011 47
- 48. - Sysmex methods (R-3000/3500) : Sysmex use supravitally-fluorescence dye , Auramine-O . Forward-scatter (measuring size) and side-fluorescence intensity(measure RNA’s content) 48 7/18/2011 48
- 49. forward light scatter & side fluorescence scattergram red cells, reticulocytes, platelets area Reticulocytes area : LFR (low fluorescence ratio) MFR (middle fluorescence ratio) HFR (high fluorescence ratio) IRF (Immature Retic.Fraction) = MFR + HFR LFR : Heilmeyer III and IV Flow-cytometry increase retics.count precision Retic. 1% -- CV manl= 47.3%; automt = 6.4% Retic. 9% -- CV manl= 27.2%; automt = 5.8% 49 7/18/2011 49
- 50. - Bayer’s method (Advia-120) : Bayer uses Oxazine-750 , a Nucleic-acid- binding dye . Erythrocyte changed into spherical-isovolumic Measurement using 3 detectors : 1. Low-angle-scatter (2-3o) 2. High-angle-scatter (5-15o) 3. Absorbance 50 7/18/2011 50
- 51. From these 3 detectors → 3 cytograms : 1. High-angle scatter vs Absorbans → LAC/MAC/HAC , IRF (MAC + HAC) 2. High-angle vs Low-angle-scatter (RBC map) → Reticulocyte’s indices 3. Volume vs Hb-concentration → CHr 51 7/18/2011 51
- 52. - Automated Reticulocyte’s Parameters : Retics # and % HAC, MAC, LAC (Absorbance/Fluorescence Ratio) IRF (Immature Retics Fraction = HAC + MAC) Retic’s indices (MCVr, MCHr, CHCMr, CHr, RDWr, HDWr) 52 7/18/2011 52
- 53. Fig. 11. CBC result ( normal patient ) 7/18/2011 53
- 54. Fig. 12. CBC results ( Reticulocytosis ) 7/18/2011 54
- 55. - Content of Retic’s Hb (CHr) : Content of retic’s Hb never changed as long as the survival of Retics and red cells The CHr’s mean : 28.5 pg CHr/CH ratio ± 1 ( range : 0.96-1.03) The meaningful of CHr : - indicate Iron availability real-time . - as a strong predictor for Iron Deficiency - Early indicator for Iron therapy in Iron Deficiency Anemia . 55 7/18/2011 55
- 56. - Reticulocyte’s Size/Volume (MCVr) : Retic’s size drastically decreased along its maturation ( Heilmeyer’s clasification ) MCVr/MCV ratio = 1.24 ( constant in normal, microcytosis or macrocytosis) Stress Retics : MCVr/MCV ratio = >1.5-3 Inverse MCVr/MCV ratio (=< 1) → seen in Vit.B12 therapy’s response in Megaloblastic Anemia . 56 7/18/2011 56
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- 58. Fig. 13. Red Cell Analisys 7/18/2011 58 58
- 59. Quantitation of : - % Microcytic and % Macrocytic - % Hypochromic and % Hyperchromic ↓ Differentiate between β-thal trait and Iron Deficiency Anemia 7/18/2011 59
- 60. - Differential Diagnosis of β-Thal trait and IDA : In β-Thal trait : The microcytosis is more significant compared with mild hypochromia . In Fe Deficiency Anemia : The hypochromia is more significant compared with mild microcytosis . 7/18/2011 60 60
- 61. Ratio % M/H (Micro/Hypo) Ratio % M/H > 0.9 → β-Thal trait Ratio % M/H < 0.9 → IDA CHr combined with Ratio % M/H give stronger differentiation ; Ratio % M/H < 0.9 with Low CHr give strong prediction for IDA 7/18/2011 61
- 62. Clinical Applications of Retic’s Indices - Retics = the first erythroid cell appeared in circulation and become mature red cell 24 hours later . - Red cell morphological begin to change in the late-stage of Fe deficiency - Retic.Indices reflects a real-time erythropoietic activities 7/18/2011 62
- 63. - When Fe-store is low and erythropoiesis is decreased , red cell indicators are still normal but marrow already release a new retic. with low Hb-content ( low-CHr ) 7/18/2011 63
- 64. - Early detection of Functional Fe Deficiency is by measuring %- Hypochromic and CHr %-Hypochromic reflects Hb concentration during 8-12 weeks . - Fe-therapy responses have already seen from the CHr in 4 days (1-2 weeks) when normally it’s only seen from the increament of Hb after 1 month therapy 7/18/2011 64
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- 67. - Hypochromic Macrocytic Cells ? Reticulocyte ? Dyserythropoiesis / Myelodysplastic syndrome / Sideroblastic anemia Fe-deficient megaloblastic anemia 7/18/2011 67 67
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- 69. Limitations - Most dyes stain also other blood components containing DNA/RNA, so assessed as reticulocytes, i.e : Howell-Jolly bodies, Cabot’s ring, Malaria parasites, white cell’s fragments in Leukemia - Retics may “mature” during storage especially if not refrigerated 7/18/2011 69
- 70. Thank U 7/18/2011 70