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In the heart of hematopoïesis

In the heart of hematopoïesis. HORIBA ABX Pentra range. 1. Leukopoïesis 2. Erythropoïesis 3. Thrombopoïesis. Leukopoïesis exploration. Leukopoïesis - Technologies. 1. WBC counting 2. Differencial leukocytes. Triangulation. WBC channel. Baso channel. DIFF channel.

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In the heart of hematopoïesis

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  1. In the heart of hematopoïesis

  2. HORIBA ABX Pentra range 1. Leukopoïesis 2. Erythropoïesis 3. Thrombopoïesis

  3. Leukopoïesis exploration

  4. Leukopoïesis - Technologies 1. WBC counting 2. Differencial leukocytes

  5. Triangulation WBC channel Baso channel DIFF channel Leukopoïesis - WBC counting

  6. QuaDimension WBC channel Baso channel Double DIFF channel ERB channel Leukopoïesis - WBC counting

  7. 3 reference principles Cytometry - Focalization with double hydrofocusing cytometer Cytochemistry - Marking with cytochemistry (Chlorazol Black) Impedance - Cell volume measurement Leukopoïesis - DIFF

  8. Leukopoïesis - DIFF Cytochemistry The Leucodiff reagent lyses the RBC, stabilizes the WBC's in their native forms and differentially stains the leukocytes. The intensity of the stain is proportional to the nucleic acid quantity, to the maturity of this nucleic acid, to the lipids and enzyme quantity. The absorbance measurement is then proportional to the stain intensity.

  9. Leukopoïesis - DIFF REACTION + WBC Chlorazol Black Eosinofix (P60, P80, P120) Leucodiff (PDX, PDF)

  10. Focused Flow Impedance Measurement of the true cellular volume by impedancemetry after incubation with stabilizing reagent. Leukopoïesis - DIFF

  11. Leukopoïesis - DIFF Double Hydrodynamic Sequential System(DHSS) Exclusive ABX patent The analysis of the white blood cells fixed in their original state by using a specific reagent (Leucodiff TM), is carried out by flow-cytometry with a double hydrodynamic focusing system, characteristic of the DHSS* technological principle. Analysis of the internal structure by measuring the cellular absorbance of light. *DHSS Analysis of cell content (optical absorbance).

  12. Why DHSS is better than single flow 2 points + timing control (co) With a double flow, there is better hydrofocusing. With sequential measurement in the case of clogging: correlation Alarm With a single flow, there is only one measuring point. In the case of clogging or poor flow : no alarm. Leukopoïesis - DIFF

  13. Leukopoïesis - DIFF Why light beam from tungsten lamp is better than Laser beam The laser is a mono-chromatic concentrated light. With its tungsten lamp the ABX optical bench covers almost all wavelengths. multi wavelength High definition screening (Cell’s complexity is explored) One wavelength Poor screening (Cell’s content is ignored)

  14. Eosinophil Neutrophil Monocyte Lymphocyte Leukopoïesis - Double DIFF Matrix Basophil

  15. Leukopoïesis - Double DIFF Matrix Abnormal Neutrophil IMG immature granular Metamyelocyte Myelocyte Promyelocyte Myeloblast

  16. Leukopoïesis - Double DIFF Matrix IMMimmature monocyte Promonocyte Monoblast

  17. Leukopoïesis - Double DIFF Matrix IML immature lymphocyte Lymphoblast Sezary cell Prolymphocyte

  18. Leukopoïesis - Double DIFF Matrix Immature Granulocytes • The slide review is not necessary because the identification of this IMG population is very accurate. • This population is present in case of: • Post-surgical intervention • Bacterial infectious • Sever infectious • AML • Myelodisplastic/Myeloproliferative diseases

  19. Leukopoïesis - Double DIFF Matrix Immature Monocytes • This population is present in case of: • Viral infectious • Infectious mononucleosis • Myelodisplastic/Myeloproliferative diseases • AML

  20. Leukopoïesis - Double DIFF Matrix Abnormal Lymphocytes • To identify the lymphoid pathologies, various flags are important: • ALY: atypical lymphocytes • IML: immature lymphocytes • NL: neutro/lympho (graph flag) • Blast: small blasts

  21. Leukopoïesis - Double DIFF Matrix • 1. Specific and very precise ALYflag (identification and counting) in case of: • Lymphoma diseases • Lymphoproliferative syndromes (ex: Sezary) • LAL diseases • Reactive lymphocytes

  22. Leukopoïesis - DIFF • 2. Specific IML flag (identification and counting) in case of: • Lymphoproliferative syndromes, examples: • CLL, B-cell prolymphocytic leukaemia

  23. Leukopoïesis - Double DIFF Matrix • 3. Specific Nl flag (morphological flag) for example in case of LGL population • 4. Specific “Blast” flag in presence of small blasts for example in case of ALL

  24. Leukopoïesis - DIFF Eosinophilia • The eosinophil population increase in case of: • Intestinal parasites: • Bihlarziose, Ascaris, Oxyure, • Taenia... • Allergies: • Asthma, Eczema... • Chronic eosinophilic leukemia

  25. Leukopoïesis - DIFF Basophilia • The basophil population increase in case of: • Acute basophilic leukaemia • CML

  26. Leukopoïesis - DIFF More information in the DIFF Matrix: Detection of platelets aggregates Detection of Nrbc population Detection of lyse resistant cells Detection of band cells

  27. Evaluation references ABX PENTRA DX 120: EVALUATION OF THE NEW PARAMETERS A side-by-side comparison with manual slide review (Double Matrix / Erythroblasts) and immunophenotyping (Erythroblasts) Dr. Jose Maria JOU Servei d’Hemostàsia i Hemoteràpia Hospital Clínico Universitario de Barcelona, C/ Villarroel nº170, 08036 Barcelona, SPAIN. Myeloid Immaturity and Instrumental Findings AM. Cenci°, B. Casolari°, M. Maconi* °Laboratory of Clinical Pathology, AUSL Agency S. Agostino Hospital, Modena; *Laboratory of Chemical Analysis Clinics, Santa Maria Nuova General Hospital, Reggio Emilia.

  28. Evaluation references Evaluation of the ABX Pentra DX 120 Dr. Francis Lacombe Laboratoire d’Hématologie, Hôpital Haut-Lévêque, France New technologies and study of blood cells: the performance of HORIBA ABX PENTRA DX 120 B. Casolaria, M. Maconib, A.M. Cencia aLaboratory of Clinical Pathology, AUSL Agency S.Agostino Hospital, Modena; bLaboratory of Clinical Pathology, O.I.R.M. Hospital Agency - S.Anna, Turin. Detection of atypical lymphocyte populations and lymphoid pathology diagnosis in adults and children (ALY alarm) P. Lemaire, Laboratoire de Longpont-Sur-Orge (91310) Georges Pompidou European Hospital (75015 Paris), France

  29. Erythropoïesis exploration

  30. Erythropoïesis - Maturation The red blood cells line maturation Proerythroblast Basophilic Erythroblast Polychromatophilic Erythroblast Acidophilic Erythroblast Erythrocyte Reticulocyte

  31. Erythropoïesis - Erythroblasts • Lyse of RBC • Thiazole orange is a fluorochrome specific to nucleic acids (reference method) • The measurement is performed with a flow cytometer (light source: argon-ion laser) • Automatic WBC correction

  32. Erythropoïesis - Erythroblasts Fluorescence Resistivity

  33. Fluorescence Volume Erythropoïesis - Erythroblasts

  34. Erythropoïesis - Erythroblasts Erythroblasts pathologies • The abnormal presence of erythroblasts in blood could indicate: • micro or macrocytic anemia • pregnancy patient monitoring: • erythroblast high value in maternal blood could indicate a • pathological situation • myelodisplasia

  35. Erythropoïesis - Erythroblasts Erythroblasts on ABX Pentra DX 120 • Reflex testing • One fluorochrome ERB + RET • 3 control levels • Good precision (separated channels)

  36. Evaluation references ABX PENTRA DX 120 : EVALUATION OF THE NEW PARAMETERS A side-by-side comparison with manual slide review (Double Matrix / Erythroblasts) and immunophenotyping (Erythroblasts) Dr. Jose Maria JOU Servei d’Hemostàsia i Hemoteràpia Hospital Clínico Universitario de Barcelona, C/ Villarroel nº170, 08036 Barcelona, SPAIN. Automated Nucleated RBC Counting: Comparison to Flow Cytometry. Bruce H. Davis*, Kathleen T. Davis*, Esther Tournier+, Karen Becker* *Trillium Diagnostics, LLC and Maine Medical Center Research Institute, Scarborough, Maine 04074 USA +HoribaABX Diagnostics, Montpellier, France

  37. Erythropoïesis Erythropoïesis - Maturation The red blood cells line maturation Proerythroblast Basophilic Erythroblast Polychromatophilic Erythroblast Acidophilic Erythroblast Erythrocyte Reticulocyte

  38. Erythropoïesis - Reticulocytes Acidophilic Erythroblast Reticulocytes Erythrocytes Enucleation process RNA +++ RNA ++ RNA + RNA -

  39. Erythropoïesis - Reticulocytes • Thiazole orange: specific RNA fluorochrome. • Flow cytometer: light source: argon-ion laser.

  40. 25 sec Erythropoïesis - Reticulocytes Reaction + Reticulocyte fluorescent molecule

  41. Acidophilic Erythroblast Reticulocyte H RNA +++ Reticulocyte M RNA ++ Reticulocyte L RNA + Erythrocyte RNA - Erythropoïesis - Reticulocytes Fluorescence Volume

  42. Erythropoïesis - Reticulocytes parameters Reticulocyte Count : RET (%) and RET (#) Corrected Reticulocyte Count: CRC (%) Maturation Classes: RETL, RETM, RETH (%) Mean Fluorescence Index : MFI (%) Mean Reticulocyte Volume : MRV (fL) Immature Reticulocyte Fraction : IRF (%)

  43. MCV < 80 fl Microcytic Anemia RET Iron MRV 2 days MCV + 3 weeks Erythropoïesis - Reticulocytes interpretation Anemia: Hb < 12 g/dl

  44. 80 fl < MCV < 100fl Normocytic Anemia 0.1x106/l < RET > 0.15x106/l No Regeneration Regeneration ??? Erythropoïesis - Reticulocytes interpretation Anemia: Hb < 12 g/dl

  45. Normocytic Anemia 0.1x106/l < RET > 0.15x106/l ??? Erythropoïesis - Reticulocytes interpretation 2% < CRC >2% Bone Marrow Abnormalities Peripheral Etiologies

  46. MCV > 100fl Macrocytic Anemia Non megaloblastic anaemia Megaloblastic anaemia or Myelodysplasic syndrome 16 % < 18 % < 129 fl < IRF MFI MRV > 16 % > 18 % > 129 fl Bone marrow & cytogenetic Erythropoïesis - Reticulocytes interpretation Anemia: Hb < 12 g/dl Evaluation reference: Cordoba, University Hospital “Reina Sofia”, Dr.Torres Publication: Haematologica 2001; 86:24-29

  47. Erythropoïesis - Reticulocytes Reticulocytes on ABX Pentra 120 range • Reflex testing (+ type) • One fluorochrome ERB + RET • Excellent precision: • 1. RET%, RET#, CRC% • Classifying / Monitoring anaemias (central/peripheral) • 2. RETH, RETM, RETL, IRF, MFI, MRV • Detection/Monitoring of erythropoietic response

  48. Evaluation references The Mean Reticulocyte Volume (MRV) on the HORIBA ABX Pentra 120/DX as an alternative measurement to the Reticulocyte Haemoglobin Content (CHr™ ) A.H. Roderick – Department of Haematology, University Hospital of Wales, Cardiff. UK J.M. Jou – Servei d’Hemoterapia, Hospital Clinic, Barcelona. Spain W.G.B. Rees, E. Thomas – Haematology Department, West Wales General Hospital, Carmarthen. UK Early diagnosis of iron deficiency anaemia by Mean Reticulocyte Volume (MRV) screening Dr.Ruud Muusze, clinical chemist, Ziekenhuis Zeeuws Viaanderen, Terneuzen, Holland.

  49. Erythropoïesis Erythropoïesis - Maturation The red blood cells line maturation Proerythroblast Basophilic Erythroblast Polychromatophilic Erythroblast Acidophilic Erythroblast Erythrocyte Reticulocyte

  50. Erythropoïesis - Red Blood Cells To identify the RBC pathologies, various quantitative or morphology flags are important.

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