1. In the heart of hematopoïesis

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

3. Leukopoïesis exploration

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

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

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

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

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

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

13. Why light beam from tungsten lamp is better than Laser beam
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. Leukopoïesis - Double DIFF Matrix
Eosinophil
Neutrophil
Monocyte
Lymphocyte
Basophil

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

16. IMM immature monocyte Leukopoïesis - Double DIFF Matrix Promonocyte
Monoblast

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

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

20. To identify the lymphoid pathologies, various flags are important:
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 ALY flag (identification and counting) in case of:
Lymphoma diseases
Lymphoproliferative syndromes (ex: Sezary)
LAL diseases
Reactive lymphocytes

22. 2. Specific IML flag (identification and counting) in case of:
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. The eosinophil population increase in case of: Intestinal parasites:
Leukopoïesis - DIFF
Eosinophilia
The eosinophil population increase in case of:
Intestinal parasites:
Bihlarziose, Ascaris, Oxyure,
Taenia...
Allergies:
Asthma, Eczema...
Chronic eosinophilic leukemia

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

26. More information in the DIFF Matrix:
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, 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. The red blood cells line maturation
Erythropoïesis - Maturation
The red blood cells line maturation
Proerythroblast
Basophilic Erythroblast
Polychromatophilic Erythroblast
Acidophilic Erythroblast
Erythrocyte
Reticulocyte

31. Automatic WBC correction
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. Erythropoïesis - Erythroblasts
Volume
Fluorescence

34. Erythroblasts pathologies
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. Erythroblasts on ABX Pentra DX 120 Reflex testing
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, 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 USA
+HoribaABX Diagnostics, Montpellier, France

37. The red blood cells line maturation
Erythropoïesis - Maturation
Erythropoïesis
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. Thiazole orange: specific RNA fluorochrome.
Erythropoïesis - Reticulocytes
Thiazole orange: specific RNA fluorochrome.
Flow cytometer: light source: argon-ion laser.

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

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

42. Reticulocyte Count : RET (%) and RET (#)
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. Erythropoïesis - Reticulocytes interpretation
Anemia: Hb < 12 g/dl
MCV < 80 fl
Microcytic Anemia
RET
Iron
In case of anemia (hemoglobin <12 g/dl), and regarding the MCV, two cases are possible:
The MCV is <80 fl, then the anemia is microcytic.
In accordance with the RET value, the treatment is iron.
After 3 weeks approximately, regarding the MCV, we could know if the treatment was good and adjust it.
Regarding the MRV, the same information is giving in 2 days: the treatment adjustment is quickly.
The MCV is >80 fl: anemia non microcytic.
If the RET value is low, the anemia is non regenerative.
If the RET value is high, the anemia is regenerative.
If the RET value is normal (but the hemoglobin low), it is necessary to wait some days to know if the anemia is or not regenerative.
MCV +
3 weeks
MRV
2 days

44. Erythropoïesis - Reticulocytes interpretation
Anemia: Hb < 12 g/dl
80 fl < MCV < 100fl
Normocytic Anemia
0.1x106/l < RET > 0.15x106/l
No Regeneration
???
Regeneration
In case of anemia (hemoglobin <12 g/dl), and regarding the MCV, two cases are possible:
The MCV is <80 fl, then the anemia is microcytic.
In accordance with the RET value, the treatment is iron.
After 3 weeks approximately, regarding the MCV, we could know if the treatment was good and adjust it.
Regarding the MRV, the same information is giving in 2 days: the treatment adjustment is quickly.
The MCV is >80 fl: anemia non microcytic.
If the RET value is low, the anemia is non regenerative.
If the RET value is high, the anemia is regenerative.
If the RET value is normal (but the hemoglobin low), it is necessary to wait some days to know if the anemia is or not regenerative.

45. Erythropoïesis - Reticulocytes interpretation
Normocytic Anemia
0.1x106/l < RET > 0.15x106/l
???
In the case of anemia with a RET normal value, the CRC give immediately the information:
< 2% the anemia is central (bone marrow abnormalities)
> 2% the anemia is peripheral (bleeding)
Conclusion:
in case of non microcytic anemia with a normal RET value, the CRC give the classification of the anemia.
2% < CRC >2%
Bone Marrow
Abnormalities
Peripheral
Etiologies

46. Publication: Haematologica 2001; 86:24-29
Erythropoïesis - Reticulocytes interpretation
Anemia: Hb < 12 g/dl
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
In case of anemia (hemoglobin <12 g/dl), and regarding the MCV, two cases are possible:
The MCV is <80 fl, then the anemia is microcytic.
In accordance with the RET value, the treatment is iron.
After 3 weeks approximately, regarding the MCV, we could know if the treatment was good and adjust it.
Regarding the MRV, the same information is giving in 2 days: the treatment adjustment is quickly.
The MCV is >80 fl: anemia non microcytic.
If the RET value is low, the anemia is non regenerative.
If the RET value is high, the anemia is regenerative.
If the RET value is normal (but the hemoglobin low), it is necessary to wait some days to know if the anemia is or not regenerative.
Evaluation reference: Cordoba, University Hospital “Reina Sofia”, Dr.Torres
Publication: Haematologica 2001; 86:24-29

47. Reticulocytes on ABX Pentra 120 range
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. The red blood cells line maturation
Erythropoïesis - Maturation
Erythropoïesis
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.

51. Quantitative & morphologic flags:
Erythropoïesis - Red Blood Cells
Quantitative & morphologic flags:
Anemia
Polyglobulia
Cold agglutinins
Micro/macrocytosis
MIC
MAC
Anisocytosis
No (noise)

52. Specific quantitative flags: Anemia: Hgb Polyglobulia: RBC
Erythropoïesis - Red Blood Cells
Specific quantitative flags:
Anemia: Hgb
Polyglobulia: RBC
Cold agglutinin: MCHC
Micro/macrocytosis: MCV

53. Specific morphology flags: MIC: presence of microcytic population
Erythropoïesis - Red Blood Cells
Specific morphology flags:
MIC: presence of microcytic population
MAC: presence of macrocytic population
Anisocytosis
No (noise): presence of lyse resistant cells

54. Red Blood Cells pathologies
Erythropoïesis - Red Blood Cells
Red Blood Cells pathologies
1. Specific and precise MIC flag (identification through 3 levels) in case of:
Anemia:
severe: anisocytosis, Joly bodies
congenital: spherocytes
hemolytic: schistocytes, poïkilocytes, Joly bodies
hypochromic: microcytes
iron deficiency: poïkilocytes

55. Red Blood Cells pathologies
Erythropoïesis - Red Blood Cells
Red Blood Cells pathologies
Myelofibrosis: tear drops
Thalassemia: poïkilocytes, sickle cells, target cells
Hemoglobinopathies:
HbC: spherocytes, target cells
HbSS: sickle cells
Microangiopathic hemolysis: poïkilocytes, schistocytes

56. Red Blood Cells pathologies
Erythropoïesis - Red Blood Cells
Red Blood Cells pathologies
2. Specific and precise MAC flag (identification through 3 levels) in case of:
Megaloblastic anemias: macrocytes, poïkilocytes, Joly bodies
Enzyme deficiency (Vit B12, folates): macrocytes, ponctuate basophilia
Lead poisoning: macrocytes, ponctuate basophilia

57. Red Blood Cells pathologies
Erythropoïesis - Red Blood Cells
Red Blood Cells pathologies
3. Specific and precise Anisocytosis flag in case of:
Double Red blood cells population: transfusion
Anemias
Thalassemias
Microcytic or marcrocytic population

58. Red Blood Cells pathologies
Erythropoïesis - Red Blood Cells
Red Blood Cells pathologies
4. Specific NO flag (noise on the Double DIFF Matrix) in case of:
Lyse resistant red blood cells population: target cells...

59. Erythrocytes on ABX Pentra range
Erythropoïesis - Red Blood Cells
Erythrocytes on ABX Pentra range
Flags: customized configuration
Abnormal population detection (MIC/MAC  MCV)
Reference principle
Choice: two lyses (with or without cyanide)

60. Thrombopoïesis exploration

61. Normal platelets population
Thrombopoïesis
Normal platelets population
Specific MIC flag

62. Thrombopoïesis
Platelet aggregates
Optical detection

63. Thrombopoïesis
Macroplatelets
Macroplatelet detection

64. Thrombocytes on ABX Pentra range
Thrombopoïesis
Thrombocytes on ABX Pentra range
Flags: customized configuration
PLT aggregates & macroplatelets detection
Reference principle

65. Thank you