In the heart of hematopoïesis

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

Leukopoïesis exploration

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

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

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

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

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.

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

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

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).

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

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)

Leukopoïesis - Double DIFF Matrix Eosinophil Neutrophil Monocyte Lymphocyte Basophil

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

IMM immature monocyte Leukopoïesis - Double DIFF Matrix Promonocyte Monoblast

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

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

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

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

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

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

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

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

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

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

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.

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

Erythropoïesis exploration

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

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

Erythropoïesis - Erythroblasts Fluorescence Resistivity

Erythropoïesis - Erythroblasts Volume Fluorescence

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

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)

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

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

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

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

Reaction + Erythropoïesis - Reticulocytes 25 sec + Reticulocyte fluorescent molecule

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

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 (%)

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

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.

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

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

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

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.

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

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

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

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

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

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

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

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

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

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...

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)

Thrombopoïesis exploration

Normal platelets population Thrombopoïesis Normal platelets population Specific MIC flag

Thrombopoïesis Platelet aggregates Optical detection

Thrombopoïesis Macroplatelets Macroplatelet detection

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

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