Automated Analysis of Hematopoietic Malignancies

1Department

of Hematopathology and Cellular Immunology, JFK Medical Center, Edison, New Jersey and 2Laboratory Testing Segment, Bayer Corporation, Tarrytown, New York

Bader Maria Pedemonte1, Loretta Catania2, Janet Hannon1, Marc Katzenberg2, Jolanta Kunicka2

INTRODUCTION
Accurate and rapid interpretation of peripheral blood samples and bone marrow aspirates is important for evaluation of patients with hematopoietic abnormalities. Prior studies have addressed the usefulness of automated hematology analyzers in the evaluation and diagnosis of hematological neoplasias performed on peripheral blood samples. Despite the increased demand for marrow analysis and the availability of automated technology throughout the clinical laboratory, automated methods for marrow aspirate analysis have not been completely developed. In this study, we evaluated peripheral blood and bone marrow aspirates performed by flow cytometry analysis using the ADVIA 120 automated Hematology System (Bayer Diagnostics, Tarrytown, New York, USA). The ADVIA 120 system can generate distinct analytical profiles for different types of hematologic abnormalities. We have documented that peripheral blood and bone marrow aspirate analysis indeed provides excellent preliminary information that effectuates optimal diagnostic strategy. The peripheral blood samples were collected in K3EDTA. Bone Marrow aspirates, obtained from the posterior superior iliac crest, were collected in K2EDTA and analyzed within 18 hours after collection. Both specimens were processed on the ADVIA 120 to obtain cytograms, histograms and CBC/differential results.

METHODS
ADVIA 120 Cell Analysis The ADVIA 120 Hematology System provides complete blood cell counts, leukocyte differential, and reticulocyte analysis. The analysis of nucleated cells is performed in two separate channels, peroxidase and baso/lobularity. Using light scatter and peroxidase staining intensity in the peroxidase channel, the ADVIA 120 provides a five-part white blood cell differential. Cellularity, nuclear density, and nuclear complexity are determined using two light-scatter measurements in the baso/lobularity channel. A combination of information from the Peroxidase and Baso/Lobularity channels was used to characterize cellular patterns and determine predominant cell lineages in peripheral blood samples and bone marrow aspirates. The RBC/PLT (platelet) channel provides information related to dyserythropoiesis. For periperal blood and bone marrow aspirate interpretation, the relevant parameters included: (1) cellularity; (2) h presence of abnormal cell populations; (3) distribution of the abnormal cells in the peroxidase channel and presence of blasts in the blast region of the baso/lobularity channel; (4) presence or absence of dyserythropoiesis by localization of erythroid cells in the RBC/platelet channel; (5) cell counts.

RESULTS
Characteristics of cell analysis patterns obtained on various hematopoietic abnormalities and malignancies on the ADVIA 120 are summarized in the accompanying figures. The acute leukemias shown except for M4 and M5 demonstrate abnormal cell populations in the LUC (large unstained cells) and blast regions of the peroxidase cytogram and baso/lobularity cytogram respectively. Monocytic leukemias and leukemias with a high component of monocytes produced an abnormal cluster in the monocyte and LUC regions of the peroxidase cytogram. Cases of RA demonstrate a hypercellular density of cells corresponding to myeloid hyperplasia and immaturity. Dyserythropoiesis is also demonstrated by the widespread distribution of red blood cells in the RBC cytogram. Chronic myelocytic leukemias show a characteristic dense distribution of varying cellular maturity on the peroxidase cytogram. Sickle Cell Anemia displays a widespread dispersed pattern typical of irreversible sickled red blood cells. The peroxidase cytogram of MPO deficiency is devoid of peroxidase containing cells while the baso/lobularity cytogram validates the presence of a PMN (polymorphonuclear) population confirming MPO deficiency.
WBC Cytograms
C e l l S i z e Peroxidase Activity

RESULTS cont.
Acute Myelomonocytic Leukemia (AML-M4) Peripheral Blood Seq. # 121 - 4/2/98 Acute Myelocytic Leukemia-M0 (AML-M0) Bone Marrow Seq. # 748 - 11/20/99

53% Blasts on manual differential

Normal Bone Marrow

Normal Peripheral Blood

ADVIA 120 Results WBC Count: 6,020 cells/L PLT Count: 20,000 cells/L 37.7% Neut 8.8% Lymph 47.3% Mono 24.2% PMN 73.1% MN 3+ Blasts Flag 3+ IG Flag

ADVIA 120 Results WBC Count: 142,860 cells/L 1.6% Neut 78.5% Lymph 18.7% LUC 1+ Blasts Flag 3+ ATYPS Flag

52 4

1 3

ADVI A 120 Pattern Significance Increased blasts cluster in the Baso/Lobularity cy togram Decreased my eloid maturation Monocyte count increased Both ery throid and megakary ocy tic precursors diminished

ADVI A 120 Pattern Significance Hy percellular marrow aspirate Decreased my eloid maturation Peroxidase-negative large LUC population Blasts cluster in Baso/Lobularity Channel Non-hemoglobin containing population on RBC map

C e l l S i z e Nuclear Complexity

Sickle Cell Anemia Peripheral Blood Seq. # 14 - 1/16/99

MPO Deficiency Peripheral Blood Seq. # 216 - 10/26/99

Chronic Lymphocytic Leukemia (CLL) Peripheral Blood Seq. # 654 - 7/4/99

Acute Lymphocytic Leukemia (ALL) Peripheral Blood Seq. # 85 - 1/29/99

ADVIA 120 Results WBC Count: 14,410 cells/L RBC Count: 1,550,000 cells/L PLT Count: 433,000 cells/L HGB: 4.8 g/dL HCT: 14.4 % MCV: 92.7 fL CHCM: 35.5 g/dL HDW: 5.13 g/dL 28.9% Retic 2+ Macro Flag 3+ Hyper Flag 3+ HC Var Flag ADVIA 120 Results WBC Count: 6,760 cells/L 1.6% Neut 21.5% Lymph 69.5% LUC 66.0% PMN 33.7% MN 3+ ATYPS Flag 1+ MPO Deficiency Flag

ADVIA 120 Results WBC Count: 140,790 cells/L 1.7% Neut 77.2% Lymph 16.4% LUC 3+ ATYPS Flag

ADVIA 120 Results WBC Count: 51,210 cells/L 1.9% Neut 12.4% Lymph 81.7% LUC 2.1% PMN 97.1% MN 3+ Blasts Flag 3+ ATYPS Flag

ADVI A 120 Pattern Significance Decreased Peroxidase positive cells Extreme leukocytosis Large LUC and ly mph population Dense Mononuclear populations in both WBC channels Non-hemoglobin containing population on RBC map

ADVIA 120 Pattern Significance Abnormal hy percellular LUC population Decreased or absent my eloid differentiation Blasts cluster in Baso/Lobularity cy togram

ADVI A 120 Pattern Significance Marked variation of RBC size and hemoglobinization Tails of hy pochromic and hyperchromic RBCs Increase in WBC and PLT count RBC cy togram pattern corresponds to irreversibly sickled red cells

ADVI A 120 Pattern Significance Significant LUC population in Perox cy togram compared to PMN population in Baso/Lobularity confirms Peroxidase negative neutrophil population

MDS/RA Bone Marrow Seq. # 1073 - 2/25/99

MDS/RA Peripheral Blood Seq. # 1076 - 2/25/99

Acute Myelocytic Leukemia-M2 (AML-M2) Peripheral Blood Seq. # 4 - 11/8/99

Promyelocytic Leukemia (AML-M3) Bone Marrow Seq. # 370 - 1/28/00

ADVIA 120 Results PLT Count: 16,000 cells/L WBC Count: 24,580 cells/L 52.6% Neut 17.2% Lymph 25.7% Mono 10.7% PMN 88.2% MN 3+ Blasts Flag 3+ IG Flag

ADVIA 120 Results WBC Count: 118,710 cells/L 47.4% Neut 11.6% PMN 76.2% MN 1+ Left Shift Flag 2+ Blasts Flag 3+ IG Flag

ADVI A 120 Pattern Significance Hy percellular marrow aspirate Decreased my eloid maturation Peroxidase-negative large cell population Abnormal ery thropoiesis

ADVIA 120 Results WBC Count: 33,250 cells/L RBC Count: 2,500,000 cells/L HGB: 9.9 g/dL HCT: 29.0 % MCV: 115.9 fL CHCM: 33.2 g/dL HDW: 3.36 41.5% PMN 44.0 % M N 3+ Macro Flag 1+ Hypo Flag 1+ Aniso Flag 3+ ATYPS Flag 2+ NRBC Flag

ADVIA 120 Results WBC Count: 2,970 cells/L ADVI A 120 Pattern Significance RBC Count: 2,500,000 cells/L Decreased my eloid maturation HGB: 9.6 g/dL Peroxidase-negative large cell population HCT: 29.4 % MCV: 117.6 fL Abnormal ery thropoiesis CHCM: 32.2 g/dL HDW: 3.07 43.6% Neut 49.4% Lymph 43.5% PMN 56.2 % M N 3+ Macro Flag 2+ Hypo Flag 1+ Aniso Flag

ADVI A 120 Pattern Significance Abnormal cell population in neutrophil, monocy te, and LUC areas Granulocy tes show vary ing maturity and differentiation Blasts cluster in Baso/Lobularity cy togram Severe thrombocy topenia

ADVI A 120 Pattern Significance Hy percellular LUC and granulocy tic populations Aty pical cellular populations with hig h Peroxidase activity Blasts cluster in the Baso/Lobularity cy togram

Chronic Myelocytic Leukemia (CML) Bone Marrow Seq. # 840 - 10/22/99

Myeloproliferative Disorder/Essential Thrombocythemia Bone Marrow Seq. # 692 - 2/18/99

Acute Myelocytic Leukemia-M5 (AML-M5) At Diagnosis - Bone Marrow Seq. # 419 - 1/28/99

Acute Myelocytic Leukemia-M5 (AML-M5) Post Therapy - Bone Marrow Seq. # 940 - 2/19/99

ADVIA 120 Results WBC Count: 51,030 cells/L PLT Count: 562,000 cells/L) 89.8% Neut 61.2% PMN 34.0% MN 3+ Left Shift Flag 3+ IG Flag

ADVIA 120 Results PLT Count: 846,000 cells/L) 1+ NRBC Flag 1+ PLT Clumps

ADVI A 120 Pattern Significance Relatively normocellular marrow aspirate Heterogeneous population of blasts Mature my eloid cells markedly decreased Monocytic differentiation Weak to moderate Peroxidase activity Increased LUCs

ADVIA 120 Results WBC Count: 28,100 cells/L 10.7% Neut 23.0% Mono 46.1% LUC 23.4% PMN 72.4% MN 3+ ATYPS Flag 2+ NRBC Flag

ADVI A 120 Pattern Significance Hy pocellular marrow aspirate Myeloid cells not present

ADVIA 120 Results WBC Count: 1,820 cells/L 61.1% PMN 27.2% MN 2+ ATYPS Flag

ADVI A 120 Pattern Significance Marked Leukocy tosis Hy perplastic maturing granulocy te population

ADVI A 120 Pattern Significance Relatively normocellular marrow aspirate Essential thrombocy themia PLT Count: 846,000 cells/L

The information provided by ADVIA 120 analysis of peripheral blood and bone marrow aspirates can serve as an effective screening tool in evaluation and choice of strategy for further diagnostic testing and patient care.

CONCLUSIONS

Although immunophenotyping by flow cytometry is a sensitive method for identifying abnormal cell populations and cell lineage, it is an expensive tool that involves extensive panels of fluorescently labeled antibodies. Microscopic examination as a screening tool is labor intensive and subject to inter-observer variability. Automated analysis of peripheral blood samples and bone marrow aspirates on the ADVIA 120-hematology analyzer appears to provide an important screening tool that can be used in determining the need for further diagnostic testing and evaluation resulting in comprehensive patient care. The preliminary classification of peripheral blood and bone marrow disorders based on ADVIA 120 pattern recognition can allow for a more cost-effective selection of monoclonal antibody panels for immunophenotypic studies and thus leads to a more cost effective disease management strategy.