A Novel Method to Assess Bone Marrow Purity is Useful in Determining Blast Percentage by Flow Cytometry in Acute Myeloid Leukemia and Myelodysplasia

Research Article

Ann Hematol Oncol. 2015;2(5): 1038.

A Novel Method to Assess Bone Marrow Purity is Useful in Determining Blast Percentage by Flow Cytometry in Acute Myeloid Leukemia and Myelodysplasia

Aldawood AM¹, Kinkade Z¹, Rosado FG¹, Esan OA¹, Gibson LF² and Vos JA¹*

¹Department of Pathology, West Virginia University School of Medicine, USA

²The Alexander B. Osborn Hematopoietic Malignancy and Transplantation Program of the Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, USA

*Corresponding author: Jeffrey A Vos, Department of Pathology, West Virginia University School of Medicine, USA

Received: April 08, 2015; Accepted: May 28, 2015; Published: June 01, 2015


Blast quantification by Flow Cytometry (FCM) may become essential in situations where morphologic evaluation is difficult or unavailable. As hemodilution invariably occurs, a means of determining Bone Marrow Purity (BMP) and normalizing FCM blast counts is essential, especially when blast percentages are diagnostically critical as in Acute Myeloid Leukemia (AML) and Myelodysplasia (MDS). By evaluating different leukocyte populations in eight initial patients, a formula to predict BMP was developed and compared to the actual BMP determined by manual counts. Performance of the formula was then validated in 86 AML/MDS patients by comparing normalized FCM blast counts to those determined by the reference manual method. A Acronym previously defined in abstract formula was empirically developed, primarily based on changes in lymphocytes which reliably correlated with the actual BMP (R2 = 0.8955). Components of the formula were derived entirely from automated lymphocyte and total leukocyte counts from the peripheral blood and FCM analyses. BMP formula was then validated in 86 AML/MDS patients. When used to normalize blast counts, the formula showed accurate correction when BMP fell between 40%-90%. In this group, correlation of normalized FCM and manual blast counts was acceptable (R² = 0.8335), being greatest at lower blast percentages. Normalization of the FCM blast count appropriately reclassified disease in 26.8% of cases. We identified a practical means of estimating hemodilution and allowing FCM blast normalization in the evaluation of AML and MDS. BMP assessment by this simple method improves the quality of the FCM data and facilitates accurate diagnosis and patient management.

Keywords: Acute myeloid leukemia; Myelodysplastic syndrome; Blast normalization; Flow cytometry; Peripheral blood dilution; Bone marrow purity


AML: Acute Myeloid Leukemia; BMP: Bone Marrow Purity; CBC: Complete Blood Count; FCM: Flow Cytometry; MDS: Myelodysplastic syndromes


Flow Cytometry (FCM) analysis has become standard of care in the evaluation of hematopoietic malignancies, offering the ability to rapidly identify, enumerate, and phenotypically characterize blast populations in such way that diagnosis can be established within hours after bone marrow procedures [1]. In conjunction with morphologic, immunophenotypic and genetic data, the classification of the Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML), in particular, requires accurate determination of the bone marrow myeloblast percentage to establish a diagnosis and to allow for appropriate patient management. Manual differential cell count of the bone marrow aspirate continues to be the gold standard for quantifying blasts, allowing classification of MDS and AML based on stepwise blast thresholds established by the World Health Organization (WHO) [2]. While obtaining an accurate blast count is typically straightforward, FCM enumeration may become critical in certain situations, such as when morphologic evaluation is difficult to interpret or not possible, increasing the reliance on other techniques to acquire this information. Moreover, in the reference laboratory setting, the bone marrow aspirate may not be available for correlation, resulting in FCM data that is essentially blinded from the morphologic impression. When performing FCM in these situations, methods to estimate inevitable hemodilution [3] and possibly normalize cell counts to prevent misleading or conflicting information are desirable.

The admixture of peripheral blood during the bone marrow aspiration process, depending on the technique used, may result in highly variable myeloblast counts as increasing numbers of mature cells from the peripheral blood contaminate the immature elements derived from the bone marrow space. This phenomenon has been well-documented in experiments performed on normal bone marrow specimens in which peripheral blood contamination was mathematically accounted for by a normalization equation [4]. Earlier, Holdrinet et al. performed a series of experiments using radioactive-labeled red blood cells and albumin to demonstrate and quantify peripheral blood dilution of bone marrow aspirate material [5]. While precise blast quantification is typically not a concern in the evaluation of normal bone marrow and the routine use of radioactivelabeled blood elements is not practical in the clinical laboratory, these studies importantly documented the effects of hemodilution during bone marrow aspiration and suggested methods in which to overcome this technical obstacle.

Earlier approaches aimed at addressing and correcting for peripheral blood contamination of bone marrow aspirate material used DNA S-phase analysis [6-8]. These studies compared DNA S-phase with percentage of lymphocytes and monocytes within the bone marrow aspirate specimen, since these latter cellular components are indicative of peripheral blood contamination. Based on the negative correlation observed between erythroid and myeloid bone marrow cells in S-phase and the percentage of lymphocytes and monocytes in the aspirates, a formula to correct for peripheral blood dilution was developed. Other studies have addressed peripheral blood contamination by evaluating the numbers of immature elements in bone marrow aspirate material compared to what is typically expected [9]. A similar approach was devised by Loken et al. who exploited the phenotypic variability of granulocytes to determine the proportion of immature neutrophils using a specific FCM gating strategy [10] and then applying these data to normalize bone marrow blast count. While promising, these methodologies were not tested in the setting of bone marrow abnormality, such as in MDS or AML, where anticipated cellular maturation rates and phenotypic profiles are inherently atypical.

Outside the setting of normal hematopoiesis, the issue of peripheral blood contamination becomes more complicated as the cellular components of the peripheral blood are often abnormal. Patients with high-grade myeloid neoplasms frequently have immature leukocytes and nucleated red blood cells within the peripheral circulation. In addition, typical antigen expression profiles in the setting of leukemia and dysplasia are often aberrant [11], and thus, relying on specific antigenic maturation patterns may not always be suitable in the setting of disease. Moreover, while data has been variable on the subject, one study showed the greatest degree of discordant results between morphological and FCM quantification of blasts around the 5% cut-off, a threshold which is critical for determining remission status of acute leukemia [12]. Given the vital role in which blast quantification plays in the diagnosis as well as monitoring of myeloid neoplasms, in addition to widespread use of FCM analysis as a part of the diagnostic process [13-16], a straightforward, reliable method for determining the degree of hemodilution in bone marrow aspirates of AML and MDS patients is necessary. While many of the previously proposed methodologies for determining Bone Marrow Purity (BMP) have offered foundational knowledge of this phenomenon, a practical approach to specifically overcome the morphologic and phenotypic abnormalities seen in these myeloid diseases, where precise blast counts become clinically relevant, is required to allow for timely and appropriate management of these patients.

Materials and Methods

Study design

Approval was obtained from the Institutional Review Board prior to commencing this study. Patients diagnosed with AML or MDS were identified through a search of the surgical pathology database at our institution between the years of 2010 and 2014. Retrospective review of the electronic medical records was conducted of the selected patients to verify the diagnosis and obtain clinical history. In order to focus on cases with elevated blast counts and the effects of hemodilution on disease classification, original diagnostic biopsies as well as cases of AML with residual/relapsed disease were included in this study. In addition, patients were excluded if archival bone marrow aspirate and biopsy slides, FCM scatterplots, Complete Blood Count (CBC) and peripheral blood smears were not available for review. Cases that met inclusion criteria were then reviewed to classify each case according to the current WHO classification system [2]. In particular, total white blood cell and lymphocyte counts from the CBC analyzer were collected and 500-cell manual differential counts of the bone marrow aspirate and peripheral blood specimens were performed on all cases in this study.

The study was conducted in two phases. First, a formula to assess BMP was developed, and second, the clinical utility of the BMP formula in determining blast percentages in MDS and AML was tested. In the initial portion of the study, a BMP gold standard was first created by performing 500-cell manual differential counts on the Peripheral Blood (PB), Bone Marrow Aspirate (BM) and Flow Cytometry (FCM) specimens on eight patients with AML or MDS. Based on the blast percentages determined from each of these differentials, the gold standard BMP (i.e., Actual BMP) was then calculated using a ratio equation as follows:

Actual BMP (%) = [BlastsFCM/(BlastsPB + BlastsBM)] x 100

Next, a formula to predict BMP was developed based on the principle that the specimen used for flow cytometric analysis is composed of a mixture of bone marrow aspirate material and peripheral blood in unknown proportions. Using manual differential counts from the eight initial patients, several different leukocyte populations, such as lymphocytes, monocytes and neutrophils, were tested to determine which population(s) best predicted the amount of peripheral blood dilution present in the bone marrow aspirate material. From these experiments, a BMP formula was empirically derived using only values obtained from the peripheral and FCM specimens. Where possible, automated leukocyte counts were then used, in place of manually-derived counts, to provide a more straightforward and objective means of implementing the formula. The accuracy of the equation was ultimately assessed by comparing the predicted BMP to the actual BMP, determined from the abovedescribed ratio.

In the second phase of the study, the BMP formula was validated by studying its performance in 86 patients with AML or MDS. The predicted BMP was used to normalize the myeloblast percentage determined by FCM and compared to the actual blast count determined from the 500-cell manual differential. Cases that challenged the diagnostic blast thresholds for AML and MDS were utilized. The limitations of the BMP formula to accurately normalize the blast count were also investigated. Finally, the cases were reviewed to assess the impact of blast normalization in disease classification, thereby determining its clinical utility in the diagnosis and monitoring of AML and MDS [17].

Bone marrow aspirates

Wright’s-Giemsa stained smears of bone marrow aspirates from eligible patients were examined by light microscopy and 500-cell manual differentials were performed. Only areas of the smear near the bone marrow particle were evaluated to decrease the effect of peripheral blood dilution and obtain the most accurate bone marrow blast count possible. Differential counts consisted of 11 components including blasts, promyelocytes, myelocytes, metamyelocytes, bands/segmented neutrophils, eosinophils, basophils, monocytes, lymphocytes, plasma cells and erythroid precursors. For the initial portion of the study (8 patients), a smear made from the FCM specimen, often obtained from a second aspirate pull, was evaluated in a similar manner including performing a 500-cell manual differential. In the latter phase of the study (86 patients), total leukocyte counts were obtained on the FCM specimens using a CELL-DYN Sapphire Automated Hematology Analyzer (Abbott Diagnostics, Abbott Park, IL).

Peripheral blood specimens and CBC analysis

Peripheral blood from each patient, obtained on the same day of the bone marrow procedure, was examined via Giemsa-stained smears. A 500-cell manual differential count was performed, recording the same cell types as those determined for the bone marrow aspirate specimens. In addition, CBC analysis with automated differential was performed using a CELL-DYN Sapphire Automated Hematology Analyzer (Abbott Diagnostics).

Multiparameter flow cytometry

Ten-color flow cytometry was performed using a FACSCanto flow cytometer (Becton Dickinson Biosciences, San Jose, CA) in conjunction with a 4-tube antibody panel designed to characterize blasts as detailed in Table 1. Briefly, 100 Μl of bone marrow aspirate specimen was prepared according to standard whole-blood lysis procedure, with the use of Becton Dickinson Lyse-Wash method. Cell solutions were stained with fluorophore-conjugated monoclonal antibodies and isotype matched controls were used to exclude nonspecific binding. Primary CD45 versus side scatter (SSC) gating was employed to discriminate blast, lymphocyte, monocyte, granulocyte and debris/erythroid populations [18]. Data was acquired via FACS Diva software (Becton Dickinson Biosciences, San Jose, CA) and analyzed using FCS Express software program (De Novo Software, Los Angeles, CA).