Laboratory Evaluation of Acute Leukemia

Etiology and Epidemiology

Etiology of Acute Leukemia

Acute leukemia, which includes both ALL and AML, results from the malignant transformation of hematopoietic progenitor cells. This transformation typically arises from a multifactorial process involving a complex interaction of genetic and environmental factors.[1]

Genetic factors: The development of acute leukemia is influenced by both inherited predispositions and acquired genetic mutations, each having a significant role in disease initiation and progression.

• Inherited predisposition: Certain inherited genetic syndromes, such as trisomy 21, neurofibromatosis type 1, Bloom syndrome, and ataxia-telangiectasia, significantly increase the risk of developing ALL.[7][8] Similarly, trisomy 21, Fanconi anemia, Li-Fraumeni syndrome, Bloom syndrome, and familial mutations in genes such as CEBPA, DDX41, and RUNX1 are strongly associated with the development of AML.[9]

• Acquired genetic mutations: Somatic mutations acquired over a person's lifetime are critical in leukemia development. These include chromosomal translocations, such as t(9;22) seen in chronic myeloid leukemia, which can progress to AML.[10] Additionally, gene mutations such as FLT3, NPM1, and CEBPA in AML, as well as epigenetic modifications, contribute significantly to disease progression.[11][12]

Environmental factors: Environmental exposures have a significant role in the development of acute leukemia, with factors such as radiation, chemicals, and viral infections contributing to the disease's onset.

• Radiation exposure: High doses of ionizing radiation from sources such as nuclear accidents or radiotherapy are established risk factors for leukemia.[13]

• Chemical exposure: Benzene, a solvent widely used in industrial processes, has been strongly linked to AML.[14][15] Additionally, certain chemotherapy drugs, such as alkylating agents, can increase the risk of secondary leukemias.[16]

• Viral infections: Certain viruses, such as HTLV-1, have been associated with the development of acute T-cell leukemia or lymphoma.[17]

Epidemiology of Acute Leukemia

The epidemiology of acute leukemia differs by subtype (ALL or AML) and is influenced significantly by age group.

Acute lymphoblastic leukemia: ALL demonstrates distinct patterns in age distribution and incidence, making it a key focus in pediatric oncology while also affecting adults.

• Age distribution: ALL primarily affects children, with peak incidence occurring between ages 2 and 5. However, it can also present in adults, with a secondary incidence peak observed after age 50.[18]

• Incidence: ALL is the most common malignancy in children, with an estimated incidence of 3.4 cases per 100,000 children in the United States.[18]

Acute myeloid leukemia: AML predominantly affects adults, with its incidence and demographic characteristics closely linked to age.

• Age distribution: AML is predominantly observed in adults, with its incidence rising steadily with age. The median age at diagnosis is 68.

• Incidence: AML is the most common type of acute leukemia in adults, with an estimated incidence of 3 to 5 cases per 100,000 adults in the United States.[19]

Table 1. International Classification of Acute Lymphoblastic Leukemia and Acute Myeloid Leukemia

Pathophysiology

Acute leukemia originates from the malignant transformation of hematopoietic stem cells (HSCs) or early progenitor cells. This process results in the accumulation of immature blasts in the bone marrow, disrupting normal hematopoiesis and impairing the production of healthy blood cells. The molecular mechanisms driving this transformation are diverse and complex, involving a combination of genetic and epigenetic alterations that deregulate key cellular processes.

Common Pathophysiological Features of Acute Leukemia

Both ALL and AML share several common pathophysiological features, as mentioned below.

Suppression of normal hematopoiesis: The proliferation of leukemic blasts within the bone marrow displaces normal hematopoietic cells, resulting in reduced production of mature blood cells and the associated clinical manifestations.[22]

Infiltration of extramedullary sites: Leukemic blasts may invade organs and tissues beyond the bone marrow, leading to organ dysfunction and additional clinical symptoms.[23]

Genetic instability: Acute leukemia cells often exhibit genetic instability, acquiring additional mutations that drive disease progression and contribute to resistance to treatment.[24]

Immune dysregulation: The immune system has a complex role in acute leukemia, exhibiting both anti-leukemic and pro-leukemic effects that influence disease development and progression.[25]

Acute Lymphoblastic Leukemia

ALL arises from lymphoid progenitor cells, primarily B-cell progenitors and, less commonly, T-cell progenitors. The accumulation of malignant blasts in the bone marrow disrupts the production of normal red blood cells, white blood cells, and platelets, resulting in anemia, increased infection risk, and bleeding tendencies. These blasts may also infiltrate organs such as the lymph nodes, spleen, liver, and central nervous system (CNS), leading to organomegaly and neurological symptoms.[3]

The pathophysiology of ALL involves diverse genetic alterations, such as chromosomal translocations, aneuploidy, and mutations in genes regulating B-cell development and signaling pathways. These genetic aberrations lead to the uncontrolled proliferation and impaired differentiation of lymphoblasts, leading to their accumulation and the characteristic clinical manifestations of ALL.[26] 

Chromosomal alterations in acute lymphoblastic leukemia: Chromosomal alterations are critical in the pathogenesis of ALL, contributing to disease initiation and progression.

• Aneuploidy: Recent studies utilizing single-cell sequencing technologies to dissect the clonal evolution of ALL have revealed that aneuploidy, particularly hyperdiploidy, can occur early in leukemogenesis, contributing to clonal diversity and the progression of ALL.[27]

• Chromosomal translocations: Recent studies have identified new fusion genes in ALL, such as DUX4 rearrangements and KMT2A mutations in infant ALL, highlighting the heterogeneity of this disease. Research is focused on understanding how these fusion genes disrupt gene expression and signaling pathways, driving leukemic transformation.[28][29]

Gene mutations: Gene mutations are a central factor in the development and progression of ALL, with both loss-of-function and gain-of-function mutations contributing to leukemogenesis.

• Loss-of-function mutations: Recent studies have identified novel tumor suppressor genes involved in ALL, such as TP53, RB1, and CREBBP.[20][30] These mutations disrupt cell cycle regulation, DNA damage repair, and epigenetic control, thereby contributing to uncontrolled cell growth and survival.

• Gain-of-function mutations: Mutations in signaling pathways, such as JAK/STAT and PI3K/AKT/mTOR, are increasingly recognized as important drivers of ALL. These mutations activate signaling cascades that promote cell proliferation, survival, and drug resistance.[20]

• Mutations in genes involved in B-cell development: Recent research highlights the role of mutations in genes such as PAX5 and IKZF1 in disrupting normal B-cell differentiation. These genetic alterations are key contributors to the pathogenesis of B-cell precursor ALL.[20][31]

Acute Myeloid Leukemia

AML arises from myeloid progenitor cells, which are responsible for the production of granulocytes, monocytes, erythrocytes, and platelets. The clonal expansion of myeloid blasts within the bone marrow impairs normal hematopoiesis, resulting in reduced production of mature blood cells. This disruption manifests clinically as anemia, increased susceptibility to infections, and a heightened risk of bleeding. In addition, leukemic blasts may infiltrate extramedullary sites such as the skin, gums, and CNS, contributing to organ-specific complications and extramedullary disease. Please see StatPearls' companion resource, "Acute Myeloid Leukemia," for more information.

The pathophysiology of AML is multifaceted and complex, driven by a multitude of genetic and epigenetic alterations. These include chromosomal abnormalities such as translocations, inversions, and deletions, as well as mutations in genes regulating myeloid cell proliferation, differentiation, and survival. These specific genetic alterations are critical in determining the disease's aggressiveness, response to treatment, and prognosis.[32] Broadly, these alterations can be classified into 2 categories based on their functional consequences, as outlined below.

Class I mutations: Mutations in genes such as IDH1, IDH2, and DNMT3A have emerged as critical contributors to AML pathogenesis. These mutations alter cellular metabolism, epigenetic regulation, and DNA methylation, thereby contributing to clonal expansion and impaired differentiation.[33]

Class II mutations: Recent studies have identified novel mutations in transcription factors such as KMT2A and NUP98, which disrupt normal hematopoietic differentiation and contribute to AML pathogenesis. These mutations alter gene expression patterns and impair myeloid cell maturation, leading to the accumulation of immature blasts.[33][34]

Microenvironmental Factors

The bone marrow microenvironment is crucial in supporting the survival and proliferation of leukemic cells. Recent research has focused on understanding how interactions between leukemic cells, stromal cells, extracellular matrix components, and inflammatory cytokines contribute to leukemogenesis and drug resistance. These interactions can promote leukemic stem cell self-renewal, suppress immune responses, and protect leukemic cells from chemotherapy-induced cell death.[35]

Specimen Requirements and Procedure

Specimen Requirements

The primary specimens for the evaluation of acute leukemia are listed below.

Peripheral blood: Collected in ethylenediaminetetraacetic acid (EDTA) (lavender-top) tubes for CBC and PBS examination. Additional samples may be collected in sodium heparin (green-top) tubes for flow cytometry and cytogenetic analysis. Please see StatPearls' companion resource, "Laboratory Tube Collection," for more information.

Bone marrow aspiration and biopsy: BMA is collected in EDTA and sodium heparin tubes for morphological assessment, flow cytometry, cytogenetic analysis, and molecular studies. BMB is collected in formalin for histopathological examination.[36]

Laboratory Procedures

The laboratory evaluation of acute leukemia involves a multifaceted approach, which is mentioned below.

Complete blood count: This test evaluates the quantity and morphology of red blood cells, white blood cells, and platelets. Findings such as anemia, thrombocytopenia, and either leukocytosis or leukopenia with circulating blasts are indicative of acute leukemia. Please see StatPearls' companion resource, "Normal and Abnormal Complete Blood Count With Differential," for more information.

Peripheral blood smear: Microscopic analysis of the PBS provides a detailed assessment of blood cell morphology. The identification of blasts is a key diagnostic feature of acute leukemia.

Bone marrow examination: Bone marrow examination is crucial for evaluating acute leukemia and involves multiple procedures to assess the presence of leukemic blasts and determine genetic and immunophenotypic features.

• Morphological assessment: BMA and BMB are examined for the presence of blasts and assessment of bone marrow cellularity and architecture. A blast count of over 20% in the bone marrow is diagnostic of acute leukemia.[37]

• Flow cytometry: Immunophenotyping of BMA cells by flow cytometry identifies the lineage and maturation stage of leukemic blasts, aiding in the classification of ALL (B-cell or T-cell) and AML (various subtypes based on immunophenotypic features).

• Cytogenetic analysis: Karyotyping and FISH of BMA cells can identify chromosomal abnormalities, such as translocations, inversions, and deletions, which are critical for the diagnosis and prognosis of acute leukemia.

• Molecular studies: PCR and NGS of BMA cells can detect gene mutations that are important for diagnosis, risk stratification, and selection of targeted therapies in acute leukemia.

Additional Tests

Several other diagnostic procedures are used to evaluate acute leukemia and its complications, as mentioned below.

• Lumbar puncture: Lumbar puncture is performed to assess CNS involvement in ALL, where cerebrospinal fluid (CSF) is examined for the presence of leukemic cells. All children diagnosed with ALL must undergo lumbar puncture to assess for CNS involvement before starting the therapy.[38][3]

• Coagulation studies: Coagulation tests are performed to assess bleeding risk and monitor for disseminated intravascular coagulation in patients with acute leukemia.

• Chemistries: Blood chemistries, including tests for electrolytes, renal and liver function, calcium, phosphate, lactate dehydrogenase, and uric acid, are performed to monitor for metabolic disturbances associated with leukemia and its treatment.

Diagnostic Tests

Complete Blood Count with Differential

The CBC is a fundamental test that evaluates the number and morphology of red and white blood cells and platelets. In acute leukemia, common findings are listed below.

• Anemia: This condition is characterized by reduced hemoglobin and hematocrit levels due to impaired red blood cell production. Please see StatPearls' companion resource, "Acute Myeloid Leukemia," for more information.

• Thrombocytopenia: This condition reveals a decreased platelet count, which increases the risk of bleeding. Please see StatPearls' companion resource, "Acute Myeloid Leukemia," for more information.

• Leukocytosis or leukopenia: These conditions show increased or decreased white blood cell counts, respectively. A hallmark feature of acute leukemia is the presence of circulating blasts (immature leukemic cells) in the peripheral blood.[37]

• Differential count: This indicates an abnormal distribution of white blood cells, with a predominance of blasts and a decrease in mature neutrophils and lymphocytes.

Peripheral Blood Smear

The PBS is an essential diagnostic tool for evaluating suspected acute leukemia. This provides detailed insights into blood cell morphology and can identify hallmark features indicative of the disease. Key findings are listed below.

• Blasts: The presence of blasts, which are immature hematopoietic cells, is the most significant finding on the PBS in acute leukemia. These blasts often constitute a significant proportion of the white blood cell count and may even be the predominant cell type. The percentage of blasts in the peripheral blood can vary significantly, ranging from a few percent to nearly 100%.[37]

• Blast morphology: The morphology of blasts on PBS can provide valuable clues to the leukemia subtype.
• Acute lymphoblastic leukemia: Lymphoblasts in ALL are generally small- to medium-sized, with a high nuclear-to-cytoplasmic ratio. They exhibit condensed chromatin, inconspicuous or absent nucleoli, and scant cytoplasm. Cytoplasmic blebbing or vacuoles may also be present.[39]
• Acute myeloid leukemia: Myeloblasts in AML are usually larger than lymphoblasts and have a moderate amount of cytoplasm, which may contain Auer rods (azurophilic, needle-like inclusions). Their nuclei are round or irregular, with fine chromatin and prominent nucleoli. Please see StatPearls' companion resource, "Acute Myeloid Leukemia," for more information.

• Additional findings: The PBS of acute leukemia patients may also reveal the conditions mentioned below.
• Anemia: A reduced number of red blood cells or decreased hemoglobin concentration.
• Thrombocytopenia: A decreased platelet count, increasing the risk of bleeding.
• Neutropenia: A decreased number of neutrophils, predisposing patients to infections.
• Dysplastic changes: Abnormalities in the morphology of red blood cells and platelets, such as anisocytosis (variation in cell size) and poikilocytosis (variation in cell shape), may also be present.

The PBS findings in acute leukemia are essential for diagnosis, risk stratification, and treatment decisions. The percentage of blasts in the peripheral blood often correlates with disease burden and prognosis. Additionally, identifying specific morphological features and immunophenotypic markers on blasts aids in classifying the leukemia subtype, which is crucial for determining the most appropriate treatment regimen.[40]

Bone Marrow Aspiration and Biopsy

BMA/BMB are essential diagnostic procedures for evaluating acute leukemia. These techniques provide vital information for disease diagnosis, classification, prognosis, and assessment of treatment response.

BMA involves collecting a liquid bone marrow sample using a needle inserted into the posterior superior iliac crest or, less commonly, the sternum. The aspirate is smeared onto slides, stained, and examined microscopically.

Key findings in BMA are mentioned below.

• Blast percentage: Quantifying blast cells is the most critical aspect of BMA. According to WHO classification criteria, a blast percentage of more than 20% confirms a diagnosis of acute leukemia. Additionally, the blast percentage provides valuable information for risk stratification, with higher levels typically indicating a poorer prognosis.[41]

• Blast morphology: BMA enables detailed evaluation of blast cell morphology. Key features to assess are listed below.
• Nuclear characteristics: Size, shape, chromatin pattern, and presence of nucleoli.
• Cytoplasmic features: Amount of cytoplasm, presence of granules, Auer rods (specific to AML).
• Cytoplasmic vacuoles: Presence and quantity.

These morphological features are instrumental in distinguishing between lymphoid and myeloid blasts and further classifying them into specific subtypes, such as B-ALL, T-ALL, AML with maturation, or AML without maturation.

Immunophenotyping

Immunophenotyping is a critical technique in diagnosing, classifying, prognosing, and monitoring acute leukemia. This laboratory technique allows for the identification and characterization of leukemic cells based on their unique surface antigen expression profiles. Common methods used for immunophenotyping include flow cytometry, immunohistochemistry (IHC), and mass cytometry (also known as cytometry by time of flight or CyTOF), each offering unique advantages in the evaluation of acute leukemia. Please see StatPearls' companion resource, "Immunophenotyping," for more information.

Flow cytometry: Flow cytometry is the most commonly utilized technique for immunophenotyping in acute leukemia. This method involves labeling cells with fluorescently tagged antibodies that target specific cell surface antigens. As the cells pass through a laser beam, scattered light and fluorescence signals are detected and analyzed, enabling the identification of distinct cell populations based on their antigen expression patterns.[42]

This method helps determine the lineage (B-cell, T-cell, and myeloid) and the maturation stage of blasts, offering critical information for leukemia subtype classification. In ALL, specific markers such as CD10, CD19, and TdT are commonly expressed on B-cell precursors, while CD2, CD3, and CD7 are indicative of T-cell lineage. AML commonly shows positivity for myeloid markers such as CD13, CD33, and myeloperoxidase. Additionally, flow cytometry detects MRD by identifying leukemic cells at very low levels, which is essential for evaluating treatment response and predicting relapse.[43]

• Advantages of flow cytometry: Flow cytometry offers several advantages, including high-throughput analysis of large cell populations, simultaneous detection of multiple antigens on individual cells, quantitative assessment of antigen expression levels, and rapid turnaround time.

• Limitations of flow cytometry: Flow cytometry has limitations, including the requirement for fresh or viable cells, restrictions on the number of markers analyzed simultaneously due to fluorochrome spectral overlap, and the need for specialized expertise to interpret complex data.

Immunohistochemistry: IHC is a technique for visualizing antigen expression in tissue sections using labeled antibodies. In acute leukemia, IHC aids in confirming the diagnosis, determining the lineage and maturation stage of leukemic cells, and identifying prognostic markers in bone marrow biopsy specimens.[44]

• Advantages of immunohistochemistry: Advantages of immunohistochemistry include its ability to be performed on formalin-fixed, paraffin-embedded tissue samples, its provision of spatial information about antigen expression within the tissue context, and its capability to assess cellular morphology.

• Limitations of immunohistochemistry: Limitations of immunohistochemistry include semi-quantitative analysis of antigen expression, the ability to analyze only a limited number of markers simultaneously, and interpretation that can be subjective and requires expertise.

Mass cytometry: Mass cytometry (CyTOF) is an advanced technique that combines flow cytometry with mass spectrometry. In this method, antibodies are tagged with heavy metal isotopes instead of fluorescent labels, enabling the simultaneous detection of a larger number of markers on a single cell without issues related to spectral overlap.[45]

• Advantages of CyTOF: Advantages of CyTOF include its higher multiplexing capability compared to flow cytometry, enabling simultaneous analysis of dozens of markers, quantitative measurement of antigen expression levels, and elimination of spectral overlap limitations. 

• Limitations of CyTOF: Limitations of CyTOF include the need for specialized equipment and expertise, higher costs compared to traditional flow cytometry, and limited integration into routine clinical practice.

Clinical Applications of Immunophenotyping in Acute Leukemia

Immunophenotyping is crucial in various aspects of acute leukemia management, as mentioned below.

• Diagnosis and classification: Immunophenotypic profiles help differentiate between ALL and AML and identify specific subtypes based on characteristic antigen expression patterns.

• Prognostic assessment: The expression of certain antigens can serve as prognostic markers for disease progression and response to therapy.

• Minimal residual disease monitoring: Immunophenotyping allows for the detection and monitoring of MRD, enabling early intervention and improved patient outcomes.

• Therapeutic target identification: Immunophenotyping can identify aberrant antigen expressions, which may serve as potential targets for targeted therapies.[46]

Cytogenetic Analysis

Cytogenetic analysis, which involves the study of chromosomes and their abnormalities, is essential in the diagnosis, prognosis, and treatment of acute leukemia. This important tool helps identify specific chromosomal aberrations, offering valuable insights into the disease subtype, risk stratification, and potential therapeutic targets.[47]

Methods of cytogenetic analysis: Several techniques are used for cytogenetic analysis in acute leukemia, each with its strengths and limitations, as mentioned below.

• Karyotyping: This traditional method involves visualizing chromosomes under a microscope to detect numerical and structural abnormalities. This method is considered the gold standard for identifying balanced translocations and large chromosomal aberrations.

• Fluorescence in situ hybridization: FISH uses fluorescent probes that bind to specific DNA sequences, enabling the detection of chromosomal abnormalities with high sensitivity and specificity. FISH is especially useful for identifying cryptic translocations and gene amplifications.

• Polymerase chain reaction: PCR-based assays can detect specific gene rearrangements and mutations associated with acute leukemia, providing additional diagnostic and prognostic information.

• Next-generation sequencing: NGS technologies enable comprehensive analysis of the leukemia genome, identifying a broad range of genetic alterations that may not be detectable by conventional methods. This approach has the potential to revolutionize the diagnosis and treatment of acute leukemia by uncovering novel biomarkers and therapeutic targets.

Role of cytogenetic analysis in diagnosis: Cytogenetic analysis is essential for the accurate diagnosis and classification of acute leukemia. Specific chromosomal abnormalities are often associated with distinct subtypes of the disease.

• Acute lymphoblastic leukemia: Common cytogenetic abnormalities in ALL include the t(9;22) translocation, also known as the Philadelphia chromosome, and the t(12;21) translocation involving the ETV6 and RUNX1 genes.[48]

• Acute myeloid leukemia: Recurrent cytogenetic abnormalities in AML include the t(8;21) translocation, inv(16), and t(15;17) translocation, each associated with distinct morphological and clinical features.

These chromosomal abnormalities not only aid in the diagnosis but also have prognostic implications, guiding risk-adapted treatment strategies.

Prognostic significance of cytogenetic abnormalities: Cytogenetic abnormalities have a significant impact on determining the prognosis of patients with acute leukemia. Certain abnormalities are associated with favorable outcomes, whereas others indicate poor prognoses (see Table 2).[49]

• Favorable prognosis: Patients with t(8;21) AML, inv(16) AML, or t(12;21) ALL generally have a better prognosis and respond well to standard chemotherapy.

• Intermediate prognosis: Patients with normal karyotype AML or those with non-recurrent cytogenetic abnormalities have an intermediate prognosis.

• Poor prognosis: Patients with complex karyotype AML, monosomal karyotype AML, or t(9;22) ALL are at high risk for treatment failure and relapse, necessitating more intensive therapeutic approaches.[49]

Therapeutic implications of cytogenetic analysis: Cytogenetic analysis provides valuable prognostic information and directly influences treatment decisions. Identifying specific chromosomal abnormalities enables targeted therapies that can improve patient outcomes.

• Philadelphia chromosome–positive acute lymphoblastic leukemia: Patients with the t(9;22) translocation benefit from tyrosine kinase inhibitors (TKIs), such as imatinib or dasatinib, in combination with chemotherapy.[50]

• Acute promyelocytic leukemia: The t(15;17) translocation in APL leads to the fusion of the PML and RARA genes. This subtype is highly responsive to all-trans retinoic acid and arsenic trioxide, resulting in high cure rates.[51]

Molecular Studies

Molecular studies, including PCR and NGS, are essential for identifying specific gene mutations and translocations that are crucial in the diagnosis, prognosis, and targeted therapy of acute leukemia. Common mutations in acute leukemia include FLT3, NPM1, and CEBPA in AML and BCR-ABL1, ETV6-RUNX1, and MLL rearrangements in ALL.

Molecular profiling aids in risk stratification and guides treatment decisions, such as the use of TKIs in BCR-ABL1–positive ALL.[50]

Genetic Testing

Genetic testing involves the analysis of specific genes or gene panels to detect mutations, translocations, or other genetic abnormalities. Techniques such as PCR, FISH, and NGS are commonly used. Key genetic alterations in acute leukemia are mentioned below.

• Acute lymphoblastic leukemia: t(12;21) (ETV6-RUNX1), t(1;19) (TCF3-PBX1), t(9;22) (BCR-ABL1), and MLL rearrangements.[52][53]

• Acute myeloid leukemia: t(8;21) (RUNX1-RUNX1T1), inv(16) (CBFB-MYH11), t(15;17) (PML-RARA), FLT3, NPM1, and CEBPA mutations.[54]

Clinical application of genetic testing in acute leukemia: Genetic testing is crucial in the diagnosis, classification, prognosis, and treatment of acute leukemia. The testing helps identify specific genetic abnormalities that guide treatment decisions and predict disease outcomes.[55]

Genetic testing methods: Genetic testing in acute leukemia is essential for detecting specific genetic alterations that inform diagnosis, prognosis, and treatment decisions. Several techniques are used, each offering unique advantages.

• Polymerase chain reaction: PCR is a molecular technique used to amplify specific DNA sequences, allowing for the detection of genetic abnormalities such as gene rearrangements, mutations, and translocations. PCR is highly sensitive and specific, making it a valuable tool for identifying MRD and monitoring treatment response.[56]

• Fluorescence in situ hybridization: FISH utilizes fluorescent probes that bind to specific DNA sequences, enabling the visualization of chromosomal abnormalities and gene fusions in interphase cells or metaphase chromosomes. FISH is commonly used to detect recurrent translocations, such as t(9;22) (BCR-ABL1) and t(12;21) (ETV6-RUNX1), which are associated with specific leukemia subtypes.[48]

• Next-generation sequencing: NGS is a high-throughput sequencing technology that allows for the comprehensive analysis of multiple genes or entire genomes. NGS identifies a broad range of genetic alterations, including point mutations, insertions, deletions, copy number variations, and structural rearrangements. NGS is increasingly used in clinical practice to provide a detailed genetic profile of leukemia, supporting precision medicine approaches.[57]

Prognostic and therapeutic implications: Genetic testing offers valuable prognostic information that can influence treatment choices and patient care.

• Acute lymphoblastic leukemia:

• Favorable prognostic markers: The presence of t(12;21) (ETV6-RUNX1) or hyperdiploidy (>50 chromosomes) is associated with a favorable prognosis and high response rates to standard chemotherapy.[58][59]
• Poor prognostic markers: The presence of t(9;22) (BCR-ABL1), MLL rearrangements, or hypodiploidy (<45 chromosomes) indicates a poor prognosis and an increased risk of relapse.[60][61]
• Targeted therapies: Patients with BCR-ABL1–positive ALL benefit from the addition of TKIs, such as imatinib or dasatinib, to standard chemotherapy regimens.[50]

• Acute myeloid leukemia:

• Favorable prognostic markers: The presence of t(8;21) (RUNX1-RUNX1T1), inv(16) (CBFB-MYH11), or biallelic CEBPA mutations is associated with a favorable prognosis and high response rates to standard chemotherapy.[62]
• Poor prognostic markers: The presence of FLT3-ITD mutations, complex karyotype (≥3 chromosomal abnormalities), or TP53 mutations is associated with a poor prognosis and increased risk of relapse.[62]
• Targeted therapies: Patients with FLT3-ITD mutations may benefit from the addition of FLT3 inhibitors (eg, midostaurin and gilteritinib) to standard chemotherapy.[63]

Minimal Residual Disease Monitoring

Genetic testing is essential for monitoring MRD, which refers to the presence of residual leukemic cells that are undetectable by conventional diagnostic methods. MRD monitoring offers valuable insights into treatment response and the risk of relapse. Patients who test positive for MRD are at a higher likelihood of experiencing a recurrence.[64][65]

Polymerase chain reaction–based minimal residual disease monitoring: PCR is a highly sensitive method for detecting specific genetic abnormalities associated with leukemia at very low levels, enabling precise MRD detection. MRD positivity following induction therapy or during remission is linked to an increased risk of relapse, potentially guiding treatment intensification or consideration of stem cell transplantation.[66]

Next-generation sequencing–based minimal residual disease monitoring: NGS offers a comprehensive analysis of multiple genetic markers, providing an in-depth assessment of MRD. NGS can detect clonal evolution and the emergence of new genetic abnormalities, facilitating early relapse detection and informing treatment adjustments.[67] 

Table 2. 2022 European LeukemiaNet (ELN) Risk Classification by Genetics at Initial Diagnosis

Reference for Table 2.[68]

Interfering Factors

While laboratory tests are crucial for diagnosing and managing acute leukemia, various factors can affect the accuracy and interpretation of results, potentially leading to misdiagnosis or improper management.

Specimen Collection and Handling

Improper collection and handling of blood and bone marrow specimens can significantly affect test results. Delays in processing, inadequate mixing of anticoagulants, or exposure to extreme temperatures can alter cell morphology and viability, leading to inaccurate results in CBC, PBS, and flow cytometry analysis.[69][70]

Pre-analytic Variables

Patient-related factors can influence laboratory findings. For instance, recent blood transfusions may obscure the presence of leukemic blasts in peripheral blood, while prior chemotherapy or radiation therapy can alter bone marrow morphology and cell counts.

Interfering Substances

Certain medications, such as heparin and specific antibiotics, may interfere with flow cytometry analysis by causing nonspecific binding or altering antigen expression. Additionally, high levels of paraproteins, as seen in conditions such as multiple myeloma, can disrupt immunophenotypic analysis.

Technical Issues

Technical errors during specimen processing, staining, or instrument calibration can affect the accuracy of laboratory results. For example, improper fixation or staining of bone marrow biopsy specimens can impair morphological assessment, while variations in flow cytometry gating strategies and antibody panels may lead to discrepancies in immunophenotypic interpretation.

Observer Variability

Microscopic interpretation of PBS and bone marrow morphology is inherently subjective, and results can vary between observers. This variability may lead to discrepancies in the identification and quantification of blasts, potentially impacting diagnosis and risk stratification.

Genetic and Epigenetic Heterogeneity

Acute leukemia is characterized by significant genetic and epigenetic heterogeneity, with different subtypes and clones exhibiting distinct molecular profiles. This heterogeneity can pose challenges in interpreting molecular test results and identifying relevant therapeutic targets.[71]

Sampling Error

BMA/BMB are invasive procedures that may not fully capture the entire spectrum of disease within the bone marrow. This can result in sampling bias and potential misinterpretation of the true disease burden.

To minimize the impact of interfering factors, it is crucial to follow standardized protocols for specimen collection, handling, and processing. Clinicians should be aware of potential pre-analytic variables and interfering substances, ensuring that this information is communicated to the laboratory. Additionally, rigorous quality control (QC) measures must be implemented throughout all laboratory procedures.

Results, Reporting, and Critical Findings

Accurate and comprehensive reporting of laboratory results is vital for the diagnosis, classification, risk stratification, and management of acute leukemia. The report should provide a concise summary of the patient's clinical information, followed by a detailed description of the laboratory findings, their interpretation, and their clinical significance.

Peripheral Blood Findings

Complete blood count: The hemoglobin, hematocrit, white blood cell count, platelet count, and differential count should be reported. Any abnormalities, such as anemia, thrombocytopenia, leukocytosis, or leukopenia, should be highlighted. If blasts are present, their percentage and morphology should be specified.

Peripheral blood smear: A detailed description of the morphology of red blood cells, white blood cells, and platelets should be provided. The presence of blasts, their percentage, and any distinctive features, such as Auer rods (in AML), should be noted. 

Bone Marrow Aspiration and Biopsy Findings

Morphological assessment: The report should include the bone marrow cellularity (hypercellular, normocellular, or hypocellular), the percentage of blasts, and their morphology (lymphoid versus myeloid and B-cell versus T-cell). The maturation pattern of hematopoietic cells should be described, along with any dysplastic changes observed.[72]

Flow cytometry immunophenotyping: The report should include the immunophenotypic profile of the leukemic blasts, detailing the expression of lineage-specific markers (eg, CD19, CD3, CD33, and CD13), maturation markers (eg, CD34 and CD117), and aberrant markers (eg, CD56 and CD123). The percentage of cells positive for each marker should also be specified.[73]

Cytogenetic analysis: The report should detail the karyotype findings, highlighting any numerical or structural chromosomal abnormalities. Specific aberrations, such as translocations, inversions, deletions, or other anomalies, should be clearly identified. If FISH analysis is performed, the results for the specific probes used should be included. Please see StatPearls' companion resource, "Genetics, Cytogenetic Testing And Conventional Karyotype," for more information.

Molecular studies: The results of PCR or NGS assays are reported, specifying the presence or absence of identified gene mutations or fusion genes. The mutant allele burden is quantified if applicable. 

Additional Tests

Lumbar puncture: The results of CSF analysis are reported, including cell count, protein and glucose levels, and cytology findings. The presence or absence of leukemic cells in the CSF is documented.

Coagulation studies: The prothrombin time, activated partial thromboplastin time, fibrinogen, and D-dimer levels are reported. Abnormal findings are interpreted in the context of the patient's clinical presentation to assess the risk of bleeding or thrombosis. 

Clinical Significance

Laboratory testing is essential for the diagnosis, classification, prognosis, and monitoring of acute leukemia. Comprehensive laboratory evaluations enable healthcare providers to accurately identify the disease, determine its subtype, and develop treatment strategies tailored to specific genetic and molecular profiles.

Diagnosis and Classification

The diagnostic process for acute leukemia typically begins with a CBC and PBS. These tests detect the presence of blasts in the peripheral blood and bone marrow—a key indicator of leukemia. The CBC quantifies the blast percentage and identifies abnormalities such as anemia, thrombocytopenia, and neutropenia, commonly associated with acute leukemia. PBS examination provides a detailed morphological evaluation of blasts, aiding in distinguishing between lymphoid and myeloid lineages and offering insights into the specific leukemia subtype.

BMA/BMB procedures are regarded as the gold standard for diagnosing acute leukemia. These procedures enable a comprehensive evaluation of bone marrow morphology, including the blast percentage, cell lineage, and maturation stage. Immunophenotyping, performed through flow cytometry or IHC, provides further classification of leukemia subtypes based on the expression of specific cell surface markers. Please see StatPearls' companion resource, "Leukemia," for more information.

Prognostic Assessment and Treatment Strategies

Extensive cytogenetic and molecular testing is crucial for determining prognosis and tailoring treatment decisions in acute leukemia. Techniques such as karyotyping, FISH, PCR, and NGS identify chromosomal abnormalities and gene mutations that categorize patients into distinct risk groups. These findings guide the selection of treatment strategies, ensuring they align with the genetic and molecular profile of leukemia.

Treatment Strategies Based on Genetic Mutations

FLT3 mutations in acute myeloid leukemia: Patients with AML who have FLT3-ITD (internal tandem duplication) mutations are at an increased risk of relapse and have a poorer prognosis. Targeted therapies, including FLT3 inhibitors such as midostaurin, quizartinib, and gilteritinib, are integrated into treatment regimens to improve outcomes. These inhibitors are used in combination with standard chemotherapy during the induction and consolidation phases and may continue as maintenance therapy.[74]

NPM1 mutations in acute myeloid leukemia: Mutations in the NPM1 gene are generally associated with a favorable prognosis in AML, particularly when not accompanied by FLT3-ITD mutations. Patients with NPM1 mutations typically undergo standard induction chemotherapy, and if they achieve complete remission, they may be candidates for less intensive post-remission therapy. If additional high-risk features are present, hematopoietic stem cell transplantation (HSCT) is considered.[75]

Philadelphia chromosome–positive acute lymphoblastic leukemia: Patients with Philadelphia chromosome–positive ALL, characterized by the BCR-ABL1 fusion gene, benefit from TKIs such as imatinib, dasatinib, or ponatinib. These TKIs are used in combination with standard chemotherapy regimens to target the BCR-ABL1 fusion protein, thereby improving remission rates and overall survival.[76]

IDH1 and IDH2 mutations in acute myeloid leukemia: Patients with AML who have IDH1 or IDH2 mutations can be treated with specific inhibitors, such as ivosidenib or olutasidenib for IDH1 mutations and enasidenib for IDH2 mutations. These targeted therapies are used in cases where patients are refractory to initial treatment or experience relapse after standard therapy.[77]

TP53 mutations: TP53 mutations are associated with poor prognosis and resistance to standard chemotherapy in both AML and ALL. Treatment strategies for patients with TP53 mutations may include the use of hypomethylating agents (eg, azacitidine and decitabine) or participation in clinical trials that explore novel therapeutic approaches.[78]

High Risk of Relapse

Certain genetic mutations and markers indicate a significantly elevated risk of relapse. For instance, AML patients with complex karyotypes, monosomy 7, or specific translocations such as t(6;9) or t(3;3) are classified as high-risk.[68] Similarly, ALL patients with hypodiploidy or the presence of MLL (KMT2A) gene rearrangements face a high risk of relapse. In these cases, aggressive treatment strategies are essential, including the consideration of early HSCT.[60][79]

Chimeric Antigen Receptor T-Cell Therapy

Chimeric antigen receptor T-cell (CAR-T cell) therapy is a form of adoptive immunotherapy that involves the transfer of genetically modified T cells to stimulate an anti-leukemic immune response. For patients at very high risk of relapse or those who have relapsed after initial treatment, CAR-T cell therapy presents a promising option. This approach modifies a patient's T cells to specifically target leukemia cells. CAR-T cell therapy has demonstrated significant success in treating relapsed or refractory ALL, especially in patients with CD19-positive B-ALL.[80] 

Although CAR-T cell therapy is primarily used in ALL, ongoing research evaluates its efficacy in AML, particularly for patients with specific genetic markers such as CD33-positive, CD123-positive, or CLL-1 leukemic cells. This approach could offer a potentially curative option for AML patients who have not responded to other treatments.[81]

Bone Marrow Transplant

Bone marrow transplant, or HSCT, is considered for patients with high-risk features or those in remission but at high risk for relapse. The decision to proceed with HSCT is influenced by factors such as genetic risk, MRD status, and overall patient health. Patients with adverse genetic markers, such as TP53 mutations, complex karyotypes in AML, or t(9;22)/BCR::ABL1 rearrangement (Philadelphia chromosome) or MLL rearrangements in ALL, are prime candidates for HSCT due to their high relapse risk. HSCT involves replacing the patient's diseased bone marrow with healthy donor cells, offering the potential for a cure.[82][83]

Monitoring and Adjusting Treatment

Monitoring treatment response and MRD is essential for timely intervention and improving patient outcomes. Sensitive techniques, such as PCR and NGS, are used to detect MRD, which signifies the persistence of a small number of leukemic cells after treatment. Early detection of MRD enables prompt adjustments to the treatment plan, helping to prevent relapse.

Flow cytometry is also used, particularly in ALL, to monitor MRD levels. By continuously evaluating the patient's response to treatment with these advanced laboratory techniques, healthcare providers can make informed decisions about modifying therapy, such as intensifying treatment, adding targeted agents, or considering HSCT, to achieve the best possible outcomes.[64]

Quality Control and Lab Safety

Implementing internal QC and external quality assessment processes in laboratories evaluating acute leukemia is crucial for ensuring diagnostic accuracy and reliability. These QC measures are instrumental in reducing errors and providing clinicians with reliable data, which is essential for effective patient care and improved outcomes.[84]

Internal QC evaluates the accuracy of a measurement procedure by routinely testing a QC sample with a known expected result. When the result of the QC sample falls within the acceptable range of the expected value, it confirms that the testing procedure is functioning correctly. This ensures that patient sample results can be reported with high confidence, supporting their suitability for clinical use. Conversely, if the QC result falls outside acceptable limits, it signals that the testing system is not functioning properly, raising concerns about the reliability of patient sample results. In such cases, corrective action is required, and patient sample measurements may need to be repeated once the procedure has been restored to stable performance. A corrected report must be issued if inaccurate results were reported before identifying the error.[85][86]

The Levey-Jennings chart is the most commonly used format for evaluating QC results. This chart displays each QC result sequentially over time, providing a quick visual assessment of performance. When the measurement procedure is stable, the mean value represents the target (or expected) value for QC results, while the SD lines indicate the expected level of imprecision.[87] 

The acceptable range and guidelines for interpreting QC results are determined by the likelihood of identifying analytical errors while minimizing false alerts. The standard method for expressing QC interpretive rules uses an abbreviation system, which is widely recognized in clinical laboratories through the work of Westgard. This nomenclature offers a concise and systematic way to communicate QC limits and criteria for identifying potential errors in laboratory analyses.[88]

Generally, 2 different concentrations of QC materials are required for effective statistical QC. For quantitative measurement procedures, it is important to select QC materials with analyte concentrations that reflect clinical decision values throughout the procedure's analytical measuring range.[89]

External quality assessment, also known as proficiency testing, evaluates the accuracy and reliability of a laboratory's measurement procedures by comparing its results with those from other laboratories analyzing the same samples. If a laboratory is not performing well in external quality assessment, it indicates potential issues that must be addressed. The laboratory must conduct a thorough investigation to identify the root cause of the poor performance, reviewing all aspects of the testing process, including sample collection, handling, analytical procedures, and reporting. Once the issues are identified, the laboratory should implement corrective actions, which may include retraining staff, recalibrating equipment, or updating standard operating procedures to ensure compliance with best practices.[90][91]

In the context of a clinical laboratory evaluating acute leukemia cases, ensuring safety is crucial. Key safety measures include establishing a formal safety program and implementing documented policies that address chemical hygiene, bloodborne pathogen exposure control, tuberculosis management, and ergonomics.[92] Identifying significant occupational hazards specific to the laboratory environment, such as biological, chemical, fire, and electrical risks, is crucial. Clear documentation of policies for managing these hazards is essential, especially regarding the handling, packaging, and shipping of diagnostic specimens and infectious materials related to acute leukemia.[93]

Additionally, the laboratory must address other critical safety concerns, such as effective waste management protocols and response plans for bioterrorism and chemical terrorism.[94] These plans are essential for preparing for potential threats or incidents involving hazardous agents that could impact laboratory personnel or patient safety. By prioritizing these safety measures, the laboratory can create a secure environment that ensures accurate diagnostics while protecting the health and well-being of both staff and patients involved in the evaluation of acute leukemia cases.[95]

Enhancing Healthcare Team Outcomes

Laboratory evaluation is crucial in managing acute leukemia by guiding diagnosis, prognosis, and treatment decisions. Optimizing laboratory procedures and adopting a multidisciplinary approach can significantly improve healthcare outcomes. Adhering to standardized protocols for specimen collection, handling, and processing, as recommended by the College of American Pathologists and the American Society of Hematology, ensures consistent and reliable results. This approach minimizes errors and improves diagnostic accuracy. Early and comprehensive testing—including CBC, PBS, BMA/BMB, flow cytometry, cytogenetics, and molecular analysis—facilitates timely diagnosis and the development of personalized treatment plans, leading to improved patient outcomes.

Effective communication and collaboration among clinicians, pathologists, and laboratory scientists are essential for optimal patient care. Regular multidisciplinary discussions support the interpretation of complex laboratory findings, promote appropriate test utilization, and ensure the implementation of personalized treatment plans. Integrating molecular testing into the diagnostic process enhances understanding of the genetic landscape of acute leukemia, identifies prognostic markers, refines risk stratification, and informs the selection of targeted therapies, ultimately enabling more effective treatment strategies.

Clear and concise reporting of laboratory results using standardized formats enhances communication among healthcare team members. Clinical decision-support tools further assist in interpreting complex data and offer evidence-based recommendations for additional testing and treatment options. Regular monitoring of laboratory performance, coupled with continuing education for healthcare professionals, ensures the delivery of accurate diagnostic information and optimal treatment recommendations. These measures collectively contribute to improved patient outcomes and quality of care.