Acute Lymphoblastic Leukemia (ALL) Workup

Updated: Apr 17, 2023
  • Author: Karen Seiter, MD; Chief Editor: Emmanuel C Besa, MD  more...
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Approach Considerations

The following studies and procedures are used in the workup for acute lymphoblastic leukemia (ALL):

  • Complete blood count (CBC) with peripheral smear
  • Coagulation studies (prothrombin time [PT], activated partial thromboplastin time [aPTT], fibrinogen)
  • Chemistry profile, including liver and kidney function studies
  • Bone marrow aspiration and biopsy – Definitive diagnostic tests
  • Cultures; in particular, blood cultures
  • Chest radiography
  • Chest computed tomography (CT) scan, as indicated by symptoms
  • Multiple-gated acquisition (MUGA) scan or echocardiogram
  • Lumbar puncture 

National Comprehensive Cancer Network (NCCN) guidelines note that diagnosis of ALL generally requires the following [20] :

  • Demonstration of ≥20% bone marrow lymphoblasts
  • Morphologic assessment of Wright/Giemsa–stained bone marrow aspirate smears
  • Hematoxylin and eosin (H&E)–stained bone marrow core biopsy and clot sections
  • Comprehensive flow cytometric immunophenotyping
  • Baseline characterization of the leukemic clone, to facilitate subsequent minimal residual disease (MRD) analysis

For optimal risk stratification and treatment planning in patients with ALL, the NCCN advises that bone marrow or peripheral blood lymphoblasts must be tested for specific recurrent genetic abnormalities, as follows [20] :

  • Cytogenetics – Karyotyping of G-banded metaphase chromosomes
  • Interphase fluorescence in situ hybridization (FISH; ALL panel to include testing for  BCR-ABL1MLLTEL/AML - ETV6/RUNX1, CEP4 and CEP10)
  • Reverse transcriptase polymerase chain reaction (RT-PCR) for fusion genes (eg,  BCR-ABL1—Philadelphia [Ph] chromosome positive), including determination of transcript size; in  BCR-ABL1–negative cases, testing for other fusions that are associated with Ph-like ALL may be considered
  • Additional assessment (array comparative genomic hybridization [cGH]) may be considered in cases of aneuploidy or failed karyotype

Next-generation sequencing is frequently performed, however the therapeutic and prognostic implicatons of the findings are still evolving in ALL.

See also Acute Lymphoblastic Leukemia Staging.


Routine Laboratory Studies

A complete blood cell (CBC) count with differential demonstrates anemia and thrombocytopenia to varying degrees in individuals with ALL. Patients with ALL can have a high, normal, or low white blood cell (WBC) count, but they usually exhibit neutropenia. The prevalence and severity of infections are inversely correlated with the absolute neutrophil count (ANC); infections are common when the ANC is less than 500/µL, and they are especially severe when it is less than 100/µL. See the Absolute Neutrophil Countcalculator.

Coagulation studies, peripheral smear, and chemistry profiles

On coagulation studies, a prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT), decreased fibrinogen levels, and the presence of fibrin split products may suggest concomitant disseminated intravascular coagulation (DIC).

A review of the peripheral blood smear confirms the findings of the CBC count. Circulating blasts are usually seen. Schistocytes are sometimes seen if DIC is present.

A chemistry profile is recommended. Most patients with ALL have an elevated lactate dehydrogenase level (LDH), and they frequently have an elevated uric acid level. In addition, liver function tests and blood urea nitrogen (BUN)/creatinine measurement to determine kidney function are necessary before the initiation of therapy.


Appropriate cultures, in particular blood cultures, should be obtained in patients with fever and in those with other signs of infection even if fever is absent.


Radiologic Studies

Chest radiographs may reveal signs of pneumonia and/or a prominent mediastinal mass in some cases of T-cell ALL.

Computed tomography (CT) scans can further define the degree of lymphadenopathy in some patients, including those with mediastinal masses.


Cardiac Studies

Multiple-gated acquisition (MUGA) scans or echocardiograms are needed when the diagnosis of ALL is confirmed, because almost all treatment regimens for acute leukemia include anthracyclines (eg, daunorubicin, doxorubicin), which are potentially cardiotoxic.



Bone Marrow Aspiration and Biopsy

Bone marrow aspiration and biopsy are the definitive diagnostic tests to confirm the diagnosis of leukemia. Immunophenotyping helps to elucidate the subtype.

Aspiration slides should be stained for morphology with either Wright or Giemsa stain. The diagnosis of ALL is made when at least 30% lymphoblasts (French-American-British [FAB] classification) or 20% lymphoblasts (World Health Organization [WHO] classification) are present in the bone marrow and/or peripheral blood.

In addition, slides should be stained with myeloperoxidase (MPO) (or Sudan black) and terminal deoxynucleotidyl transferase (TdT), unless another method is used, such as flow cytometry.

Bone marrow samples should also be sent for flow cytometry and cytogenetics. Approximately 15% of patients with ALL have a t(9;22) translocation (ie, Philadelphia [Ph] chromosome), but other chromosomal abnormalities may also occur, such as t(4;11), t(2;8), and t(8;14). Abnormalities of chromosome number are common in ALL.


Histologic Features

The older, traditional classification of acute lymphoblastic leukemia (ALL) is the French-American-British (FAB) classification. This has now been replaced by the newer World Health Organization (WHO) classification but the FAB system is listed for historical purposes, as follows:

  • L1 – Small cells with homogeneous chromatin, regular nuclear shape, small or absent nucleolus, and scanty cytoplasm; subtype represents 25-30% of adult cases

  • L2 – Large and heterogeneous cells, heterogeneous chromatin, irregular nuclear shape, and nucleolus often large; subtype represents 70% of cases (most common); see the image below

  • L3 – Large and homogeneous cells with multiple nucleoli, moderate deep blue cytoplasm, and cytoplasmic vacuolization that often overlies the nucleus (most prominent feature); subtype represents 1-2% of adult cases

Acute lymphoblastic leukemia (ALL): Bone marrow sh Acute lymphoblastic leukemia (ALL): Bone marrow shows proliferation of large and heterogeneous lymphoblasts consistent with pre–B-cell ALL (French-American-British L2 morphology).

The WHO classifies the L1 and L2 subtypes of ALL as either precursor B lymphoblastic leukemia/lymphoblastic lymphoma (see the following image) or precursor T lymphoblastic leukemia/lymphoblastic lymphoma depending on the cell of origin. The L3 subtype of ALL is included in the group of mature B-cell neoplasms, as the subtype Burkitt lymphoma/leukemia.

In 2016 the World Health Organization Classification published a revised classification of ALL. [21]  This classification included 2 new provisional entities for B-ALL. The first, B-lymphoblastic leukemia/lymphoma, BCR-ABL1–like, was originally reported as a subtype of poor-prognosis childhood ALL with a gene expression profile similar to Philadelphia chromosome–positive ALL. [22, 23]  Some cases of this subtype of ALL respond to therapy with tyrosine kinase inhibitors.

The second, B-ALL with intrachromosomal amplification of chromosome 21, is characteristically detected by FISH with a probe for RUNX1 that reveals 5 or more copies of that gene. [24, 25]  This subtype occurs in older children with a low WBC and is associated with a poor prognosis. Currently there is no subdivision of T-ALL, with the exception of two new provisional subtypes. Early T-cell precursor lymphoblastic leukemia is a subtype with only limited early T-cell differentiation with retention of some myeloid and stem cell characteristics. [26, 27]  The prognosis of this subtype is variable from report to report. Natural killer cell lymphoblastic leukemia/lymphoma is another newly described subtype.

Current World Health Organization Classification of ALL

B-lymphoblastic leukemia/lymphoma

  • B-lymphoblastic leukemia/lymphoma, NOS
  • B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities
  • B-lymphoblastic leukemia/lymphoma with t(9;22)(q34.1;q11.2);  BCR-ABL1
  • B-lymphoblastic leukemia/lymphoma with t(v;11q23.3);  KMT2A rearranged
  • B-lymphoblastic leukemia/lymphoma with t(12;21)(p13.2;q22.1);  ETV6-RUNX1
  • B-lymphoblastic leukemia/lymphoma with hyperdiploidy
  • B-lymphoblastic leukemia/lymphoma with hypodiploidy
  • B-lymphoblastic leukemia/lymphoma with t(5;14)(q31.1;q32.3);  IL3-IGH
  • B-lymphoblastic leukemia/lymphoma with t(1;19)(q23;p13.3);  TCF3-PBX1
  • Provisional entity: B-lymphoblastic leukemia/lymphoma,  BCR-ABL1–like
  • Provisional entity: B-lymphoblastic leukemia/lymphoma with  iAMP21

T-lymphoblastic leukemia/lymphoma

  • Provisional entity: Early T-cell precursor lymphoblastic leukemia
  • Provisional entity: Natural killer (NK) cell lymphoblastic leukemia/lymphoma

Immunohistochemistry and Cytogenetics

A negative myeloperoxidase (MPO) stain and a positive and terminal deoxynucleotidyl transferase (TdT) is the hallmark of the diagnosis of most cases of ALL. However, positive confirmation of lymphoid (and not myeloid) lineage should be performed by flow cytometric demonstration of lymphoid antigens, such as CD3 (T-lineage ALL) or CD19 (B-lineage ALL).

Although more than 95% of cases of the L1 or L2 subtype of ALL are positive for terminal deoxynucleotidyl transferase (TdT), TdT is not specific for ALL; TdT is absent in L3 (mature B-cell) ALL. However, TdT helps to distinguish ALL from malignancies of more mature lymphocytes (ie, non-Hodgkin lymphoma [NHL]).

Flow cytometry helps to distinguish B-ALL from T-ALL. A classic phenotype for B-ALL is seen in the image below. Flow cytometry can also identify whether patients are eligible for certain therapies, such as agents directed against CD20 (rituximab) or CD22 (inotuzumab). Some patients with ALL have aberrant expression of myeloid markers, such as CD13 or CD33.

Pre–B-cell acute lymphoblastic leukemia: Flow cyto Pre–B-cell acute lymphoblastic leukemia: Flow cytometry of bone marrow shows that the cells are positive for CD10, CD19, CD22, CD34, and terminal deoxynucleotidyl transferase.

Cytogenetic abnormalities occur in approximately 70% of cases of ALL in adults (see Table 2, below). These abnormalities include balanced translocations as occur in cases of AML. However, abnormalities of chromosome number (hypodiploidy, hyperdiploidy) are more common in ALL than in AML.

Table 2. Common Cytogenetic Abnormalities in ALL (Open Table in a new window)


Genes Involved

3-Year Event-Free Survival





































* Traditional regimens

 Hyper-CVAD (cyclophosphamide, vincristine, doxorubicin [Adriamycin], dexamethasone) with rituximab

 Hyper-CVAD with imatinib

Eighty-five percent of cases of ALL are derived from B cells. The primary distinction is among the following (see also Table 3, below):

  • Early (pro-B) ALL, which is TDT positive, CD10 (CALLA) negative, surface immunoglobulin (Ig) negative
  • Precursor B ALL, which is TDT positive, CD10 (CALLA) positive, surface Ig negative
  • Mature B cell (Burkitt) ALL, which is TdT negative, surface Ig positive. Fifteen percent of these cases are derived from T cells.

Table 3. Immunophenotyping of ALL Cells – ALL of B-Cell Lineage (85% of cases of adult ALL) (Open Table in a new window)

ALL Cells






Early B-precursor ALL






Pre–B-cell ALL






B-cell ALL






ALL = acute lymphoblastic leukemia; Cylg = Cytoplasmic immunoglobulin; SIg =Surface immunoglobulin; TdT = terminal deoxynucleotidyl transferase.

These cases are subclassified into different stages corresponding to the phases of normal thymocyte development. The early subtype is surface CD3 negative, cytoplasmic CD3 positive, and either double negative (CD4-, CD8-) or double positive (CD4+, CD8+). The latter subtype is surface CD3 positive, CD1a negative, and positive for either CD4 or CD8, but not both. See Table 4, below.

Table 4. Immunophenotyping of ALL Cells – ALL of T-Cell Lineage (15% of cases of adult ALL) (Open Table in a new window)

ALL Cells


Surface CD3


Early T-precursor ALL



+/+ or -/-

T-cell ALL



+/- or -/+

Studies for BCR-ABL analysis by polymerase chain reaction (PCR) or cytogenetics may help distinguish patients with Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL) from those with the lymphoid blastic phase of chronic myelogenous leukemia (CML). Most patients with Ph+ ALL have the p190 type of BCR-ABL, whereas patients with lymphoid blastic CML have the p210 type of BCR-ABL.


Lumbar Puncture

Lumbar puncture (LP) is used to evaluate CNS involvement. In pediatric patients, LP is typically included in the diagnostic workup. National Comprehensive Cancer Network (NCCN) guidelines advise that timing of LP should be consistent with the chosen treatment regimen, and recommend performing LP concurrently with initial intrathecal therapy. [20]

CNS status is classified as follows, on the basis of cerebrospinal fluid (CSF) findings [20] :

  • CNS-1: No lymphoblasts in CSF, regardless of white blood cell (WBC) count
  • CNS-2: WBC < 5/mcL in CSF with presence of lymphoblasts
  • CNS-3: WBC ≥5/mcL in CSF with presence of lymphoblasts

If the patient has leukemic cells in the peripheral blood and the LP is traumatic and WBC ≥5/mcL in CSF with blasts, CNS status is determined by comparing the WBC/red blood cell (RBC) ratio in the CSF to the WBC/RBC ratio in the blood. If the CSF ratio is at least two-fold greater than the blood ratio, the classification is CNS-3; if not, it is CNS-2.