https://www.lls.org/leukemia/chronic-lymphocytic-leukemia/diagnosis

An accurate diagnosis of the type of leukemia is important. The exact diagnosis helps the doctor to

Estimate how the disease will progress
Determine the appropriate treatment

Blood Tests

After your doctor or clinician takes your blood, he or she sends it to a lab for a complete blood count (CBC), which shows the number of red cells, white cells and platelets in your blood.

A person with CLL will have increased numbers of lymphocytes (a type of white blood cell). Low platelet counts and low red blood cell counts may also be present; these counts are usually only slightly decreased in the early stage of the illness.

Immunophenotyping

Immunophenotyping of lymphocytes is an important test used to diagnose CLL, by comparing the cancer cells to normal immune cells. The test results indicate whether or not the person’s lymphocytes are derived from a single cancer cell (leukemia) or from other noncancerous conditions. This test is especially important if the number of lymphocytes in the blood is only slightly elevated. Immunophenotyping also determines whether the abnormal cells are from a change in either B-cell or T-cell development. If the abnormal cells are of the B-cell type, the disease is CLL. If the abnormal cells are T-cells, the disease is called “T-cell prolymphocytic leukemia.”

Immunophenotyping is done with an instrument called a “flow cytometer.” A sample of cells from blood or marrow is tagged with an antibody that is specific for a site on the cell surface. The cells, stained with a light-sensitive dye, go through the flow cytometer, passing through a laser beam; if they have the antibody-specific surface feature, the cells light up and these cells are counted. This test can measure the number of cells in a sample and specific characteristics of the cells, including size, shape and the presence of specific markers on the cell surface. The diagnosis of CLL requires the presence of 5,000 abnormal B-cells per microliter of blood (5,000/uL).

Bone Marrow Tests

Generally, if the red blood cells and platelets are normal, a bone marrow aspiration and biopsy are not needed to make a diagnosis of CLL. However, these tests may be recommended before treatment begins. The test results can help rule out other diseases during the diagnostic stage and they can also be used later, during treatment, to evaluate the effectiveness of therapy.

Bone marrow testing involves two steps usually done at the same time in a doctor's office or a hospital:

A bone marrow aspiration to remove a liquid marrow sample
A bone marrow biopsy to remove a small amount of bone filled with marrow

Quantitative Immunoglobulin Test

This test provides the measurement of the concentration of immunoglobulins in the blood. Immunoglobulins are proteins, called “antibodies,” which are made by B cells in healthy individuals to protect the body from infection. CLL cells do not make effective antibodies. CLL cells also interfere with the ability of the normal lymphocytes to make antibodies. As a result, people with CLL often have low levels of immunoglobulins, causing immune deficiency, which increases their risk of getting infections.

Prognostic Factors

The following tests are not essential to diagnoses CLL, but may help predict the likely outcome (called "prognosis") for the patient, assess the extent of the disease and determine if the patient is ready for certain treatments. Numerous markers have been identified that can help segregate patients who have different rates of disease progression requiring therapy. See the tables below the test descriptions to learn more about these markers.

Fluorescence In Situ Hybridization (FISH). This test studies chromosomes in tissue using DNA probes tagged with molecules that emit light of different colors. The DNA probes then bind to specific genes or areas in the chromosomes within the cells and light up when viewed under a microscope with a special light.

About 80 percent of CLL patients who are tested with FISH have cytogenetic abnormalities in their leukemia cells. These cytogenetic abnormalities can help the doctor identify those people with CLL who are more likely to progress to the point of requiring therapy or those who may benefit most from use of certain types of therapy.

Karyotyping. This test provides a snapshot of the chromosomes by pairing and arranging all the chromosomes of a cell in order. The test can show if there is an abnormality in the size, shape or number of chromosomes. A blood or bone marrow sample from the patient can be used. In CLL, a "complex karyotype," the presence of three or more unrelated defects in chromosomes that occur in more than once cell, is associated with a poorer prognosis.

DNA Sequencing. This lab test uses blood or marrow samples to look for mutations in genes. In CLL, DNA sequencing is used to test for mutation in the immunoglobulin heavy chain variable (IGHV) region, and NOTCH1, SF3B1, and TP53 genes. Mutations or lack of mutations in these genes can help determine a patient's prognosis.

Beta-2 Microglobulin. This small protein is made by many types of cells, including CLL cells. The protein can be measured through a blood chemistry test. High levels of beta-2 microglobulin (B2M) are associated with a type of CLL that is harder to treat.

Common Cytogenetic Abnormalities in CLL

Abnormality	Features	Frequency	Associated Risk
Del(13q)	Deletion in the long arm of chromosome 13	55%	Favorable outcome if not associated with any other abnormality
Trisomy 12	Three copies of chromosome 12	16%	If by itself, associated with intermediate-risk CLL
Del(11q)	Deletion in the long arm of chromosome 11 Often associated with extensive lymph node involvement	18%	High risk
Del(17p)	Deletion in short arm of chromosome 17 Critical TP53 gene in the region is deleted Does not respond well to chemotherapy or chemoimmunotherapy	<10% at diagnosis Up to 30% in relapse/refractory cases	High risk

Some Factors Associated with Higher-Risk CLL

Factor	Features and Associated Outcomes
Blood Lymphocyte Doubling	People with CLL whose lymphocyte number doubles in 1 year have higher-risk CLL and may need closer follow-up care. A lymphocyte number that remains stable generally indicates a relatively lower risk.
CD38	CD is an abbreviation for “cluster designation,” a term that identifies a specific molecule on the surface of an immune cell. CD38 expression is an indicator of higher-risk CLL
Beta₂ - microglobulin (B₂M)	Beta₂-microglobulin (B₂M) is a protein that is shed from CLL cells It is associated with a greater extent of disease.
CD49d	CD is an abbreviation for “cluster designation,” a term that identifies a specific molecule on the surface of an immune cell. CD49d expression is an indicator of higher-risk disease.
IGHV	The unmutated immunoglobulin heavy chain variable (IGHV) region gene is associated with higher-risk disease. About 40% of CLL patients at diagnosis have unmutated IGHV status while the other 60% have the more favorable IGHV-mutated disease. Testing for IGHV mutational status is necessary for treatment when considering chemoimmunotherapy. IGHV is a stable marker (does not change over time), so this mutation should only need to be checked once.
ZAP-70	ZAP-70 (zeta-chain-associated protein kinase 70) Protein expressed near the surface membrane of T cells Plays a key role in T-cell signaling Increased expression of ZAP-70 may be associated with higher-risk disease.
NOTCH1 Gene Mutations	NOTCH1 is a gene involved in the development of different types of blood cells. Approximately 10%–15% of CLL patients have this mutation. CLL patients who have NOTCH1 gene mutations may have a faster progression of disease and a less favorable outcome. Associated with increased risk of transformation to diffuse large B-cell lymphoma (Richter's transformation).
SF3B1 Gene Mutations	The SF3B1 gene is involved in the forming of select proteins in CLL and other blood cancers. Approximately 10%–15% percent of CLL patients have this mutation, resulting in dysfunctional protein processing. CLL patients who have SF3B1 gene mutations may have a faster progression of disease and a less favorable outcome. Associated with resistance to treatment with fludarabine.
TP53 Gene Mutations	The TP53 gene is considered the gatekeeper that protects cell DNA from damage. In cancer cells this mutation leads to increased cell growth and resistance to chemotherapy. Mutation of the TP53 gene is very commonly seen in patients who also have deletion 17p (del17p). CLL patients who have this mutation may have a faster progression of disease, resistance to traditional therapy and a less favorable outcome. Select newer therapies (venetoclax and ibrutinib) have been approved to treat patients who have del(17p) or TP53 gene mutations.

Diagnosis

Leukemia