Chronic Lymphocytic Leukemia
Frequently Asked Questions
Diagnosis, Diagnostic Procedures & Other Tests
Frequently Asked Questions
How is CLL diagnosed?
Should I seek a second opinion?
What are the blood count basics in CLL?
What do laboratory references ranges mean?
What is the difference between percentage of lymphocytes and absolute lymphocyte count (ALC)?
When do neutrophil counts become a problem?
When do platelet counts become a problem?
What are smudge cells?
What are immunoglobulins?
What questions should I ask in early visits with my hematologist?
What is flow cytometry?
What is polymerase chain reaction (PCR)?
What is the significance of IgVH gene mutational status in CLL?
What is the significance of CD38 in CLL?
What is the significance of ZAP-70 in CLL?
What is the significance of bcl-2 in CLL?
Will I need a bone marrow biopsy and how is it performed?
What information is detected in a bone marrow biopsy and aspirate?
What is the significance of beta-2-microglobulin in CLL?
What is fluorescence in situ hybridization (FISH)?
What does trisomy mean?
What are some of the main prognostic indicators in CLL?
What is tissue typing?
What is DiSC assay?
 
CLL is often discovered by chance through routine blood tests, and many people have no symptoms at the time of diagnosis. CLL is suspected when blood tests reveal an absolute lymphocyte count in excess of 5,000 lymphocytes per microliter of blood. This threshold is frequently expressed as 5.0 x 10 raised to the 9th power per liter of blood or 5.0 x 10 raised to the 3rd power in a microliter of blood. The lymphocyte count itself is frequently expressed in the complete blood count (CBC) report as LY# or LY-ABS.

Diagnosis is usually confirmed via a blood test called flow cytometry or via a bone marrow biopsy (BMB). Symptoms that may be present at diagnosis include fatigue, a general feeling of ill health, loss of appetite, enlarged lymph nodes, enlarged spleen, low-grade fever, weight loss, anemia, frequent infections, bruising/bleeding, bone or joint pain, and night sweats.

It never hurts to seek a second opinion. Most CLL patients will be initially diagnosed and cared for by community hematologists/oncologists whose experience with CLL will likely be limited. Very few CLL patients will be fortunate enough to have immediate access to a CLL specialist.

For those who don’t have access to CLL specialists in their communities, it may be worthwhile to travel to see a specialist. Many specialists will work with local physicians in the treatment and management of CLL. A directory of CLL specialists can be found on-line at, http://cll.acor.org/DRdoctors.html.

Perhaps the most important times to seek a second opinion are at the critical junctures in CLL: initial diagnosis, the point of requiring treatment, and when treatment fails and the patient must look at other options.

Understanding changes in basic blood counts is important in monitoring and managing CLL. There are three major types of blood cells: red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes).

Red blood cells (RBCs) are the major component of blood. They carry oxygen and carbon dioxide throughout the body. The percentage of red blood cells in the blood is called the hematocrit. The part of the red blood cells that carries oxygen in a protein is called hemoglobin.

White blood cells (WBCs) are the main component of the immune system, the body’s defence mechanism that fights and destroys such foreign substances as bacteria and viruses. There are several types of white blood cells, each with its own function in protecting the body from germs. Three major types of white blood cells are granulocytes (neutrophils, eosinophils, and basophils), monocytes, and lymphocytes.

Platelets prevent excessive bleeding by helping blood clot at the site of an injury. An abnormally low platelet count (thrombocytopenia) may result in small vessel bleeding (petechiae) or in excessive bleeding from wounds in mucous membranes, skin, or other tissues (hematomas).

Normal blood counts are difficult to state as they vary with the age and sex of the patient as well as where they live. Higher values are usually seen in people who live at higher elevations. The reference ranges for each test will be printed on the laboratory report form and will be specific to your locale. (see also: What do laboratory reference ranges mean?)

On the whole, adult values are typically:

  • Red blood cells:
  • In males, 4.6 to 6.2 million per microliter of blood or 4.6 – 6.2x1012/L
    In females, 4.2 to 5.4 million per microliter of blood or 4.2 – 5.4x1012/L
  • White blood cells:
  • 4,000 to 11,000 per microliter of blood or 4.0 – 11.0x109/L
  • neutrophils:
  • 50% to 60% of total white blood cells
  • monocytes:
  • 3% to 10% of total white blood cells
  • lymphocytes:
  • 25% to 35% of total white blood cells
  • eosinophils:
  • 0% to 3% of total white blood cells
  • basophils:
  • 0% to 2% of total white blood cells
  • Platelets:
  • 150,000 to 350,000 per microliter of blood or 150. – 350.x109/L
  • Hemoglobin:
  • In males, 14 to 18 grams per 100 milliliters of blood or 14 – 18g/dL. (In some countries, these values are given in liters, so they read 140-180 g/L.)
    In females, 12 to 15 grams per 100 milliliters of blood or 12 – 15g/dL.
  • Hematocrit:
  • The percentage of red blood cells per microliter of blood:
    In males 40% to 54%
    In females 35% to 47%

    When speaking about these values, many practitioners use a shortened language by omitting adjectives such as grams. For example, suppose the white cell count is 10.5x109L. Since all white cell counts end in 109L, the number spoken is just the 10.5. A hemoglobin value would be 14 instead of 14 grams per deciliter.

    One calculation that is very important is the absolute cell count. This is achieved by multiplying the total white cell count by the percent of a specific cell reported. Based on these figures, the normal lymphocyte count is in the range of 1,000 to 3,850 per microliter of blood or 1.0 – 3.8x109L. Often the first symptom of CLL is an above normal lymphocyte count, which is discovered in a routine blood test.

    Reference range values are for apparently healthy individuals. Every laboratory should have developed its own reference ranges for all of the procedures it performs. While all laboratories’ ranges will be close, there will be variations due to collection, storage, transport, preparation techniques, types of instruments used, and the specific patients in the laboratory’s population.

    Reference ranges are calculated by performing the same test on a number of “assumed healthy” people that mirror the age or gender of the population that is of interest. The greater the number of individuals in the pool, the more valuable the range will be. Once the results are finalized, an average (mean) is calculated. The next step is similar to a teacher putting a class’s grades onto a Normal (Gaussian) Curve. The calculation is called a standard deviation. The range of +/-2 standard deviations should be wide enough that 95% of all of the test results should be included. This is the reference range. Since the ranges that are developed are averages, not a definition of “normal”, the best way to look at these values is as a reference—something with which to compare yourself against others in your situation. The best comparison, however, is against your own previous reports.

    In the US, the reference range for total white blood cell counts is 4,000 to 11,000 or 4. – 11. x 10 raised to the 9th power per liter or 4. – 11. x 10 raised to the 3rd power per microliter. The various types of white blood cells are often expressed as a percentage of the total white blood cell count. Usual percentage ranges are as follows:

    • Basophils – 0 to 2%
    • Eosinophils – 0% to 3%
    • Lymphocytes – 25% to 35%
    • Monocytes – 3% to 10%
    • Neutrophils – 50% to 60%
    These percentages are derived from 1) a microscopic examination of blood performed manually in which some hundreds of cells are differentiated from each other (this procedure is called a differential or diff) or 2) a machine scored differentiation based on cell patterns.

    Absolute counts are calculated by multiplying the total white blood cell count by the percent of the specific cell line in which you are interested. A percentage of 50% neutrophils in a total white blood cell count of 6,000 equals an absolute neutrophil count of 3,000. While percentage reports are considered adequate for most patients, absolute values are more important for patients with hematologic disorders.

    In order to minimize variations based on laboratory differences, CLL patients who are tracking their counts may want to always use the same lab for their blood count evaluations. It’s important to remember that these statistics work only on populations, not on individuals. You need to compare yourself to you, not to a scale.

    Absolute counts are extremely important. If lymphocyte or other counts are reported as percentages of the total white blood count (wbc), the absolute values can be calculated as follows: Total wbc x % cell type reported ÷ 100. This formula can be used for calculating the absolute lymphocyte count, absolute neutrophil count, etc.

    By way of example, if the total white count reported is 25,000 and the percentage of lymphocytes reported is 80%, the calculation is as follows: 25,000 x 80 ÷ 100. The result is an absolute lymphocyte count of 20,000.

    If the total white blood count minus the total lymphocytes is less than 2000, the patient becomes increasingly at risk of infection. This is one of the reasons it is essential to monitor absolute counts, not percentages.

    As CLL progresses excess lymphocytes in the bone marrow compromise the production of other blood cells. Chemotherapy aimed at reducing lymphocytes also reduces other blood cell counts.

    One of the cell types that is important for CLL patients to monitor is neutrophils. Neutrophils are a type of white blood cell and are an important defense against infection, especially bacterial infection. Neutrophils are also referred to as granulocytes, polys, bands, PMNs, segs, and nonsegs. When neutrophil counts become too low (neutropenia), patients are at risk of infection.

    Normal neutrophil counts are between 2,000 and 8,000 (2.0 to 8.0) per microliter of blood. Many laboratories will also report this as 2.0 - 8.0 x 10.09/L. The numbers 2.0 - 8.0 remain the same but have been adjusted to reflect the larger volume of blood. Experience has shown that neutrophil counts above 2.0 will keep almost everyone safe from infections. Patients with counts between 2.0 and 1.0 should take precautions such as staying away from crowds, children with runny noses, etc. Between 1.0 and .5, patients are in jeopardy of infections from everyday bacteria found in salads, fresh unpeeled fruit, shellfish, rarely cooked foods, etc. Below .5 most physicians consider that patients already have infections, usually from their own bacteria, in the gastrointestinal tract, nose, etc. (see also: What is a neutropenic diet?)

    When neutrophil counts fall too low because of advancing disease and/or chemotherapy, physicians may administer granulocyte colony-stimulating factors (G-CSF) to boost neutrophil counts. G-CSF is also known as filgrastrim or by its trade name Neupogen™.

    If neutrophil counts are reported as percentages, patients can calculate absolute neutrophil counts (ANC) by multiplying the total white blood count by the percentage of neutrophils. While percentage reports are considered adequate for most patients, absolute values are more important for patients with blood (hematologic) disorders.

    Platelets prevent excessive bleeding by helping blood to clot at the site of an injury. An abnormally low platelet count (thrombocytopenia) may result in small vessel bleeding that can cause small red dots on the body (petechiae), or in excessive bleeding from wounds in mucous membranes, skin, or other tissues (ecchymoses and hematomas). Platelet counts in CLL patients can become compromised by excessive lymphocytes, by treatment, and by related conditions such as Immune Thrombocytopenic Purpura (ITP).

    Normal platelet counts are between 150,000 and 350,000 (150 – 350 x 103μL or 150 – 350 x 109L) per microliter of blood. There are no clear-cut numbers for when platelet counts become a concern. As oncologists gain more experience with decreased platelet counts, the threshold of concern has dropped. It used to be that anyone with a platelet count of lower than 75 was a candidate for platelet transfusion; now it is down into the 10’s and 20’s - lower yet if there is no sign of bleeding.

    Platelet counts below 20 are of serious concern, but anyone is at risk of bleeding episodes if the platelets are decreased below 75.

    Patients often see a reference to smudge cells in their complete blood count (CBC) reports. Smudge cells are cells that are probably damaged during the CBC process. The cell walls rupture, and when seen under the microscope, they look like a smudge; hence the term smudge cells. These cells are probably lymphocytes and are so distorted they can't be given a "real" name.

    Smudge cells are not unique to CLL. However, they are seen much more frequently and in much higher numbers in CLL than in any other condition. For example, in normal specimens, there may be .01 percent. In patients with severe infections or burns, there may be 0.1 to 0.3 percent. In patients with acute leukemias, there may be as many as 1 to 3 percent, but in CLL patients, smudge cells can be up to 20 percent of all cells, or higher. The percentage of smudge cells present in a complete blood count may also be an independent prognostic marker in patients with early stage CLL. Less than 30 percent smudge cells appears to be predictive of a less favorable clinical course; whereas, greater than 30 percent smudge cells suggests a more favorable prognosis.

    Immunoglobulins are proteins called globulins, specifically immunoglobulins (Ig) or antibodies (Abs). They are made by B-lymphocytes and appear on the outside of cells when seen under the microscope. The antibodies that these cells produce are in response to the presence of foreign substances called antigens.

    B-lymphocytes make five different types of antibodies: IgA, IgG, IgM, IgE, and IgD. The type of antibody produced is based on the type of antigen detected and the method of stimulation. IgE, for example, is the response of choice for allergic reactions.

    For the most common antigens, (flu, viruses, and the like) the first antibody made is usually IgM. How the antigen got into the patient's system also plays a role. IgA is usually made if the antigen was found in body fluids such as saliva while IgM usually responds to GI tract antigens. IgG is the first responder for blood borne items and the final choice for more antibodies.

    Eventually, the antibody destroys the antigen, and a small number of cells settle into a memory state. They are no longer making the antibody, but the next time they recognize that specific antigen, they will react faster and more efficiently. This "memory" cell is the cell responsible for the protection we have from vaccinations.

    Normal levels of immunoglobulins vary somewhat, but are typically as follows:

    • IgG: 5.5 – 19.0 g/L (grams per liter of blood)
    • IgA: 0.60 – 3.3 g/L (grams per liter of blood)
    • IgM: 0.45 - 1.5 g/L (grams per liter of blood
    • IgD: 5 - 30 mg/L (milligrams per liter of blood)
    • IgE: < 500 ug/L (micrograms per liter of blood)
    When responding to antigens, these levels will rise.

    CLL patients—particularly those with progressive disease—may not be capable of producing sufficient quantities of immunoglobulins in which case their ability to fight infections may be diminished. In these cases, patients are sometimes given immunoglobulins intravenously to help them fight infections.

    Following are some of the questions to ask in early visits to your hematologist:
    • What do the blood tests show? How is this different from “normal”?
    • What stage am I in?
    • Will I need further tests? How often will they be?
    • Will you mail or fax copies of my test results, or should I come in to pick them up? If I pick them up, when will they be ready?
    • What symptoms should I watch for?
    • What changes should I make to my daily routine to avoid complications?
    • When do I see you and when do I see my family Doctor?
    • When do you anticipate that I will require treatment?
    • How do you anticipate treating my CLL?
    • How many CLL patients do you have?
    • What is the long-term outlook for my CLL?
    Flow cytometry is a diagnostic technique that is used to measure the chemical or physical characteristics of cells in suspension. Flow cytometry can also determine the types and the quantities of antigens expressed on cell membranes through a process called immunophenotyping. Antigens are substances that are capable of activating the immune system. Each antigen category is called a cluster of differentiation (CD) and is numbered.

    This is very much an evolving field. Isolation of CD markers is continuing and new ones are being found monthly. As a result, marker panels will change, and more specific information will be available. As a general rule, it can be said that,

    • Cells that are positive for CD2, 3, 4, 5, 8, 45RA and/or 45RO are T-lymphocytes.
    • Cells that are positive for CD10, 19, 20, 21, 23, 35, 40, and sometimes 77 are B-lymphocytes (CD5 can also be found on a specific subset of B-lymphocytes).
    • CD28 can be positive with T or B-cells and NK (natural killer) cells, which are another lymphocyte subset.
    • CD34 is one of the most important markers for it is seen in the hematopoietic pluripotential stem cell (PSC) which is the cell that is "wanted" in bone marrow or peripheral blood stem cell transplants.
    • CD38 is a receptor that is found in plasma cells, some thymocytes (early lymphocytes still in the thymus), NK cells, and in very early B-cells. Increased presence of CD38 has been found in cells in multiple myeloma, and certain acute lymphoblastic and myeloblastic leukemias. CD38 is also thought to be of predictive value in determining the clinical course that CLL will take.

    In CLL cells, a distinct pattern of antigens is expressed. CLL lymphocytes coexpress the B-cell antigens CD19 and CD20 along with the T-cell antigen CD5. The analysis of a blood sample by flow cytometry is therefore very useful in confirming the diagnosis of CLL.

    The following diagram illustrates how CD markers are used in the diagnosis of disorders that are characterized by elevated lymphocyte counts (lymphocytosis). It is presented here with permission of the copyright holder, Dr. Margaret Uthman.

    The terms kappa and lambda, which appear on flow cytometry reports, refer to portions of the immunoglobulin or antibody molecule. Kappa and lambda are long chains of amino acids. While there are heavy chains and light chains, kappa and lambda are both light chains. Most people should be able to make either of these light chains in the amounts appropriate for antibody activity. Over expression of one of the chains is often seen in CLL patients and usually means a loss of control by the cells.

    When the leukemias were first discovered, the only tool available was the microscope. Now we have learned how to identify cells by their cell membranes. It is hoped that we will soon be able to identify cells by their DNA.

    Flow cytometry is very time consuming to perform. Cells cannot be tested for every known antigen—it’s too expensive, requires too much time, and there is too great a possibility of error. For these reasons, every lab makes a series of decisions concerning which tests will be run on which samples.

    In CLL, malignant lymphocytes derive from cells gone berserk (monoclonal expansion). If we can find out what type of cell it is (for example, does it have a compound on its membrane called CD20), then it is possible to design a drug (monoclonal antibody) to fight only CD20+ cells. This is the underlying principle of medications like rituximab (Rituxan).

    Polymerase Chain Reaction, (PCR) is a laboratory process that was developed in 1985. In PCR, a particular DNA segment from a mixture of DNA chains is rapidly replicated, (between 10,000 and 1,000,000 copies can be made, depending on the procedure) producing a large, readily analyzed sample of a piece of DNA. The process is sometimes called DNA amplification. PCR has had an immense impact on biology and medicine, especially genetic research.

    PCR is commonly used in CLL to test for minimal residual disease (MRD) in patients who have achieved complete remissions. This technique is very sensitive and is capable of detecting a single CLL cell in 100,000 cells.

    PCR can be performed on any number of specimens: tissue, bone marrow, peripheral blood, and fluids. It is usually performed on the specimen that is thought to harbor the cell line of interest. In CLL, PCR is typically performed using the bone marrow or the peripheral blood (a normal blood draw). Because the leukemias arise in the marrow, some physicians prefer to perform PCR using a marrow specimen.

    The presence of minimal residual disease in patients who have achieved complete remissions is predictive of a shorter event-free remission. For this reason, when minimal residual disease is detected, further treatment aimed at eradicating residual CLL cells may be recommended.

    The mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes in the leukemic cells of chronic lymphocytic leukemia patients is thought to be the gold standard for prognosis.

    The division of B-CLL into stable and progressive disease was noted in the 1960’s, and today, it is generally agreed that there are two subsets of CLL based on IgVH gene mutational status. Patients with unmutated immunoglobulin V genes (approximately 40%) form one subset while patients with mutated immunoglobulin V genes form the other subset. There is no evidence that the subsets change from one into the other. The unmutated subset is three times as common in men while the mutated subset is equally common in men and women.

    These two subsets of CLL have separate and distinct natural histories. Early stage patients with unmutated immunoglobulin V genes have a median life expectancy of 8 years, while those with mutated immunoglobulin V genes have a median survival of twenty-five years. It is not correct to assume that all CLL patients with mutated immunoglobulin V genes are smoldering, since some cases do progress to advanced stage disease. However, it is very likely that all cases of smoldering CLL are confined to patients with mutated immunoglobulin V genes.

    The best specimens for testing IgVH gene mutational status are the peripheral blood and the bone marrow. To be absolutely certain, both should be evaluated since there are patients who express this mutation in the marrow, but not in the peripheral blood. The best test for IgV mutational status involves sequencing of IgVH genes. Another option is single-cell reverse transcription-polymerase chain reaction (RT-PCR). Both of these procedures are expensive and are not readily available. For this reason, researchers look for surrogate markers that are strong predictors of IgVH mutational status.

    Some researchers believe CD38 is an accurate surrogate for IgVH mutational status—patients with less than 30 percent CD38+ B-CLL cells are likely to have mutated immunoglobulin V genes while patients with greater than 30 percent CD38+ B-CLL cells are likely to have unmutated immunoglobulin genes. This is a matter of ongoing debate as it appears that there is approximately a 30% discordance between the assays. Moreover, in 25% of cases the expression of CD38 changes during the course of the disease. Serum thymidine kinase level is thought to be another surrogate marker where >15 U/l has proved to be a strong predictor of mutational status. ZAP-70 is yet another surrogate marker for IgVH gene mutational status where absence of ZAP-70 suggests mutated genes, and expression of ZAP-70 is indicative of unmutated genes.

    The presence of the antigen CD38 on B-CLL cells is a much discussed prognostic indicator in CLL. Whether it is a truly independent prognostic indicator or simply a reflection of IgVH gene mutational status, CD38 clearly seems to have some relevance in predicting whether a patient’s CLL is likely to have a favorable or unfavorable clinical course. CD38 is detected by flow cytometry, a diagnostic technique frequently used in confirming CLL.

    Patients with less than 30 percent CD38+ B-CLL cells are likely to have a favorable clinical course requiring minimal or no therapy. Patients with equal to or greater than 30 percent CD38+ B-CLL cells are more likely to have an unfavorable clinical course requiring earlier and ongoing treatment. Significant differences in survival are also thought to exist between these two groups. CD38 expression remains stable over time in the majority of patients, but it is known to change in approximately 25 percent of cases. Its level of expression does not seem to be influenced by chemotherapy.

    The connection between CD38 expression and IgVH gene mutational status is not well understood. It appears that patients with less that 30 percent CD38+ B-CLL cells are likely to have mutated IgVH genes while patients with greater than 30 percent+ B-CLL cells are more likely to have unmutated IgVH genes. While this is often the case, there is approximately a 30 percent discordance between assays for CD38 and IgVH mutational status (see also: What is the significance of IgVH gene mutational status in CLL?)

    Both CD38 and IgVH gene mutation are thought to be useful prognostic indicators in B-CLL, but because of the relative ease of testing for CD38, it is a much more convenient test.

    CD38 and IgVH mutational status are just two of a number of prognostic indicators in CLL. Others include, circulating levels of beta-2-microglobulin and soluble CD23, lymphocyte doubling time, serum thymidine kinase levels, bone marrow histology, and chromosome abnormalities.

    ZAP-70 is an abbreviation for Zeta-chain-associated protein kinase 70. This protein is a member of the protein-tyrosine kinase family and when expressed on B-CLL cells is surrogate marker for IgVH gene mutational status. The presence of ZAP-70 can be detected by flow-cytometric analysis, and the level of expression is thought to correlate with mutational status.

    CLL patients with less than 20 percent ZAP-70 positive B-CLL cells are likely to have mutated immunoglobulin V genes, predictive of a more favorable clinical course, while patients with greater than 20 percent positive B-CLL cells are likely to have unmutated immunoglobulin V genes, predictive of a less favorable clinical course. (see also: What is the significance of IgVH gene mutational status in CLL?).

    Bcl-2 is one of several proteins that positively and negatively regulate cell death. Bcl-2 inhibits programmed cell death and is consistently over-expressed in B-CLL patients. This over-expression of bcl-2 that occurs in many forms of leukemia contributes to the relentless accumulation of lymphocytes that fail to die and to their resistance to chemotherapy.

    In the laboratory, Antisense drugs such as Genasense™ have demonstrated an ability to inhibit bcl-2 expression in CLL, thereby reducing resistance to programmed cell death and to chemotherapy. Antisense treatment is currently being tested in clinical trials, followed by state-of-the art anticancer therapy in an effort to improve patient outcome.

    Yes, in all likelihood, you will be asked to undergo a bone marrow biopsy at some point in your diagnosis or treatment. In some major cancer centers, bone marrow biopsies are not performed until treatment begins.

    There are two procedures used for obtaining bone marrow samples: the bone marrow aspirate which is used to obtain a small amount of marrow from inside the bone, and the bone marrow biopsy which is used to obtain a sample from the bone showing the structure of the bone marrow cavity.

    Aspiration works extremely well when there is little or no fibrosis (when the cells in the marrow are not tightly packed) and when some cells are individual (not so tightly bound to each other that they look like a single entity). This is because of the need to force single cells to come into the syringe by applying a vacuum. Biopsies work well when there are decreased numbers of cells or the cells form tight packets. Many facilities perform both in the same procedure. The aspirate is done first and then the “core” biopsy is performed. This provides the best of both worlds with only one needle insertion.

    These procedures are useful in confirming CLL, determining the extent of the disease, and deciding on treatment. The pattern of lymphoid infiltration in the biopsy specimen of the marrow also provides useful prognostic information—diffuse involvement correlates with progressive or advanced disease, while nodular or interstitial (non-diffuse) patterns predict a better prognosis.

    The samples are usually obtained from the back of the hip bone, although the breast bone (sternum) may be used instead for bone marrow aspirates only. These procedures cause some brief and usually mild discomfort. They are usually carried out with local anaesthetic, although oral or intravenous sedation may also be available.

    Before the invention of immunophenotyping, examination of the bone marrow was the required method of diagnosing CLL. By agreed definition, anyone whose lymphocytes made up more than 30% of the blood cells in the marrow was presumed to have CLL. That, together with the signs, symptoms, and a complete blood count (CBC) of the peripheral blood made the diagnosis.

    Bone marrow biopsies are no longer required to diagnose CLL, but biopsies and aspirates are regularly used to assess disease extent, degree of marrow involvement, effect of treatment, and readiness for transplant.

    Bone marrow biopsies and aspirates look very much alike. They are drawn from the same sites; they even use the same insertion needle. They are different in the type of specimen that they withdraw. Biopsies take a solid core of marrow with all of its structures such as capillaries and collagen fibers intact. Aspirates sacrifice the architecture of the marrow in order to spread the cells out more thinly so that the individual cells can be evaluated. Some conditions can be evaluated just by biopsy; others just by aspirate. Many physicians prefer to obtain both types of specimens so that a more complete picture of the marrow can be seen. It is a difficult specimen collection, so it makes sense to get as much information as possible with a single needle.

    The bone can be thought of as a commercial honeybee hive. The object is to get through the outer structure of the hive without damaging it and take some of the honeycomb and some of the honey. The marrow, which is represented as the honeycomb, is three-dimensional and contains significant numbers of support structures such as collagen, arteries, veins, capillaries, and lymphatics. The aspirate, which is the honey, contains the developing blood (hematopoietic) cells.

    Bone Marrow Biopsy

    In bone marrow biopsies, an extremely sharp but hollow needle-like tool is placed into the marrow and twisted. The piece of marrow inside the hollow is removed as a unit with all of its cells and structures untouched. This is the biopsy material and it is treated in the histology laboratory the same way that one treats biopsies of lymph nodes, of breast tissue, or any other tissue.

    This specimen is placed into hot wax (paraffin), which permeates the entire specimen giving it strength and rigidity. It is then cut into extremely thin sections (approximately 4-6 millionths of an inch). These sections are then stained and the physician examines them under the microscope. It is then possible to see if there is any damage to the structure of the marrow. Are there, for example, increased amounts of scarring (fibrosis)? Is there damage to the vessels (arteries, veins, and capillaries) that provide the marrow with nutrients? Is there sufficient iron storage in the tissue cells? It is in this sample that one can evaluate cellularity. As one ages, the number of hematopoietic cells lessens, and the amount of fat increases. Cellularity can only be evaluated if you know for sure that you have a sample that contains all of the cells in a given volume, so cellularity is only reported on a biopsy or clot section. Overall numbers of cell types can be identified and how they are grouped together can be viewed as well.

    Bone Marrow Aspirate

    One of the drawbacks to a biopsy is that many of the cells cannot be seen well. This is primarily because the stain used is not specific to hematopoietic cells, but also there is the issue of the damage that is done to the cells in the treatment process. While individual cells are seen, the processing concentrates on structure and relationship, not on individual cells. To see individual cells requires a clear separation of cells. In order to accomplish this, a drop or two of liquid marrow is placed on a slide, and another slide is placed on top of it, and the specimen is squeezed between the two slides, causing it to spread out. This spreading destroys any structural components, but it provides a thin, well-separated coating of marrow across the slide. When this is stained with the usual stains found in a hematology laboratory, one can view the smear with a microscope and identify individual cells and assess their quality.

    It is from the aspirate that a differential report is produced showing percentages of different cell lines. The differential section of biopsy/aspirate lab report looks similar to a complete blood count (CBC) report, but it is performed by identifying between 500 and 1000 cells instead of the traditional 100 cells used in the peripheral blood differential. The percentage of lymphocytes shown in this section of the report represents the percentage of marrow involvement that is often quoted in CLL. This percentage is a key indicator in determining the extent of disease, the efficacy of treatment, and in preparing patients for transplant. Most transplant centers want this percentage to be below ten percent prior to transplant.

    Common Processes

    Multiple slides of each type of specimen are evaluated and each type of material will have its place in the report. As a consequence, it may appear that the report contains the same thing over and over. It does. It is possible for a report to contain 2 different slide reports from the biopsy, 2 reports from the aspirate and reports from additional or special staining techniques. Why multiple slides? Many conditions are very focal, and appear in small discrete places. If only one slide was evaluated, the condition might be missed entirely. In some institutions, marrows may be checked by a minimum of 4 slides from each type of specimen.

    Each slide is evaluated and the total impression is reported. The steps listed are performed on each slide. Different steps are performed with differing levels of magnification. For some steps, a small amount of magnification is needed; for others a very high amount of magnification is needed.

    At the lowest level of magnification, the first step is to confirm that there are spicules of real bone marrow present. It is possible to get just fat deposits or just blood. Without the spicules, you cannot tell if what you are looking at is really a reflection of the marrow or just material that was picked up by the aspiration. So you will see on the report some comment about the quality of the aspirate that mentions spicules. If you or your physician sees that the specimen was poor or good, then you can interpret the results in the light of that comment.

    The next step in the evaluation assesses the relative cellularity of the specimen. This is best done on the biopsied material and not the aspirate but many times there will be a confirmatory comment about cellularity on an aspirate's report. In general, as one ages, the cellularity of the marrow declines and that space is used by fat cells. Newborns have essentially no fat and 100% cellularity while an eighty year old will have approximately 60% fat and 40% blood producing cells. If the marrow is under stress and can increase the number of blood producing cells, then the cellularity will be increased. The next step is to scan the slide for the presence of any large abnormal cells or clumps of cells. Malignant cells tend to adhere to each other so they may be in abundance in one portion of the slide and not in another. The cells from which platelets arise (megakaryocytes) are also evaluated. These cells are quite large and relatively few in number so you scan the entire slide to get a sense of how many are present. It is important for megakaryocytes to be present in adequate numbers and to be seen to be producing platelets.

    At the next magnification level, the Myeloid to Erythroid (M:E) ratio is performed. Since red cells live significantly longer than white blood cells, usually there are three to four times the number of white cell precursors to red cell precursors. On the slides containing biopsied material, comments about the support structure, the vessels, bone cells, and collagen are made.

    At the highest level of magnification, individual cells are evaluated and comments are made about their numbers and quality. At this point comments about the exact appearance of the abnormal lymphocytes are made. In CLL, commonly used words are "monotonous in appearance" or "clonal expansion". The red cell precursors are evaluated to see if there are problems with hemoglobin synthesis (most typically from iron deficiency) or if there are problems with the nucleus (most typically from folic acid deficiency) or if they are adequate in number. These comments support the CBC's hemoglobin, hematocrit, and red cell indices results. Finally, the platelet production is evaluated. By looking at megakaryocytes, one can see if they are producing platelets and if the platelets being produced are appropriate looking.

    Special testing

    One test that is routinely performed on bone marrow biopsy material is a test for iron deposits. This requires the use of a different stain so while it is a routine test it is usually thought of as a special stain. In this stain, iron deposits are stained blue while the cells themselves stain pink. The absence of any blue deposits confirms that the person lacks adequate iron and is either iron deficient or barely able to keep up with need. In addition to certain inherited conditions, there are a number of ways in which a person may have too much iron stored. One of the more common ways is by repeated transfusions. Another is by using too little iron because there is a suppression of red cell production since hemoglobin production accounts for over 90% of all iron in the body. Iron stores will not tell which process is involved but will provide the physician with a needed piece of information for treatment decisions.

    Additionally, marrow cells can be tested by flow cytometry, cytogenetics, FISH, or PCR techniques. This is important because there are situations in which malignant cells will be found in the marrow only or in the nodes only or in the peripheral blood only. Close monitoring of where the malignant cells are is important in prognosis as well as treatment.

    Beta-2-microblobulin (β2M) is a protein found on the surface of all cells. Small amounts are shed into the serum and are usually filtered out by the kidneys. Increased amounts are found in the serum of patients with kidney disease, lymphomas, some leukemias, and myelomas.

    Of interest is the prognostic connection that is sometimes seen with this protein, especially with myeloma, but in the other conditions as well. People diagnosed with these diseases and who have levels of beta-2-microglobulin below 2.0 (2mg/L) seem to have at least 2 more relatively complication-free years. Beta-2-microglobulin is evaluated via a normal blood draw.

    Fluorescence In Situ Hybridization (FISH) is a technique that is used to detect chromosomal abnormalities in cells.

    In CLL, FISH is used to analyze lymphocytes for chromosome defects. It is estimated that more than 50 percent of patients with CLL will have detectable chromosomal abnormalities, some of which are associated with disease progression and survival.

    Examples of chromosomal abnormalities commonly found in CLL include, trisomy 12 and deletions in chromosomes 6, 11, 13, and 17. These are not the only chromosomal abnormalities that can occur in CLL, and combinations of abnormalities are also possible.

    FISH can be performed on any number of specimens: tissue, bone marrow, peripheral blood, and fluids. It is usually performed on the specimen that is thought to harbor the cell line of interest. For most of the leukemias, the marrow is typically used, but other specimens that are sometimes evaluated include cells from the cerebrospinal fluid and the lymph nodes.

    Tests are often carried out on leukemia patients to detect any chromosomal abnormalities associated with their disease. Trisomy is a chromosomal defect that involves the presence of an additional whole chromosome.

    Every cell in the human body (with the exception of ovum and sperm) has 46 chromosomes. There are 22 pairs of chromosomes called somatic chromosomes plus the 2 sex chromosomes. As each cell prepares to undergo the usual process of cell reproduction (mitosis), it doubles the number of chromosomes and then splits into 2 cells, each with 46 chromosomes. Or at least that is how it is supposed to work.

    Every now and again, three copies of a chromosome will go into one cell giving the cell a trisomy of a specific chromosome, and the remaining copy goes into the other cell resulting in a monosomy. Usually both of these circumstances result in the death of both cells, but occasionally, a cell can live to produce a cell line that is unique, and unfortunately, unique cell lines can be malignant.

    Trisomy 12, the presence of an additional 12th chromosome, is a trisomy that is frequently found in CLL patients. The defect responsible for trisomy 12 in CLL is not known. Studies suggest that CLL patients with such chromosomal abnormalities carry a poorer prognosis than patients with normal chromosomes.

    We don’t know exactly what causes these trisomies, and much more important, regardless of what the Human Genome project press releases say, we really don’t know what information is where or what it means. So, all we can do at this stage is note that certain diseases seem to have certain trisomies, or monosomies.

    When a person has just been diagnosed with early stage CLL, it is important to exercise caution in making long-term predictions. The course of CLL is highly variable. Some patients remain without symptoms for years while in others the disease progresses much more rapidly. Only after a physician has had an opportunity to monitor a newly diagnosed, early stage patient for a period of approximately one year can a more reliable long-term prognosis be given.

    A number of prognostic indicators are available to CLL patients and their physicians. Very few early stage patients will be tested for all of the indicators listed below, but they are some of the main indicators that are available. One should never rely on a single indicator, but should use as many of the indicators as possible in determining the prognosis. Most physicians experienced in treating individuals with CLL have developed their own set of indicators/tests that they use at different points in time after the initial diagnosis.

    Newly diagnosed patients who are in early-stage disease will find these indicators particularly helpful in determining how their CLL might progress. Patients with more advanced disease will also find these indicators useful. These patients are often already aware of the indicators and their impact on prognosis.

    Indicator Prognostic implications
    Lymphocyte doubling time Peripheral lymphocyte doubling times of less than one year are indicative of aggressive CLL, whereas, doubling times of greater than one year suggest a more indolent situation.
    Absolute lymphocyte count Once an abnormal cell population is noted, the traditional method of reporting (the percentage) is no longer valid. Only the absolute counts correctly reflect the white cell population in the peripheral blood. The lymphocyte doubling can be seen here and together with the absolute granulocyte count, the general state of the individual’s immune system can be seen. The absolute lymphocyte count is usually listed as either #LY or ABS LY on a differential report.
    Hemoglobin level Hemoglobin levels of greater than 13 grams per deciliter of blood (13g/dL) are desired. Levels of less than 13g/dL may indicate aggressive or advanced disease. Some laboratories report hemoglobin levels in liters and not in deciliters. In that situation the desired level is greater than 130 grams.
    Bone marrow histology Diffuse bone marrow involvement correlates with progressive or advanced disease, while nodular or interstitial (non-diffuse) patterns in the bone marrow indicate a better prognosis.
    Beta-2-microglobulin Beta-2-microglobulin (β2m) is a piece of the cell membrane. When cells are particularly active or are damaged easily, this piece is broken off and remains in the plasma where it can be quantified. The slower the cell’s activity or the less damage within the cell, the lower the amount of β2m in the plasma. Patients with β2m values below 2.0 mg/L seem to have a more favorable outlook than those with values above 2.0.
    CD38 expression Patients with less than 20 percent CD38+ CLL cells are likely to have a more favorable clinical course requiring minimal or no therapy, whereas, patients with greater than 20 percent CD38+ B-CLL cells are more likely to have an unfavorable clinical course requiring earlier and ongoing treatment. CD38 seems to be closely linked with IgV gene mutation; however, this correlation is a matter of debate.
    Serum lactate dehydrogenase (LD or LDH) Most cells in the body require this enzyme in their production of energy. As cells are damaged or die inappropriately, they will spill the contents of their cytoplasm into the peripheral blood where it can be quantified. There are five major types of LD and each can be identified through a procedure known as isoenzyme electrophoresis. Increases in LD can be seen when there is increased cell death due to chemotherapy or when a person is relapsing from remission.
    Soluble CD23 CD23 is supposed to be found on all B-cells and a few other cells. B-cells are supposed to constitute less than 25% of the total number of lymphocytes in the peripheral blood. If CD23 levels begin to increase in the peripheral blood, it can be assumed that the number of B-cells in the blood stream is greater than expected (or wanted) in the blood stream.
    Smudge Cells Less than 30 percent smudge cells appears to be predictive of a less favorable clinical course; whereas, greater than 30 percent smudge cells suggests a more favorable prognosis.

    Chromosome and gene abnormalities – normal chromosome profiles (karyotypes) are predictive of a more stable situation. However, chromosome abnormalities are found in a large percentage of CLL patients. Using molecular testing techniques, it is estimated that abnormalities can now be identified in approximately 80 percent of CLL cases. Prior to the availability of current molecular testing techniques, conventional testing detected abnormalities in 40 to 50 percent of cases. These abnormalities are helpful in predicting the course of CLL. Following are some of the most important disease-associated abnormalities and their implications for disease progression:

    IndicatorPrognostic implications
    17p deletion Aggressive disease progression.
    11q deletion Associated with a poor prognosis and often accompanied by bulky lymph node involvement. This deletion also identifies patients who are at high risk for disease persistence after high-dose therapy and autologous transplantation.
    trisomy 12 Predictive of a shorter treatment free interval.
    13q deletion 13q deletion is a positive indicator that predicts indolent disease progression.
    p53 gene mutations These mutations predict for non-response to treatment with alkylating agents and purine analogs.
    p53 gene deletions Patients with p53 deletions are thought to have a shorter survival time and to be more resistance to treatment than those without this deletion.
    IgV gene mutation status Unmutated IgV genes suggest an inferior prognosis; whereas, mutated IgV genes indicate a much better prognosis

    Some patients will have combinations of these abnormalities. When looking at chromosomal abnormalities, it is also useful to consider the IgV gene mutational status. Patients with chromosomal abnormalities and mutated IgV genes have a better outlook than those with chromosomal abnormalities and unmutated IgV genes. Checking for IgV gene status is currently available only for research purposes. It is anticipated that it will soon be available in the clinical laboratory.

    Tissue typing is the name given to the test that identifies an individual's Human Leukocyte Antigens (HLA). HLA's are a set of six antigens that define "self". The concepts of "self" and "nonself" explain how lymphocytes tell the difference between what to attack and what to ignore. These antigens appear on the white blood cells as well as cells of almost all other tissues. They are analogous to red blood cell antigens, types A, B, O, etc. This test is used to match a blood or bone marrow donor to a recipient. By typing for HLA antigens, donors and recipients can be matched to ensure good performance and survival of transfused and transplanted cells. A perfect HLA match occurs only between identical twins.

    HLA's are inherited from our parents, and it is therefore possible to determine which set was inherited from which parent (if tissue typing has been performed on both parents). The following example illustrates how HLA's are inherited:

    Antigens are inherited from each parent as a group, and each set of antigens is called a haplotype. One haplotype must be inherited from each parent. Each number represents a separate inherited antigen. Other possible combinations arising from the parents above are, (1,8,10/10,16,8); (2,7,11/2,14,17); and (3,14,17/10,16,8).

    Should two siblings have exactly the same HLA, it is referred to as an "identical match". Siblings who share one-half of the same HLA are referred to as a "one-haplotype match". Siblings who do not share any of the same HLA are referred to as a "two-haplotype mismatch". There are many Human Leukocyte Antigens in the general population, and for this reason, you may share HLA with someone who is not even related to you.

    After tissue typing is completed and a potential donor is identified, a crossmatch test is performed to determine if there is specific immune reactivity between the donor and the recipient. If the patient has antibodies which react to the donor's HLA's, the donor's cells will be injured. This is referred to as a "positive crossmatch" and is a contraindication to transplant. A negative crossmatch indicates that the patient does not have antibodies against the donor's HLA, and a transplant can be performed. This type of crossmatch should not be confused with the traditional crossmatch used to test the compatibility of donor red blood cells and the patient's naturally occurring antibodies (e.g. anti-A, anti-B).

    Lastly, a test called "antiboody screening" determines whether or not the patient has antibodies to other Human Leukocyte Antigens. This enables the avoidance of those antigens when selecting an appropriate donor. Again, this antibody screening is not the only antibody screen that can be performed. For example, prior to any blood transfusion, a patient's blood will be tested to see if there are any unusual antibodies that would interfere with the donor red blood cells.

    Integrating information from tissue typing, crossmatching, and antibody screening is extremely valuable in predicting compatibility between the recipient and the potential donor.

    DiSC assay stands for Differential Staining Cytotoxicity. It is a technique that assesses the cytotoxic drug sensitivity of fresh human cells from patients with leukemia, lymphoma, and other cancers. It is carried out outside the body (ex vivo) and has had most use with chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML or ANLL), and non-Hodgkin’s lymphoma. Because individual differences in drug sensitivity are considerable, the DiSC assay attempts to assess how patients will respond to various chemotherapies prior to actually administering chemotherapy.

    The center for DiSC assay research is Bath Cancer Research, Wolfson Centre, Royal United Hospital, Bath, BA1 3NG, England. The work is headed by Dr. Andrew G. Bosanquet, BSc, PhD, CBiol, MIBiol, CChem, FRSC. Bath Cancer Research provides an international service for consultant hematologists/oncologists, testing cells from patients for drug response against a panel of up to 35 cytotoxic drugs. Reports are then sent to consulting physicians to aid in the choice of optimum therapies. The information is intended to maximize the likelihood of response and reduce the risk of causing patient toxicity with no clinical benefit.

    Critics of the DiSC assay procedure contend that cells do not necessarily react the same in the laboratory (in vitro) as they do in the body (in vivo). While there have been no comparative studies yet published that determine an increased efficacy of treatment based on this test, its proponents see it as a step toward tailor-made treatment.

    Additional information on DiSC assay and Bath Cancer Research can be found on-line at http://caltri.org.