CLINICAL LABORATOTY DIAGNOSTIC OF ANEMIAS

CLINICAL LABORATOTY DIAGNOSTIC OF ANEMIAS.

Anemia may be defined as any condition resulting from a significant decrease in the total body erythrocyte mass. Measurement of total body rbc mass requires special radiolabeling techniques that are not amenable to general medical diagnostic work. Measurements typically substituted for rbc mass determination take advantage of the body's tendency to maintain normal total blood volume by dilution of the depleted rbc component with plasma. This adjustment results in decrease of the total blood hemoglobin concentration, the rbc count, and the hematocrit. Therefore, a pragmatic definition of anemia is a state which exists when the hemoglobin is less than 12 g/dL or the hematocrit is less than 37 cL/L. Anemia may exist as a laboratory finding in a subjectively healthy individual, because the body can, within limits, compensate for the decreased red cell mass

Classification of anemias

Anemias can be classified by cytometric schemes (i.e., those that depend on cell size and hemoglobin-content parameters, such as MCV and MCHC), erythrokinetic schemes (those that take into account the rates of rbc production and destruction), and biochemical/molecular schemes (those that consider the etiology of the anemia at the molecular level.

An example: sickle cell anemia

         Cytometric classification: normochromic, normocytic

         Erythrokinetic classification: hemolytic

         Biochemical/molecular classification: DNA point mutation producing amino acid substitution in hemoglobin beta chain

A. Cytometric classification

Because cytometric parameters are more easily and less expensively measured than are erythrokinetic and biochemical ones, it is most practical to work from the cytometric classification, to the erythrokinetic, and then (hopefully) to the biochemical. Your first job in working up a patient with anemia is to place the case in one of three major cytometric categories:

1.     Normochromic, normocytic anemia (normal MCHC, normal MCV).

These include:

1.     anemias of chronic disease

2.     hemolytic anemias (those characterized by accelerated destruction of rbc's)

3.     anemia of acute hemorrhage

4.     aplastic anemias (those characterized by disappearance of rbc precursors from the marrow)

2.     Hypochromic, microcytic anemia (low MCHC, low MCV).

These include:

1.     iron deficiency anemia

2.     thalassemias

3.     anemia of chronic disease (rare cases)

3.     Normochromic, macrocytic anemia (normal MCHC, high MCV).

These include:

1.     vitamin B12 deficiency

2.     folate deficiency

B. Erythrokinetic classification

You would now want to proceed with classifying your case based on the rate of rbc turnover. If this is high, a normoregenerative anemia exists. Such anemias are seen in hemolysis (excess destruction of rbc's) or hemorrhage (loss of rbc's from the vascular compartment. In these cases, the marrow responds appropriately to anemia by briskly stepping up the production of rbc's and releasing them into the bloodstream prematurely. There are several lab tests that allow you to determine if increased rbc turnover exists:

1.                 Reticulocyte count

A sample of blood is stained with a supravital dye that marks reticulocytes. An increased number of reticulocytes is seen when the marrow is churning out rbc's at excessive speed (presumably to make up for those lost to hemolysis or hemorrhage). Most labs will report the result of the reticulocyte count in percent of all rbc's counted. A typical normal range is 0.5-1.5 %. Making clinical decisions based on this raw count is somewhat fallacious.

For instance: A normal person with an rbc count of 5,000,000 /microliter and an absolute reticulocyte count of 50,000 /microliter would have a relative retic count of 1.0%. An anemic person with 2,000,000 rbc's/microliter and the same 50,000 retics/microliter would have an apparently "abnormal" relative retic count of 2.5 % and could be misdiagnosed as having high turnover.

Clearly, one needs to find some way to correct the raw retic count so as to avoid this problem. One can easily calculate the absolute retic count (in cells/microliter) by multiplying the rbc count by the relative retic count. The normal range for the absolute retic count is 50,000-90,000 /microliter.

2.     Bone marrow biopsy

This can be used to directly observe any accelerated production of rbc's. The ratio of the number of myeloid to erythroid precursors (the M:E ratio) tends to decrease in high-production states, and the marrow becomes hypercellular. Marrow biopsy is not usually performed just to measure the M:E ratio, but to answer other hematologic questions that have been raised.

The normoregenerative anemias are in contrast to those characterized by inadequate marrow response to the degree of anemia. These are the hyporegenerative anemias. In such cases, the reticulocyte production index is decreased. The classic example is aplastic anemia, in which there is primary marrow failure to produce enough erythrocyte mass. As you have probably come to expect, the distinction of these categories is not always absolute. For instance, in thalassemia major there is a degree of hemolysis (generally associated with the normoregenerative states) and inadequate marrow response to the degree of anemia.

C. Biochemical classification

Finally, one should attempt to determine the etiology of the anemia as specifically as possible. In some cases (e.g., iron deficiency), etiologic classification is easily attained; in others (e.g.. aplastic anemia) the biochemical mechanism of disease may be hopelessly elusive. Generally, biochemical tests are aimed at identifying a depleted cofactor necessary for normal hematopoiesis (iron, ferritin, folate, B12), an abnormally functioning enzyme (glucose-6-phosphate dehydrogenase, pyruvate kinase), or abnormal function of the immune system (the direct antiglobulin [Coombs'] test).

 

Table 1. Laboratory Tests in Anemia Diagnosis

I. Complete blood count (CBC)

A. Red blood cell count

1. Hemoglobin

2. Hematocrit

B. Red blood cell indices

1. Mean cell volume (MCV)

2. Mean cell hemoglobin (MCH)

3. Mean cell hemoglobin concentration (MCHC)

4. Red cell distribution width (RDW)

C. White blood cell count

1. Cell differential

2. Nuclear segmentation of neutrophils

D. Platelet count

E. Cell morphology

1. Cell size

2. Hemoglobin content

3. Anisocytosis

4. Poikilocytosis

5. Polychromasia

II. Reticulocyte count

III. Iron supply studies

A. Serum iron

B. Total iron-binding capacity

C. Serum ferritin, marrow iron stain

IV. Marrow examination

A. Aspirate

1. E/G ratioa

2. Cell morphology

3. Iron stain

B. Biopsy

1. Cellularity

2. Morphology

a E/G ratio, ratio of erythroid to granulocytic precursors.

 

Iron metabolism and iron deficiency anemia

A. Iron and its metabolism

The fourth most abundant element in the earth's crust, iron is only a trace element in biologic systems, making up only 0.004% of the body's mass. Yet it is an essential component or cofactor of numerous metabolic reactions. By weight, the great proportion of the body's iron is dedicated to its essential role as a structural component of hemoglobin. Hemoglobin without iron is totally useless (in fact, hemoglobin with Fe+++ instead of the normal Fe++ is the ugly brown methemoglobin and is also worthless as an oxygen carrier). Without sufficient iron available to the rbc precursors, normal erythropoiesis cannot take place, and anemia develops. On the other hand, iron is a toxic substance. Too much iron accumulating in vital structures (especially the heart, pancreas, and liver) produces a potentially fatal condition, hemochromatosis. Clearly, iron, like oxygen, is another of the deleterious substances that evolution has led biologic systems into flirtation with.

Most of the iron not circulating in the rbc's is stored in the Fe+++ (ferric) oxidation state. This iron is stored in marrow histiocytes in the form of hemosiderin. When iron is needed by the erythron, the hemosiderin gives up its iron to nearby rbc precursors who line up around the histiocyte like pigs around a trough. Hemosiderin is easy to see microscopically in smear or section preparations of marrow, due to the ferric iron's ability to produce an intense blue color in the Prussian blue stain. This reaction is the basis of the routine "iron stain" done on bone marrow specimensto assess adequacy of depot iron. Erythrocytes would not be expected to stain positively, since they contain ferrous iron. Because the body is dealing with such an essential but dangerous and biologically rare substance (and because you have become resigned to endless memorization in your hazing as medical students), you would expect that there would be some kind of complicated mechanism for the absorption and transport of iron.

Iron is present in greatest concentration in meat and dark green vegetables. The U.S. Recommended Daily Allowance for adults is 10 mg for males, 18 mg for menstruating females. The average daily American diet contains about 10 mg iron, of which only about 1 mg is absorbed. What goes in must come out, and in the adult male, the 1 mg/day iron loss occurs almost exclusively in the stool. For reproductive-aged females, an additional route is the menstrual flux, which accounts for a wildly variable incremental loss. While the average monthly menstrual blood loss is 40 mL (equivalent to 16 mg iron), some women who consider themselves healthy may lose up to 495 mL blood (about 200 mg iron) per menstrual period, or an average of about 7 mg iron per day (200 mg iron ÷ 28 days/cycle). It is not surprising that iron deficiency anemia is relatively common in women of this age group.

Following ingestion, iron is absorbed primarily in the duodenum, although any portion of the small bowel is efficient at iron absorption (in contrast to the situation with B12, as noted below). Only ferrous iron can be absorbed. The normal gastric acidity provides an optimal environment for the reduction of any ferric iron to the ferrous version. In states of iron depletion, a greater proportion of iron is absorbed than in states of normal iron depots. After uptake, the ferrous iron is transported to the subepithelial capillaries (possibly by intracellular transferrin), and released into the bloodstream. There it is oxidized to Fe+++ and again taken up by plasma transferrin. It is then conveyed to the erythron (and reduced again to the ferrous version) or to marrow histiocytes for eventual incorporation into hemoglobin. Storage iron exists as part of a ferric iron-apoprotein complex called ferritin. Ferritin molecules are water-soluble and are present in plasma in concentration equilibrium with ferritin molecules in histiocytes. Therefore, decreased iron stores (as is seen in impending iron deficiency anemia) are reflected by decreased serum concentration of ferritin, a substance easily measured in clinical laboratories. In marrow histiocytes, most of the ferritin molecules glom up into visible (through the microscope, that is) blobs of cytoplasmic inclusions rich in iron and poor in apoprotein; this substance is called hemosiderin. Hemosiderin is easily seen with the Prussian blue stain but can even be observed in unstained preparations of marrow, if present in sufficient quantities, due to the natural golden brown color of iron itself. Since hemosiderin is not soluble, it does not float around in the plasma with ferritin.

B. Iron deficiency anemia

When there is insufficient iron available for the normal production of hemoglobin, anemia results. The cells which are produced are small and pale, and indices from such specimens show low values for MCHC and MCV. Therefore, the classic anemia that occurs in iron deficiency is hypochromic, microcytic. Early or mild cases of iron deficiency anemia (IDA) show microcytosis without hypochromia. Since this is a hyporegenerative anemia, the retic count would be expected to be low; however, because so many cases of IDA are due to chronic bleeding, it is not uncommon to see patients with episodes of hemorrhage that have produced an elevated RPI on clinical presentation. It would appear that the marrow is able to produce a transient response to bleeding, but over the long haul it is a day late and a dollar short. Another finding commonly seen on clinical presentation is thrombocytosis, again probably reflecting marrow response to bleeding. The sine qua non of IDA is the observation that there is essentailly no iron in the marrow (that's zero, zilch, nada), since erythropoiesis can occur normally as long as at least some storage iron is present. In iron-deficient states, one of the body's clever reactive phenomena is the increase in production of transferrin. This is sometimes measured as total iron binding capacity of serum (TIBC). Without the availability if iron, a heme precursor, protoporphyrin, and a porphyrin side-reactant, zinc protoporphyrin, accumulate in the red cell. These may also be measured. In summary, the laboratory features of IDA are:

Hypochromic, microcytic anemia

Variable retic count

Increased erythrocyte zinc protoporphyrin

Increased free erythrocyte protoporphyrin

Decreased serum iron

Increased TIBC

Decreased serum ferritin

Absent marrow storage iron

Variable platelet count

 

 

Occurrence of macrocytes and microcytes

C. Causes of IDA

Iron stores can be depleted either through insufficient intake or excessive loss. In America, the combination of our meat-rich diet and fortification of our staples (such as Wonder Bread, Quaker Instant Grits, and Kellogg's 40% Bran Flakes) with added iron makes dietary insufficiency a very rare condition. The one exception to this is the case with milk-fed infants. Bovine milk has almost no iron. An iron-deficient state in such babies often is sown in the fertile soil of an antenatal life in a mother who was also overtly or borderline iron-deficient (iron requirements are markedly increased in pregnancy due to the demands of developing the fetal tissues). Fortunately most infant formulas are fortified with iron now. Moreover, today's parents are so paranoid about iron deficiency that it is surprising that the typical child of the 1990's can get through an airport without setting off any alarms. Still, nutritional cases of IDA do occur.

Although dietary deficiency of iron is rare, individuals with gastrointestinal lesions producing malabsorption syndromes may fail to assimilate sufficient iron to maintain the erythron, even in the face of adequate iron intake.

The much more important cause of iron depletion is chronic blood loss. In females, this is usually due to menses. Other more sinister causes include chronically bleeding lesions of the gastrointestinal tract, from reflux esophagitis, to peptic ulcers, to gastric or colorectal adenocarcinomas. Because these bad guys may be lurking asymptomatically, spilling erythrocytes here and there for months, all cases of iron deficiency anemia must be thoroughly investigated for the presence of bleeding sites. This is especially true in cases involving females who are not of reproductive age and in all males. In these demographic groups, to simply treat IDA with iron and not investigate for bleeding lesions is unequivocal gross negligence.

 

II. Anemia of Chronic Disease (ACD)

This is a condition seen in individuals suffering from chronic infections, noninfectious inflammatory diseases (such as rheumatoid arthritis), and neoplasms. The following pathogenetic observations have been made to help characterize the anemia:

         Decreased rbc life span. This appears to be due to a factor or factors extrinsic to the red cell. The chemical nature of such factor(s) is completely unknown.

         Impaired iron metabolism. Iron accumulates in the marrow histiocytes, but its uptake into rbc precursors is impaired. Therefore the marrow shows decreased sideroblastic iron in the face of increased histiocytic iron. This is probably because lactoferrin (an iron-containing compound made by neutrophils to employ in destroying bacteria) competes with transferrin for surface receptors on macrophages. The iron in lactoferrin is not available for use by developing red cell precursors.

         Refractoriness to erythropoietin is an effect of lymphokines that are secreted by turned-on immune cells. This anti-growth effect of inflammation is not limited to erythroblasts; even hair and nails grow more slowly in times of inflammation.

The anemia is usually said to be normochromic/normocytic, but most patients actually have a slightly decreased MCHC (thus hypochromia). A minority of patients will be microcytic as well. The serum iron is decreased, as is the transferrin (or TIBC) in contrast to iron deficiency anemia, where transferrin is elevated. The absolute retic count is normal or slightly elevated. Bone marrow biopsy shows increased histiocytic iron and decreased sideroblastic iron, but no other morphologic findings are characteristic of this condition.

Megaloblastic anemias

These are a number of conditions which have in common the failure to synthesize adequate amounts of normal DNA. The anemias are macrocytic, since hemoglobinization is allowed, but cells mature more slowly in the marrow; therefore, the cells vegetate in the marrow, slowly maturing but stuffing their greedy little figurative mouths with iron, making hemoglobin, and getting larger as a result. Although some of these obese cells make it out of the marrow, many more never mature properly and eventually are destroyed before they have tasted the thrill of the extramedullary hunt. This phenomenon is referred to as ineffective erythropoiesis. Such marrows are packed with erythroid precursors, even in the face of severe anemia. The rbc precursors are notable morphologically for their immature, sometimes even blast-like chromatin in large nuclei. Such cells are called megaloblasts. Megaloblastic changes are not limited to the erythroid precursors, but are also seen in myeloid precursors. In some cases of megaloblastic anemia, there is concomitant leucopenia and thrombocytopenia, reflecting the troubled development of granulocytes and platelets as well.

 

Acute posthemorrhagic anemia

or acute blood loss anemia - a disease associated with loss of a large volume of hemoglobin.

Etiology. Developed as a result of acute blood loss during injury or disease complicated with hemorrhage.

Symptoms and flow. Actually anemia with concomitant hypoxia, hemodynamic symptoms (collapse). Immediately after bleeding red blood is usually not sharply reduced due to a reflex decrease in total vascular channel and compensatory revenues deposited into the circulation of blood. After 1-2 days, upon receipt of a blood flow of tissue fluid and restore the original volume of the vascular channel, there is a uniform decline in hemoglobin and red blood cells. This anemia is classified as normochromic, normocytic, regenerative.

After 4-5 days, there are signs of regeneration of blood: reticulocytosis, neutrophilic leukocytosis with a shift of leukocyte counts to myelocytes and mild thrombocytosis. In bone marrow is determined by the increase in red growth of 30-40% (normal 16-20%) with predominance in it oxyphilic erythroblasts and normoblasts.

Recognition in most cases is not difficult. Difficulties arise when suddenly fledged internal bleeding (eg, rupture of fetal-receptacle with ectopic pregnancy).

Aplastic Anemia (Hypoplastic Anemia)

: http://www.merck.com/site_images/mm/s.gifAplastic anemia is a normocytic-normochromic anemia that results from a loss of blood cell precursors, causing hypoplasia of bone marrow, RBCs, WBCs, and platelets. Symptoms result from severe anemia, thrombocytopenia (petechiae, bleeding), or leukopenia (infections). Diagnosis requires demonstration of peripheral pancytopenia and the absence of cell precursors in bone marrow. Treatment is equine antithymocyte globulin and cyclosporine. Erythropoietin, granulocyte-macrophage colony-stimulating factor, and bone marrow transplantation may also be useful.

The term aplastic anemia commonly implies a panhypoplasia of the marrow with associated leukopenia and thrombocytopenia. In contrast, pure RBC aplasia is restricted to the erythroid cell line. Although both disorders are uncommon, aplastic anemia is more common.

Etiology

True aplastic anemia (most common in adolescents and young adults) is idiopathic in about ½ of cases. Recognized causes are chemicals (eg, benzene, inorganic arsenic), radiation, and drugs (eg, antineoplastic drugs, antibiotics, NSAIDs, anticonvulsants, acetazolamide , gold salts, penicillamine , quinacrine). The mechanism is unknown, but selective (perhaps genetic) hypersensitivity appears to be the basis.

Fanconi's anemia is a very rare, familial form of aplastic anemia with bone abnormalities, microcephaly, hypogonadism, and brown pigmentation of skin. It occurs in children with abnormal chromosomes. Fanconi's anemia is often inapparent until some illness (especially an acute infection or inflammatory disorder) supervenes, causing peripheral cytopenias. With clearing of the supervening illness, peripheral values return to normal despite reduced marrow mass.

Pure RBC aplasia may be acute and reversible. Acute erythroblastopenia is a brief disappearance of RBC precursors from the marrow during various acute viral illnesses (particularly human parvovirus infection), especially in children. The anemia lasts longer than the acute infection. Chronic pure RBC aplasia has been associated with hemolytic disorders, thymomas, and autoimmune mechanisms and, less often, with drugs (eg, tranquilizers, anticonvulsants), toxins (organic phosphates), riboflavin deficiency, and chronic lymphocytic leukemia. A rare congenital form, Diamond-Blackfan anemia, usually occurs during infancy but has also been reported in adulthood. Diamond-Blackfan anemia is associated with bony abnormalities of the thumbs or digits and short stature.

Symptoms and Signs

Although onset of aplastic anemia usually is insidious, often occurring over weeks or months after exposure to a toxin, occasionally it is acute. Signs vary with the severity of the pancytopenia. Symptoms and signs of anemia (eg, pallor) usually are severe.

Severe thrombocytopenia may cause petechiae, ecchymosis, and bleeding from the gums, into the conjunctivae, or other tissues. Agranulocytosis commonly causes life-threatening infections. Splenomegaly is absent unless induced by transfusion hemosiderosis. Symptoms of pure RBC aplasia are generally milder and relate to the degree of the anemia or to the underlying disorder.

Diagnosis

         CBC

         Bone marrow examination

Aplastic anemia is suspected in patients, particularly young patients, with pancytopenia (eg, WBC < 1500/μL, platelets < 50,000/μL). Pure RBC aplasia (including Diamond-Blackfan anemia) is suspected in patients with bony abnormalities and normocytic anemia but normal WBC and platelet counts. If either diagnosis is suspected, bone marrow examination is done.

In aplastic anemia, RBCs are normochromic-normocytic (sometimes marginally macrocytic). The WBC count reduction occurs chiefly in the granulocytes. Platelets are often far below 50,000/μL. Reticulocytes are decreased or absent. Serum iron is elevated. The bone marrow is acellular. In pure RBC aplasia, normocytic anemia, reticulocytopenia, and elevated serum iron are present, but with normal WBC and platelet counts. Bone marrow cellularity and maturation may be normal except for absence of erythroid precursors.

 

Hemolytic Anemias

Previously we have looked at nutritional anemias and the anemia of chronic disease, in which the metabolic needs of erythrocyte development are not met. The result is failure to produce enough healthy red cells. Now we turn to conditions in which the erythrocyte construction industry is healthy, but where the red cells produced are incapable of surviving the normal 120-day life span. These hemolytic anemias may be due to either intrinsic defects in rbc structure/function or a hostile external environment in which the cells are forced to live. To start with a few definitions:

         Hemolysis: Any condition characterized by a significantly decreased erythrocyte life span.

         Compensated hemolytic state: A state of hemolysis in which the resulting increased erythrocyte production is able to keep up with accelerated rbc destruction, thus averting any anemia.

         Hemolytic anemia: A state of hemolysis in which increased erythrocyte production is insufficient to keep up with accelerated rbc destruction, thus producing anemia. This anemia is characterized as normochromic/normocytic, except when sufficient outpouring of the larger reticulocytes produces a resulting elevation of the MCV.

II. Diagnosis of hemolytic anemia

Diagnosis of hemolytic anemia is performed in four steps:

1.     Establish that anemia exists.

The diagnosis of anemia has been previously covered.

2.     Look for marrow response

The sine qua non for the diagnosis of hemolysis is demonstration of an attempted marrow response to erythrocyte destruction. The classic way to do this is with the reticulocyte count. Remember that you must correct the count for the degree of anemia to prevent overdiagnosis of hemolysis. The absolute retic count (in cells/L) or, better, the reticulocyte production index (RPI) can be used to avoid this pitfall. Even so, one should never take a positive result out of context . A classic cause of reticulosis is recovery from a nutritional anemia (esp. iron and folate). For this reason, you also need corroborating evidence of erythrocyte destruction, thus:

3.     Look for erythrocyte detritus

We have previously discussed the fate of destroyed red cells and their component catabolites, such as free hemoglobin, methemoglobin, methemalbumin, bilirubin, and urobilinogen, as well as the specific binding proteins for these catabolites, such as haptoglobin and hemopexin. Laboratory measurement of some or all of these assists in the diagnosis of hemolysis.

4.     Establish the pathophysiological mechanism of hemolysis

The first distinction to make is to determine whether the hemolysis is taking place in the sinusoids of the reticuloendothelial system (extravascular hemolysis) or in the bloodstream proper (intravascular hemolysis). Both types produce indirect hyperbilirubinemia, urobilinogen in stool and urine, decreased serum haptoglobin, and reticulocytosis. In addition, assuming hemolysis is brisk enough to overwhelm the haptoglobin hemoglobin salvage mechanism, intravascular hemolysis produces hemosiderin in the urine sediment, free hemoglobin in the serum (which may be grossly visible), and free denatured hemoglobin in the urine. Some intravascular hemolytic conditions due to mechanical destruction of rbc's produce the helmet-shaped schizocytes (or "schistocytes"), which can be seen on the routine peripheral blood film. Extravascular hemolytic anemias may produce spherocytes, which are the result of an rbc having a narrow escape from the clutches of the RES.

The next determination to make is the mechanism of rbc destruction. Performing a thorough history and physical (including family and drug history), examining the peripheral blood film, and ordering a few inexpensive laboratory tests, such as the direct antiglobulin (Coombs') test for autoantibodies directed against the rbc membrane antigens and the hemoglobin electrophoresis, will lead you into Diagnosisland in 95+% of the cases. Rare cases will require labor-intensive, costly tests that have to be sent away somewhere like King of Prussia, Pennsylvania, or that are batched for six months in a dusty, coffee-stained research lab tucked away in a closet into which the Medicare inspector has yet to stumble.

III. Specific Conditions

Let us consider selected hemolytic anemias individually. These particular diseases are covered either because they are common or because they illustrate important pathophysiologic features (or both).

A. Mechanical hemolytic anemias

These are certainly the easiest to understand, even to the most concrete of thinkers. Red cells are destroyed due to hydrodynamic turbulence when they are forced over gross obstructions (such as an artificial heart valve) or "clotheslined" by innumerable fibrin strands in such microangiopathic conditions as disseminated intravascular coagulation (better known by the machonym "DIC," covered later in the heme bloc) or thrombotic thrombocytopenic purpura (TTP), an uncommon and mysterious disease of unknown etiology. The hallmark of microangiopathic hemolytic anemia is the presence of schizocytes on the routine blood film.

 

Schizocytes

 

 

 

: Schizocytes photo

 

 

B. Immunohemolytic anemias

In autoimmune hemolytic anemias, the body discourteously mounts an immune attack against its own rbc membrane antigens. This condition not surprisingly tends to occur in states characterized by systemic autoimmunity, such as lupus erythematosus. If the autoantibody is of the IgG class, hemolysis will usually occur at any temperature ( "warm autoimmune hemolytic anemia" ). Several drugs are known to produce warm autoimmune hemolytic anemia which goes away after withdrawal of the drug. Typically the antibody in warm hemolysis is one directed against a universal component of the Rh system absent only in individuals (usually of native Australian blood) with the extremely rare Rh-null rbc membrane phenotype

Autoantibodies of the IgM class typically produce cold agglutinin syndrome, in which the patient is at greater risk of symptoms in a low-temperature environment. Cold agglutinin syndrome may occasionally occur transiently in cases of Mycoplasma pneumonia and rarely infectious mononucleosis. Most cold autoagglutinins are directed against the I antigen, found in almost all adults. The rare infectious mono cold agglutinin has been characterized as anti-i.

Paroxysmal cold hemoglobinuria is a very rare syndrome in which intravascular hemolysis is produced upon exposure to cold temperature by an IgG autoantibody directed against the P antigen found on the red cells of nearly all individuals.

In alloimmune hemolytic anemia, the body synthesizes antibodies against red cell antigens foreign to the host. These antibodies may be naturally occurring (such as those directed against ABO blood group antigens) or acquired as a result of blood transfusion (including that from a fetus to its pregnant mother). Acquired antibodies include those directed against the Rh, Kell, Duffy, and Kidd system antigens. Clinical hemolysis occurs 1) when maternal antibodies send a raiding party across the placenta to raise a little hell in Fetusville (to produce hemolytic disease of the newborn "erythroblastosis fetalis"), and 2) when host antibodies destroy transfused red blood cells in a hemolytic transfusion reaction (which fortunately is very rare with modern blood banking practices).

Diagnosis of immunohemolytic anemia is made by demonstrating (after having proved hemolysis is occurring, as discussed above) a positive result on a simple agglutination test to demonstrate that antibodies are present on the surface of the patient's rbc's. This test is properly called the direct antiglobulin test. All but the most pedantic eschew this term, preferring the eponymous designation direct Coombs' test. Another term for immunohemolytic anemia is, therefore, "Coombs'-positive hemolytic anemia." The diagram below illustrates the principle of the Coombs' test:

: Diagram: direct Coombs' test

A blood sample from the patient has the plasma poured off and the cells washed. The rbc's are resuspended in an aqueous medium. Antiserum containing antibodies against human immunoglobulin (prepared by diabolically injecting cute, innocent little animals with doses of human immunoglobulin) is then added to the patient's cells. A positive result is indicated grossly (or occasionally microscopically) by a visible agglutination reaction.

 

The Lymphatic System

The lymphatic system and the blood cell-forming system in the marrow are closely related. Most lymphocytes are in the lymph nodes and other parts of the lymphatic system such as the skin, spleen, tonsils and adenoids (special lymph nodes), intestinal lining, and in young people, the thymus. The lymphocytes circulate through channels called lymphatics that connect the lymph nodes scattered throughout the body. The lymphatic channels collect into large ducts that empty into a blood vessel. The lymphocytes enter the blood via these ducts. There are three types of lymphocytes. T lymphocytes (T cells) originate in the thymus, hence the designation "T." The B lymphocytes (B cells) originate in the marrow in bone. (The "B" comes from the word "bursa," an organ in birds that was first found to be the source of B lymphocytes.) B lymphocytes make antibodies in response to foreign antigens, especially microbes. Collections of B lymphocytes are present in the marrow, which is an important site for their function.

The T lymphocytes have several functions, including assisting B lymphocytes to make antibodies against invading bacteria, viruses, or other microbes. The antibodies attach to the microbe and in so doing make it possible for other white cells to ingest and kill them. The white cells recognizes the antibody and pull (ingest) it into the cell with its attached microbe. The cells can then kill and digest the microbes.

The third type of lymphocyte, natural killer or NK cells, attack virus-infected cells as a natural function without requiring antibody or other mediation. T cells and NK cells have other functions as well, and are important elements in studies that are designing immunotherapies to treat leukemia and other cancers.


Leukemia

 

Acute leukemia is a rapidly progressing disease that affects mostly cells that are unformed or immature (not yet fully developed or differentiated). These immature cells cannot carry out their normal functions.

nAcute forms of leukemia can occur in children and young adults. (In fact, it is a more common cause of death for children than any other type of malignant disease.)

nImmediate treatment is required in acute leukemias due to the rapid progression and accumulation of the malignant cells, which then spill over into the bloodstream and spread to other organs of the body. If left untreated, the patient will die within months or weeks.

 

Chronic leukemia progresses slowly and permits the growth of greater numbers of more developed cells. In general, these more mature cells can carry out some of their normal functions.

Typically taking months to years to progress, the cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood.

Chronic leukemia mostly occurs in older people, but can theoretically occur in any age group. Whereas acute leukemia must be treated immediately, chronic forms are sometimes monitored for some time before treatment to ensure maximum effectiveness of therapy.

 

nFurthermore, the diseases are classified according to the type of abnormal cell found most in the blood.

When leukemia affects lymphoid cells (lymphocytes and plasma cells), it is called lymphocytic leukemia.

nWhen myeloid cells (eosinophils, neutrophils, and basophils) are affected, the disease is called myeloid or myelogenous leukemia.

 

The major forms of leukemia are divided into four categories. The terms myelogenous or lymphocytic denote the cell type involved. Myelogenous and lymphocytic leukemia each have an acute or chronic form. Thus, the four major types of leukemia are acute or chronic myelogenous and acute or chronic lymphocytic leukemia.

 

Acute leukemias

Acute leukemias are clonal diseases of hematopoietic precursors with molecular genetic abnormalities. All hematopoietic cell lines may be affected. Proliferation of the leukemic cell clone replaces normal hematopoiesis in varying degrees. In acute myeloid leukemia (AML), it is most common for granulocytopoiesis and monocytopoiesis to be affected. Erythropoiesis is less frequently affected, and megakaryopoiesis rarely so. The distribution of the subtypes varies according to age. Acute lymphocytic leukemia (ALL) occurs predominantly in children, while AML has its peak in adults. The involvement of several myeloid cell lines is relatively common, but the simultaneous involvement of myeloid and lymphoid cell lines is very rare (hybrid and bilinear acute leukemias). WHO has recently proposed that the percentage of blasts in the bone marrow must be approximately 20 % to justify a diagnosis of acute leukemia. Examination of the peripheral blood is not essential for diagnosis but can provide important additional information. The diagnosis and classification (subtype assignment) always rely on the bone marrow. The most widely accepted system at present for the classification of AML is based on the criteria of the French-American- British (FAB) Cooperative Group (Table see below) and of the WHO.

 

 

 

 

Table 1. FAB classification of akute myeloid leukemia (AML), modified

 

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Table 2. WHO classification of acute myeloid leukemias

 

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Because the quantity and quality of the blasts is of central importance in this classification, they should be defined as accurately as possible. The leukemic blasts include myeloblasts, monoblasts, and megakaryoblasts. The cells of erythropoiesis are generally counted separately, although in acute erythroleukemia (M6 subtype) the erythroblasts account for a preponderance of the leukemic cells. Blasts are traditionally defined as immature cells that do not show signs of differentiation. But since French hematologists have always accepted blasts with granules, and cells having the nuclear and cytoplasmic features of classic blasts but containing granules are known to occur in leukemias, a distinction is now drawn between type I blasts, which are devoid of granules, and type II blasts, which contain scattered granules or Auer rods and do not show perinuclear pallor. Occasional reference is made to type III blasts, but we consider this a needless distinction except for the abnormal cells of promyelocytic leukemia (M3), which may be heavily granular but are still classified as blasts even though they do not strictly meet the criteria. Besides bone marrow and blood smears, histologic sections are required in cases where aspiration yields insufficient material. The standard stains (Pappenheim, Giemsa) are useful for primary evaluation, but a more accurate classification of the cells requires cytochemical analysis and immunophenotyping (flow cytometry and/or immunochemistry). In order to make a prognosis or define biological entities, it is further necessary to perform cytogenetic and molecular genetic studies, fluorescence in situ hybridization (FISH), and combine FISH technology with immunocytochemistry. As mentioned, the classification of AML essentially follows the recommendations of the FAB and the WHO. However, the experience of recent years in cooperative and prospective studies using modern methods supports the notion of a future-oriented, prognostic classification that takes into account the results of therapies and patient follow-ups. This will allow for a more individually tailored approach. At present this biological classification, or subdivision into biological entities, does not cover all the subtypes of AML or ALL, but in the future these gaps will be filled through the application of more sensitive methods. Acute lymphocytic leukemias (ALL) are currently classified according to immunologic criteria. The classification into three subtypes (L1 L3) originally proposed by the FAB no longer has clinical importance today except for the L3 subtype, which is virtually identical to the immunologic B4 subtype (mature BALL). Morphology and cytochemistry form the basis of diagnosis in ALL. The immunologic classification of ALL is based on two principal groups the B-cell line and T-cell line with their subtypes. Cytogenetic and/or molecular genetic studies can also be used to define prognostic entities. These include c-ALL with t(9;22) and pre-B-cell ALL with t(4;11), which has a characteristic immunophenotype. Besides morphologic analysis, the peroxidase technique is the most important basic method because lymphoblasts are always peroxidase-negative, regardless of their immunophenotype.

 

 

Acute lymphoblastic leukemia

 

Acute lymphoblastic leukemia (ALL) is a malignant (clonal) disease of the bone marrow in which early lymphoid precursors proliferate and replace the normal hematopoietic cells of the marrow. ALL may be distinguished from other malignant lymphoid disorders by the immunophenotype of the cells, which is similar to B- or T-precursor cells. Immunochemistry, cytochemistry, and cytogenetic markers also may aid in categorizing the malignant lymphoid clone.

Pathophysiology: The malignant cells of ALL are lymphoid precursor cells (ie, lymphoblasts) that are arrested in an early stage of development. This arrest is caused by an abnormal expression of genes, often as a result of chromosomal translocations. The lymphoblasts replace the normal marrow elements, resulting in a marked decrease in the production of normal blood cells. Consequently, anemia, thrombocytopenia, and neutropenia occur to varying degrees. The lymphoblasts also proliferate in organs other than the marrow, particularly the liver, spleen, and lymph nodes.

 

Lab Studies:

         A CBC count with differential demonstrates anemia and thrombocytopenia to varying degrees. Patients with ALL can have a high, normal, or low WBC count, but usually exhibit neutropenia.

          

 

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Pict.1 Small blasts. These may closely resemble lymphocytes but are distinguished by their finer chromatin structure and the occasional presence of nucleoli

 

 

 

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Pict. 2 Different case showing blasts of varying sizes, some with pleomorphic nuclei. Panels a and b illustrate B-lineage ALL

 

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Pict. 3. Peroxidase reaction. All lymphoblasts are negative and are interspersed with residual cells of granulocytopoiesis, whose proportion is more clearly demonstrated by the peroxidase reaction

 

 

         Abnormalities in the prothrombin time/activated partial thromboplastin time/fibrinogen/fibrin degradation products may suggest concomitant DIC, which results in an elevated prothrombin time, decreased fibrinogen levels, and the presence of fibrin split products.

         A review of the peripheral blood smear confirms the findings of the CBC count.

o    Circulating blasts are usually seen.

o    Schistocytes are sometimes seen if DIC is present.

         A chemistry profile is recommended.

o    Most patients with ALL have an elevated lactic dehydrogenase level and frequently have an elevated uric acid level.

o    Liver function tests and BUN/creatinine determinations are necessary prior to the initiation of therapy.

         Appropriate cultures, in particular blood cultures, should be obtained in patients with fever or with other signs of infection without fever.

Imaging Studies:

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

         Multiple gated acquisition scan or ECG is needed when the diagnosis is confirmed because many chemotherapeutic agents used in the treatment of acute leukemia are cardiotoxic.

Other Tests:

         ECG is recommended prior to treatment.

Procedures:

         Bone marrow aspiration and biopsy are the definitive diagnostic tests to confirm the diagnosis of leukemia (see pict. 20-34). Immunophenotyping helps elucidate the subtype.

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

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

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

 

         A negative myeloperoxidase stain and a positive TdT is the hallmark of the diagnosis of most cases of ALL. However, positive confirmation of lymphoid (and not myeloid) lineage should be sought by flow cytometric demonstration of lymphoid antigens, such as CD3 (T-lineage ALL) or CD19 (B-lineage ALL), in order to avoid confusion with some types of myeloid leukemia (eg, M0, acute monocytic leukemia), which also stain negative with myeloperoxidase. Although more than 95% of cases of the L1 or L2 subtype of ALL are positive for TdT, TdT is not specific for ALL. TdT is present in some subtypes of AML such as M0. Additionally, TDT is absent in cases of L3 type ALL. However, TdT helps distinguish ALL from malignancies of more mature lymphocytes (ie, NHL).

         In cases of acute leukemia that are MPO negative, TdT positive, the distinction between AML and ALL is made based on the analysis of flow cytometry results. Patients with AML demonstrate myeloid markers such as CD33, whereas patients with ALL demonstrate lymphoid markers. Further confusion arises because some patients with ALL have aberrant expression of myeloid markers, such as CD13. However, if the cells are TdT-positive, myeloperoxidase-negative, and CD33-negative and demonstrate lymphoid markers, the leukemia is considered ALL.

         Studies for bcr-abl analysis by polymerase chain reaction or cytogenetics may help distinguish patients with Philadelphia chromosome positive ALL from those with the lymphoid blastic phase of chronic myelogenous leukemia. 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.

         Newer studies are analyzing ALL subtypes by gene expression profiling. In children with ALL, Bogni et al distinguished 3 groups of patients. Interestingly, one of these groups had a significantly increased risk of developing treatment-related AML following chemotherapy for their ALL.

Histologic Findings:

French-American-British Classification

         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)

         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

The WHO classifies the L1 and L2 subtypes of ALL as either precursor B lymphoblastic leukemia/lymphoblastic lymphoma 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.

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

 

 

 

Table 3. Common Cytogenetic Abnormalities in ALL

Abnormality

Genes Involved

Three-Year, Event-Free Survival

t(10;14)(q24;q11)

HOX11/TCRA

75%

6q

Unknown

47%

14q11

TCRA/TCRD

42%

11q23

MLL

18-26%

9p

Unknown

22%

12

TEL

20%

t(1;19)(q23;p13)

PBX1/E2A

20%

t(8;14)(q24;q23)

t(2;8)(p12;q24)

t(8;22)(q24;q11)

c-myc/IGH

IGK/c-myc

c-myc/IGL

17%

t(9;22)(q34;q11)

bcr-abl

5-10%

t(4;11)(q21;q23)

AF4-MLL

0-10%

 

 

Table 4. Effect of Chromosome Number on Prognosis

Chromosome Number

Three-Year, Event-Free Survival

Near tetraploidy

46-56%

Normal karyotype

34-44%

Hyperdiploidy >50

32-59%

Hyperdiploidy 47-50

21-53%

Pseudodiploidy

12-25%

Hypodiploidy

11%

 

 

Eighty-five percent of cases of ALL are derived from B cells. The primary distinction is between (1) early (pro-B) ALL, which is TDT positive, CD10 (CALLA) negative, surface Ig negative; (2) precursor B ALL, which is TDT positive, CD10 (CALLA) positive, surface Ig negative; and (3) mature B cell (Burkitt) ALL, which is TdT negative, surface Ig positive. Fifteen percent of cases are derived from T cells. 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.

 

 

Table 5. Immunophenotyping of ALL Cells - ALL of B-Cell Lineage (85% of cases of adult ALL)

ALL Cells

TdT

CD19

CD10

CyIg*

SIg

Early B-precursor ALL

+

+

-

-

-

PreB-cell ALL

+

+

+

+

-

B-cell ALL

-

+

+/-

+/-

+

*Cytoplasmic immunoglobulin
Surface immunoglobulin


 

: Click to see larger picture

Pict. 4. Diagnostic workup of a patient with preB-cell acute lymphoblastic leukemia. Bone marrow aspiration revealed French-American-British L2 morphology.

 

 

Table 6. Immunophenotyping of ALL Cells - ALL of T-Cell Lineage (15% of cases of adult ALL)

ALL Cells

TdT

surface CD3

CD4/CD8

Early T-precursor ALL

+

-

+/+ or -/-

T-cell ALL

+

+

+/- or -/+

 

 

Acute Myelogenous Leukemia

 

Acute myelogenous leukemia (AML) is a malignant disease of the bone marrow in which hematopoietic precursors are arrested in an early stage of development. Most AML subtypes are distinguished from other related blood disorders by the presence of more than 20% blasts in the bone marrow.

Pathophysiology: The underlying pathophysiology consists of a maturational arrest of bone marrow cells in the earliest stages of development. The mechanism of this arrest is under study, but in many cases, it involves the activation of abnormal genes through chromosomal translocations and other genetic abnormalities.

This developmental arrest results in 2 disease processes. First, the production of normal blood cells markedly decreases, which results in varying degrees of anemia, thrombocytopenia, and neutropenia. Second, the rapid proliferation of these cells, along with a reduction in their ability to undergo programmed cell death (apoptosis), results in their accumulation in the bone marrow, blood, and, frequently, the spleen and liver.

Lab Studies:

         CBC count with differential demonstrates anemia and thrombocytopenia to varying degrees. Patients with acute myelogenous leukemia (AML) can have high, normal, or low WBC counts.

         Prothrombin time/activated partial thromboplastin time/fibrinogen/fibrin degradation products

o    The most common abnormality is disseminated intravascular coagulation (DIC), which results in an elevated prothrombin time, a decreasing fibrinogen level, and the presence of fibrin split products.

o    Acute promyelocytic leukemia (APL), also known as M3, is the most common subtype of AML associated with DIC.

         Peripheral blood smear

o    Review of peripheral blood smear confirms the findings of the CBC count.

o    Circulating blasts are usually seen.

o    Schistocytes are occasionally seen if DIC is present.

 

 

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Pict. 5 Two type I blasts. The cytoplasm is devoid of granules

 

 

 

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Pict. 6 Blood smear in AML. Undifferentiated blasts with scant cytoplasm

 

 

 

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Pict. 7 Peroxidase reaction in the same patient. All blasts in the field are strongly positive

 

 

 

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Pict. 8 Bone marrow from the same patient shows pronounced maturation (more than 10 %)

 

 

 

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Pict. 9 Very strong peroxidase reaction in the same patient. This case demonstrates that bone marrow examination is necessary for an accurate classification

 

 

 

         Chemistry profile

o    Most patients with AML have an elevated lactic dehydrogenase level and, frequently, an elevated uric acid level.

o    Liver function tests and BUN/creatinine level tests are necessary prior to the initiation of therapy.

o    Appropriate cultures should be obtained in patients with fever or signs of infection, even in the absence of fever.

         Perform HLA or DNA typing in patients who are potential candidates for allogeneic transplantation.

         Bone marrow aspiration (see pict. 20-34)

o    A blast count can be performed with bone marrow aspiration. Historically, by French-American-British (FAB) classification, AML was defined by the presence of more than 30% blasts in bone marrow. In the newer World Health Organization (WHO) classification, AML is defined as the presence of greater than 20% blasts in the marrow.

o    The bone marrow aspirate also allows evaluation of the degree of dysplasia in all cell lines.

         Flow cytometry (immunophenotyping) can be used to help distinguish AML from acute lymphocytic leukemia (ALL) and further classify the subtype of AML. The immunophenotype correlates with prognosis in some instances.

         Cytogenetic studies performed on bone marrow provide important prognostic information and are useful to confirm a diagnosis of APL, which bears the t(15;17) and is treated differently.

 

 

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Pict. 10 Schematic diagram and partial karyotype of the translocation t(8;21)(q22;q22), found predominantly in the M2 subtype of AML

 

 

 

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Pict. 11 Schematic diagram and partial karyotype of the t(3;12)(q26;p13), which is found in AML and MDS. Changes in the short arm of chromosome 12 (12p), like t(6;9) [below t(3;12)], may be associated with increased basophilic granulocytes in the bone marrow. A similar increase is occasionally found in the M4 subtype in the absence of detectable anomalies

 

 

 

         Recently, several molecular abnormalities that are not detected with routine cytogenetics have been shown to have prognostic importance in patients with AML. When possible, the bone marrow should be evaluated for the following abnormalities:

o    Fms-like tyrosine kinase 3 (FLT3) is the most commonly mutated gene in persons with AML and is constitutively activated in one third of AML cases. Internal tandem duplications (ITDs) in the juxtamembrane domain of FLT3 exist in 25% of AML cases. In other cases, mutations exist in the activation loop of FLT3. Most studies demonstrate that patients with AML and FLT3 mutations have a poor prognosis.

o    Mutations in CEBPA are detected in 15% of patients with normal cytogenetics findings and are associated with a longer remission duration and longer overall survival.

o    Mutations in nucleophosmin (NPM) are associated with increased response to chemotherapy in patients with a normal karyotype.

         Gene-expression profiling is a research tool that allows a comprehensive classification of AML based on the expression pattern of thousands of genes.

Imaging Studies:

         Chest radiographs help assess for pneumonia and signs of cardiac disease.

         Multiple gated acquisition (MUGA) scan is needed once the diagnosis is confirmed because many chemotherapeutic agents used in treatment are cardiotoxic.

Other Tests:

         Electrocardiography should be performed prior to treatment.

Procedures:

         Bone marrow aspiration and biopsy are the definitive diagnostic tests.

o    Aspiration slides are stained for morphology with either Wright or Giemsa stain.

o    To determine the FAB type of the leukemia, slides are also stained with myeloperoxidase (or Sudan black), terminal deoxynucleotidyl transferase (TdT) (unless performed by another method [eg, flow cytometry]), and double esterase

         Bone marrow samples should also be sent for cytogenetics testing and flow cytometry.

         Patients with APL should have their marrow evaluated for the PML/RARa genetic rearrangement.

         When possible, the bone marrow should be evaluated for FLT3 mutations.

Histologic Findings: The older, more traditional, FAB classification is as follows:

         M0 - Undifferentiated leukemia

         M1 - Myeloblastic without differentiation

         M2 - Myeloblastic with differentiation

         M3 - Promyelocytic

         M4 - Myelomonocytic

o    M4eo - Myelomonocytic with eosinophilia

         M5 - Monoblastic leukemia

o    M5a - Monoblastic without differentiation

o    M5b - Monocytic with differentiation

         M6 - Erythroleukemia

         M7 - Megakaryoblastic leukemia

The newer WHO classification is as follows:

         AML with recurrent genetic abnormalities

o    AML with t(8;21)(q22;q22), (AML1/ETO)

o    AML with abnormal bone marrow eosinophils and inv(16)(p13q22) or t(16;16)(p13)(q22), (CBFB/MYH11)

o    APL with t(15;17)(q22;q12), (PML/RARa) and variants

o    AML with 11q23 (MLL) abnormalities

         AML with multilineage dysplasia

o    Following myelodysplastic syndrome (MDS) or MDS/myeloproliferative disease (MPD)

o    Without antecedent MDS or MDS/MPD but with dysplasia in at least 50% of cells in 2 or more lineages

         AML and MDS, therapy related

o    Alkylating agent or radiation-related type

o    Topoisomerase II inhibitor type

o    Others

         AML, not otherwise classified

o    AML, minimally differentiated

o    AML, without maturation

o    AML, with maturation

o    Acute myelomonocytic leukemia

o    Acute monoblastic or monocytic leukemia

o    Acute erythroid leukemia

o    Acute megakaryoblastic leukemia

o    Acute basophilic leukemia

o    Acute panmyelosis and myelofibrosis

o    Myeloid sarcoma

 

 

Table 7. Common Cytogenetic Abnormalities in AML

Abnormality

Genes Involved

Morphology

Response

t(8;21)(q22;q22)

AML/ETO

M2

Good

inv(16)(p13;q22)

CBFb/MYH11

M4eo

Good

Normal

Multiple

Varies

Intermediate

-7

Multiple

Varies

Poor

-5

Multiple

Varies

Poor

+8

Multiple

Varies

Intermediate-poor

11q23

MLL

Varies

Intermediate-poor

Miscellaneous

Multiple

Varies

Intermediate-poor

Multiple complex*

Multiple

Varies

Poor

*Refers to 3-5 different cytogenetic abnormalities, depending on the classification used

 

 

Table 8. Cytogenetic Abnormalities in APL

Translocation

Genes Involved

All-Trans-Retinoic Acid Response

t(15;17)(q21;q11)

PML/RARa

Yes

t(11;17)(q23;q11)

PLZF/RARa

No

t(11;17)(q13;q11)

NuMA/RARa

Yes

t(5;17)(q31;q11)

NPM/RARa

Yes

t(17;17)

stat5b/RARa

Unknown

 

 

Table 9. Immunophenotyping of AML Cells

Marker

Lineage

CD13

Myeloid

CD33

Myeloid

CD34

Early precursor

HLA-DR

Positive in most AML, negative in APL

CD11b

Mature monocytes

CD14

Monocytes

CD41

Platelet glycoprotein IIb/IIIa complex

CD42a

Platelet glycoprotein IX

CD42b

Platelet glycoprotein Ib

CD61

Platelet glycoprotein IIIa

Glycophorin A

Erythroid

TdT

Usually indicates acute lymphocytic leukemia, however, may be positive in M0 or M1

CD11c

Myeloid

CD117 (c-kit)

Myeloid/stem cell

CD56

NK-cell/stem cell

 

 

 

 

 

Chronic Lymphocytic Leukemia

 

Chronic lymphocytic leukemia (CLL) results from an acquired injury to the DNA of a single cell, a lymphocyte, in the marrow. This injury is not present at birth. Scientists do not yet understand what produces this change in the DNA of CLL patients.

This change in the cell's DNA confers a growth and survival advantage on the cell, which becomes abnormal and malignant (leukemic). The result of this injury is the uncontrolled growth of lymphocytic cells in the marrow, leading invariably to an increase in the number of lymphocytes in the blood. The leukemic cells that accumulate in the marrow in chronic lymphocytic leukemia do not impede normal blood cell production as profoundly as in the case of acute lymphocytic leukemia. This important distinction from acute leukemia accounts for the less severe early course of the disease.

Chronic lymphocytic leukemia (CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. It is the most common form of leukemia found in adults in Western countries.

 

Pathophysiology

        The cells of origin in the majority of patients with CLL are clonal B cells arrested in the B-cell differentiation pathway, intermediate between pre-B cells and mature B cells. Morphologically in the peripheral blood, these cells resemble mature lymphocytes. B-CLL lymphocytes typically show B-cell surface antigens, as demonstrated by CD19, CD20, CD21, and CD24 monoclonal antibodies. In addition, they express CD5, which is more typically found on T cells. Because normal CD5+ B cells are present in the mantle zone (MZ) of lymphoid follicles, B-cell CLL is most likely a malignancy of an MZ-based subpopulation of anergic self-reactive cells devoted to the production of polyreactive natural autoantibodies.

        B-CLL cells express extremely low levels of surface membrane immunoglobulin, most often immunoglobulin M (IgM) or IgM and immunoglobulin D (IgD). Additionally, they also express extremely low levels of a single immunoglobulin light chain (kappa or lambda).

        Recent studies have demonstrated that bcl2, a protooncogene, is overexpressed in B-CLL. The protooncogene bcl2 is a known suppresser of apoptosis (programmed cell death), resulting in a long life for the involved cells. Despite the frequent overexpression of bcl-2 protein, genetic translocations that are known to result in the overexpression of bcl2, such as t(14;18), are not found in patients with CLL.

        An abnormal karyotype is observed in the majority of patients with CLL. The most common abnormality is deletion of 13q, which occurs in more than 50% of patients. Patients showing 13q14 abnormalities have a relatively benign disease that usually manifests as stable or slowly progressive isolated lymphocytosis. The presence of trisomy 12, which is observed in 15% of patients, is associated with atypical morphology and progressive disease. Deletions of bands 11q22-q23, observed in 19% of patients, are associated with extensive lymph node involvement and aggressive disease. More sensitive techniques have demonstrated abnormalities of chromosome 12. Approximately 2-5% of patients with CLL exhibit a T-cell phenotype.

        CLL also should be distinguished from prolymphocytic leukemia, in which more than 65% of the cells are morphologically less mature prolymphocytes.

 

Frequency

        In the US: More than 17,000 new cases are reported every year.

        Internationally: Unlike the incidence of CLL in the Western countries, which is similar to that of the United States, the disease is extremely rare in Asian countries (ie, China, Japan), where it is estimated to comprise only 10% of all leukemias.

 

Mortality/Morbidity

        The natural history is heterogeneous.

        Some patients die rapidly, within 2-3 years of diagnosis, because of CLL complications.

        The majority of patients live 5-10 years, with an initial course that is relatively benign but followed by a terminal progressive and resistant phase lasting 1-2 years. During the later phase, morbidity is considerable, both from the disease and from complications of therapy.

 

Race: The incidence is higher among whites compared to African Americans.

 

Sex: The incidence is higher in males than in females, with a male-to-female ratio of 1.7:1.

 

Age:

        CLL is a disease that primarily affects elderly individuals, with the majority of cases reported in individuals older than 55 years.

        The incidence continues to rise in those older than 55 years.

        Recently, individuals aged 35 years or younger are being diagnosed more frequently.



 

Symptoms and Signs

Early in the disease, chronic lymphocytic leukemia often has little effect on a person's well being. The disease may be discovered after finding an abnormal blood count during the course of a periodic medical examination or while the patient is under care for an unrelated condition. The report of an elevated white cell count is the most common clue that leads a physician to consider the diagnosis of chronic lymphocytic leukemia.

The symptoms of chronic lymphocytic leukemia usually develop gradually. Patients tire more easily and may feel short of breath when physically active, as a result of anemia. They may lose weight. The leukemic lymphocytes (white cells) can accumulate in the lymphatic system and the lymph nodes and spleen may become enlarged. Patients may experience infections, sometimes recurrent, of the skin, lungs, kidneys, or other sites.

 

Bypass of CLL has three stages:

1. Initial (slight increasing of lymphatic nodes one or two groups, leucocytosis no more than 30 - 50 109/l, working capacities preserved.

2. Unrolled (increasing leucocytosis, progressing generalized enlargement of lymph nodes, relapsing infections, and autoimmune cytopenia).

3. Terminal - malignant transformation with expanding of leucosis process out of the borders of hemopoetic system.

 

Basis of clinical diagnostics of CLL is lymphadenopathy, splenomegaly, hepatomegaly, infiltration by tumoral lymphocytes of pleura, gastrointestinal tract (with simulation of stomach tumor, intestinal polyposis), prostate, bones and joint with development of osteoporosis and osteolisis of vertebra and pelvic bones; perivascular infiltration of retine of eye, middle ear, vestibular apparatus, nervous system (hemiplegia, meningisms, paralysis of cranial nerves), skin (lymphocytic tumoral erythema, macropapules, exematous placodes, rarely - specific skins leukemias, erythrodermia, prurigo). From general features - hyperhydrosis, weight loss, undue fatigue.

 

Physical:

        Localized or generalized lymphadenopathy

o   Splenomegaly (30-40% of cases)

o   Hepatomegaly (20% of cases)

        Petechiae

        Pallor


Diagnosis

To diagnose the disease, the blood and, in most cases, the marrow cells are examined. The white cell count is increased in the blood. The increase is the result of an increase in blood lymphocytes. A marrow examination also will show an increase in the proportion of lymphocytes, often accompanied by some decrease in the normal marrow cells. In addition, a sample of marrow cells is examined to determine if there is an abnormality of chromosomes. The examination of cells to determine if an abnormality of chromosomes is present is referred to as a cytogenetics analysis.

Low platelet counts and low red cell counts (anemia) may be present but are usually only slightly decreased in the early stage of the illness.

 

Determining the immunophenotype of the lymphocytes in the blood or marrow is important. This distinguishes whether the lymphocytes that accumulate are derived from a malignant transformation of a lymphocyte in the B cell developmental pathway or the T cell developmental pathway (see Figure 3). The T cell type of disease, called T cell chronic lymphocytic leukemia, is very infrequent. It affects the skin, nervous system, and lymph nodes more often and may be more rapidly progressive than is the B cell type. (See also Other Related Lymphocytic Leukemias). Immunophenotyping also permits assessment of whether the lymphocytes in the blood are derived from a single malignant cell (in other words, whether they are monoclonal). The test for monoclonality is important because it distinguishes leukemia from the very infrequent increase in the blood lymphocytes in adults that is not the result of a malignant transformation characteristic of cancer. This test is especially important if the lymphocytes in the blood are only slightly elevated.

Another very important test that is performed is the measurement of the concentration of gamma globulins (immunoglobulins) in the blood. Immunoglobulins are proteins called antibodies that the B cells of healthy individuals make to protect themselves from infection. They are often deficient in persons with chronic lymphocytic leukemia. The leukemic B lymphocytes do not make protective antibodies effectively. At the same time, the leukemia acts to prevent remaining normal lymphocytes from doing so. This inability to make antibodies efficiently causes CLL patients to be susceptible to infections.

 

Lab Studies

 

        CBC count with differential shows absolute lymphocytosis with more than 5000 lymphocytes/mL. Some authors consider this to be a prerequisite for the diagnosis of CLL and classify cases that would otherwise meet the criteria as small lymphocytic lymphoma/diffuse well-differentiated lymphoma.

        Microscopic examination of the peripheral blood smear is indicated to confirm lymphocytosis. It usually shows the presence of smudge cells, which are artifacts due to damaged lymphocytes during the slide preparation.

        Peripheral blood flow cytometry is the most valuable test to confirm CLL.

        It confirms the presence of circulating clonal B-lymphocytes expressing CD5, CD19, CD20(dim), CD 23, and an absence of FMC-7 staining.

        Consider obtaining serum quantitative immunoglobulin levels in patients developing repeated infections because monthly intravenous immunoglobulin administration in patients with low levels of immunoglobulin G (<500 mg) may be beneficial in reducing the frequency of infectious episodes.

        The differential diagnosis of CLL includes several other entities, such as hairy cell leukemia, which is moderately positive for surface membrane immunoglobulins of multiple heavy-chain classes and typically negative for CD5 and CD21. Prolymphocytic leukemia has a typical phenotype that is positive for CD19, CD20, and surface membrane immunoglobulin and negative for CD5. Large granular lymphocytic leukemia has a natural killer cell phenotype (CD2, CD16, and CD56) or a T-cell immunotype (CD2, CD3, and CD8). The pattern of positivity for CD19, CD20, and the T-cell antigen CD5 is shared only by mantle cell lymphoma.

         

 

 

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Pict. 16 Peripheral blood smear showing CLL cells

 

 

 

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Pict. 17 This is a microscopic view of bone marrow from a person with chronic lymphocytic leukemia; it shows predominantly small, mature lymphocytes

 

 

 

 

Imaging Studies:

        Liver/spleen scan may demonstrate splenomegaly.

        Computed tomography of chest, abdomen, or pelvis generally is not required for staging purposes. However, be careful to not miss lesions such as obstructive uropathy or airway obstruction that are caused by lymph node compression on organs or internal structures.

 

 

 

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Picture 18. Peripheral smear of a patient with chronic lymphocytic leukemia, small lymphocytic variety.

 

 

 

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Picture 19. This is a peripheral smear of a patient with chronic lymphocytic leukemia, showing the large lymphocytic variety. Smudge cells also are observed. Smudge cells are the artifacts produced by the lymphocytes damaged during the slide preparation.

 

 

 

Procedures:

        Bone marrow aspiration and biopsy with flow cytometry is not required in all cases but may be necessary in selected cases to establish the diagnosis and to assess other complicating features such as anemia and thrombocytopenia. For example, bone marrow examination may be necessary to distinguish between thrombocytopenia of peripheral destruction (in the spleen) and that due to marrow infiltration.

        Consider a lymph node biopsy if lymph node(s) begin to enlarge rapidly in a patient with known CLL to assess the possibility of transformation to a high-grade lymphoma. When such transformation is accompanied by fever, weight loss, and pain, it is termed Richter syndrome.

 

 

Bone marrow aspiration and biopsy

 

 

Pict. 20. Bone marrow aspiration and biopsy. Patient position (superior posterior iliac crest).

 

Pict. 21. Bone marrow aspiration and biopsy. Bone marrow tray.

 

Pict. 22. Bone marrow aspiration and biopsy. Skin preparation.

 

Pict. 23. Bone marrow aspiration and biopsy. Site preparation.

 

Pict. 29. Bone marrow biopsy specimen.

 

Chronic myelogenous leukemia

 

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by increased proliferation of the granulocytic cell line without the loss of their capacity to differentiate. Consequently, the peripheral blood cell profile shows an increased number of granulocytes and their immature precursors, including occasional blast cells. CML is a tumor arisen from one mutate predecessors-cells of mielopoiesis, morphological substrate of which, as a rule, is three- sprouts proliferation, which displays that with prevalent surplus excrescence of cells of granulocytes row, also has a place moderate cell proliferation of erytrocytes and megacariocytes sprouts. This peculiarity is associated with that these sprouts develop from one matter predecessors-cells of mielopoiesis.

 

Pathophysiology: CML is an acquired abnormality that involves the hematopoietic stem cell. It is characterized by a cytogenetic aberration consisting of a reciprocal translocation between the long arms of chromosomes 22 and 9; t(9;22). The translocation results in a shortened chromosome 22, an observation first described by Nowell and Hungerford and subsequently termed the Philadelphia (Ph) chromosome after the city of discovery.

Pict. 35 The Philadelphia chromosome as seen by metaphase FISH

 

This translocation relocates an oncogene called abl from the long arm of chromosome 9 to the long arm of chromosome 22 in the BCR region. The resulting BCR/ABL fusion gene encodes a chimeric protein with strong tyrosine kinase activity. The expression of this protein leads to the development of the CML phenotype through processes that are not yet fully understood.

The presence of BCR/ABL rearrangement is the hallmark of CML, although this rearrangement has also been described in other diseases. It is considered diagnostic when present in a patient with clinical manifestations of CML.

 

Causes:

        The initiating factor of CML is still unknown, but exposure to irradiation has been implicated, as observed in the increased prevalence among survivors of the atomic bombing of Hiroshima and Nagasaki.

        Other agents, such as benzene, are possible causes.

Among etiological factors the most essential are physical and chemical mutagens, viruses, congenital or acquireed defects of immune defense. However in majority of cases (87 %) the cause of beginnings of leucocytosis growth is chromosomal pathology (Philadelphia chromosome).

In some cases the conditions of "slip out" are created of mutant from under immune control. As a result, happened to be outside of organism control, a mutant cell continues uncontrolledly to "reproductive", that brings to fast accumulation of tumoral tissues and forcing out healthy haemopoiesis.

 

Frequency:

        In the US: CML accounts for 20% of all leukemias affecting adults. It typically affects middle-aged individuals. Although uncommon, the disease also occurs in younger individuals.

        Internationally: Increased incidence was reported among individuals exposed to radiation in Nagasaki and Hiroshima after the dropping of the atomic bomb.

 

Mortality/Morbidity: Generally, 3 phases of the disease are recognized. The general course of the disease is characterized by an eventual evolution to a refractory form of acute myelogenous or, occasionally, lymphoblastic leukemia. The median survival of patients using older forms of therapy was 3-5 years.

 

        Most patients present in the chronic phase, characterized by splenomegaly and leukocytosis (see Image below) with generally few symptoms. This phase is easily controlled by medication. The major goal of treatment during this phase is to control symptoms and complications resulting from anemia, thrombocytopenia, leukocytosis, and splenomegaly. Newer forms of therapy aim at delaying the onset of the accelerated or blastic phase.

 

 

 

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Picture 36. Blood film at 400X magnification demonstrates leukocytosis with the presence of precursor cells of the myeloid lineage. In addition, basophilia, eosinophilia, and thrombocytosis can be seen. Courtesy of U. Woermann, MD, Division of Instructional Media, Institute for Medical Education, University of Bern, Switzerland.

 

 

 

        After an average of 3-5 years, the disease usually evolves into the blast crisis, which is marked by an increase in the bone marrow or peripheral blood blast count or by the development of soft tissue or skin leukemic infiltrates. Typical symptoms are due to increasing anemia, thrombocytopenia, basophilia, a rapidly enlarging spleen, and failure of the usual medications to control leukocytosis and splenomegaly. The manifestations of blast crisis are similar to those of acute leukemia. Treatment results are unsatisfactory, and most patients succumb to the disease once this phase develops. In approximately two thirds of cases, the blasts are myeloid. However, in the remaining one third of patients, the blasts exhibit a lymphoid phenotype, further evidence of the stem cell nature of the original disease. Additional chromosomal abnormalities are usually found at the time of blast crisis, including additional Ph chromosomes or other translocations.

        In many patients, an accelerated phase occurs 3-6 months before the diagnosis of blast crisis. Clinical features in this phase are intermediate between the chronic phase and blast crisis.

 

Age:

        In general, this disease occurs in the fourth and fifth decades of life.

        Younger patients aged 20-29 years may be affected and may present with a more aggressive form, such as in accelerated phase or blast crisis.

        Uncommonly, CML may appear as a disease of new onset in elderly individuals.

 

o   an increase in bone marrow fibrosis, are harbingers of the last phase.

 

 

 

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Picture 37. Blood film at 1000X magnification shows a promyelocyte, an eosinophil, and 3 basophils. Courtesy of U. Woermann, MD, Division of Instructional Media, Institute for Medical Education, University of Bern, Switzerland.

Picture 23. Chronic Myelogenous Leukemia (A) Myeloblasts,(B) Neutrophilic Myelocyte. (C) Neutrophilic Metamyelocyte. (D) Band neutrophil. (E) Basophil.

 

o   Acute phase, or blast crisis, is similar to acute leukemia, and survival is 3-6 months at this stage. Bone marrow and peripheral blood blasts of 30% or more are characteristic. Skin or tissue infiltration also defines blast crisis. Cytogenetic evidence of another Ph-positive clone (double) or clonal evolution (other cytogenetic abnormalities such as trisomy 8, 9, 19, or 21, isochromosome 17, or deletion of Y chromosome) is usually present.

        In some patients who present in the accelerated, or acute, leukemia phase of the disease (skipping the chronic phase), bleeding, petechiae, and ecchymoses may be the prominent symptoms. In these situations, fever is usually associated with infections.

 

In CML flowing they pick out the next stages initial, unroll and terminal.

        For todays understanding initial stage is that disease stage, when only small part of cells of granulocytes sprout is tumoral, and majority are the cells of normal hemopoiesis. As a rule, this stage has never diagnosed, because specific clinical disease symptoms of disease are absents.

        Unroll stage is manifestation of total generalisation of tumoral cells in marrow with forcing out of healthy haemopoiesis sprout. Clinical symptoms in this stage are crescent general weakness, rapid fatigue, hyperhydrosis, weight loss, increase of temperature, osseous and articulate pains, spleen and livers enlargement which one can be combined into syndrome of tumoral intoxication.

        Terminal stage starts when a monotonously flowing monoclone tumor turns into policlone. Under this sharp there is increase of amount of tumoral cells, that with each following mutation lose ability to differentiatie, the manifestation of what is sharp increasing of cells amount of granulocytes row of different ripening degrees. Metastatic spreading of these cells, adapted to survival for boundary paths of haemopoietic system, brings about appearance of metaplastatic hearths of tumoral growth in liver, skin, bone, lymph node and oth., with clinical features of dysfunctions of these organs and systems. The most threatful features of terminal stage is blastogenic crises. A clinical picture of CML consists from tumoral intoxication syndrome, syndrome of tumoral metaplasy, syndrome of metabolitic distebance. The cells substrate of tumor are leucocytosis for a counting of immature cells of granulocytes row, metaplastic anaemia and tromcytopenia, pletora of marrow for a counting of tumoral granulocytes sprout.

 

 

 

Stages of CML

 

 

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Physical:

        Splenomegaly is the most common physical finding in patients with CML.

        In more than half the patients with CML, the spleen extends more than 5 cm below the left costal margin at time of discovery.

        The size of the spleen correlates with the peripheral blood granulocyte counts (see Image below), with the biggest spleens being observed in patients with high WBC counts.

 

 

 

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Picture 38. Blood film at 1000X magnification demonstrates the whole granulocytic lineage, including an eosinophil and a basophil. Courtesy of U. Woermann, MD, Division of Instructional Media, Institute for Medical Education, University of Bern, Switzerland.

 

 

 

        A very large spleen is usually a harbinger of the transformation into an acute blast crisis form of the disease.

        Hepatomegaly also occurs, although less commonly than splenomegaly. Hepatomegaly is usually part of the extramedullary hematopoiesis occurring in the spleen.

        Physical findings of leukostasis and hyperviscosity can occur in some patients, with extraordinary elevation of their WBC counts, exceeding 300,000-600,000 cells/mL. Upon funduscopy, the retina may show papilledema, venous obstruction, and hemorrhages.

Lab Studies:

        Peripheral blood findings show a typical leukoerythroblastic blood picture, with circulating immature cells from the bone marrow (see Image below).

 

 

 

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Picture 39. Bone marrow film at 400X magnification demonstrates clear dominance of granulopoiesis. The number of eosinophils and megakaryocytes is increased. Courtesy of U. Woermann, MD, Division of Instructional Media, Institute for Medical Education, University of Bern, Switzerland.

 

 

 

        The increase in mature granulocytes and normal lymphocyte counts (low percentage due to dilution in the differential count) results in a total WBC count of 20,000-60,000 cells/mL. A mild increase in basophils and eosinophils is present and becomes more prominent during the transition to acute leukemia.

o   These mature neutrophils, or granulocytes, have decreased apoptosis (programmed cell death), resulting in accumulation of long-lived cells with low or absent enzymes, such as alkaline phosphatase. Consequently, the leukocyte alkaline phosphatase stains very low to absent in most cells, resulting in a low score.

o   Early myeloid cells such as myeloblasts, myelocytes, metamyelocytes, and nucleated red blood cells are commonly present in the blood smear, mimicking the findings in the bone marrow. The presence of the different midstage progenitor cells differentiates this condition from the acute myelogenous leukemias, in which a leukemic gap (maturation arrest) or hiatus exists that shows absence of these cells.

o   A mild-to-moderate anemia is very common at diagnosis and is usually normochromic and normocytic.

o   The platelet counts at diagnosis can be low, normal, or even increased in some patients (>1 million in some).

        Bone marrow is characteristically hypercellular, with expansion of the myeloid cell line (eg, neutrophils, eosinophils, basophils) and its progenitor cells. Megakaryocytes (see Image above) are prominent and may be increased. Mild fibrosis is often seen in the reticulin stain.

        Cytogenetic studies of the bone marrow cells, and even peripheral blood, should reveal the typical Ph1 chromosome, which is a reciprocal translocation of chromosomal material between chromosomes 9 and 22. This is the hallmark of CML, found in almost all patients with CML, and is present in CML throughout its entire clinical course.

o   The Ph translocation is the translocation of the cellular oncogene c-abl from the 9 chromosome, which encodes for a tyrosine protein kinase, with a specific breakpoint cluster region (bcr) of chromosome 22, resulting in a chimeric bcr/c-abl messenger RNA that encodes for a mutation protein with much greater tyrosine kinase activity compared with the normal protein (see Image below). The latter is presumably responsible for the cellular transformation in CML. This m-RNA can be detected by polymerase chain reaction (PCR) in a sensitive test that can detect it in just a few cells. This is useful in monitoring minimal residual disease (MRD) during therapy.

 

 

 

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Picture 40. The Philadelphia chromosome, which is a diagnostic karyotypic abnormality for chronic myelogenous leukemia, is shown in this picture of the banded chromosomes 9 and 22. Shown is the result of the reciprocal translocation of 22q to the lower arm of 9 and 9q (c-abl to a specific breakpoint cluster region [bcr] of chromosome 22 indicated by the arrows). Courtesy of Peter C. Nowell, MD, Department of Pathology and Clinical Laboratory of the University of Pennsylvania School of Medicine.

 

 

 

o   Karyotypic analysis of bone marrow cells requires the presence of a dividing cell without loss of viability because the material requires that the cells go into mitosis to obtain individual chromosomes for identification after banding, which is a slow, labor-intensive process. The new technique of fluorescence in situ hybridization (see Image below) uses labeled probes that are hybridized to either metaphase chromosomes or interphase nuclei, and the hybridized probe is detected with fluorochromes. This technique is a rapid and sensitive means of detecting recurring numerical and structural abnormalities.

Picture 41. Fluorescence in situ hybridization using unique-sequence, double-fusion DNA probes for bcr (22q11.2) in red and c-abl (9q34) gene regions in green. The abnormal bcr/abl fusion present in Philadelphia chromosomepositive cells is in yellow (right panel) compared with a control (left panel). Courtesy of Emmanuel C. Besa, MD.

o   Two forms of the BCR/ABL mutation are present, depending on the location of their joining regions on bcr 3' domain. Approximately 70% of patients who have the 5' DNA breakpoint have a b2a2 RNA message, and 30% of patients have a 3'DNA breakpoint and a b3a2 RNA message. The latter is associated with a shorter chronic phase, shorter survival, and thrombocytosis.

o   CML should be differentiated from Ph-negative diseases with negative PCR results for BCR/ABL m-RNA. These diseases include other myeloproliferative disorders and chronic myelomonocytic leukemia, which is now classified with the myelodysplastic syndromes.

o   Additional chromosomal abnormalities, such as an additional or double Ph-positive chromosome or trisomy 8, 9, 19, or 21; isochromosome 17; or deletion of the Y chromosome, have been described as the patient enters a transitional form or accelerated phase of the blast crisis as the Ph chromosome persists.

o   Patients with conditions other than chronic-phase CML, such as newly diagnosed acute lymphocytic leukemia or nonlymphocytic leukemia, also may be positive for the Ph chromosome. Some consider this the blastic phase of CML without a chronic phase. The chromosome is rarely found in patients with other myeloproliferative disorders, such as polycythemia vera or essential thrombocythemia, but these are probably misdiagnosed CML. It is rarely observed in myelodysplastic syndrome.

        Other laboratory abnormalities include hyperuricemia, which is a reflection of high bone marrow cellular turnover and markedly elevated serum vitamin B-12binding protein (TC-I). The latter is synthesized by the granulocytes and reflects the degree of leukocytosis.

 

Imaging Studies:

        Typical hepatomegaly and splenomegaly may be imaged by using a liver/spleen scan. Most often, these are so obvious that radiological imaging is not necessary.

        Histologic Findings: Diagnosis is based on the histopathologic findings in the peripheral blood and the Ph1 chromosome in the bone marrow cells.

 

 

 

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Pict.42 Oil immersion field demonstrating myeloid cells of all degrees of maturity

 

 

 

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Pict. 43 This high-power microscopic view of a blood smear from a person with classical CML shows predominantly normal-appearing cells with intermediate maturity

 

 

 

 

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Pict. 44 Low power view showing marked hypercellularity with a broad-spectrum of myeloid and erythroid cell types and marked myeloid hyperplasia