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The Anemia of Lymphomas and Leukemias
The Anemia of Lymphomas and Leukemias DWIGHT J. HOTCHKISS, JR., M.D.*
One of the most frequent and challenging problems requiring hematologic evaluation is the anemia associated with the lymphomas and leukemias. Not infrequently, depression of the erythropoietic capacity of the bone marrow by lymphomatous or leukemic tissue is accepted as an adequate explanation for the anemia, thus precluding further investigation into other possible causes. Needed forms of therapy, therefore, may be overlooked. It will be the purpose of this paper to define briefly the known mechanisms involved in the anemia of lymphomas and leukemias, to outline a practical clinical approach to the evaluation of the anemia, and finally to illustrate the value of this approach with several case reports. The diseases to be included are the acute leukemias, chronic lymphocytic leukemia, chronic granulocytic leukemia, lymphosarcoma, reticulum cell sarcoma, and Hodgkin's disease. The problem of anemia in malignant disease in general has recently been thoroughly reviewed by Hyman14 and by Price and Greenfield. 22 MECHANISMS INVOLVED IN THE ANEMIA OF LYMPHOMAS AND LEUKEMIAS
Anemia develops during the course of the disease in most patients with a lymphoma or leukemia. In a study of 158 patients with neoplastic disease, Samuels and Bierman26 found anemia in 77 per cent of patients with lymphoma, 75 per cent with chronic lymphocytic leukemia, 95 per cent with chronic granulocytic leukemia and 97 per cent with acute leukemia. These figures have limited value, however, since the presence and severity of an anemia varies as the disease evolves and as various forms of therapy are employed. * Assistant Director of Internal Medicine, Hunterdon Medical Center, Flemington,
N. J.; Clinical Instructor in Internal Medicine, New York University School of Medicine, New York, N. Y.
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Table 1.
Mechanisms
rif Anemia in Lymphoma
and Leukemia
1. Decrease in rate of red cell production
a. Myelophthisis b. Ineffective erythropoiesis c. Associated disease-infection, uremia, liver disease, arthritis, etc. d. Radiotherapy and chemotherapy e. Nutritional state including folic acid deficiency 2. Increase in rate of red cell destruction a. Overt hemolysis b. Occult hemolysis 3. Blood loss
Anemia may be considered to result from: (1) a decrease in the rate of red cell production, (2) an increase in the rate of red cell destruction, (3) blood loss, or (4) a combination of these factors. Table 1 lists the factors involved in the anemia of the lymphomas and leukemias subdivided under these general headings. The time-honored explanation for anemia in leukemia has been a "crowding-out" effect of the erythroblasts by the leukemic tissue. This arose from the fact that examination of the bone marrow in many patients with leukemia revealed a decrease in erythroblastic tissue concomitant with the progressive proliferation of leukemic cells. That this was not the sole explanation for the anemia of leukemia was first suggested by Jaffe,15 who found adequate erythroblastic tissue in the bone marrow of some anemic patients with acute leukemia, as well as an increase in iron in the cells of the reticulo-endothelial system. The role of hemolysis, therefore, was stressed by Jaffe and supported by recent investigators. His studies emphasized that a combination of factors is usually involved, thus complicating the evaluation of the anemia.
Decrease in the Rate of Red Cell Production Myelophthisis is an important factor in the production of the anemia, especially in the acute leukemias, where replacement of erythroblasts may occur relatively early in the disease. This may also occur in chronic granulocytic leukemia and chronic lymphocytic leukemia, particularly during the later stages of the disease, although by this time hemolysis may also play a role. In a recent study of the anemia of chronic lymphocytic anemia, Wasi and Block32 employed the bone marrow section technique to demonstrate a normal amount erythroblastic tissue in nonanemic patients. In a group of six patients with hemoglobin values from 9.3 to 12.7 gm. per 100 ml., the degree of developing anemia could be roughly correlated with the decrease in erythroblastic tissue in the marrow. Hemolysis complicated the anemia only in the patient with the hemoglobin of 9.3 gm. In a similar study of bone marrow sections in anemic patients with chronic granulocytic leukemia,11 a significant decrease in erythroblastic tissue was observed in 11 of 15
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sections obtained prior to therapy. An increase in the amount of erythroblastic tissue was observed following successful therapy and was associated with an increase in hemoglobin. With relapse, erythroblastic tissue again decreased markedly in amount. Hemolysis aggravated the anemia only in those patients with marked splenomegaly. In a recent study of lymphosarcoma and reticulum cell sarcoma by Rosenberg et aL,24 16 per cent of patients were found to have marrow replaced by a sufficient amount of abnormal tissue to contribute significantly to an anemia. The availability of isotopic Fe69 has permitted an estimation of the rate of red cell production by determination of the plasma iron clearance and turnover and the incorporation of radio-iron into red cells and the red cell iron turnover.12 These methods, however, are subject to misinterpretation because not all of the radio-iron is represented in red cell hemoglobin. 21 In spite of their limitations, these techniques have demonstrated a decrease in red cell production in many patients, although in some normal erythropoiesis seemed apparent. 6, 7, 18, 33 The decrease in red cell production probably could be explained in those instances in which the marrow was invaded by lymphomatous or leukemic tissue and the amount of erythroblastic tissue decreased. This was particularly true in many cases of acute leukemia as well as late in the course of chronic lymphocytic leukemia, chronic granulocytic leukemia, and occasionally the lymphomas. A normal rate of production was often present early in the chronic leukemias and in the lymphomas. In patients with leukemias and lymphomas with apparently normal erythropoiesis as determined by the radio-iron techniques, or in whom marrow replacement by lymphomatous or leukemic tissue has not occurred, an anemia may develop when even a mild degree of hemolysis is present. A normal marrow may increase its rate of red cell production six to eight times in response to hemolysis or blood loss. This group of patients is unable to compensate fully for the hemolytic process. This form of ineffective erythropoiesis has been termed "relative bone marrow failure" by MooreY A similar mechanism is applicable to the anemias of infection, rheumatoid arthritis, liver disease and renal disease. Since infection, rheumatoid arthritis, liver disease and renal disease in themselves may result in anemia, their association in leukemias and lymphomas may induce or further aggravate an anemia. Radiotherapy and tumor chemotherapy excluding the adrenal steroid hormones are known marrow depressants,34 and thus may aggravate an anemia. The all,:ylating agents, antimetabolites and folic acid antagonists have all been incriminated. The folic acid antagonists may induce a megaloblastic anemia when usage is prolonged. A poor nutritional state may contribute to anemia by failure of adequate intake of proteins. In chronic lymphocytic leukemia and lymphosarcoma a malabsorption syndrome presumably related to small bowel involvement has been described resulting in poor absorption of folic acid, vitamin B12 and iron. 19 In addition, a relative deficiency of folic acid as well
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as a rare case of megaloblastic anemia has been described in association with leukemia. The problem has been discussed by Chanarin et aI. 6 with the thought that a decrease in folic acid may result from an increased demand for folic acid by the leukemic cells. Increase in the Rate of Red Cell Destruction
An increase in the rate of red cell destruction may become an important feature of the anemia of the leukemias and lymphomas. Descriptively, the hemolytic process may be overt or occult. An overt hemolytic anemia may occur in approximately 15 to 20 per cent of patients with chronic lymphocytic leukemia and lymphosarcoma. 9, 25, 28 In these patients the hemolytic process may be relatively severe and associated with a reticulocytosis, hyperbilirubinemia, spherocytosis and, usually but not invariably, a positive Coombs' test. This type of hemolysis may occur at any stage of the disease process and, indeed, may be the insult initiating medical attention. An overt hemolytic anemia is less common in other forms of leukemia and lymphoma, but isolated cases have been reported in acute leukemia, reticulum cell sarcoma, and Hodgkin's disease. With the application of the Ashby and Cr51 techniques to determine accurately the rate of red cell destruction, it has become apparent that an occult hemolytic process occurs commonly in the lymphomas and leukemias, particularly late in the course of the disease. Berlin,! utilizing the Ashby technique, found a decrease in red cell survival in 12 of 15 patients with chronic granulocytic leukemia and in six of nine patients with chronic lymphocytic leukemia. Many other authors have reported a similar increase in the rate of red cell destruction. 6, 7, 9, 13, 18, 31, 33 In these cases, the reticulocyte count, serum bilirubin and fecal urobilinogen may be normal or only slightly elevated. In the early stages of these diseases the rate of red cell destruction may be within normal limits. The mechanisms involved in the increase in the rate of red cell destruction are still being investigated. In many patients, particularly those with an overt hemolytic anemia and those with a positive Coombs' test, an autoimmune process is responsible. 9 , 25, 28 In others, hemolysis may be related to a dysproteinemic state in which the red cells become "coated" by gamma globulin, not necessarily an auto-antibody,' or by a non-gamma globulin. 10 One source of the "coating" material has been suggested by Pirofsky,20 who extracted hemolysins and agglutinins from leukemic white cells which were found to be active against normal erythrocytes. An increase in splenic sequestration of red cells is present in most patients with hemolysis. 29 but is probably more apparent in those with splenomegaly. I, 7, 11 In the lymphomas and leukemias, particularly chronic lymphocytic leukemia and lymphosarcoma, an overt and often severe hemolytic episode has been observed following treatment with x-ray or drugs. 9 , 26 This phenomenon has been reported after the use of alkylating agents, particu-
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larly nitrogen mustard, chlorambucil and triethylene melamine, as well as following total body and splenic irradiation. Whether the hemolysis is precipitated by the therapy or is merely coincidental remains to be ascertained.
Blood Loss Active or occult bleeding may contribute to the anemia of the leukemias and lymphomas. The coexistence of peptic ulceration, particularly in patients who have been receiving corticosteroids, or of an associated neoplasm must not be overlooked. 14 • 28 Bleeding may also result from an accompanying thrombocytopenia and/or other clotting abnormalities. The bleeding may remain fairly well localized, as with epistaxis or excessive menstrual flow, or may be a diffuse capillary bleeding into many tissues. 22 TECHNIQUES OF STUDY OF THE ANEMIA OF LYMPHOMAS AND LEUKEMIAS
The clinical laboratory evaluation of anemia has been reviewed thoroughly in the literature. 8 • 27. 34 The emphasis here will concern those techniques which may be easily applied in the average community hospital (Table 2). The importance of an extremely thorough history and a very complete physical examination cannot be overemphasized. The evaluation of red cell production usually can be approximated from the reticulocyte count and examination of the bone marrow. 27 Red cell destruction can be assessed by measurement of the retiCUlocyte count, bilirubin, evaluation of the peripheral blood smear and bone marrow, and, if necessary, the Cr51 red cell survival. Coombs' test, warm and cold agglutinins and autohemolysis28 may be obtained, if indicated. An increase in transfusion requirement beyond one unit per week is excellent evidence for hemolysis. An evaluation of iron deficiency involves utilization of the hemo-
Table 2. Evaluation of Anemia, a Practical Approach 1. Thorough history 2. Complete physical examination 3. Laboratory evaluation: a. Hemoglobin and hematocrit b. Stools for blood c. Reticulocyte count, corrected d. Peripheral blood smear e. Bilirubin, direct and indirect f. Bone marrow biopsy, smear and section; iron stain g. Serum iron and unbound iron binding capacity h. Coombs' test, if indicated 4. Rarely required studies: a. Cr"1 red cell survival b. Warm and cold agglutinins, autohemolysis c. Fecal urobilinogen
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JR,
globin and hematocrit (mean corpuscular hemoglobin concentration34), the peripheral blood smear, the iron stain of the bone marrow, the serum iron and unbound iron binding capacity, and examination of stools for blood. The RETICULOCYTE COUNT is perhaps the simplest of the readily available laboratory tests in evaluating erythropoiesis, and, if red cell production is not impaired, is a reliable measure of red cell destruction. By the usual slide technique utilizing a brillant cresyl blue stain,34 reticulocytes are recorded as a percentage compared to the total number of red cells. This will result in an overestimation of reticulocytes if the total number of circulating red cells is decreased. Therefore, Finch and Noyes 8 stress the importance of employing a corrected reticulocyte count: .. patient's hematocrit PatIent's retIculocyte count X Ih t 't norma ema oen
The retiCUlocyte count will also be falsely high if there is a premature release of reticulocytes from the marrow, which probably obtains when circulating nucleated red cells are found. In these instances, the reticulocyte count should be divided by two, owing to the fact that the number of reticulocytes in the marrow is equal to the number of circulating reticulocytes. It also must be realized that the reticulocyte count may rise modestly with active bleeding. A consistently low reticulocyte count of 0.1 to 0.2 per cent may suggest a decrease in red cell production. 27 In evaluating the BILIRUBIN LEVEL it must be recalled that the indirect bilirubin (unconjugated), a product of hemoglobin catabolism, must be roughly equated with the amount of red cell hemoglobin. A patient with a hemoglobin which is half of normal, and whose indirect bilirubin is the upper limit of "normal," may be catabolizing twice the expected amount of hemoglobin. Adequate evaluation of the PERIPHERAL BLOOD SMEAR has been discussed in the literature. 3 • 34 With decreased red cell production, mild hemolysis, and/or early iron deficiency, the red cell morphology may well be normal. A decrease in polychromatophilic cells may occur as red cell production decreases. With active hemolysis, polychromatophilia, anisocytosis, poikilocytosis, spherocytosis, occasional macrocytosis and occasional normoblastemia may occur. The microcytic, hypochromic red cells of chronic iron deficiency may not be seen until the hemoglobin becomes less than 8 gm. per 100 ml. Preceding these changes may be poikilocytosis with "cigar" and "tear-drop" forms, red cell fragmentation, and modest increase in target cells. The BONE MARROW EXAMINATION is important in the evaluation of red cell production. Most commonly used is the bone marrow smear in which the granulocytic/erythroblastic (G/E) ratio is determined. However, this ratio is valid only if the granulocytic mass remains unaltered. 8 Also, there is a wide variability of cell counts even when taken from the same area at the
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same time due, in part, to contamination by peripheral blood. 23 The bone marrow section technique enables a more accurate evaluation of the number
and distribution of erythroblasts. Block3 states that if an adequate sample of marrow is obtained, the number of erythroblasts in the marrow gives a semiquantitative estimation of red cell production. This estimation is not valid, however, if there is an arrest of maturation of the erythroblasts (an increase in the number of immature to mature red cell precursors) or if there is karyorrhexis (death) of erythroblasts. In addition, estimates of erythropoiesis are invalid in the presence of iron deficiency (when erythropoiesis is often less than would be expected from the number of erythroblasts present), in the presence of extramedullary hematopoiesis, and in children (when excessive erythroblastic tissue is present). It previously has been estimatedl l that erythropoiesis is probably decreased if the erythroblastic tissue comprises less than 5 per cent of the total marrow section, realizing that the active marrow contains from 20 to 40 per cent myeloid * tissue with the remainder as fat. It must be emphasized, however, that attempts to equate a dynamic process such as erythropoiesis with a static picture of a segment of bone marrow can be only semiquantitative at best. However, combined with the peripheral smear, reticulocyte count and other studies mentioned, a reasonable estimation of erythropoiesis can be made. It is a common misconception that the bone marrow section technique is too difficult for both physician and technician. A technician capable of preparing ordinary pathology slides can prepare marrow sections with little difficulty.t The marrow particles are fixed in Zenker's solution (9 parts) to which neutral formalin (1 part) is added. The particles are then embedded in paraffin. A Giemsa-hematoxylin stain is preferable to the routine hematoxylin-eosin stain since it permits staining of the granules of the granulocytes. With several particles of marrow, smears are also made. Perhaps the most reliable method in the evaluation of iron deficiency is the study of the STAINABLE IRON IN THE BONE MARROW either in the smear or in the section. 2 Iron is stored in the marrow in reticulum cells or in the more immature erythroblasts (siderblasts). The SERUM IRON AND UNBOUND IRON-BINDING CAPACITY may be difficult to evaluate in the anemia associated with leukemias and lymphomas. As is the case of the anemia associated with most chronic diseases, both the serum iron and iron-binding capacity may be low. 16 Therefore a low serum iron may not be indicative of iron deficiency, and conversely, iron deficiency may be present without an elevation of iron-binding capacity. However, the serum iron and iron-binding capacity may be helpful to confirm an iron deficiency, particularly early in the disease process before the total iron-binding protein (transferrin) has begun to diminish. ISOTOPE TECHNIQUES are often not available in the average community hospital. Anemia can be fairly well delineated without their useY Occa-
* Myeloid is used to mean red and white cell precursors and megakaryocytes. t Laboratory technique available upon request.
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sionally a red cell survival test utilizing the radiochromium technique is useful since this is perhaps the most accurate measurement of red cell life span. However, its usefulness is limited by the length of time required for the determination. Body-surface scanning performed concomitantly with the radio chromium red cell survival test may enable more accurate prediction of the results of a contemplated splenectomy.3o An increase in radioactivity over the spleen as compared with the activity over the precordium or liver may indicate increased splenic sequestration of red cells. The FECAL UROBILINOGEN determination is of limited value in the average hospital because of (1) difficulty in accurate and complete stool collections, (2) wide variability in the limits of normal, and (3) difficulties in interpretation since it is a measure of total hemoglobin destruction rather than a measure of red cell destruction.
SPECIFIC PROBLEMS ENCOUNTERED IN THE EVALUATION AND MANAGEMENT OF THE ANEMIA OF LYMPHOMAS AND LEUKEMIAS
Myelophthisis-Depression of Marrow Leukemia
with Chemotherapy-Acute
CASE I. A 3 year old boy was admitted to the Hunterdon Medical Center on 11/8/63 with a 2- to 4-week history of progressive ease of bruisability. The examination revealed a well developed, well nourished boy, who appeared moderately pale and sallow. Multiple ecchymoses were scattered throughout the body; numerous petechiae appeared over the face and upper thorax. Small 1- to 2-cm. lymph nodes were palpable in the anterior cervical, axillary and inguinal regions bilaterally. The liver was palpable 2 cm. below the right costal margin; the spleen was 3 cm. enlarged below the left costal margin. The remainder of the examination was unremarkable. The hemoglobin was 8 gm. per 100 ml. and the hematocrit 24 per cent; the white count was 10,000 with 98 per cent mononuclear cells; the urinalysis and chest x-ray were normal. In the peripheral blood smear the majority of cells were stem cells although a few polymorphonuclear cells and a few lymphocytes were also seen. The red cells were normal in morphology; the platelets were markedly decreased. The bone marrow smear and section were typical of an acute stem cellleukemia. The bone marrow section (Fig. 1) was solidly cellular. Ninety-five per cent of cells seen were stem cells. An occasional erythroblast and granulocyte was noted. Megakaryocytes were not seen. A small amount of iron was present in the iron-stained sections. Treatment with 40 mg. a day of prednisone was instituted, but with little success. Because of a moderately severe episode of epistaxis associated with a progression of the anemia to a hemoglobin of 5.6 gm. per 100 ml., the patient received 1 unit of blood approximately 7 days after institution of steroids. 6-Mercaptopurine at a dosage of 50 mg. a day was then added. By 12/9/63 a complete remission was apparent. The hemoglobin was 12.2 gm. per 100 ml.; the hematocrit 37 per cent; the white count 4000 with a normal peripheral blood smear. A repeat bone marrow examination was essentially normal. The prednisone was tapered and then discontinued. On 1/7/64, Methotrexate
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20 mg. intramuscularly (30 mg.jsq.m. body surface) biweekly was begun and the 6-mercaptopurine discontinued. Clinically the patient was essentially normal and displayed no abnormal physical signs. However, by 2/19/64 the white count had fallen to 2300; the hemoglobin remained at 12.1 gm. per 100 ml. The peripheral blood smear revealed a moderate neutropenia with 70 per cent of leukocytes as lymphocytes and 30 per cent as polymorphonuclear cells. The red cells displayed a 2+ anisocytosis. Platelets were reduced to one-half their normal number. The bone marrow smear suggested grossly a normal cellularity. The G/E ratio was 2/1. Maturation arrest was noted in the granulocytic series at the myelocyte and metamyelocyte stages, and in the erythroblastic series at the polychromatophilic stage. Megakaryocytes were decreased in number. A rare blast cell was noted. There was insufficient material for a bone marrow section. The Methotrexate was discontinued for one week, then resumed at 15 mg. intramuscularly biweekly. The white count has risen to 5500 and the peripheral smear is now essentially normal.
This case demonstrates a fairly typical onset and course of childhood acute leukemia. The anemia at the onset was largely myelophthisic in type resulting from a marked decrease in red cell production due to overwhelming proliferation of leukemic cells. Erythroblastic tissue comprised well below 5 per cent of the total marrow section. Although there was no evidence of overt hemoly~i~, an occult process cannot be excluded. Blood loss had become a contributing factor in this patient at the time of the epistaxis. The depressive influence of drug therapy upon the marrow elements was also observed in this patient. The neutropenia and thrombocytopenia which developed after the institution of Methotrexate were undoubtedly related to the effect of the drug upon the marrow. This was not clear, however, until a marrow was examined, since a progression in the leukemic process might also have been responsible. Although, anemia did not occur from Methotrexate in this patient, it can occur, and indeed, may be megaloblastic in type.
Figure 1 (Case I). Acute leukemia. Solidly cellular marrow due to proliferation of stem cells; erythroblasts (arrows) markedly decreased in number. X400. (This and the following photomicrographs were taken from sections of aspirated marrow-biopsies.)
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Hemolytic Anemia-Hodgkin's Disease
CASE n. A 53 year old white man was first diagnosed as having Hodgkin's disease in August, 1959 following a biopsy of a right anterior cervical lymph node. Other areas of involvement included the left axilla and the mediastinum. There was no hepatosplenomegaly. He was given x-irradiation therapy to the involved areas. In July, 1960, x-ray therapy was given to both groins because of the development of weight loss, edema of the scrotum, and edema of the right leg, associated with the presence of bilateral inguinal nodes. The left axilla, left cervical and left supraclavicular areas were re-treated in November of 1960. In April, 1961, the patient began to complain of night sweats but no fever, and noted an increase in the edema of the scrotum and right leg. At this time a spleen was palpable 10 cm. below the left costal margin. There was no hepatomegaly and no significant adenopathy. A hemoglobin of 6.2 gm. per 100 ml., and reticulocyte count of 5.1 per cent (2.1 per cent corrected) was present. The white count was 6700 with a normal differential; the platelet count was 154,000. The total serum bilirubin was 0.2 mg. per 100 ml. with 0.12 mg. direct. A bone marrow examination was not performed. A Coombs' test, direct and indirect, was negative. The patient was treated with prednisone with subsequent rise in hemoglobin to 10.2 gm. per 100 ml. Forty milligrams of prednisone was required daily to maintain the hemoglobin at this level. Under this program the patient's edema regressed, and his strength improved considerably. An attempt to decrease the steroids resulted in a fall in hemoglobin to 7 gm. per 100 ml. requiring an increase in prednisone dosage. The hemoglobin subsequently rose to 8.2 gm. In the fall of 1961, splenectomy was advised, but refused by the patient owing to his general feeling of good health. By November, 1961, the spleen had enlarged to 18 cm. and the liver was felt 3 cm. below the costal margin. In January, 1962, the patient developed pneumonitis of the left lower lung and bilateral dependent edema, followed by generalized anasarca, requiring hospital admission. Hepatosplenomegaly was present as in November, 1961. Only bilateral shotty inguinal lymphadenopathy was observed. The hemoglobin was 5.7 gm. per 100 ml. and the hematocrit 16.5 per cent; the white count was 7300 with a left shift. A reticulocyte count was 1.8 per cent (0.68 per cent corrected). The serum bilirubin was 4.8 mg. total per 100 ml. with 3.4 mg. direct; and the alkaline phosphatase was 18.3 Shinowara units. The peripheral blood smear revealed normal platelets and white cells. The red cells showed a 2+ anisocytosis. An adequate bone marrow specimen could not be obtained from the sternum, presumably because of previous x-irradiation therapy to that area. A marrow section from the posterior iliac crest (Fig. 2) obtained with a Westerman-Jensen needle* was normal in cellularity. A moderate degree of serous fat atrophy was present indicative of severe chronic disease. 3 The G/E ratio was 1/2 (normal G/E-1/1) and maturation of granulocytic and erythroblastic series appeared normal. Megakaryocytes were normal in number and maturation. Plasma cells were moderately elevated in number. A few small lymphoid nodules were also noted. Iron was increased moderately in the ironstained section. At this point, the patient was transfused and treated with antibiotics but he rapidly progressed to his demise. The autopsy revealed extensive infiltration of liver, spleen, lung, lymph nodes and bone by Hodgkin's disease in the sarcomatous stage. Compression of the superior and inferior vena cava had produced the generalized anasarca. Hemosiderosis of the spleen was severe.
This patient demonstrates the hemolytic anemia not infrequently seen in patients with Hodgkin's disease. 6 There well may have been an
* A fortified Vim-Silverman needle made by Becton, Dickinson Company.
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Figure 2 (Case II). Hodgkin's disease. Cellularity of 40 per cent (normal); moderate serous fat atrophy; G/E ratio 1/2; erythroblasts (arrows) adequate in number and normal in maturation. X 400.
impairment of erythropoiesis as the disease progressed. Although there was moderate erythroblastic activity observed in the marrow section during his terminal admission, the marked hyperplasia usually seen in an otherwise normal marrow compensating for hemolysis was not observed. The failure of the reticulocyte count to increase later in his disease also suggests that compensatory erythropoiesis was impaired. He, therefore, displayed a "relative bone marrow failure" with inability to compensate for hemolysis with an adequate increase in erythropoiesis. There was no evidence of blood loss in this patient.
Blood Loss-Chronic Lymphocytic Leukemia CASE Ill. A 75 year old white man was first seen in December, 1961. One month prior to admission he had developed transient constipation associated with abdominal "gaseousness." In addition, he had developed progressive tiredness and lethargy, occasional dizziness, and a weight loss of from 172 to 164 pounds. The physical examination revealed a well developed, well nourished male with obvious pallor. The tongue was normal. There was no significant lymphadenopathy other than a I-cm. node in the left axilla. The liver was palpable 4 cm. below the right costal margin; the spleen, 3 cm. below the left. The remainder of the examination was unremarkable. The hemoglobin was 5.1 gm. per 100 ml., the hematocrit 18 per cent. A white count was 13,600 with 25 per cent polymorphonuclear cells, 3 per cent bands, 76 per cent lymphoxytes, 1 per cent monocytes, and 1 per cent basophils. The reticulocyte count was 5.6 per cent (1.9 per cent corrected). The peripheral blood smear reflected the modest increase in white count with 60 to 70 per cent of leukocytes being lymphocytes. One-third of these were smudge cells. Platelets were normal in number. The red cells displayed a 2+ anisocytosis, a slight polychromatophilia, and a modest increase in "cigar" forms, target forms, and red cell fragments. There were no hypochromic cells. The bone marrow smear suggested a modest increase in cellularity with approximately 80 per cent of cells as mature lymphocytes. The remaining cells were normal granulocytes and erythroblasts. The marrow section (Figs. 3, 4)
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Figure 3 (Cll8e Ill). Chronic lymphocytic leukemia and blood loss. Cellularityof 50 to 60 per cent slightly increll8ed; a small area of diffuse infiltration by mature lymphocytes; erythroblasts (arrows) in this area markedly decreased. X400.
Figure 4 (Cll8e Ill). Area of essentially normal myeloid tissue except for approximately 10 per cent mature lymphocytes; erythroblll8ts (arrows) are adequate in number and normal in maturation. Many areas of marrow section comparable to this. X400.
displayed an increased cellularity of 50 to 60 per cent (normal 20 to 40 per cent.) Four to five large lymphatic nodules were seen. The remaining myeloid tissue contained from 10 to 50 per cent mature lymphocytes. The remaining cells were of the granulocytic, erythroblastic and megakaryocytic series which were adequate in number and normal in maturation. The G/E ratio was 1/1. No increase in plasma cells was seen. No iron was present in the iron stain. Further studies revealed a serum bilirubin of 0.29 mg. per 100 ml. total, 0.05 mg. direct; serum iron 50 /-Lg. and iron-binding capacity 372 /-Lg. per 100 m!.; the stool was positive for occult blood. Sigmoidoscopy and barium enema were normal. An upper gastrointestinal series revealed the presence of a gastric carcinoma. A subtotal gastrectomy was subsequently performed. The pathologist reported an adenocarcinoma of the stomach metll8tatic to mesenteric lymph nodes. The patient did fairly well for approximately 6 months, then began to deteriorate rapidly and expired in June, 1962.
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The initial reaction was to attribute the severe anemia to depression of erythropoiesis due to marrow replacement as a result of chronic lymphocytic leukemia, whose presence was apparent at the completion of the initial blood count and study of the peripheral blood smear. The elevated reticulocyte count suggested a hemolytic component. However, subsequent evaluation did not bear this out. The bone marrow revealed an adequate amount of erythroblastic tissue-at least 10 to 15 per cent of the marrow section. Myelophthisis was not a factor in producing anemia. The absence of iron in the marrow in association with a moderate poikilocytosis in the peripheral blood smear indicated an iron deficiency as the most likely cause of the anemia. The serum iron and iron-binding capacity lent further evidence for iron deficiency. The corrected reticulocyte count was 5.1 (is X 5.6) = 1.9, which is just above the upper limits of normal and could reflect blood loss or a mild hemolytic process. However, occult hemolytic anemia is usually seen later in the course of chronic lymphocytic leukemia. 9 , 32 The pursuit of the source of blood loss led to the discovery of the gastric carcinoma. The anemia, therefore, was due to blood loss from the gastric malignancy. The early chronic lymphocytic leukemia probably did not contribute significantly to the anemia. SUMMARY
The anemia often found in association with the lymphomas and leukemias frequently involves several mechanisms. Thorough evaluation employing readily available techniques will enable the physician to ascertain the relative importance of the degree of the decrease in the rate of red cell production, increase in the rate of red cell destruction, and blood loss. Adequate therapy will obtain only after the mechanisms producing the anemia are understood in each patient. REFERENCES 1. Berlin, R.: Red cell survival studies in normal and leukaemic subjects. Acta med.
scandinav., Suppl. 252, 1951. 2. Beutler, E., Robson, M. J. and Buttenwieser, E.: A comparison of the plasma. iron, iron-binding capacity, sternal marrow iron and other methods in the clinical evaluation of iron stores. Ann. Int. Med. 48: 60, 1958. 3. Block, M.: Morphologic aspects of erythropoiesis. In Mechanisms of Anemia. (1. Weinstein and E. Beutler, Eds.) New York, N. "¥., McGraw Hill Book Co., 1962. 4. Brody, J. 1. and Finch, S. C.: Serum factors of acquired hemolytic anemia in leukemia and lymphoma. J. Clin. Invest. 40: 181, 1961. 5. Chanarin, 1., Mollin, D. L. and Anderson, B. B.: Folic acid deficiency and the megaloblastic anaemias. Proc. Roy. Soc. Med. 51: 757, 1958. 6. Cline, M. J., and Berlin, N.!.: Anemia in Hodgkin's disease. Cancer 18: 526, 1963.
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7. Cline, M. J. and Berlin, N. I.: Patterns of anemia in chronic myelocytic leukemia. Cancer 16: 624, 1963. 8. Finch, C. A. and Noyes, W. D.: ErythrokineticB in diagnosis of anemia. J.A.M.A. 175: 1163, 1961. 9. Freymann, J. G., Burrell, S. B. and Marler, E. A.: Role of hemolysis in anemia secondary to chronic lymphocytic leukemia and certain malignant lymphomas. New England J. Med. 259: 847, 1958. 10. Green, H. N., Wakefield, J. and Littlewood, G.: The nature of cancer anaemia and its bearing on the immunological theory of cancer. Brit. M. J. 2: 779, 1957. 11. Hotchkiss, D. J., Jr. and Block, M. H.: Effect of splenic irradiation on systemic hematopoiesis. Arch. Int. Med. 109: 695, 1962. 12. Huff, R. L., Hennessy, T. G., Austin, R. E., Garcia, J. F., Roberts, B. M. and Lawrence, J. H.: Plasma and red cell iron turnover in normal subjects and in patients having various hematopoietic disorders. J. Clin. Invest. 29: 1041, 1950. 13. Hyman, G. A.: Studies on anemia of disseminated malignant neoplastic disease. I. The hemolytic factor. Blood 9: 911,1954. 14. Hyman, G. A.: Anemia in malignantneoplastic disease. J. Chron. Dis.16: 645,1963. 15. Jaffe, R. H.: Erythropoiesis in leukemia. Folia haemat. 49: 51, 1933. 16. Laurell, C. B.: Studies on the transportation and metabolism of iron in the body. Acta physioI. scandinav., Suppl. 46, 1947. 17. Moore, C. V.: The concept of relative bone marrow failure (Editorial). Am. J. Med. 23: 1, 1957. 18. Nathan, D. G. and Berlin, N. I.: Studies of the rate of production and life span of erythrocytes in acute leukemia. Blood 14: 935, 1959. 19. Pitney, W. R., Joske, R. A. and MacKinnon, N. L.: Folic acid and other absorption tests in lymphosarcoma, chronic lymphocytic leukaemia, and Borne related conditions. J. Clin. Path. 13: 440, 1960. 20. Pirofsky, B.: Studies on the hemolysin and agglutinin of leukemic leukocytes. Blood 12: 620, 1957. 21. Pollycove, M. and Mortimer, R.: The quantitative determination of iron kinetics and hemoglobin synthesis in human subjects. J. Clin. Invest. 40: 753, 1961. 22. Price, V. E. and Greenfield, R. E.: Anemia in cancer. Advances Cancer Res. 5: 199, 1958. 23. Reich, C. and Kolb, E. M.: A quantitative study of the variations in multiple sternal marrow samples taken simultaneously. Am. J. M. Sc. 204: 496,1942. 24. Rosenberg, S. A., Diamond, H. D., Jaslowitz, B. and Craver, L. F.: Lymphosarcoma: A review of 1269 cases. Medicine 40: 31, 1961. 25. Rosenthal, M. C., Pisciotta, A. V., Komninos, Z. D., Goldenberg, H. and Dameshek, W.: The auto-immune hemolytic anemia of malignant lymphocytic disease. Blood 10: 197, 1955. 26. Samuels, A. J. and Bierman, H. R.: Anemia in patients with neoplastic diseases. California Med. 84: 180, 1956. 27. Tocantins, L. M. (Ed.): Modern methods for quantitation of red blood cell production and destruction: Erythrokinetics. Blood 18: 225, 1961. 28. Troup, S. B., Swisher, S. N. and Young, L. E.: The anemia of leukemia. Am. J. Med. 28: 751, 1960. 29. Ultmann, J. E.: Role of the spleen in the hemolytic anemia of cancer patients. Cancer Res. 18: 959, 1958. 30. Veeger, W., Woldring, M. G., van Rood, J. J., Ernisse, J. G., Leeksma, C. H. W., Verloop, M. C. and Nieweg, H. 0.: The value of the determination of the site of red cell sequestration in hemolytic anemia as a prediction test for splenectomy. Acta. med. scandinav. 171: 507, 1962. 31. Waggener, R. E., Pratt, P. T. and Hunt, H. B.: Erythrocyte survival in leukemia, Hodgkin's disease, and malignant lymphoma as determined by radiochromate. Am. J. Roentgenol. 79: 1045, 1958. 32. Wasi, P. and Block, M.: The mechanism of the development of anemia in untreated chronic lymphatic leukemia. Blood 17: 597, 1961. 33. Wetherley-Mein, G., Epstein, I. S., Foster, W. D. and Grimes, A. J.: Mechanisms of anaemia in leukaemia. Brit. J. Haemat. 4: 281, 1958. 34. Wintrobe, M. M.: Clinical Hematology. 5th.Ed. Philadelphia, Lea & Febiger, 1961.
One of the most frequent and challenging problems requiring hematologic evaluation is the anemia associated with the lymphomas and leukemias. Not infrequently, depression of the erythropoietic capacity of the bone marrow by lymphomatous or leukemic tissue is accepted as an adequate explanation for the anemia, thus precluding further investigation into other possible causes. Needed forms of therapy, therefore, may be overlooked. It will be the purpose of this paper to define briefly the known mechanisms involved in the anemia of lymphomas and leukemias, to outline a practical clinical approach to the evaluation of the anemia, and finally to illustrate the value of this approach with several case reports. The diseases to be included are the acute leukemias, chronic lymphocytic leukemia, chronic granulocytic leukemia, lymphosarcoma, reticulum cell sarcoma, and Hodgkin's disease. The problem of anemia in malignant disease in general has recently been thoroughly reviewed by Hyman14 and by Price and Greenfield. 22 MECHANISMS INVOLVED IN THE ANEMIA OF LYMPHOMAS AND LEUKEMIAS
Anemia develops during the course of the disease in most patients with a lymphoma or leukemia. In a study of 158 patients with neoplastic disease, Samuels and Bierman26 found anemia in 77 per cent of patients with lymphoma, 75 per cent with chronic lymphocytic leukemia, 95 per cent with chronic granulocytic leukemia and 97 per cent with acute leukemia. These figures have limited value, however, since the presence and severity of an anemia varies as the disease evolves and as various forms of therapy are employed. * Assistant Director of Internal Medicine, Hunterdon Medical Center, Flemington,
N. J.; Clinical Instructor in Internal Medicine, New York University School of Medicine, New York, N. Y.
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Table 1.
Mechanisms
rif Anemia in Lymphoma
and Leukemia
1. Decrease in rate of red cell production
a. Myelophthisis b. Ineffective erythropoiesis c. Associated disease-infection, uremia, liver disease, arthritis, etc. d. Radiotherapy and chemotherapy e. Nutritional state including folic acid deficiency 2. Increase in rate of red cell destruction a. Overt hemolysis b. Occult hemolysis 3. Blood loss
Anemia may be considered to result from: (1) a decrease in the rate of red cell production, (2) an increase in the rate of red cell destruction, (3) blood loss, or (4) a combination of these factors. Table 1 lists the factors involved in the anemia of the lymphomas and leukemias subdivided under these general headings. The time-honored explanation for anemia in leukemia has been a "crowding-out" effect of the erythroblasts by the leukemic tissue. This arose from the fact that examination of the bone marrow in many patients with leukemia revealed a decrease in erythroblastic tissue concomitant with the progressive proliferation of leukemic cells. That this was not the sole explanation for the anemia of leukemia was first suggested by Jaffe,15 who found adequate erythroblastic tissue in the bone marrow of some anemic patients with acute leukemia, as well as an increase in iron in the cells of the reticulo-endothelial system. The role of hemolysis, therefore, was stressed by Jaffe and supported by recent investigators. His studies emphasized that a combination of factors is usually involved, thus complicating the evaluation of the anemia.
Decrease in the Rate of Red Cell Production Myelophthisis is an important factor in the production of the anemia, especially in the acute leukemias, where replacement of erythroblasts may occur relatively early in the disease. This may also occur in chronic granulocytic leukemia and chronic lymphocytic leukemia, particularly during the later stages of the disease, although by this time hemolysis may also play a role. In a recent study of the anemia of chronic lymphocytic anemia, Wasi and Block32 employed the bone marrow section technique to demonstrate a normal amount erythroblastic tissue in nonanemic patients. In a group of six patients with hemoglobin values from 9.3 to 12.7 gm. per 100 ml., the degree of developing anemia could be roughly correlated with the decrease in erythroblastic tissue in the marrow. Hemolysis complicated the anemia only in the patient with the hemoglobin of 9.3 gm. In a similar study of bone marrow sections in anemic patients with chronic granulocytic leukemia,11 a significant decrease in erythroblastic tissue was observed in 11 of 15
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sections obtained prior to therapy. An increase in the amount of erythroblastic tissue was observed following successful therapy and was associated with an increase in hemoglobin. With relapse, erythroblastic tissue again decreased markedly in amount. Hemolysis aggravated the anemia only in those patients with marked splenomegaly. In a recent study of lymphosarcoma and reticulum cell sarcoma by Rosenberg et aL,24 16 per cent of patients were found to have marrow replaced by a sufficient amount of abnormal tissue to contribute significantly to an anemia. The availability of isotopic Fe69 has permitted an estimation of the rate of red cell production by determination of the plasma iron clearance and turnover and the incorporation of radio-iron into red cells and the red cell iron turnover.12 These methods, however, are subject to misinterpretation because not all of the radio-iron is represented in red cell hemoglobin. 21 In spite of their limitations, these techniques have demonstrated a decrease in red cell production in many patients, although in some normal erythropoiesis seemed apparent. 6, 7, 18, 33 The decrease in red cell production probably could be explained in those instances in which the marrow was invaded by lymphomatous or leukemic tissue and the amount of erythroblastic tissue decreased. This was particularly true in many cases of acute leukemia as well as late in the course of chronic lymphocytic leukemia, chronic granulocytic leukemia, and occasionally the lymphomas. A normal rate of production was often present early in the chronic leukemias and in the lymphomas. In patients with leukemias and lymphomas with apparently normal erythropoiesis as determined by the radio-iron techniques, or in whom marrow replacement by lymphomatous or leukemic tissue has not occurred, an anemia may develop when even a mild degree of hemolysis is present. A normal marrow may increase its rate of red cell production six to eight times in response to hemolysis or blood loss. This group of patients is unable to compensate fully for the hemolytic process. This form of ineffective erythropoiesis has been termed "relative bone marrow failure" by MooreY A similar mechanism is applicable to the anemias of infection, rheumatoid arthritis, liver disease and renal disease. Since infection, rheumatoid arthritis, liver disease and renal disease in themselves may result in anemia, their association in leukemias and lymphomas may induce or further aggravate an anemia. Radiotherapy and tumor chemotherapy excluding the adrenal steroid hormones are known marrow depressants,34 and thus may aggravate an anemia. The all,:ylating agents, antimetabolites and folic acid antagonists have all been incriminated. The folic acid antagonists may induce a megaloblastic anemia when usage is prolonged. A poor nutritional state may contribute to anemia by failure of adequate intake of proteins. In chronic lymphocytic leukemia and lymphosarcoma a malabsorption syndrome presumably related to small bowel involvement has been described resulting in poor absorption of folic acid, vitamin B12 and iron. 19 In addition, a relative deficiency of folic acid as well
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as a rare case of megaloblastic anemia has been described in association with leukemia. The problem has been discussed by Chanarin et aI. 6 with the thought that a decrease in folic acid may result from an increased demand for folic acid by the leukemic cells. Increase in the Rate of Red Cell Destruction
An increase in the rate of red cell destruction may become an important feature of the anemia of the leukemias and lymphomas. Descriptively, the hemolytic process may be overt or occult. An overt hemolytic anemia may occur in approximately 15 to 20 per cent of patients with chronic lymphocytic leukemia and lymphosarcoma. 9, 25, 28 In these patients the hemolytic process may be relatively severe and associated with a reticulocytosis, hyperbilirubinemia, spherocytosis and, usually but not invariably, a positive Coombs' test. This type of hemolysis may occur at any stage of the disease process and, indeed, may be the insult initiating medical attention. An overt hemolytic anemia is less common in other forms of leukemia and lymphoma, but isolated cases have been reported in acute leukemia, reticulum cell sarcoma, and Hodgkin's disease. With the application of the Ashby and Cr51 techniques to determine accurately the rate of red cell destruction, it has become apparent that an occult hemolytic process occurs commonly in the lymphomas and leukemias, particularly late in the course of the disease. Berlin,! utilizing the Ashby technique, found a decrease in red cell survival in 12 of 15 patients with chronic granulocytic leukemia and in six of nine patients with chronic lymphocytic leukemia. Many other authors have reported a similar increase in the rate of red cell destruction. 6, 7, 9, 13, 18, 31, 33 In these cases, the reticulocyte count, serum bilirubin and fecal urobilinogen may be normal or only slightly elevated. In the early stages of these diseases the rate of red cell destruction may be within normal limits. The mechanisms involved in the increase in the rate of red cell destruction are still being investigated. In many patients, particularly those with an overt hemolytic anemia and those with a positive Coombs' test, an autoimmune process is responsible. 9 , 25, 28 In others, hemolysis may be related to a dysproteinemic state in which the red cells become "coated" by gamma globulin, not necessarily an auto-antibody,' or by a non-gamma globulin. 10 One source of the "coating" material has been suggested by Pirofsky,20 who extracted hemolysins and agglutinins from leukemic white cells which were found to be active against normal erythrocytes. An increase in splenic sequestration of red cells is present in most patients with hemolysis. 29 but is probably more apparent in those with splenomegaly. I, 7, 11 In the lymphomas and leukemias, particularly chronic lymphocytic leukemia and lymphosarcoma, an overt and often severe hemolytic episode has been observed following treatment with x-ray or drugs. 9 , 26 This phenomenon has been reported after the use of alkylating agents, particu-
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larly nitrogen mustard, chlorambucil and triethylene melamine, as well as following total body and splenic irradiation. Whether the hemolysis is precipitated by the therapy or is merely coincidental remains to be ascertained.
Blood Loss Active or occult bleeding may contribute to the anemia of the leukemias and lymphomas. The coexistence of peptic ulceration, particularly in patients who have been receiving corticosteroids, or of an associated neoplasm must not be overlooked. 14 • 28 Bleeding may also result from an accompanying thrombocytopenia and/or other clotting abnormalities. The bleeding may remain fairly well localized, as with epistaxis or excessive menstrual flow, or may be a diffuse capillary bleeding into many tissues. 22 TECHNIQUES OF STUDY OF THE ANEMIA OF LYMPHOMAS AND LEUKEMIAS
The clinical laboratory evaluation of anemia has been reviewed thoroughly in the literature. 8 • 27. 34 The emphasis here will concern those techniques which may be easily applied in the average community hospital (Table 2). The importance of an extremely thorough history and a very complete physical examination cannot be overemphasized. The evaluation of red cell production usually can be approximated from the reticulocyte count and examination of the bone marrow. 27 Red cell destruction can be assessed by measurement of the retiCUlocyte count, bilirubin, evaluation of the peripheral blood smear and bone marrow, and, if necessary, the Cr51 red cell survival. Coombs' test, warm and cold agglutinins and autohemolysis28 may be obtained, if indicated. An increase in transfusion requirement beyond one unit per week is excellent evidence for hemolysis. An evaluation of iron deficiency involves utilization of the hemo-
Table 2. Evaluation of Anemia, a Practical Approach 1. Thorough history 2. Complete physical examination 3. Laboratory evaluation: a. Hemoglobin and hematocrit b. Stools for blood c. Reticulocyte count, corrected d. Peripheral blood smear e. Bilirubin, direct and indirect f. Bone marrow biopsy, smear and section; iron stain g. Serum iron and unbound iron binding capacity h. Coombs' test, if indicated 4. Rarely required studies: a. Cr"1 red cell survival b. Warm and cold agglutinins, autohemolysis c. Fecal urobilinogen
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JR,
globin and hematocrit (mean corpuscular hemoglobin concentration34), the peripheral blood smear, the iron stain of the bone marrow, the serum iron and unbound iron binding capacity, and examination of stools for blood. The RETICULOCYTE COUNT is perhaps the simplest of the readily available laboratory tests in evaluating erythropoiesis, and, if red cell production is not impaired, is a reliable measure of red cell destruction. By the usual slide technique utilizing a brillant cresyl blue stain,34 reticulocytes are recorded as a percentage compared to the total number of red cells. This will result in an overestimation of reticulocytes if the total number of circulating red cells is decreased. Therefore, Finch and Noyes 8 stress the importance of employing a corrected reticulocyte count: .. patient's hematocrit PatIent's retIculocyte count X Ih t 't norma ema oen
The retiCUlocyte count will also be falsely high if there is a premature release of reticulocytes from the marrow, which probably obtains when circulating nucleated red cells are found. In these instances, the reticulocyte count should be divided by two, owing to the fact that the number of reticulocytes in the marrow is equal to the number of circulating reticulocytes. It also must be realized that the reticulocyte count may rise modestly with active bleeding. A consistently low reticulocyte count of 0.1 to 0.2 per cent may suggest a decrease in red cell production. 27 In evaluating the BILIRUBIN LEVEL it must be recalled that the indirect bilirubin (unconjugated), a product of hemoglobin catabolism, must be roughly equated with the amount of red cell hemoglobin. A patient with a hemoglobin which is half of normal, and whose indirect bilirubin is the upper limit of "normal," may be catabolizing twice the expected amount of hemoglobin. Adequate evaluation of the PERIPHERAL BLOOD SMEAR has been discussed in the literature. 3 • 34 With decreased red cell production, mild hemolysis, and/or early iron deficiency, the red cell morphology may well be normal. A decrease in polychromatophilic cells may occur as red cell production decreases. With active hemolysis, polychromatophilia, anisocytosis, poikilocytosis, spherocytosis, occasional macrocytosis and occasional normoblastemia may occur. The microcytic, hypochromic red cells of chronic iron deficiency may not be seen until the hemoglobin becomes less than 8 gm. per 100 ml. Preceding these changes may be poikilocytosis with "cigar" and "tear-drop" forms, red cell fragmentation, and modest increase in target cells. The BONE MARROW EXAMINATION is important in the evaluation of red cell production. Most commonly used is the bone marrow smear in which the granulocytic/erythroblastic (G/E) ratio is determined. However, this ratio is valid only if the granulocytic mass remains unaltered. 8 Also, there is a wide variability of cell counts even when taken from the same area at the
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1485
same time due, in part, to contamination by peripheral blood. 23 The bone marrow section technique enables a more accurate evaluation of the number
and distribution of erythroblasts. Block3 states that if an adequate sample of marrow is obtained, the number of erythroblasts in the marrow gives a semiquantitative estimation of red cell production. This estimation is not valid, however, if there is an arrest of maturation of the erythroblasts (an increase in the number of immature to mature red cell precursors) or if there is karyorrhexis (death) of erythroblasts. In addition, estimates of erythropoiesis are invalid in the presence of iron deficiency (when erythropoiesis is often less than would be expected from the number of erythroblasts present), in the presence of extramedullary hematopoiesis, and in children (when excessive erythroblastic tissue is present). It previously has been estimatedl l that erythropoiesis is probably decreased if the erythroblastic tissue comprises less than 5 per cent of the total marrow section, realizing that the active marrow contains from 20 to 40 per cent myeloid * tissue with the remainder as fat. It must be emphasized, however, that attempts to equate a dynamic process such as erythropoiesis with a static picture of a segment of bone marrow can be only semiquantitative at best. However, combined with the peripheral smear, reticulocyte count and other studies mentioned, a reasonable estimation of erythropoiesis can be made. It is a common misconception that the bone marrow section technique is too difficult for both physician and technician. A technician capable of preparing ordinary pathology slides can prepare marrow sections with little difficulty.t The marrow particles are fixed in Zenker's solution (9 parts) to which neutral formalin (1 part) is added. The particles are then embedded in paraffin. A Giemsa-hematoxylin stain is preferable to the routine hematoxylin-eosin stain since it permits staining of the granules of the granulocytes. With several particles of marrow, smears are also made. Perhaps the most reliable method in the evaluation of iron deficiency is the study of the STAINABLE IRON IN THE BONE MARROW either in the smear or in the section. 2 Iron is stored in the marrow in reticulum cells or in the more immature erythroblasts (siderblasts). The SERUM IRON AND UNBOUND IRON-BINDING CAPACITY may be difficult to evaluate in the anemia associated with leukemias and lymphomas. As is the case of the anemia associated with most chronic diseases, both the serum iron and iron-binding capacity may be low. 16 Therefore a low serum iron may not be indicative of iron deficiency, and conversely, iron deficiency may be present without an elevation of iron-binding capacity. However, the serum iron and iron-binding capacity may be helpful to confirm an iron deficiency, particularly early in the disease process before the total iron-binding protein (transferrin) has begun to diminish. ISOTOPE TECHNIQUES are often not available in the average community hospital. Anemia can be fairly well delineated without their useY Occa-
* Myeloid is used to mean red and white cell precursors and megakaryocytes. t Laboratory technique available upon request.
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DWIGHT J. HOTCHKISS, JR.
sionally a red cell survival test utilizing the radiochromium technique is useful since this is perhaps the most accurate measurement of red cell life span. However, its usefulness is limited by the length of time required for the determination. Body-surface scanning performed concomitantly with the radio chromium red cell survival test may enable more accurate prediction of the results of a contemplated splenectomy.3o An increase in radioactivity over the spleen as compared with the activity over the precordium or liver may indicate increased splenic sequestration of red cells. The FECAL UROBILINOGEN determination is of limited value in the average hospital because of (1) difficulty in accurate and complete stool collections, (2) wide variability in the limits of normal, and (3) difficulties in interpretation since it is a measure of total hemoglobin destruction rather than a measure of red cell destruction.
SPECIFIC PROBLEMS ENCOUNTERED IN THE EVALUATION AND MANAGEMENT OF THE ANEMIA OF LYMPHOMAS AND LEUKEMIAS
Myelophthisis-Depression of Marrow Leukemia
with Chemotherapy-Acute
CASE I. A 3 year old boy was admitted to the Hunterdon Medical Center on 11/8/63 with a 2- to 4-week history of progressive ease of bruisability. The examination revealed a well developed, well nourished boy, who appeared moderately pale and sallow. Multiple ecchymoses were scattered throughout the body; numerous petechiae appeared over the face and upper thorax. Small 1- to 2-cm. lymph nodes were palpable in the anterior cervical, axillary and inguinal regions bilaterally. The liver was palpable 2 cm. below the right costal margin; the spleen was 3 cm. enlarged below the left costal margin. The remainder of the examination was unremarkable. The hemoglobin was 8 gm. per 100 ml. and the hematocrit 24 per cent; the white count was 10,000 with 98 per cent mononuclear cells; the urinalysis and chest x-ray were normal. In the peripheral blood smear the majority of cells were stem cells although a few polymorphonuclear cells and a few lymphocytes were also seen. The red cells were normal in morphology; the platelets were markedly decreased. The bone marrow smear and section were typical of an acute stem cellleukemia. The bone marrow section (Fig. 1) was solidly cellular. Ninety-five per cent of cells seen were stem cells. An occasional erythroblast and granulocyte was noted. Megakaryocytes were not seen. A small amount of iron was present in the iron-stained sections. Treatment with 40 mg. a day of prednisone was instituted, but with little success. Because of a moderately severe episode of epistaxis associated with a progression of the anemia to a hemoglobin of 5.6 gm. per 100 ml., the patient received 1 unit of blood approximately 7 days after institution of steroids. 6-Mercaptopurine at a dosage of 50 mg. a day was then added. By 12/9/63 a complete remission was apparent. The hemoglobin was 12.2 gm. per 100 ml.; the hematocrit 37 per cent; the white count 4000 with a normal peripheral blood smear. A repeat bone marrow examination was essentially normal. The prednisone was tapered and then discontinued. On 1/7/64, Methotrexate
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20 mg. intramuscularly (30 mg.jsq.m. body surface) biweekly was begun and the 6-mercaptopurine discontinued. Clinically the patient was essentially normal and displayed no abnormal physical signs. However, by 2/19/64 the white count had fallen to 2300; the hemoglobin remained at 12.1 gm. per 100 ml. The peripheral blood smear revealed a moderate neutropenia with 70 per cent of leukocytes as lymphocytes and 30 per cent as polymorphonuclear cells. The red cells displayed a 2+ anisocytosis. Platelets were reduced to one-half their normal number. The bone marrow smear suggested grossly a normal cellularity. The G/E ratio was 2/1. Maturation arrest was noted in the granulocytic series at the myelocyte and metamyelocyte stages, and in the erythroblastic series at the polychromatophilic stage. Megakaryocytes were decreased in number. A rare blast cell was noted. There was insufficient material for a bone marrow section. The Methotrexate was discontinued for one week, then resumed at 15 mg. intramuscularly biweekly. The white count has risen to 5500 and the peripheral smear is now essentially normal.
This case demonstrates a fairly typical onset and course of childhood acute leukemia. The anemia at the onset was largely myelophthisic in type resulting from a marked decrease in red cell production due to overwhelming proliferation of leukemic cells. Erythroblastic tissue comprised well below 5 per cent of the total marrow section. Although there was no evidence of overt hemoly~i~, an occult process cannot be excluded. Blood loss had become a contributing factor in this patient at the time of the epistaxis. The depressive influence of drug therapy upon the marrow elements was also observed in this patient. The neutropenia and thrombocytopenia which developed after the institution of Methotrexate were undoubtedly related to the effect of the drug upon the marrow. This was not clear, however, until a marrow was examined, since a progression in the leukemic process might also have been responsible. Although, anemia did not occur from Methotrexate in this patient, it can occur, and indeed, may be megaloblastic in type.
Figure 1 (Case I). Acute leukemia. Solidly cellular marrow due to proliferation of stem cells; erythroblasts (arrows) markedly decreased in number. X400. (This and the following photomicrographs were taken from sections of aspirated marrow-biopsies.)
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DWIGHT J. HOTCHKISS, JR.
Hemolytic Anemia-Hodgkin's Disease
CASE n. A 53 year old white man was first diagnosed as having Hodgkin's disease in August, 1959 following a biopsy of a right anterior cervical lymph node. Other areas of involvement included the left axilla and the mediastinum. There was no hepatosplenomegaly. He was given x-irradiation therapy to the involved areas. In July, 1960, x-ray therapy was given to both groins because of the development of weight loss, edema of the scrotum, and edema of the right leg, associated with the presence of bilateral inguinal nodes. The left axilla, left cervical and left supraclavicular areas were re-treated in November of 1960. In April, 1961, the patient began to complain of night sweats but no fever, and noted an increase in the edema of the scrotum and right leg. At this time a spleen was palpable 10 cm. below the left costal margin. There was no hepatomegaly and no significant adenopathy. A hemoglobin of 6.2 gm. per 100 ml., and reticulocyte count of 5.1 per cent (2.1 per cent corrected) was present. The white count was 6700 with a normal differential; the platelet count was 154,000. The total serum bilirubin was 0.2 mg. per 100 ml. with 0.12 mg. direct. A bone marrow examination was not performed. A Coombs' test, direct and indirect, was negative. The patient was treated with prednisone with subsequent rise in hemoglobin to 10.2 gm. per 100 ml. Forty milligrams of prednisone was required daily to maintain the hemoglobin at this level. Under this program the patient's edema regressed, and his strength improved considerably. An attempt to decrease the steroids resulted in a fall in hemoglobin to 7 gm. per 100 ml. requiring an increase in prednisone dosage. The hemoglobin subsequently rose to 8.2 gm. In the fall of 1961, splenectomy was advised, but refused by the patient owing to his general feeling of good health. By November, 1961, the spleen had enlarged to 18 cm. and the liver was felt 3 cm. below the costal margin. In January, 1962, the patient developed pneumonitis of the left lower lung and bilateral dependent edema, followed by generalized anasarca, requiring hospital admission. Hepatosplenomegaly was present as in November, 1961. Only bilateral shotty inguinal lymphadenopathy was observed. The hemoglobin was 5.7 gm. per 100 ml. and the hematocrit 16.5 per cent; the white count was 7300 with a left shift. A reticulocyte count was 1.8 per cent (0.68 per cent corrected). The serum bilirubin was 4.8 mg. total per 100 ml. with 3.4 mg. direct; and the alkaline phosphatase was 18.3 Shinowara units. The peripheral blood smear revealed normal platelets and white cells. The red cells showed a 2+ anisocytosis. An adequate bone marrow specimen could not be obtained from the sternum, presumably because of previous x-irradiation therapy to that area. A marrow section from the posterior iliac crest (Fig. 2) obtained with a Westerman-Jensen needle* was normal in cellularity. A moderate degree of serous fat atrophy was present indicative of severe chronic disease. 3 The G/E ratio was 1/2 (normal G/E-1/1) and maturation of granulocytic and erythroblastic series appeared normal. Megakaryocytes were normal in number and maturation. Plasma cells were moderately elevated in number. A few small lymphoid nodules were also noted. Iron was increased moderately in the ironstained section. At this point, the patient was transfused and treated with antibiotics but he rapidly progressed to his demise. The autopsy revealed extensive infiltration of liver, spleen, lung, lymph nodes and bone by Hodgkin's disease in the sarcomatous stage. Compression of the superior and inferior vena cava had produced the generalized anasarca. Hemosiderosis of the spleen was severe.
This patient demonstrates the hemolytic anemia not infrequently seen in patients with Hodgkin's disease. 6 There well may have been an
* A fortified Vim-Silverman needle made by Becton, Dickinson Company.
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1489
Figure 2 (Case II). Hodgkin's disease. Cellularity of 40 per cent (normal); moderate serous fat atrophy; G/E ratio 1/2; erythroblasts (arrows) adequate in number and normal in maturation. X 400.
impairment of erythropoiesis as the disease progressed. Although there was moderate erythroblastic activity observed in the marrow section during his terminal admission, the marked hyperplasia usually seen in an otherwise normal marrow compensating for hemolysis was not observed. The failure of the reticulocyte count to increase later in his disease also suggests that compensatory erythropoiesis was impaired. He, therefore, displayed a "relative bone marrow failure" with inability to compensate for hemolysis with an adequate increase in erythropoiesis. There was no evidence of blood loss in this patient.
Blood Loss-Chronic Lymphocytic Leukemia CASE Ill. A 75 year old white man was first seen in December, 1961. One month prior to admission he had developed transient constipation associated with abdominal "gaseousness." In addition, he had developed progressive tiredness and lethargy, occasional dizziness, and a weight loss of from 172 to 164 pounds. The physical examination revealed a well developed, well nourished male with obvious pallor. The tongue was normal. There was no significant lymphadenopathy other than a I-cm. node in the left axilla. The liver was palpable 4 cm. below the right costal margin; the spleen, 3 cm. below the left. The remainder of the examination was unremarkable. The hemoglobin was 5.1 gm. per 100 ml., the hematocrit 18 per cent. A white count was 13,600 with 25 per cent polymorphonuclear cells, 3 per cent bands, 76 per cent lymphoxytes, 1 per cent monocytes, and 1 per cent basophils. The reticulocyte count was 5.6 per cent (1.9 per cent corrected). The peripheral blood smear reflected the modest increase in white count with 60 to 70 per cent of leukocytes being lymphocytes. One-third of these were smudge cells. Platelets were normal in number. The red cells displayed a 2+ anisocytosis, a slight polychromatophilia, and a modest increase in "cigar" forms, target forms, and red cell fragments. There were no hypochromic cells. The bone marrow smear suggested a modest increase in cellularity with approximately 80 per cent of cells as mature lymphocytes. The remaining cells were normal granulocytes and erythroblasts. The marrow section (Figs. 3, 4)
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Figure 3 (Cll8e Ill). Chronic lymphocytic leukemia and blood loss. Cellularityof 50 to 60 per cent slightly increll8ed; a small area of diffuse infiltration by mature lymphocytes; erythroblasts (arrows) in this area markedly decreased. X400.
Figure 4 (Cll8e Ill). Area of essentially normal myeloid tissue except for approximately 10 per cent mature lymphocytes; erythroblll8ts (arrows) are adequate in number and normal in maturation. Many areas of marrow section comparable to this. X400.
displayed an increased cellularity of 50 to 60 per cent (normal 20 to 40 per cent.) Four to five large lymphatic nodules were seen. The remaining myeloid tissue contained from 10 to 50 per cent mature lymphocytes. The remaining cells were of the granulocytic, erythroblastic and megakaryocytic series which were adequate in number and normal in maturation. The G/E ratio was 1/1. No increase in plasma cells was seen. No iron was present in the iron stain. Further studies revealed a serum bilirubin of 0.29 mg. per 100 ml. total, 0.05 mg. direct; serum iron 50 /-Lg. and iron-binding capacity 372 /-Lg. per 100 m!.; the stool was positive for occult blood. Sigmoidoscopy and barium enema were normal. An upper gastrointestinal series revealed the presence of a gastric carcinoma. A subtotal gastrectomy was subsequently performed. The pathologist reported an adenocarcinoma of the stomach metll8tatic to mesenteric lymph nodes. The patient did fairly well for approximately 6 months, then began to deteriorate rapidly and expired in June, 1962.
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The initial reaction was to attribute the severe anemia to depression of erythropoiesis due to marrow replacement as a result of chronic lymphocytic leukemia, whose presence was apparent at the completion of the initial blood count and study of the peripheral blood smear. The elevated reticulocyte count suggested a hemolytic component. However, subsequent evaluation did not bear this out. The bone marrow revealed an adequate amount of erythroblastic tissue-at least 10 to 15 per cent of the marrow section. Myelophthisis was not a factor in producing anemia. The absence of iron in the marrow in association with a moderate poikilocytosis in the peripheral blood smear indicated an iron deficiency as the most likely cause of the anemia. The serum iron and iron-binding capacity lent further evidence for iron deficiency. The corrected reticulocyte count was 5.1 (is X 5.6) = 1.9, which is just above the upper limits of normal and could reflect blood loss or a mild hemolytic process. However, occult hemolytic anemia is usually seen later in the course of chronic lymphocytic leukemia. 9 , 32 The pursuit of the source of blood loss led to the discovery of the gastric carcinoma. The anemia, therefore, was due to blood loss from the gastric malignancy. The early chronic lymphocytic leukemia probably did not contribute significantly to the anemia. SUMMARY
The anemia often found in association with the lymphomas and leukemias frequently involves several mechanisms. Thorough evaluation employing readily available techniques will enable the physician to ascertain the relative importance of the degree of the decrease in the rate of red cell production, increase in the rate of red cell destruction, and blood loss. Adequate therapy will obtain only after the mechanisms producing the anemia are understood in each patient. REFERENCES 1. Berlin, R.: Red cell survival studies in normal and leukaemic subjects. Acta med.
scandinav., Suppl. 252, 1951. 2. Beutler, E., Robson, M. J. and Buttenwieser, E.: A comparison of the plasma. iron, iron-binding capacity, sternal marrow iron and other methods in the clinical evaluation of iron stores. Ann. Int. Med. 48: 60, 1958. 3. Block, M.: Morphologic aspects of erythropoiesis. In Mechanisms of Anemia. (1. Weinstein and E. Beutler, Eds.) New York, N. "¥., McGraw Hill Book Co., 1962. 4. Brody, J. 1. and Finch, S. C.: Serum factors of acquired hemolytic anemia in leukemia and lymphoma. J. Clin. Invest. 40: 181, 1961. 5. Chanarin, 1., Mollin, D. L. and Anderson, B. B.: Folic acid deficiency and the megaloblastic anaemias. Proc. Roy. Soc. Med. 51: 757, 1958. 6. Cline, M. J., and Berlin, N.!.: Anemia in Hodgkin's disease. Cancer 18: 526, 1963.
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7. Cline, M. J. and Berlin, N. I.: Patterns of anemia in chronic myelocytic leukemia. Cancer 16: 624, 1963. 8. Finch, C. A. and Noyes, W. D.: ErythrokineticB in diagnosis of anemia. J.A.M.A. 175: 1163, 1961. 9. Freymann, J. G., Burrell, S. B. and Marler, E. A.: Role of hemolysis in anemia secondary to chronic lymphocytic leukemia and certain malignant lymphomas. New England J. Med. 259: 847, 1958. 10. Green, H. N., Wakefield, J. and Littlewood, G.: The nature of cancer anaemia and its bearing on the immunological theory of cancer. Brit. M. J. 2: 779, 1957. 11. Hotchkiss, D. J., Jr. and Block, M. H.: Effect of splenic irradiation on systemic hematopoiesis. Arch. Int. Med. 109: 695, 1962. 12. Huff, R. L., Hennessy, T. G., Austin, R. E., Garcia, J. F., Roberts, B. M. and Lawrence, J. H.: Plasma and red cell iron turnover in normal subjects and in patients having various hematopoietic disorders. J. Clin. Invest. 29: 1041, 1950. 13. Hyman, G. A.: Studies on anemia of disseminated malignant neoplastic disease. I. The hemolytic factor. Blood 9: 911,1954. 14. Hyman, G. A.: Anemia in malignantneoplastic disease. J. Chron. Dis.16: 645,1963. 15. Jaffe, R. H.: Erythropoiesis in leukemia. Folia haemat. 49: 51, 1933. 16. Laurell, C. B.: Studies on the transportation and metabolism of iron in the body. Acta physioI. scandinav., Suppl. 46, 1947. 17. Moore, C. V.: The concept of relative bone marrow failure (Editorial). Am. J. Med. 23: 1, 1957. 18. Nathan, D. G. and Berlin, N. I.: Studies of the rate of production and life span of erythrocytes in acute leukemia. Blood 14: 935, 1959. 19. Pitney, W. R., Joske, R. A. and MacKinnon, N. L.: Folic acid and other absorption tests in lymphosarcoma, chronic lymphocytic leukaemia, and Borne related conditions. J. Clin. Path. 13: 440, 1960. 20. Pirofsky, B.: Studies on the hemolysin and agglutinin of leukemic leukocytes. Blood 12: 620, 1957. 21. Pollycove, M. and Mortimer, R.: The quantitative determination of iron kinetics and hemoglobin synthesis in human subjects. J. Clin. Invest. 40: 753, 1961. 22. Price, V. E. and Greenfield, R. E.: Anemia in cancer. Advances Cancer Res. 5: 199, 1958. 23. Reich, C. and Kolb, E. M.: A quantitative study of the variations in multiple sternal marrow samples taken simultaneously. Am. J. M. Sc. 204: 496,1942. 24. Rosenberg, S. A., Diamond, H. D., Jaslowitz, B. and Craver, L. F.: Lymphosarcoma: A review of 1269 cases. Medicine 40: 31, 1961. 25. Rosenthal, M. C., Pisciotta, A. V., Komninos, Z. D., Goldenberg, H. and Dameshek, W.: The auto-immune hemolytic anemia of malignant lymphocytic disease. Blood 10: 197, 1955. 26. Samuels, A. J. and Bierman, H. R.: Anemia in patients with neoplastic diseases. California Med. 84: 180, 1956. 27. Tocantins, L. M. (Ed.): Modern methods for quantitation of red blood cell production and destruction: Erythrokinetics. Blood 18: 225, 1961. 28. Troup, S. B., Swisher, S. N. and Young, L. E.: The anemia of leukemia. Am. J. Med. 28: 751, 1960. 29. Ultmann, J. E.: Role of the spleen in the hemolytic anemia of cancer patients. Cancer Res. 18: 959, 1958. 30. Veeger, W., Woldring, M. G., van Rood, J. J., Ernisse, J. G., Leeksma, C. H. W., Verloop, M. C. and Nieweg, H. 0.: The value of the determination of the site of red cell sequestration in hemolytic anemia as a prediction test for splenectomy. Acta. med. scandinav. 171: 507, 1962. 31. Waggener, R. E., Pratt, P. T. and Hunt, H. B.: Erythrocyte survival in leukemia, Hodgkin's disease, and malignant lymphoma as determined by radiochromate. Am. J. Roentgenol. 79: 1045, 1958. 32. Wasi, P. and Block, M.: The mechanism of the development of anemia in untreated chronic lymphatic leukemia. Blood 17: 597, 1961. 33. Wetherley-Mein, G., Epstein, I. S., Foster, W. D. and Grimes, A. J.: Mechanisms of anaemia in leukaemia. Brit. J. Haemat. 4: 281, 1958. 34. Wintrobe, M. M.: Clinical Hematology. 5th.Ed. Philadelphia, Lea & Febiger, 1961.