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Page 1: Hematology for primary care physicians

Disease-a-Month” Volume XLII Number 3 March 1996

HEMATOLOGY FOR PRIMARY CARE PHYSICIANS

Earl W. Campbell, Jr., MD, FACP Professor Department of Internal Medicine Medical College of Ohio Toledo, Ohio

Mary R. Smith, MD Professor Departments of Internal Medicine and Pathology

Medical College of Ohio Toledo, Ohio

h’if Mosby m ATimes Mirror u Company

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Disease-a-Month” Volume XLII Number 3 March 1996

HEMATOLOGY FOR PRIMARY CARE PHYSICIANS

CONTENTS

132 Foreword

133 Abstract

133 In Brief

137 Erytbrocyte Disorders 137 Erythrocyte Laboratory Studies 138 Anemia

163 Leukocyte Disorders 163 The Basic Leukocyte Report 164 Identifying Features of Leukocyte Cell Populations 166 Abnormalities of Leukocytes 176 The Leukemias

180 Myeloproliferative Disease 180 Polycythemia Rubra Vera 182 Agnogenic Myeloid Metaplasia, Myelofibrosis 184 Thrombocythemia 185 Paroxysmal Nocturnal Hemoglobinuria

185 185 186 186 189 190 191 192

Hemostasis Medical History Physical Examination Coagulation Profile Problem-Solving Strategies in Hemostasis Molecular Markers of Hemostasis Disseminated Intravascular Coagulation Laboratory Evaluation of Increased Clotting Risk (Hyperco-

agulability States)

194 References

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FOREWORD

The blood has been an object of fascination from earliest times. The ancient Hebrews believed that the blood was the seat of the soul, and the Romans viewed blood as the source of a warrior’s cour- age and ability. The first understanding of the content, function, and circulation of the blood, De Motu Cordis et Sanguinis, was writ- ten by William Harvey and published around 1650. Whereas Aristotle had pronounced the heart to be the center of life, Harvey maintained that the blood was the core of existence and that the heart served as a pump to keep the blood moving.

The early advancement of the discipline of hematology was in- exorably linked to the development of the microscope, which al- lowed visualization, identification, and ultimately, study of the con- tents of the blood. Hematology is still very much linked to labora- tory studies. Whereas primary hematologic diseases are relatively uncommon, hematologic symptoms secondary to other illnesses oc- cur frequently, and laboratory studies provide valuable clues about the next step in discerning the cause of a patient’s symptoms.

The authors of this issue of DISEASE-A-MONTH set as their goals help- ing primary care physicians make the best use of basic laboratory tests and providing primary care physicians with the information necessary to decide whether further laboratory tests are needed and which ones would be most helpful. To facilitate decision making, authors Earl Campbell, Jr., and Mary Smith have divided the issue into four sections that cover erythrocyte disorders, disorders of leu- kocytes, myeloproliferative diseases, and coagulation system disor- ders. The liberal use of tables contributes to the overall usefulness of information.

Keep this issue handy. I know it’s one you will use again and again.

Roger C. Bone, MD Editor

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HEMATOLOGY FOR PRIMARY

CARE PHYSICIANS

ABSTRACT.-Many hematologic disorders present minimal physical signs and symptoms in the early state. For example, chronic myelogenous leukemia may not manifest splenomegaly or any obvious physical signs, yet the laboratory report may demonstrate leukocytosis, eosinophilia, basophilia, and thrombocytosis. Although the anemic condition of a patient with a hemoglobin level of 7 gm/dl may be readily apparent to the clinician, a he- moglobin level of 10.5 gm/dl may be difficult to discern during a brief visit that is focused on another organ sys- tem. The same laboratory report, however, may contain valuable clues about unsuspected anemia related to mean corpuscular volume or morphology. Information from sup- porting chemistry studies often may be helpful in inter- pretation of the diagnosis. An elevated uric acid level, for example, may indicate hyperkinetic cytogenesis related to myeloproliferative or lymphoproliferative neoplastic disorders.

This monograph is designed to be useful to busy physi- cians who want to use basic hematologic studies in a cost- effective manner. Hematology is viewed in a problem-ori- ented way; the laboratory report is used as the problem generator.

IN BRIEF The need to be cost effective and the need to accommodate the

HMO-related influx and exit of patients in the practice of the pri- mary care physician necessitate efficient use of basic laboratory tests. Hematologic laboratory studies are unique because they constitute direct biopsy of a major organ system. In fact, when a physician or technologist views a peripheral smear, a cytologic assessment oc- curs. The hematopoietic system often reflects disease of other organ

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systems through perturbations of blood cell kinetics and the coagu- lation system.

Unlike in earlier times, a primary care physician may be presented with laboratory reports that show hematologic abnormalities, some- times quite important, without the benefit of detailed historical and physical findings. One example is chronic myelogenous leukemia, which may be insidious. Thus, screening laboratory tests may be the first clue to a hematologic disease. Scrutiny of preliminary he- matologic findings prompts the physician to review historical and physical events in the patient’s chart that might support further in- vestigation of the unusual hematologic result. At the same time, the primary care physician is able to use time well, deciding quickly whether the test in question should be repeated or if other tests should be ordered.

To aid the primary care physician in selecting the next laboratory test, this monograph is divided into sections related to anemia, dis- orders of leukocytes, myeloproliferative diseases, and dysfunction of the coagulation system. In each section, the goal is to guide deci- sion making about further evaluation with minimum use of labora- tory studies. The supporting text in each section is designed to help the physician ask pertinent questions to find the most effective an- swer. It is always better to ask the right question than to mistakenly believe that one has the right answer.

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Earl TN Campbell, Jr, MD, FACR is Professor and Vice Chairman of the Department of Medicine at the Medical College of Ohio at Toledo. Dr Campbell received his bach- elor of arts degree from Harvard University and earned his doctor of medicine degree at the University of Roch- ester, where he also completed his internship. After a tour of duty with the U.S. Air Force, Dr Campbell com- pleted his residency at Henry Ford Hospital in Detroit, followed by a fellowship iu hematology at the Univer- sity of Utah.

Dr Campbell was the founding chief of the Division of General Internal Medicine and Geriatrics at the Medi- cal College of Ohio at Toledo. Dr. Campbell has been instrumental in developing the educational programs at the Medical College of Ohio and, is currently director of the internal medicine residency program.

Dr Campbell was a member of the Acute Leukemia Group B Study Group and the Polycythemia Rubra Vera Study Group. Among his publications is an article de- scribing the index case of required factor VII hemophilia. Dr Campbell is a graduate of the Stanford University ficulty Development Program. He has been repeatedly recognized for excellence in teaching and is a recipient of the American College of Physicians, Ohio Region, Mas- ter Teacher Award.

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Mary R. Smith, MD, is Professor of Clinical Medicine and Arrthology and Associate Dean for Medical Education in the School of Medicine at the Medical College of Ohio at Toledo. Dr Smith received a bachelor of arts degree from the University of Western Ontario and earned her doctor of medicine degree at the University of Ottawa, Ontario. She completed a residency in internal medicine and a fellowship in hematology at the Royal Victoria Hospital of McGill University, Montreal, Quebec.

In addition to maintaining an active practice in hema- tology and oncology, Dr. Smith is Director of the Coagu- lation Laboratory at Medical College Hospital, Toledo. In her role as Associate Dean for Medical Education, Dr Smith is involved in the development of innovative meth- ods for the education of medical students and the evahz- a tion of their progress.

DE Smith maintains an active interest in research, es- pecially in disorders of hemostasis associated with dis- orders of platelet function. She is actively involved in research directed at identifying causes of and therapy for cerebral vascular thromboembolic disease.

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HEMATOLOGY FOR PRIMARY

CARE PHYSICIANS

ERYTHROCYTE DISORDERS ERYTHROCYTE LABORATORY STUDIES

Frequently, anemia is first noted in a laboratory report and not in a history or during a physical examination. Of the erythrocyte studies available on laboratory reports, four are of use to the clinician: he- moglobin level (Hgb), mean corpuscular volume (MCV), red blood cell distribution width (RDW), and morphology. Other tests, includ- ing the actual red blood cell count (RBC), are much less frequently useful.

Hemoglobin The proportion of red blood cells to plasma is reflected by the

hemoglobin pigmentation concentration (that is, hemoglobin and the packed volume of red blood cells, or hematocrit [Hct]). Auto- mated cytometry allows direct, highly reproducible measurement of hemoglobin, and hematocrit is a derived measurement obtained with automated devices. The normal range of hemoglobin level var- ies according to age and sex.

Mean Corpuscular Volume The MCV is measured in femtoliters (fl) or cubic micrometers bm3).

MCV is derived from the RBC and the hematocrit, as follows:

MCV = Hct (%)

RBC (xlO’Y/~l)

The MCV is of great value in determining the cause of anemia.

Red Cell Distribution Width Red cell distribution width reflects perturbation of red blood cell

maturation and hemoglobinization (for example, vitamin or iron deficiencies) as well as disorders that affect mature red blood cells in the peripheral blood (for example, fragmentation or loss of mem- brane). These phenomena result in increased anisocytosis or in- creased numbers of cells with varying sizes. A greater than normal RDW may reflect a hematologic disorder in which there is wide varia- tion in cell size.

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TABLE 1. Basic ervthrocvte values Test Men Women

Hemoglobin (gm/dl) 13-18 12-16 Hematocrit 0.40-0.52 0.35-0.47 Mean coruuscular volume Ifll 80-100 80-100

TABLE 2. Basic classification of anemia MCV Class Normocytic

Hemoglobin (gm/dl) Cl2 Corrected <2 reticulocyte count

1 Production CR Class Hypoproliferative

Macrocytic or microcytic

<12 <2

Ineffective Maturation defect

Normocytic

Cl2 <3-5

1‘Destruction Hemolytic or hemorrhaeic

CR, corrected reticulocyte count; MCV, mean corpuscular volume.

Reficulocyte Corn t Although basic to hematologic diagnosis, the reticulocyte count

requires separate preparation and is not as cost effective as a screen- ing test, although it is most useful in hematologic diagnosis. The reticulocyte count is expressed in terms of percentage of immature cells. Hypoproliferation or hyperproliferation is better understood with calculation of a corrected reticulocyte count (CR). The formula for CR is as follows:

CR =Reticulocyte % x Observed Hct

45

Morphology More often than in the past, physicians are relying on laboratory

reports for information about erythrocyte morphology. Information frequently includes commentary on the nature of red blood cell de- formation (poikilocytosis) or size variation (anisocytosis). At times, specific forms may correlate with disease states (for example, mac- rocyte and vitamin B,, deficiency, microcyte and thalassemia, schizocyte and disseminated intravascular coagulation [DE], acanthocytosis and a beta lipoproteinemia).

ANEMLA

Although authors of articles in the literature agree that anemia may be defined as a decrease in red blood cell mass to below-normal values, the application of the definition must be determined in each case on the basis of patient-centered data. The numbers in Table 1, for example, are for adults only. Values for newborns, infants, and

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TABLE 3. Subclassification of anemia Hypoproliferative Maturation abnormality Hemolytic or

hemorrhagic

Iron deficiency Marrow damage Loss of

erythropoietin effect

Nuclear defect Folate deficiency Vitamin B,, deficiency Refractory anemia Cytoplasmic defects Thalassemia Iron deficiency Sideroblastic

Blood loss Intravascular hemolysis Autoimmune

disease Hemoglobinopathy Metabolic Membrane

pregnant women vary from the generally accepted standards for adults. A reliable laboratory will provide the physician with values specific for age and sex. Some cautions are in order, however. When considering the diagnosis of anemia in pregnancy, keep in mind that plasma volume may increase by SO%, with the result that Hgb will decrease without an actual decrease in the red blood cell mass. In dehydrated patients with anemia, the Hgb may appear normal when the red blood cell mass is accompanied by a proportional decrease in plasma volume (Table 1).

Clinical Clues to Anemia Anemia, like congestive heart failure, is a symptom of a disease.

Although this discussion emphasizes the use of laboratory tests, valuable information can be derived from historical data related to symptoms, heredity, exposures, habits, lifestyle, and physical signs such as alteration in skin color, weight, bowel habits, or the devel- opment of organ enlargement or tumors. The clinician must ask, “What other disorders does the patient have that may lead to an anemia?” Abnormalities in red blood cells often occur in company with hematopoietic disorders of platelets and leukocytes. Cost-ef- fective, quality care requires a thorough review of the patient record and, often, re-examination of the patient.

Classification of Anemia Anemia can be classified, according to MCV, into microcytic, mac-

rocytic, or normocytic types (Table 2). The corrected reticulocyte count can be used for further identification of the disorder as hypo- proliferative, maturational, or hemolytic. Once the anemia has been classified with preliminary data, one should incorporate informa- tion from the history and physical examination to reach a diagnosis. Further tests, carefully selected, can be used to confirm the diagno- sis. The categories used are not discrete. For example, iron deficiency most frequently is normocytic, but when severe, may be microcytic. Anemias may be multideterminant. For example, sickle cell disease,

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TABLE 4. Laboratory studies and values definitive for iron deficiency anemia

Studv SI value Conventional value

Serum iron Total iron&&ingcapaoity Percentage saturation Ferritin Marrow iron stain

< 7 mmol/L > 7 L%mTnaUL 0.15 <15mg/L 0

~40 mgidl 3r 59@rnddl Cl5 cl5 rig/ml 0

TABLE 5. Causes of marrow iniurv anemia

Stem cell damage

I d

Structural damage

Uncertain causation

Drugs Chemotherapy Radiation therapy Chemicals Toxins Infection Leukemia Lymphoma Myeloproliferative disorder Fanconi anemia

Malignant metastasis

Granulomatous disorder

Radiation Myelofibrosis Lipidosis

Collagen- vascular

Infection Graft vs.

host disease Aplasia

Pure erythroid aplasia

Diamond- Blackfan syndrome

Idiopathic

which is hemolytic, may be quite intense, exhausting folate stores and becoming macrocytic. Classification of anemia allows cost-ef- fective laboratory evaluation and ultimately both cost-effective and quality treatment. Amplification of the functional classification al- lows subclassification (Table 3).

Hypoproliferative Anemias Hypoproliferative anemias generally have in common reti-

culocytopenia and cells of normal volume, or if severe, decreased MCV The mechanisms vary. As with all anemias, patient history and physical examination may offer important clues.

Iron Deficiency Anemia.-In addition to having the general fea- tures of anemia, patients with iron deficiency may have stomatitis, angular cheilitis, and glossitis with loss of papillae of tongue. In- frequently, koilonychia (spooned nails), dysphagia, and pica (crav- ing to eat substances such as dirt, clay, ice, or laundry starch) are seen.

Iron deficiency anemia may be related to chronic gastrointestinal bleeding, which may be either obvious or occult. If the iron defi- ciency is severe, the cells may be microcytic. Iron stores are reflected in serum iron, total iron binding capacity (TIBC), percentage satura- tion, ferritin levels, and ultimately by means of direct assessment of iron stains on bone marrow. Iron stores can be evaluated with any of these methods in a continuum without discrete cut-off values to

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TABLE 6. Medications that cause marrow injury

Group Medication

Antimicrobials

Antidepressants

Anti-inflammatory drugs

Anti-arrhythmics Antithyroid drugs Diuretics Antihypertensives Anti-uricemics Hypoglycemics Tranquilizers Antirheumatics

Chloramphenicol,* penicillins, sulfonamides, .oxytetracychne, ampbotericin B, dap.sone,.arsenicals. * quinacrine*

Lithium, tricyclics, insecticides (DDT, parathion, chlor- dane, pentachlorophenol)

Phenylbutazone,* acetylsalicylic acid, indomethacin, carbamazepine (Tegretol)

Lidocaine, quinidine, procainamide Propylthiouracil Thiazides, pyrimethamine Captopril Allopurinol, colchicine Tolbutamide Meprobamate, chlordiazepoxide (Librium), methyprylon Quinicrine,* chloroquine, gold compounds,* d penicil-

lamine, acetylsalicylic acid, indomethacin, phenyl- butazone, low-dose methotrexate

*Associated with 20 or mere cases.

indicate deficiency of iron. Bone marrow sampling, the most invasive diagnostic method, is seldom used. The characteristics of clinically significant iron deficiency are shown in Table 4. Coexisting disor- ders, such as liver disease, may elevate the ferritin level.

Correction of the underlying pathologic condition is imperative for patients with iron deficiency anemia. Replacement iron should provide 100 to 200 mg of elemental iron daily. An easily remem- bered scheme for adults is to prescribe 300 mg of non-enteric-coated ferrous sulfate tablets to be taken during meals three times a day until the hemoglobin level is normal, followed by one tablet a day for 1 year from the inception of iron therapy. A search for the cause of iron deficiency must always precede therapy. The diagnosis must be unequivocal because the administration of excess iron produces hemosiderosis, which has dreadful complications.

Morrow Zn@ry &ilure.-Marrow-injury anemia may occur with perturbation of the stem cell pool or with disruption of the marrow structure itself. In adults, the marrow is located in the axial skeleton or the proximal long bones. Marrow dysfunction is anticipated dur- ing chemotherapy. Anemia caused by drug toxicity or tumor infil- tration may be mild at first. The most severe of these anemias are aplastic anemia and the anemia that occurs with leukemia. In some populations, anemia may be the result of chemicals in the work- place or the environment.

Aplastic anemia is characterized by a rapid onset without a pre- cipitating illness. There are exceptions to this mode of onset, how- ever. Viral hepatitis is one example. Pure red blood cell aplasia has a

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TABLE 7. Agents that induce dose-related aplasia

Benzene, benzene derivatives (trinitrotoluene), and related agents

Ionizing radiation Chemotherapeutic agents Alkylating agents Antimitotics Some antibiotics (doxorubicin hydrochloride [Adriamycin], daunorubicin HCl)

similar presentation, often associated with thymoma in adults or with parvovirus.

Myeloproliferative disorders with anemia become apparent with other hematologic clues [for example, leukemias with organ enlarge- ment and white blood cell abnormalities). Myelofibrosis and thrombocythemia exhibit diagnostic blood cell morphologic changes. In patients undergoing chemotherapy, anemia is anticipated. Per- haps the most challenging diagnostic marrow failure anemias are those related to drugs and toxic chemicals. The 1:30,01X1 incidence per patient use ratio of aplastic anemia resulting from chlor- amphenicol administration is well known. Much less is known, how- ever, about the relationship between aplastic anemia and the in- creasing environmental exposure to uncontrolled chemical contami- nation. Except in cases of pure red blood cell aplastic anemia, bone morrow elements (that is, platelets and white blood cells) are often perturbed. Tables 5 through 7 present the causes of marrow failure.

Laboratory findings are characterized by low hemoglobin level, low reticulocyte count, increased serum iron, and normal TIBC (Table 8). Findings in the peripheral smear may contribute to the diagno- sis, possibly demonstrating nucleated erythrocytes and abnormal or precursor white blood cells and platelets. A bone marrow smear and a biopsy are necessary for confirmation of the diagnosis and often define the origin of the marrow damage. Bone marrow studies should be performed early to clarify an uncertain diagnosis.

Management of marrow-injury anemia may be simple or complex. Removal of the influencing agent may, at times, be the effective treat- ment. Often, aplastic anemias are much more complex, necessitat- ing consultation for further diagnosis. Transfusion therapy may in- duce iron storage disease. Further diagnosis and management of au- toimmune conditions is complex. Some disorders, particularly he- reditary ones, may necessitate bone marrow transplantation. Re- sponse to therapy with exogenous erythropoietin is unpredictable.

Anemias Related to Loss of Erythropoietin Efiect.-These anemias, perhaps the most common of all anemias, occur in patients with renal insufficiency, inflammatory diseases, and endocrine disorders. These anemias have in common a relative loss of response to eryth-

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TABLE 8. Laboratory features of clinically significant marrow injury Test

Red blood cell morphology

Reticulocytic index Ferritin level Serum iron level Total iron-binding capacity

Finding

Normocytic Nucleated cells may or may not be present <l

: t

Marrow morphology Often provides enough information to allow diagnosis ?, increased.

TABLE 9. Hypometabolic states associated with anemia

1 Protein* 1 Testosterone 1 Parathormone 1 Thyroid hormonet 1 Pituitary function 1 Adrenal function 1, decreased. *May be associated with other dietary deficiencies. tMay be associated with macrocytic or microcytic changes.

ropoietin. The erythropoietin level frequently is disproportionately low for the level of anemia. Erythropoietin, which stimulates pro- duction of red blood cells, is produced in the region of the peritubular cells of the glomeruli in response to hypoxia.

RENAL DISEASE ANEMIA.-&IXtl disease anemia shares the following features with other anemias in this category: failure of the normal erythropoietin response and, unless the renal disease anemia is se- vere, normochromia, normocytosis, normal MCV, and a low reticu- locyte count. The severity of anemia correlates with worsening re- nal function. With end-stage renal disease, the life span of erythro- cytes is much shortened and uncompensated. In diabetic renal dis- ease, the severity of anemia may be in excess of that predicted by renal function, because erythrocyte hypoproliferation occurs quite early. Patients with uremia may suffer blood loss; thus iron studies in the baseline state are helpful. Erythropoietin therapy may fail if iron stores are low.

HYPOMETABOLIC STAT-es.-Anemia secondary to protein deprivation, which is seen in elderly hospitalized patients and after surgical pro- cedures, is mild and responds spontaneously with nutrition and normalization of albumin level. Other deficiencies in these situa- tions, such as vitamin B, and riboflavin deficiencies, may contrib- ute to anemia. Deficiencies of thyroid hormone, testosterone, par- athormone, and adrenal and pituitary hormones may be associated with hypoproliferation of erythrocytes (Table 9). The mechanisms

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TABLE 10. Anemias of chronic inflammation*

Collagen diseases AIDS and related disorders Malignant tumors Chronic infections * Viral disorders may produce anemias through direct in- jury to stem cells 01 induction of autoimmune dysfunc- tion.

of induction of anemia are not clearly identified in all instances. In the aforementioned states, kidney sensor cells appear to slow regu- lar erythropoietin production.

ACUTE AND CHRONIC INFLAMMATION.-Acute bacterial, viral, and col- lagen vascular diseases can produce anemia in which there is a rapid fall in hemoglobin level within 48 hours of onset. This relates to the demise of erythrocytes nearing the end of the life span and accelera- tion of red blood cell senescence from stress. This phase is soon made more severe by the failure of erythropoietin and erythroid pro- liferation. Concomitantly, serum iron level and TIBC fall and the ferritin level rises as a reaction to the acute phase. Cytokines (for example, tumor necrosis factor [TNF], interleukin-1 [IL-l], and 13- interferon related to infection) play a role in inhibiting erythropoi- etin release and suppressing the erythropoietic response.

Chronic disease or inflammatory response anemia results in part from an inflammatory cytokine effect (from, for example, fi-inter- feron, TNF, and a-interferon) (Table 10). If the process is of long duration and great intensity, the normocytic pattern may switch to hypochromia and microcytosis. Markedly increased iron stores ap- pear as large granules in the reticuloendothelioid cells of the mar- row. Table 11 presents laboratory values for anemias that are related to loss of erythropoietin effect, which are a subset of the hypoproliferative anemias,

Therapy varies according to disorder, thus there is a great need for diagnostic accuracy. Transfusion over time may induce hemosiderosis and autoimmune responses directed against blood cells. The risks of transfusion-induced hepatitis, human immunodeficiency virus (HIV) infection, and cytomegalovirus (CMV) infection are well known. When possible, correction of the underlying disorder is im- perative. The use of recombinant erythropoietin has proved effec- tive in the treatment of patients with end-stage renal disease. The starting dose is 50 to 150 U/kg subcutaneously three times a week, which is reduced when the hemoglobin level reaches 10 gm/dl to avoid erythrocytosis. The average dose is 75 U/kg subcutaneously three times a week. Erythropoietin is used to balance the suppres- sive effect of zidovudine (AZT) and other chemotherapeutic agents in the management of acquired immunodeficiency syndrome (AIDS).

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TABLE 11. Anemia related to low erythropoietin level Laboratory value Renal disease Inflammation

Anemia Varying severity Minimal Mean corpuscular Normocytic Normocytic

Hypometabolism

Minimal Usually normocytic

volume Reticulocyte index Serum iron level*

Total iron-binding capacity

Percentage saturation Ferritin level Marrow iron*

<2 Normal*

Normal*

Normal* Normal* Normal*

to microcytic <2 < 9 mmol/I. (<50mg/dl) < 54 mmol/L (<soopg/dl) 10-20 t t

<2 Normal

Normal

Normal Normal Normal

?, increased. *Applies if the patient has no related causes of anemia.

Anemias of1neffctive Maturation.-This group of disorders is di- verse in origin and cell size, but the disorders have in common ab- normalities of nuclear or cytoplasmic components. A high propor- tion of erythrocyte precursors are destroyed in the marrow (ineffec- tive erythropoiesis), and mature cells in the circulation have a short- ened life span, resulting in failure to maintain a normal erythrocyte mass. Cytoplasmic defects lead to reduction in cell size (microcytosis), and nuclear maturation defects lead to enlargement (macrocytosis or megaloblastosis) .

Anemias Secondary to Cytoplasmic Defects Hereditary failure to produce normal amounts of hemoglobin re-

sults in diminished cytoplasm and decreased erythrocyte volume. This can be a consequence of insufficiency of any one of the compo- nents of the hemoglobin molecule: iron, heme porphyrin, or protein globin chains.

Iron Deficiency Anemia.-Iron deficiency anemia is discussed in the previous section. When iron availability becomes severely re- stricted, microcytosis results. The mechanisms discussed in the pre- vious section apply here as well.

Thalassemia.-Thalassemia, although microcytic in most of its forms, contrasts with iron deficiency in that it is hereditary, results from insufficient globin molecule synthesis, and is often associated with toxic increases in iron stores. In general, l3 thalassemia is found in persons of African and Mediterranean ancestry, whereas a thalassemia and hemoglobin E occur more frequently in persons from Southeast Asia. Thalassemia may be found sporadically in other populations. This disorder must be differentiated from iron defi- ciency through the use of iron studies. Inappropriate therapy with iron is dangerous and, therefore, contraindicated.

Some of the differences between a and p thalassemia relate to the

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TABLE 12. Normal adult hemoglobin values Hemoglobin Chain Value (%)

Hgb A aaPP 96-97 Hgb F aaYY -cl Hgb A.. aa 1

fact that each of the adult hemoglobin molecules is one-half an a chain. Hemoglobin A, which composes 96% to 97% of adult hemo- globin, contains equal numbers of a and j3 chains. Hemoglobin A, comprises two a and two p chains and accounts for 1% of hemoglo- bin in adults, whereas hemoglobin F, or fetal hemoglobin, formed of two a and two (3 chains, normally accounts for less than 1%. Table 12 presents normal adult hemoglobin values.

At birth, the percentage of hemoglobin F falls from near 90% and the percentage of hemoglobin A rises inversely. A failure of 6 or y chain synthesis in the normal adult has little effect on total hemoglo- bin. Therefore, pure thalassemia of y or 6 chains is clinically inappar- ent in an adult. Identification of the quantity of each chain and its related hemoglobin is the basis for the diagnosis of thalassemia.

Fetal hemoglobin chain production, two a and two y chains, de- creases near birth. Suppression of y chains after birth results in a rise of a and 6 chains and, therefore, a rise in hemoglobins A and A, to adult levels.

CLINICAL MANIFESTATIONS.-The spectrum of clinical features varies according to the type of thalassemia and chain insufficiency (Table 13). Insufficiency may result from the absence of chain production (that is, p”) or the partial deficiency of production (that is, j3’). The best way to understand this complex concept is to consult a major hematology text.1-3 The symptoms of the disease (for example, bone changes, splenomegaly, hepatomegaly) vary with severity. Most pa- tients are heterozygous and have minimal or moderate disease. A small number of patients may have thalassemia related to crossing over between gene sequences at the DNA level (for example, hemo- globin Lepore).

THALASSEMIA MINIM.-These are often described as silent-carrier thalassemias because they generally escape clinical detection. Al- though thalassemia minimae are microcytic and hypochromic, pa- tients may have normal hemoglobin levels.

THALASSEMIA INTERMEDIA.-This anemia is clinically apparent and symptomatic. Hepatosplenomegaly, cardiomegaly, and variable skel- etal changes are found, and iron storage problems occur. Hemoglo- bin H disease, a term used to describe a chain deletions that clini- cally resemble certain p chain disorders, is characterized by hemo- globin Bart’s (r) and hemoglobin H (p4), which are formed in the absence of the a chain. Hemoglobin Bart’s and hemoglobin H are dysfunctional hemoglobins and precipitate easily. After the first year

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TABLE 13. Clinical forms of thalassemia and genotype Phenotype Severity Genotype

Thalassemia minima

Thalassemia minor a-Thal-1 a-Thal-2 Hgb constant spring p Thalassemia trait Lepore trait r-p Thal

Thalassemia intermedia p Thalassemia cc-p Thalassemia p Thal major with high y(Hgb F) Thalassemia major (p)

Hgb H (p,) disease

Hydrops fetalis Absence of a chains

Microcytosis -alaa Hypochromia Silent/p Hgb may or may not be normal Microcytosis Hypochromia --laa Mild anemia -a/-a Hgb lo-14 gm/dl aaCs/aacS

p” or P+/p P/P Lepore p/p+ S’

Microcytosis Hypochromia p+Afr@+Afr Moderate anemia p/p’-al-a Hgb 6-10 gm/dl p/p with ry Microcytosis Lw/p Hgb 3-5 gm/dl +medlt,p+msdit

;‘Wl3 Skeletal distortion WP) lqmre (qpm Jaundice, frontal bossing, cranial enlargement, hepa- tosplenomegaly, cardiomegaly

Microcytosis --l-a, H Anisopoikilocytosis __ Iaa’, H Moderate to severe --/acsa, H

anemia --iaathat, H Severe anemia --/--, Bart’s (y,) Neonatal death No a chains

CS-Hemoglobin Constant Spring

hemoglobin Bart’s is found in very small quantities as a result of the decline in neonatal hemoglobin E

THALASSEMIA MAJOR.--This severe, life-threatening anemia appears in the first or second year of life. Without transfusion therapy, which itself induces iron storage problems, the patient may die or may suf- fer extreme skeletal changes, growth retardation, or failure of sexual maturation.

HEMOGLOBIN H DISEASE.-Hemoglobin H is frequently a double het- erozygote, resulting from the interaction of an a0 gene and either a deletion or non-deletion a+ thalassemia gene (-a+/aa). Less fre- quently, it may result from a%” (represented as --) and normal a genes (--/aa). Some patients with this disease are as severely affected as those with thalassemia major, and others are less affected. Ane- mia, variable splenomegaly, and variable bone changes are always present.

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TABLE 14. Thalassemia laboratory values contrasted with iron deficiency values Laboratorv value Thalassemia Iron deficiencv

Anemia Mean corpuscular volume (fl) Red blood cell morphology

Reticulocyte index Marrow E:G Serum iron/TIBC Percentage saturation Marrow iron Serum ferritin level (&L)

Variable severity Variable severity <70 ~80 Microcytic, hypochromic, Normal to microcytic- targeting hypochromic, pencil forms <2 <2 >2:1 1.1-1.3 Increased/Normal Very low/ t >50 110 7 0 >lOO 112

?, increased: E:G, erythrocyte-to-granulocyte ratio; TIBC, total iron-binding capacity.

TABLE 15. Hemoglobin levels in i3 thalassemia Condition Hb A HbF Hb A, Hb

Lenore

Normal 0.97 <O.Ol 0.02-0.03 0 j3 Thal minor P/p” or + 0.90-0.95 0.01-0.05 0.04-0.07 - p/pm-e 0.80 0.01-0.05 0.02-0.03 0.15 j3 Thal intermedia p+ Afr@+Afr 0.30-0.50 0.50-0.70 o-0.05 - j3 Thal major

Y +medit,+medit 0 0.10-0.20 0.95-1.00 0.70-0.80 o-o.5 o-o.5 - - ppX~,p*OE 0 0.80 0 0.20

HYDROPS FETALIS.-II1 hydrops fetalis, there is an absence of a chains. The fetus is stillborn or dies soon after birth.

LABORATORY STUDIES.-Thalassemia is characterized by microcytosis and hypochromia. The silent and intermediate variants have eryth- rocytes that are geometrically uniform and have normal RDW num- bers. The more severe forms are associated with a rise in RDW and clinically significant microcytosis and anisocytosis.

Marrow studies are rarely needed, except in cases of differentia- tion-form iron deficiency (Table 14). Ineffective erythropoiesis is noticeable; marrow hyperplasia occurs and the erythrocyte-to-granu- locyte ratio is abnormal at 1:l or 2:l. Concomitant findings include elevated bilirubin levels, reflecting hemolysis, and elevated lactate dehydrogenase (LDH) levels.

The patient’s hereditary background may be a guide to test selec- tion Patients whose background is uncomplicated, require quanti- tative measurements of hemoglobin A, AZ, R and H (Tables 15, 16). Hemoglobin electrophoresis may help identify hemoglobin variants. Cellulose acetate electrophoresis can be used to quantitate hemo- globin A,, and hemoglobin F can be measured with the acid hemoly-

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TABLE 16. Hemoglobin values with CI thalassemia Condition HbA HbH Hb Hb

(p”) Bart’s (x4) Constant Spring

Normal 0.97 0 0 0 CL Thal silent -alaa 0.98-1.00 0 0 0 a Thai trait Red blood 0 -al-a 0.85-0.95 cell 0 -4aa 0.85-0.95 inclusions 0 acP/aaCs 0.85-0.95 0 H disease --l-a 0.75-0.95 0.05-0.30 Trace 0 --IacP 0.60-0.90 5-30 Trace 5-10

Note: In hydrops fetalis, no hemoglobin A is found, hemoglobin H is 0.05-0.10, and hemoglobin Bart’s is 0.90-0.95.

sis test. Hemoglobin H can be quantitated with starch cell electro- phoresis. Patients with p thalassemia minor have a 4% to 7% eleva- tion in hemoglobin F and 6 hemoglobin levels (low iron may de- crease hemoglobin A, level). Patients with j3 thalassemia major and thalassemia intermedia have a marked increase in the hemoglobin F proportion.

The physician ‘must be aware that there may be double heterozy- gotes, particularly with the p chain. Some patients may be double heterozygotes for j3 Thal and S, C, or E variants of the j3 chain.

Genetic counseling is part of management of hemoglobinopathy. Prenatal diagnosis can be made by means of fetal blood or chorionic villous biopsy or direct fetal DNA analysis. For a fetus at risk for one of the severe forms of thalassemia, early diagnosis allows consider- ation of therapeutic abortion.

THERAPY.-htientS with minimal symptoms require no therapy. Those with moderate anemias and a MCV less than 70 fl are at risk for bone marrow expansion, splenomegaly, and cardiomegaly. These patients are also at risk for iron overload. The severe, life-threaten- ing thalassemia major is associated with striking anemia, and marked targeting. Death may occur from heart failure. Severe iron overload can occur even if transfusion is not performed. Splenectomy for hypersplenism may be indicated. Management of severe thalassemia is quite complex, relying on transfusion and chelation, and is best undertaken in consultation. Without careful treatment, the patient likely will not live beyond 20 years of age and will suffer from ana- tomic abnormalities, growth and sex retardation, and the complica- tions of iron overload.

Other Microcytic Anemias-Lead poisoning produces anemia through more than one mechanism. Decreased production of nor- mal heme is associated with lead-induced alterations of porphyrin molecule metabolism. As a result, there are striking increases in the

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TABLE 17. Macrocytosis Status Morphology Mean

COT uscular vo P ume (fl)

Normal Reticulocytosis Liver disease

Cytometric artifact

Megaloblastosis

Normocytic Polychromasia Uniform macrocyte Leptocytosis Agglutination Leukocytosis Macro-ovalocytes Poikilocytes

90 90-l 10 95-110

100-130

100-130

levels of 6 aminolevulinic acid (ALA), coproporphyrin, and free pro- toporphyrin molecules. Other postulated abnormalities include cell wall damage, resulting from lead interference with the cation pump, and failure of RNA depolymerization at the ribosomal level. The morphologic result is microcytic hypochromic cells with baso- philic stippling in the peripheral blood and ringed siberoblastosis in the marrow. Occupational and environmental contamination, espe- cially among inner-city children, are prime factors. Apart from the anemia, the varied presentation of lead poisoning’ may include at- tacks of abdominal pain, motor neuropathy, a lead line on the gums, renal damage, and neuropsychiatric abnormalities.

LABORATORY DIAGNOSIS.-urinary porphyrin evaluation will show in- creased levels of ALA. Stippled microcytic red blood cells are char- acteristic. Blood or urine lead levels indicate the severity of expo- sure and confirm the diagnosis.

THsr&&y.-Therapy is removal of the source of lead. The patient is given a slow intravenous infusion of ethylenediaminetetraacetic acid; EDTA), 50 mg/kg a day, in two divided doses, for 5 days. The patient also should receive ample amounts of fluids. The treatment is re- peated in 3 to 4 weeks depending on lead levels.

Anemias Secondary to Maturation Disorders These anemias result from impaired DNA synthesis, marrow

megaloblastosis, dysynchrony between nuclear and cytoplasmic maturation, ineffective erythropoiesis, and macrocytosis. The bone marrow maturation abnormalities affect all three cell lines. Vitamin B,, and folate deficiencies are the most common examples of this mechanism.

Vitamin B,, or folate deficiency results in deficient DNA synthesis through failure to convert deoxyuridylate to thymidylate. In food, vitamin B,, is bound to salivary binding protein until pancreatic proteases catalyze the binding to intrinsic factor, which binds it to mucosal cells of the ileum, where it is transferred from the gas-

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TABLE 18. Congenital defects: An infrequent cause of vitamin B,, defi- ciency

Intrinsic factor defects Transcobalamin II defects Imerslund disease Vitamin B,,-deficient diet Malabsorption

trointestinal tract to the circulation. Intrinsic factor, produced by gastric cells, is a crucial metabolic intermediary. In the general cir- culation, transcobalamin II is the principal transporter of vitamin B,, to the liver, where it is stored, and to the tissues.

Folate from dietary sources undergoes hydrolysis of dietary polyglutamate to methyltetrahydrofolate monoglutamate. This pro- cess requires the presence of mucosal cell carboxypeptidase and in- tracellular mucosal dihydrofolate reductase in the duodenum and jejunum. A functional enterohepatic folate cycle is necessary for folate hemostasis.

Folate supplies may be depleted by dietary insufficiency, blockage of folate metabolism, or interference with hepatic folate excretion or enteric uptake. Folate is particularly abundant in green leafy veg- etables, although the amount of folate may be much reduced by ex- cessive heating. Pregnancy, hemolysis, and the presence of rapidly growing tumors increase the need for folate. Persons with chronic alcoholism are at risk for dietary folate insufficiency (Table 17). Vi- tamin BJ2, unlike folate, is derived mostly from meat, and usually only persons who are strictly vegetarian experience dietary deficiency of vitamin B,,. Congenital defects that result in vitamin B,, deficiency are shown in Table 18.

Symptoms related to anemia, fatigue, exercise intolerance, and cardiac failure are not uniquely defining. Neurologic features may include paraesthesia of hands and feet, unsteadiness of gait, memory loss, and alterations in personality. Objective findings of vitamin B,, deficiency include distal loss of vibratory sense, loss of propriocep- tion, positive Romberg test, and unsteady gait. Neuropathic features may precede anemia. Mucosal changes range from soreness of the mouth to beefy, red tongue with absent papillae. Vitiligo and early graying may be found in j3,, deficiency. In the United States, the disorder is most frequently autoimmune in origin. It is derived from antibodies to gastric parietal cells, intrinsic factor, or the cobalamin- intrinsic factor complex. Other causes include gastrectomy, gastric bypass, intestinal resection, pancreatic insufficiency, and intestinal bacterial overgrowth. In patients with alcoholism, folate deficiency is often coupled with dietary insufficiency and may persist as low- grade anemia, which is easily reversed with a hospital diet.

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TABLE 19. Causes of folate deficiency

Poor intake Malabsorption Anticonvulsant drugs Hemolytic anemia Vitamin C deficiency Dialysis Sprue Tumors Psoriasis, exfoliation Pregnancy Alcohol Folate reductase inhibitors Methotrexate Sulfamethoxazole Trimethoprim

Folate and vitamin B.,, deficiencies are increasingly managed in the outpatient setting, m which bone marrow testing is not practi- cal. Initial laboratory results that include an MCV greater than 115 fl coupled with hypersegmented neutrophils provide a strong clue to the diagnosis.

Serum vitamin B,, levels and serum and red blood cell folate lev- els should be measured. The diagnosis should be confirmed with assessment of blood transcobalamin levels, serum antiparietal, and anti-intrinsic factor levels and a Schilling test (used to define the cause of vitamin B,, malabsorption). Serum and urine methylmalonate and homocystine levels (homocysteine increased in folate and vitamin B,, deficiencies, methylmalonate increased in vitamin B,, deficiency) may be helpful at times but are expensive.

Macrocytosis can be a result of reticulocytosis, liver disease, or an automated cytometric artifact (increased white blood cell count [WBC], increased red blood cell agglutinins, hyperglycemia) (Table 19). Megaloblastosis and a MCV of 100 to 130 fl are frequently caused by a lack of vitamin B,, or folate.

Although the means of replacing vitamin B,, are simple, the pro- cess may not be. The clinician must be aware of the following: 1. Vitamin B,, neuropathy is often irreversible if not treated in a

timely manner. 2. Vitamin B,, neuropathy may worsen irreversibly if treated inap-

propriately with folate only. Treatment with folate alone must . be avoided if the cause of the deficiency is not clear. 3. Potassium deficiency may occur, related to the exuberant

hyperplasia induced by folate or vitamin B,, replacement. 4. Evaluation for malabsorption can be concomitant with the ad-

ministration of vitamin B,, or folate. In patients with severe macrocytic anemia, urgent replacement is

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needed. Vitamin B 2, i

100 pg cyanocobalamin, and folic acid, 1 to 5 mg, intramuscular y should be given as replacement therapy. Pa- tients with established vitamin B, deficiency should be given cyano- cobalamin 100 pg intramuscular y for 10 days followed by 100 to I 1000 ug intramuscularly monthly thereafter.

Patients with established folate deficiency caused by pregnancy take folic acid 1 mg by mouth once a day. Those with established folate deficiency resulting from chronic hemolysis or dermatitis take 1 to 2 mg of folic acid a day. The dose may be as high as XI mg a day. For those with a dietary folate deficiency, the amount in most multi- vitamin preparations (1 mg) is adequate.

Sideroblastic Anemia and Myelodysplasia Sideroblastic anemia and myelodysplasia are uncommon. They

may transform to leukemia and may derive from stem cell matura- tion abnormalities. Sideroblastic anemias are characterized by ringed sideroblasts on iron stain and may occur with varying erythrocyte morphology. Myelodysplastic disorders are characterized by abnor- mal marrow precursor morphology and ineffective erythropoiesis. They comprise several different forms of leukemia and throm- bocytopenia. The key to diagnosis in this instance is review of the morphology of a marrow aspirate by a colleague. Growth abnormali- ties may include distorted mature forms, an abnormal increase in precursor forms, and evidence of ineffective erythropoiesis. Although indium scans can be performed to determine the extent of the bone marrow increase, these scans are usually not cost effective, except in experimental situations.

The clinical presentation of sideroblastic anemia often relates to the patient’s age. Young patients may experience fatigue, and eld- erly patients may experience congestive heart failure. At times, the clinical effects of thrombocytopenia may be striking (petechiae, purpura). The onset is insidious, and not infrequently, the diagnosis is suggested in a peripheral smear morphology report, which is ab- normal. A painstaking search must be made for the inciting agents. These may include lead, alcohol, chloramphenicol, isoniazid, radia- tion, autotoxic drugs, and toxic chemicals. Chromosome analysis may be helpful in making the diagnosis. Performance of the sugar- water test of Hartman or the Ham acid hemolysis test, often used in the paroxysmal nocturnal hemoglobinuria variant, may help eluci- date the nature and severity of the disorder.

Bone marrow erythrocyte evaluation is productive. Siderotic (iron- containing) granules are normally found in the cytoplasm. In sideroblastic disorders, the mitochondria are arranged in a perinuclear design and become filled with ferruginous micelles be- tween the mitochondrial cristae. This produces the ringed sideroblast identified with Prussian blue staining when mitochondrial iron over-

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load is present. No single defect in heme synthesis has been found for this group of disorders. Sideroblastic anemias may be idiopathic, secondary, or hereditary, in which case the patterns may be X-linked, autosomal, or mitochondrial. All presentations are characterized by ineffective erythropoiesis and, in most cases, splenomegaly.

Most patients with hereditary forms of sideroblastic anemias re- spond to high oral doses of pyridoxine (50 to 200 mg a day). How- ever, in such patients, mild anemia remains and worsens with discon- tinuation of the medication. Iron overload may be a cause of death. Cell kinetics in this disorder dictate the use of chelation and desferrioxamine to remove iron.

Myelodysplastic Syndromes.-Myelodysplastic syndromes include a diverse spectrum of rare disorders, which are variously classified. A very terse description follows. For these rare disorders, consulta- tion with a specialist is in the patient’s best interest.

REFRACTORY ANEMIA.-This anemia has no apparent cause, and the marrow is hyperplastic. Bone marrow precursor maturation, mar- row hyperplasia, red blood cell macrocytosis, and other variants are found. Point mutations of ras oncogenes are infrequent.

Refractory Anemia with Ringed Sideroblasts. These anemias may be considered a subset of the sideroblastic anemias. Refractory ane- mia with ringed sideroblasts is seen chiefly in older men and is of long duration. Marrow is often megaloblastoid and hyperplastic, with lobulated nuclei of red blood cell precursors. Patients are at risk for severe iron overload. This disorder must be differentiated from other presentations of sideroblastosis.

Refractory Anemia with Excess Blasts. The granulocyte cell line has considerable involvement in this disorder. Peripheral smears demonstrate a low percentage of myeloblasts and bilobed granulo- cytes (pseudo-Pelger-Huet cells). Monocyte precursors may be in- creased. One variant with less than 5% blasts in the peripheral blood and less than 20% blasts in the marrow has been recognized; addi- tional features are suggestive of acute myelogenous leukemia. Mul- tiple chromosomal abnormalities may be found and are associated with a poor prognosis.

CHRONIC MYELOMONOCXTIC LEUKEMIA.-In chronic myelomonocytic leukemia (CML), the blood morphology resembles that of refractory anemia with excessive blasts, but there is an increase in monocyte precursors. The blast count of marrow is less than 20%.

CONGENITAL DYSERYTHROPOIETIC ANEMIAS.-Congenital dysery- thropoietic anemias tend to occur in childhood and may be mild. The only clue may be macrocytosis with anisopoikilocytosis. Dis- tinctive abnormal morphology of the bone marrow is diagnostic. Presentations of this anemia are shown in Table 20. Chromosome abnormalities are rare, but iron overload can be a serious problem.

THERAPY.-Refractory anemia and refractory anemia with ringed

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TABLE 20. Congenital dyserythropoietic anemias (WA) Class MCV Ifll Marrow

CDA I 95-110

CDA II hempas 90

CDA III go-100

CDA IV 80-90

Megaloblastic Binucleated erythroblasts Normoblastic Binucleated erythroblasts (acid hemolysis present)

Multinucleated Gigantic erythroblasts Normoblastic Binucleated erythroblasts

(no acid hemolysis)

sideroblasts evolve slowly, and 10% of cases may become leukemia. Patients with refractory anemia with excess blast forms have short- ened survival times and a 50% rate of leukemia transformation. Pa- tients with deletions of long arms of chromosomes 5, 7, 20, or 8 trisomy have a worse prognosis. The c-ferns mutation on chromo- some 5 may result in disruption of growth factor or growth factor receptor.

Although blood transfusions may be required by patients with he- moglobin levels less than 7 gm/dl, transfusions should be given judi- ciously in view of the resultant iron overload and risk of associated human leukocyte (HLA) antibody formation. Platelet transfusions should be given only for hemorrhage and as surgical preparation. Pa- tients may tolerate very low platelet counts without bleeding.

In general, vitamin therapies are not effective. An exception is pyridoxine therapy for the sideroblastic forms of anemia. Iron is to be avoided. Androgens, steroids, and immunosuppressive medica- tions are not effective, and cytometric therapy has limited efficiency. Leukemic therapy with three-drug regimens is not tolerated by eld- erly persons and is variably successful in others. Overall survival is not enhanced with use of low-dose cytarabine or cis-retinoic acid, although transient responses have been noted with each drug.

Sporadic responses with erythropoietin have been noted in pa- tients with low erythropoietin levels (150 to 300 &kg subcutane- ously per week). The colony-stimulating factors (G-C% GM-CSF) have been used successfully in granulopenic states. It is not known whether long-term administration of colony-stimulating factors will enhance leukemic transformation.

Anemias of Increased Erythrocyte Destruction The two main components of this classification, hemolysis and

acute hemorrhage, result in a threefold increase in the reticulocyte index, reflecting a marrow with an erythrocyte-to-granulocyte ratio greater than 1:l. Erythropoietin is greatly increased in response to

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TABLE 21. Laboratory values that indicate hemolysis Value Intravascular Extravascular

Reticulocyte index > 3 J J t LDH J J TIndirect bilirubin J J I Haptoglobin J J Methemalbumin 0 J Free hemoglobin 0 J Urinary hemosiderin 0 J ?, increased: 1, decreased: J. present; 0, not present.

these anemias. It manifests itself in polychromatophilic erythrocytes, often with increased diameters, in the peripheral blood.

The following are the important causes of hemolysis. They set the stage for the selection of appropriate tests. Laboratory values that indicate extravascular and intravascular hemolysis are shown in Table 21.

Iktravascular HemoIysis.-Monocytes and macrophages in the spleen and other organs release carbon monoxide, unconjugated bilirubin, and iron from hemoglobin, which becomes bound to transferrin.

Intravascular Hemolysis.-Erythrocytes free in the plasma lyse, with the following effect: Dissociated hemoglobin dimers bind to haptoglobin and are removed by the liver, or hemoglobin may be oxidized to methemoglobin, later binding with albumin or hemopexin. This ultimately results in recovery of iron and bilirubin by the liver.

Severe Hemolysis.-When the hemoglobin clearance mechanisms are exceeded, free hemoglobin becomes present in plasma. It is then excreted in the urine, and methemalbumin levels rise. Hemo- siderinuria (siderotic renal tabular granules) may persist for several dhys after hemolysis subsides.

Confounding Features of These Anemias.-Confounding features of anemias with increased erythrocyte destruction include iron de- ficiency (decreased MCV), folate deficiency (increased MCV), very high reticulocyte counts (increased MCV), and an increased indi- rect bilirubin level (Gilbert’s disease, Crigler-Najjar syndrome).

Hemoglobinopathies.- In most instances, these anemias result from a single amino acid substitution in the globin chain, with resultant cellular structural changes and enhanced erythrocyte destruction. Hemoglobinopathies occur with highest frequency in Africa, Asia, the Indian subcontinent, and in areas of the Mediterranean. Treat- ment of patients with a hemoglobinopathy depends on accurate di- agnosis and knowledge of the related clinical manifestations. Often, the heterozygous forms of the condition have few manifestations. Genetic counseling should be considered.

LABORATORY STUDIES.--Red blood cell morphology of the peripheral

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TABLE 22. Common hemoglobinopathies Severe Mild Minimal

S/S (disease) SIC SifPThal’ S/E

S@+Thal’ CIC SiLepore’ SD S/O Arab

AIS AK+ ND’

*Thalassemic syndromes. tOften described as a trait.

smear should be evaluated. Screening tests for sickling, including the sodium metabisulfate test and the hemoglobin solubility test (sickle dex), should be conducted. Hemoglobin electrophoresis should be performed by means of the cellulose acetate method.

COMMON HEMOGLOBINOPATHIES AND SICKLE CELL DIsiWsE.--In the case of frequently occurring hemoglobinopathies (that is, S, C, D, and E), recognizable, severe disease manifests itself in the homozygous form or the double heterozygous form (Table 22). Sickle cell disease, de- scribed here only briefly, is the most frequently occurring example of hemoglobinopathy.

Homozygous sickle disease (S/S) occurs in childhood with severe hemolysis and vaso-occlusive involvement of marrow, the central nervous system, the kidneys, and the spleen. Hemolytic crises are accompanied by extreme pain, which may become severely disabling. Loss of splenic function and recurrent infarction of bone and mar- row engender infections with Streptococcus pneumoniae, Salmonella species, and Haemophilus infhenzae. Renal concentrating ability falters; hematuria and renal failure can occur. Sepsis and central nervous system infarction also can occur. In adults with sickle cell disease, chronic pulmonary vascular stasis can result in pulmonary hypertension and fatal cardiac failure. Long-term transfusion can result in disabling hemosiderosis.

Double heterozygous disease may occur with patterns similar to but less severe than those of the homozygous form. For example, sickling occurs with splenomegaly but not with infarct-induced asplenia. The distribution of hemoglobin F may, when increased, favorably modify the pattern of severity.

Comprehensive management of sickle cell disease must be re- viewed in detail. The following is intended to be an overview. Het- erozygotes should undergo screening tests. Counseling is strongly recommended and should include the prenatal fetal blood and tis- sue digenetic techniques. From their child’s infancy, the family must understand the potential complications and management of this dis- ease. Pain must be controlled during crises, and a sudden fall in hemoglobin must be evaluated closely. Acute infection, priapism, pigmented gallstones, osteomyelitis, and infections with encapsu-

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TABLE 23. Hereditary membrane defect anemias Osmotf~sffagility

Anemia

Hereditary spherocytosis -I-++ Hereditary elliptocytosis Variable Stomatocytic hereditary elliptocytosis Variable Hereditary pyropoikilocytosis +++

Thermal instability

Variable +++

lated bacteria are constant health threats. The patient may have con- siderable psychological needs.

Transfusion therapy of as much as one-third of the red blood cell mass will reduce crises but increases the risk of antibody formation and development of hemosiderosis, which may require prolonged chelation therapy with desferrioxamine.

Hydroxyurea, a chemotherapeutic drug, can induce the produc- tion of cells with a higher proportion of hemoglobin F and therefore a lower proportion of hemoglobin S. Hydroxyurea must be given in daily doses, but the amount of the dose should not be enough to induce leukopenia or thrombocythemia. The number of crises will diminish in response to elevation of hemoglobin F level.

UNSTABLE HEMOGLOBINS .-Certain globin mutations yield hemoglo- bins that are unstable, precipitate easily, and when stained, produce Heinz bodies. Some mutations result in hemoglobins that easily trans- form to methemoglobin, in which solubility is decreased. Hemolysis results from loss of soluble hemoglobin, The incidence of these disor- ders is quite low, and inheritance is frequently autosomal dominant. The anemia is frequently well compensated, with a slightly lowered hemoglobin level and a high reticulocyte count. MCV may vary, and hemolysis may be increased by exposure to oxidant medications.

Basophilic stippling and Heinz bodies may be seen with Giemsa stain. Heat stability and isopropanol tests should be performed. Eryth- rocyte stains with brilliant cresyl blue and methyl violet may help identify inclusions from denatured hemoglobin. Hemoglobin elec- trophoresis is often definitive, and globin chain purification, pep- tide analysis, and gene sequencing may be necessary.

Patients with unstable hemoglobinopathy may benefit from the administration of 1 to 2 mg of folate a day. Exposure to oxidant drugs and febrile illness may accelerate hemolysis, and infection with vi- ruses, particularly parvovirus, may induce pure red blood cell aplasia.

Hereditary Membrane Defect Anemias-This group of disorders is characterized by membrane abnormalities and distinct morphologic features, often with a specific hereditary pattern.

HEREDITARY SP~Rocy’rosrs.-Hereditary spherocytosis is common. Hemolysis is of varying severity, osmotic fragility is increased, and the response to splenectomy results in a decrease in gallstone for-

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TABLE 24. Clinical characteristics of autoimmune hemolytic anemia (AIHA) Clinical finding Cold AIHA Warm AIHA

Onset Insidious Abrupt JaQn&Ge Frequent Less frequent Splenomegaly Absent Present Related diseases Mycoplasma Systemic lupus

Histiocytic lymphoma erythematosus Chronic lymphocytic leukemia Lymphoma

mation and a decrease in hemolytic response to illness. Spherocytosis is a response to a variety of membrane structure abnormalities re- lated to genetic miscoding of spectrin, ankyrin, and certain other erythrocyte membrane components. The osmotic fragility test is the definitive laboratory study.

HERFII~ARY Emocrrosrs.-This name designates a diverse group of disorders of varying genetic origin and inheritance. There are spherocytic- elliptocytic variants and stomatocytic hereditary elliptocytosis (Melanesian or Southeast Asian ovalocytosis), in which there is a longi- tudinal ridge. Diagnosis is usually established with a family history of elliptocytosis and the appropriate morphologic picture.

HEREDITARY P~oPoIkILocYrosrs.-This disorder demonstrates severe hemolysis with thermal instability and an a spectrin abnormality. Acquired stomatocytosis may be seen in patients with neoplasia and cardiovascular diseases. Acanthocytosis-speculated erythrocytes are seen with hereditary a-P lipoproteinemia, acquired severe liver dis- ease, and other disorders.

Laboratory features of this anemia include marked fragmentation and poikilocytosis, a positive heat denaturation test of 45” to 46” C. The abnormalities may overlap with the hereditary elliptocytosis anemias (Table 23).

Metabolic Intracellular Defects. Metabolic intracellular defects oc- cur with variable degrees of inheritance and involve abnormalities of the hexose-monophosphate shunt on the Embden-Meyerhof path- way. They are inherited and normocytic and are worsened by expo- sure to oxidant drugs. Mediterranean forms of glucose-6-phosphate dehydrogenase (GGPD) deficiency and severe y-glutamylcyste- inylglycine (GSH) deficiency can occur as somewhat severe chronic hemocytic anemias. Pyruvate kinase (PK) and hexokinase deficiency may be readily detectable as well.

Laboratory Studies. Osmotic fragility and autohemolysis tests are positive, but morphologic features are not distinctive. Specialized laboratory enzyme analysis is required.

Autoimmune Hemolytic Anemias.-Autoimmune hemolytic ane- mias are caused by self-induced antibodies to erythrocyte surface antigens. Ninety percent of cases of autoimmune hemolytic anemia

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TABLE 25. Laboratory studies in autoimmune hemolytic anemia (AIHA) Test Cold AIHA Warm AIHA

Direct Coombs’ test Monospecific sera

Anti IgG Anti IgG+ C’ Anti C’

Serum antibody Specificity Technique

Cold agglutinin titer Serum complement Osmotic fragility Thermal range Red blood cell morphology

2-4 + 2-4-k

0 1+ 0 1+ 1+ Rare I@ I& Anti I/i, Pr(sp)I’ Anti-e, C, c, LW, U, Wright Saline, enzyme Indirect antibodies, enzymes >256 Normal Decreased May be normal Normal Increased Positive at 4°C Positive at 37°C Normal Microspherocyte

(AIHA) are related to another disease. The characteristic clinical fea- tures of these anemias are outlined in the following section and in Table 24.

COLD AIHA.-Cold AIHA is frequently associated with an IgM an- tibody that initiates the entire complement cascade and results in osmotic lysis.

WARM AIHA.-Warm AIHA is initiated by an IgG antibody and sometimes is initiated by complement. Piecemeal destruction of red blood cells occurs when macrophage receptors in the spleen attach to the IgG Fc fragment. IgGl and IgG3, when combined with comple- ment absorption, may cause considerable red blood cell destruction. Destruction by complement alone is negligible.

PAROXYSMAL COLD HnMocLosINuRIA.-Paroxysmal cold hemoglobinuria erythrocytes, when incubated in the presence of complement, are ly- sed by the action of a thermally active antibody, which attaches to cells at 4°C and induces lysis when cells are at 37°C. In addition to this biphasic reaction, the antibody is auto-P specific. The Donath- Landsteiner test is used to define the presence of this anemia.

LABORATORY Srun~~s IN AIHA.-Table 25 lists the clinical features of specific laboratory tests for ARIA. Drug-induced autoimmune hemoly- sis can be defined by the following three mechanisms (Table 26): 1. Hapten or drug absorption. The offending drug (for example, peni-

cillin) induces an antibody against itself and, having a high affin- ity for erythrocytes, attracts the antibody, causing splenic seques- tration and hemolysis.

2. Ternary complex formation. The instigating drug (for example, quinidine) induces an antibody that recognizes drug and mem- brane-drug binding site together (so-called innocent bystander reaction).

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TABL

E 26

. Me

chan

isms

of dr

ug-in

duce

d he

molys

is

Penic

illin

Drug

Qu

inidin

e a-

Methy

ldopa

Mech

anism

Ha

pten

Antib

ody

to dr

ug

Pres

ent

Antib

ody

class

I&

Pr

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de

tected

wi

th

direc

t an

tiglob

ulin

test

IgG,

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TABLE 27. Signs and symptoms of acute blood loss in adults PefcE-rtage Volume

0 (ml) Symptoms Signs

<20 <lOOO Anxiety May or may not have vasovagal symptoms

~20-30 1000-2000 Anxiety, exercise Orthostatic hypotension and tachycardia intolerance, with exertion orthostatic syncope

30-40 1500-2000 Syncope with sitting Orthostatic hypotension, rest or standing tachycardia

140 2000 Anxiety, restlessness, Shock, fall in supine blood pressure, confusion tachvcardia. cool diauhoresis

3. Autoantibody. The drug (for example, a-methyldopa) induces an antibody that reacts to cellular antigen.

A fourth autoimmune mechanism occurs when the drug (for ex- ample, cephalosporin) induces erythrocyte membrane- changes that allow various plasma proteins to attach to membrane surfaces. A positive immune test may result, but hemolysis is not found. This mechanism complicates blood cross matching.

LABORATORY TESTS FOR AUTOIMMUNE HEMoLYsrs.-The direct antiglobu- lin test is used to detect autoimmune hemolysis. Therapy for autoim- mune hemolysis is discontinuation of the offending medication,

HEMOLYSIS SECONDARY TO MILIEU.-In hemolysis secondary to milieu, mechanical fragmentation of erythrocytes may occur with mechani- cal heart valves or as a result of the presence of fibrin strands in DIC, thrombotic thrombocytopenic purpura (TTP), or the hemocytic uremic (Evans) syndrome.

Blood Loss Anemias.-These anemias may be viewed as acute or chronic. In acute blood loss anemias, hemorrhage may induce car- diovascular failure and death, although patients have nearly nor- mal hemoglobin levels. A volume loss of two to three units of blood (1000 to 1500 ml) can require 20 to 60 hours for volume restoration. Hemoglobin levels are not reliable guides to blood loss in acute situ- ations. Other signs and symptoms are more reliable (Table 27). The bone marrow response is characterized by the presence of young hematopoietic cell forms in the peripheral blood, including nucle- ated erythrocytes if the bleeding is severe.

Acute blood loss should be managed as follows: 1. Application of military antishock trousers (MAST) to induce a

500 to 1000 ml autotransfusion 2. Administration of crystalloids as first line of defense, or another

volume expanders if necessary 3. Red blood cell transfusion 4. Administration of fresh frozen plasma if associated coagulation

defects are present

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TABLE 28. Commonlv used leukocvte reference values for adults White blood cell count 4500-10,100/mm3 (4.5-10.1 x log/L) Differential Relative I%) Absolute (cells/mm31

Neutrophils 50-70 2400-7560 Bands 2-6 96-248 Lymphocytes 20-44 960-4752 Monocytes 2-9 96-972 Eosinophils o-4 0432 Basophils o-2 O-216

The presentation of chronic blood loss is variable, but it is gener- ally characterized by iron deficiency. Effective cessation of the ane- mia results from identification and elimination of the bleeding site and adequate iron replacement.

Chronic blood loss should be managed as follows: 1. Identification of the bleeding site and treatment if possible 2. Iron replacement (see Iron Deficiency Anemia). 3. Avoidance of transfusion unless physiologically necessary

LEUKOCYTE DISORDERS

To make an effective diagnosis in problems deriving from leuko- cyte abnormalities, the clinician must use basic laboratory tests ef- fectively, know the role of leukocyte subpopulations and their iden- tifiers, understand the differential diagnosis of these disorders, and order the most appropriate second-level studies.

THE BASIC LEUKOCYIE REPORT

The value of the leukocyte count and differential diagnosis is en- hanced with careful analysis of the basic report. In this section, the normal report for adults is reviewed. The application of absolute values is emphasized (Table 28).

Absolute values, if not printed on the computer report from the laboratory, can be easily calculated by multiplying the leukocyte count (WBC) by the percentage of a given cell type, as follows.

Example: Laboratory report: WBC = 4500/mm3 (4.5 x log/L); lymphocyte

percentage is 20 Calculation: 4500 X .20 = 900/mm3 The importance of this calculation becomes readily if one notes

that although the reported 20% is “normal,” the absolute example, 900/mm3, is lower than the normal range (960 to 4752/mm3). The patient has lymphopenia that could have been missed. In the post- operative treatment of an elderly patient, this lymphocyte count may

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be associated with progressive malnutrition, with an associated fall in T lymphocyte numbers, and a predisposition to infection. This technique is useful in the treatment of patients with AIDS in its various stages. It is cost effective to remember the absolute range for neutrophils (2400 to 7000/mm3 [2.0 to 7.0 x log/L]) and for lympho- cytes (950 to 4000/mm3 [9.5 x log/L]). This same technique can be applied to other leukocyte subpopulations.

Exceptions to the commonly used or “normal” leukocyte report are worth noting. African-American adults have a slightly lower leu- kocyte count (3600 to 10,000/mm3 [3.6 to 100 x log/L]) and therefore a lower neutrophil count (1300 to 7400 mm3 [1.3 to. 7.4 x log/L]). Some studies show a slight difference between white North Ameri- cans and western Europeans. If there is a question about borderline values, watching the patient for a time and repeating the measure- ment may allow for variations of minimal importance.

IDENTZFYING FEATURES OF LEUKOCYTE CELL POPULATIONS

This section contains a summary of the physiologic role of each cell population and a description of the identifying features. Detec- tion of immunologic markers by use of cell cytometry with fluores- cent tagged antibodies is being used increasingly to identify discrete diagnoses.

Leukocytosis Leukocytosis refers to elevation of the total leukocyte count above

10,000/mm3 [lo0 x log/L]. Often, one specific cell line demonstrates an elevation above the normal range, resulting in categorization of the finding nonspecifically as leukocytosis. Although somewhat awkward, -OS& and -philia are used to indicate absolute elevations of cell subpopulations (for example, neutrophilia, eosinophilia, monocytosis, lymphomatosis). The -emia ending was used to des- ignate disorders that had an unknown cause and that were malig- nant or paraneoplastic (for example, lymphocytic leukemia, polycythemia [rubra Vera]). A third ending, -emoid, as in leukemoid reaction, is ambiguous and is used variously to describe a leuko- cyte count greater than 50,000/mm3 [SO0 x log/L], or hematologic findings that resemble those of leukemia but that may be a specific response to infection, nonhematologic malignant disease, hemor- rhage, or hemolysis.

Myeloid Cell Function Myeloid cells include the granulocytes (neutrophils, eosinophils,

basophils) and the monocyte-macrophage series. They are derived from a common precursor colony-forming cell. Myeloid cells per- form diapedesis from circulation into tissues at the site of infection

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or inflammation; they perform phagocytosis and degradation of in- gested particles; and they accomplish exocytosis of granules that contain elliptocytic enzymes, chemotactic factors, activators of the coagulation and fibrinolytic system, and substances that affect vas- cular permeability.

Lysozyme (muramidase), esterases, cathepsin G, and proteinase are used to identify the pathogen degradative role. Peroxidase is the basis of the oxidant role in the destruction of pathogens. The pres- ence of elastase and collagenase suggests the role in tissue degrada- tion.

Identifying features of myeloid cells are immunologic markers CD13, CDllb, and CD16. Morphologically, myeloid cells are 1.2 to 16 urn, twice the size of red blood cells, with a segmented bilobed or trilobed nucleus. Azurophilic granules in cytoplasm contain myeloperoxidase. Specific granules are smaller and contain lactoferrin, lysozyme, vitamin B,, binding proteins, and other sub- stances.

Increased numbers of lobes occur with vitamin B and folic acid deficiency. Cells are often quite large (macrocyt;:). Pelger-Huet anomaly is characterized by dumbbell- or spectacle-shaped nuclei with reduced numbers of lobes. A hereditary form is benign. An acquired variant of Pelger-Huet anomaly is seen with a variety of infections and neoplastic disorders and as a reaction to drug toxic- ity. Alder-Reilly anomaly is characterized by the presence of larger than normal azurophil granules and may be confused with a toxic state. Alder-Reilly anomaly may be seen with Hurler and Hunter syndromes and polydystrophic dwarfism. Toxic granulation is seen with infections and drug reactions.

The function of eosinophils in myeloid cells is to kill parasites by means of phagosome formation or exocytosis. Eosinophils also have a role in immediate-type medication hypersensitivity reactions. When eosinophils are present in excessive numbers, granule release in- duces tissue damage and fibrosis. With most stains, the granules are reddish and abundant. The nucleus is bilobed.

Basophils have an important role in immediate hypersensitivity reactions. Granules contain proteoglycans and histamine. Basophils have a high affinity for Fc receptors of IgE. Basophils may release leukotrienes, but, unlike mast cells, they do not release heparin. Excessive granule release induces urticaria, rhinitis, asthma, and anaphylaxis.

Monocfles are similar to neutrophils. In addition, they process and present antigens to T lymphocytes on a surface loosely bound to HLA modules. Monocytes are effective at killing intracellular patho- gens (that is, Listeria monocytogenes, mycobacteria, yeasts), and they kill tumor cells where appropriately activated. Monocytes are a source of plasminogen activator.

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TABLE 29. Common causes of absolute neutrophil count increase

Acute Physical stimuli: Heat, cold, exercise, convulsion, pain, labor, anesthesia, surgery Emotional stimuli: Rage, stress,, panic Infection: Bacterial, mycotic, rickettsial, spirochetes, some viruses Inflammation or tissue necrosis: Burns, electrical trauma, infarction, gout, vasculitis, anti g-aby complexes, complement activation

Drugs, hormones, toxins: Epinephrine, etiocholanolone, endotoxin, glucocorticoids, venoms, vaccines, colony-stimulating factors

Chronic Persistent infection Inflammation: Rheumatic fever, rheumatoid arthritis, gout, chronic vasculitis, myositis, colitis, pancreatitis, dermatitis, thyroiditis, drug sensitivity, periodontitis, Sweet’s syndrome

Tumors: Gastric, bronchogenic, heart, renal, hepatic, pancreatic, uterine, squamous cell Drugs: Hormones, toxins, lithium, glucocorticoids Metabolic and endocrinologic: Eclampsia, thyroid stem, corticotropin (ACTH), or glucocorticoid excess

Hematologic disorders: Rebound from a granulopenia, therapy for megaloblastic anemia, chronic hemolysis or hemorrhage, asplenia, myeloproliferative disorders, chronic idiopathic leukophilia

Hereditary and congenital causes: Down syndrome, congenital neutrophilia

The monocyte nucleus is often obscured by numerous large, dark granules, but it is bilobed or trilobed. Immunologic markers are HLA- DR, CD13, CD33, CD14, CDllb, and CD in small amounts. Mono- cytes are larger than neutrophils, with an irregular cell border and a nucleus that is ovoid to horseshoe shaped. Chromatin is less dense than that in neutrophils. Staining often reveals tiny rose-red gran- ules in the grayish blue cytoplasm. Monocytes stain strongly for nonspecific esterase and less strongly for peroxidase and acid phos- phatase. Muramidase (lysozyme) is present in large amounts. CD4, which is present in minimal amounts on the monocyte surface, is a target for HIV.

Macrophages may be mononucleated or, less frequently, polynucleated. They represent a differentiated monocyte located in tissues. The function of macrophages is very similar to that of mono- cytes. Surface antigens are similar to those of monocytes. Macroph- ages are larger than monocytes and contain more granules than monocytes. Macrophages show morphologic variations related to both location and function. The surface of the macrophage has a broad variety of receptors.

ABNORiULXi7ES OF LEUKOCYTES

Neutrophils Neutrophilio-Table 29 demonstrates the importance of careful

history taking and physical examination to help in removing the

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TABLE 30. Causes of neutropenia

Infection Certain bacteria, typhoid, salmonella, Pasteurella tularensis Debilitated condition: Oirei%vhehning sepsis in-patients with malnutrition or alcoholism Rickettsial infection: In certain rickettsial infections, such as typhus and Rocky Mountain spotted fever, neutropenia may occur during the first week

Protozoa1 infections: Neutropenia may accompany this type of infection, with increasing parasitemia in malaria and kala-azar

Immune Disorders Isoimmune disease: May be transient in infants born to mothers with autoimmune disease, hypertension, or drug ingestion

Lupus erythematosus and rheumatoid arthritis with Felty syndrome Hypersplenism: Seen with abnormal disorders but also seen with lymphoma, myelopro- liferative disease, and liver disease with portal hypertension

Deficiencies Vitamin B,,, folate, copper Dl-llgs Analgesics, antibiotics, antihistamines (HZ blockers), anticonvulsants, antidepressants, anti-inflammatories, antimalarials, antithyroid drugs, cardiovascular drugs, diuretics, hypoglycemic agents, hypnotics, and phenothiazine Other: Penicillamine, ticlopidine, levamisole

patient from neutrophilic stimuli or in determining whether a spe- cific test or a limited number of tests will be useful. Certain disor- ders, such as vasculitis, may be difficult to diagnose, and early pan- creatic carcinoma or apathetic hyperthyroidism may be elusive ini- tially. Bone marrow examination is rarely, if ever, helpful in the di- agnosis of neutrophilia. Because neutrophilia, unlike leukemia, is secondary to other conditions, identification and removal of the pri- mary cause is therapeutic.

Neutropenia.-The term agranulocytosis historically was used to describe the sudden reduction or absence of granulocytes from the peripheral blood, coupled with severe sore throat, sepsis, and death. It was initially associated with aminopyrine administration; how- ever, lately it has been reported with administration of chloram- phenicol, indomethacin, and phenylbutazone. Agranulocytosis may be viewed as an acute variant of neutropenia. The effects of neutropenia are often not noted until the absolute count falls to less than 5OO/cu mm [0.5 x log/L]. Mechanisms of neutropenia include decreased production, increased destruction, or increased utiliza- tion of granulocytes (Table 30).

Toxic exposure, particularly from medication, is an increasing prob- lem. Table 31 provides a list of medications associated with neutropenia. Those associated with the highest frequency of neutropenia are marked with an asterisk. The list should not be con- sidered definitive.

Patients with chronic neutropenia should undergo antinuclear an-

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TABLE 31. Classification of widely used drugs associated with idiosyncratic neutropenia

Analgesic and anti-inflammatory agents Indomethacin* Gold salts Pentazocine Para-aminophenol derivatives*

Acetaminophen Phenacetin (acetophenetidin)

Pyrazolon derivatives* Aminopyrine Dipyrone Oxyphenbutazone Phenylbutazone

Antibiotics Cephalosporins Chloramphenicol* Clindamycin Gentamycin Isoniazid Para-aminosalicylic acid Penicillins and semisynthetic penicillins* Rifampin Streptomycin Sulfonamides* Tetracyclines Trimethoprim-sulfamethoxazole Vancomycin

Anticonvulsants Carbamazepine Mephenytoin Phenytoin

Antidepressants Amitriptyline Amoxapine Desipramine Doxepin Imipramine

Antihistamines-HZ blockers Cimetidine Ranitidine

Antimalarials Amodiaquine Chloroquine Dapsone Pyrimethamme Quinine

Antithyroid drugs Carbimazole* Methimazole* Propylthiouracil*

continued on next page

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TABLE 31. Continued

Cardiovascular drugs Captopril Disopyramide Hydralazine Methyldopa Procainamide Propranolol Quinidine Tocainide Diuretics Acetazolamide Chlorthalidone Chlorothiazine Ethacrynic acid Hydrochlorothiazide Hypoglycemic agents Chlorpropamide Tolbutamide Hypnotics and sedatives Chlordiazepoxide and other benzodiazepines Meprobamate Phenothiazines Chlorpromazine* Phenothiazine (thiodiphenylamine) Other drugs Allopurinol Clozaril (clozapine) Levamisole Penicillamine Ticlopidine * Most frequently reported.

tibody, rheumatoid factor, and other studies for collagen vascular dis- ease. A history of neutropenia suggesting a cyclical phenomenon is important. Examination of bone marrow is of particular importance to rule out infection, myelodysplastic syndromes, and “aleukemic” (with minimal peripheral blood manifestations) leukemia.

For a patient with neutropenia of acute onset, signs of fever, sore throat, or evidence of inflammation, the following sequence of treat- ment is indicated. For patients with chronic neutropenia, the evalu- ation may be similar. I. Prompt antimicrobial culture 2. Administration of intravenous fluids, broad-spectrum coverage

against gram-positive, gram-negative, and associated organisms 3. History-taking with attention to exposure to drugs, insecticides,

and pesticides 4. History-taking with attention to problems with the lymph nodes

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TABLE 32. Causes of eosinophilia

Infection Protozoans, amebae Toxoplasma gondii Metazoa Nematodes, trematodes, cestodes Sarcoptes scabiei (scabies) Allergic disorders Asthma Vasculitides Urticaria Drug reactions Multiple drugs Hypereosinophilia Hypereosinophilic syndromes Loeffler’s syndrome Interleukin-2 therapy Dermatologic disorders Eczema Psoriasis Dermatitis herpetiformis Pemphigus Gastrointestinal disorders Ulcerative colitis Regional enteritis Eosinophilic enteritis Neoplasia Myeloproliferative disorders Carcinomatosis Eosinophilic granuloma Hodgkin’s disease, lymphocytic neoplasia Musculoskeletal disorders Rheumatoid arthritis Fasciitis

5. Blood and bone marrow studies 6. Possible short-term support with granulocyte transfusion 7. Possible treatment with colony-stimulating factors (this therapy

is considered investigational)

Eosinophils Eosinophilia.-Eosinophilia is often described as mild, 400 to 1500

cells/mm3 (0.4 to 1.5 x log/L); moderate, 1500 to 5000 cells/mm3 (1.5 to 5.0 x log/L); or severe, more than 5000 cells/mm3 (5.0 x log/L). Severe eosinophilia can cause organ damage, and irreversible endomyocardial fibrosis can occur with prolonged, severe eosinophilia. On a worldwide basis, the most common cause of eosinophilia is infestation with helminthic parasites. In industrial-

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TABLE 33. Disorders associated with basophilia

Allergy Drug, food, inhalers Hypersensitivity Erythrodermia Urticaria Inflammation Colitis Rheumatoid arthritis Iron deficiency Endocrinopathy Diabetes mellitus Estrogens Myxedema Infection Chicken pox Influenza Small pox Tuberculosis Neoplasia Basophilic leukemia Carcinoma Myeloproliferative disease

ized countries, eosinophilia is most frequently associated with atopy, drug allergies, seasonal rhinitis, and asthma (Table 32). The Churg- Strauss syndrome and allergic bronchopulmonary aspergillosis are often linked to moderate eosinophilia. The recently reported eosinophilia-myalgia syndrome has receded in frequency with rec- ognition of its genesis from a contaminant. Reactive hyper- eosinophilia may be an important manifestation of chronic myelo- cytic leukemia.

The appropriate diagnosis of eosinophilia requires a detective-like investigation of the following: I. Patient history for travel, drug exposure, prior illness, contacts,

ingestion of unprocessed food, or symptoms of neoplasia 2. Physical examination with emphasis on skin, lymph nodes, spleen,

and neurologic findings 3. Laboratory diagnostic procedures based on selective features of

the history and examination may include stool for ova and para- sites, cultures, bone marrow examinations, sural nerve biopsy (rarely), and arterial biopsy (rarely)

Treatment depends on identification of a cause. Severe hypereosinophilia can be quite damaging to end organs. Consider- ation may be given to therapy with steroids, leukapheresis, or hydroxyurea.

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TABLE 34. Disorders linked to monocytosis or macrophage increase Infection Cytomegalovirus Varicella zoster ‘Ibberculosis Subacute bacterial endocarditis Syphilis Collagen disease Rheumatoid arthritis Systemic lupus erythematosus Temporal arteritis Myositis Polyarteritis nodosa Gastrointestinal disorders Alcoholic liver disease Ulcerative colitis Regional enteritis Sprue Sarcostosis Nonhematopoietic malignant disease Hematologic disorders Myelopoietic disorders Chronic neutropenia Preleukemia Acute leukemia Monocytic malignant disease Myelomonocytic malignant disease Polycythemia vera Lymphopoietic disorders Chronic lymphocytic leukemia Lymphoma Hodgkin’s lymphoma Multiple myeloma Histiocytosis Gaucher’s disease Niemann-Pick disease Gangliosidosis Sea-blue histiocyte disease Miscellaneous Exposure to tetrachlorethylene Langerhans histiocytosis Glucocorticoid excess Parturition Drug reaction Depression

Eosinopenk-Rarely commented on in the literature, eosinopenia is transient. It is usually associated with steroid use and can also occur with the use of epinephrine or with infection.

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TABLE 35. Diseases linked to monocytopenia

Pancytopenia Certain acute leukemias Hairy cell leukemia Chronic lymphocytic leukemia Cyclic neutropenia Human immunodeficiency virus infection Systemic lupus erythematosus Rheumatoid arthritis -

Basophils Although figures are available for normal basophil values, the low

percentage of basophils and variations in the number according to age, sex, and season complicate precise determination of the normal range. Basophilia is infrequent and is usually associated with other diseases (Table 33). A thorough physical assessment and review of the patient’s history are essential if basophilia persists. Basophilic leukemia (quite rare) and myeloproliferative diseases are diagnoses supported with findings of bone marrow examination and other tests (see Leukemias and Myeloproliferative Diseases). The treatment of patients with basophilia depends entirely on the diagnosis of the basic disease.

Monocytes and Macrophages Histiocyte and macrophage are viewed as synonymous in this dis-

cussion. Current knowledge has invalidated earlier concepts related to terminology (that is, histiocytic lymphoma is most likely to be a large B-cell lymphoma). The increase and decrease in monocytes- macrophages is rarely a primary process; it is usually a reflection of other diseases. Monocyte-macrophage system dysfunction may oc- cur in patients with solid tumors and is correlated with decreased expression of monocytic HLA-DR antigen and a decrease in tumor necrosis factor and interleukin-1P production.

Monocytosis.-Table 34 lists the disorders associated with monocytosis, or macrophage increase.

Monocytopenia.-As with other perturbations of the blood cells, monocytopenia is frequently a sign of disease and supports a known or discovered diagnosis (Table 35). A thorough historical review and physical examination with the foregoing diagnoses in mind are im- portant. On the basis of carefully derived conclusions, a bone mar- row examination and other studies sometimes may be important. As with other disorders, treatment relies on careful diagnosis and de- pends on the underlying cause.

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TABLE 36. Absolute lymphocytosis

Reactive Lymphocytosis Mononucleosis syndromes Epstein-Barr virus infection Cytomagalovirus infection Human immunodeficiency virus infection Bordetella pertussis infection Toxoplasma gondii infestation Persistent lymphocytosis Autoimmune disorder Rheumatoid arthritis Wegener’s granulomatosis Cancer Cigarette smoking Chronic inflammation Sarcoid tumors Thymoma Stress lymphocytosis Cardiovascular collapse Drug reaction Septic shock Hypersensitivity reaction Major surgical procedure Trauma Primary Lymphocyotosis Lymphocytic malignant disease Acute lymphocytic leukemia Chronic lymphocytic leukemia Large granular lymphocytic leukemia Prolymphocytic leukemia Hairy cell leukemia Certain lymphomas

TABLE 37. Congenital lymphopenia Disease Cellular abnormality

Severe combined immune deficiency Thymic aplasia (DiGeorge syndrome) Ataxia-telangiectasia Wiskott-Aldrich syndrome

No or very low level of T and B cells Decreased number of T cells Decreased number of T cells, low IgA level Abnormal amount of CD,, antigen on

lymphocytes and platelets

Lymphocytes Lymphocytosis. -Absolute elevation in lymphocyte level may be

primary, may be related to disorders of the lymphocyte population, or may be reactive. Differentiation of these causes of lymphocytosis is, at times, difficult. Table 36 presents the importance of history and physical examination in determination of the most likely diag- nosis to pursue. Removal of culprits such as cigarette smoking or

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TABLE 38. Acquired lymphopenia

Aplastic anemia

Infectious disease Viral HIV, AIDS CD, deficiency Bacterial infection Tuberculosis Typhoid fever Pneumonia Sepsis Nutritional or dietary disorder Adult kwashiorkor Ethanol abuse Zinc deficiency Iatrogenic factor Antilymphocyte globin Use of glucocorticoids Chemotherapy for neoplasia Platelet apheresis Thoracic duct drainage Systemic disease Renal failure Autoimmune disorder Rheumatoid arthritis Systemic lupus erythematosus Myasthenia gravis Hodgkin’s disease Sarcoid tumor

drug use may be simple and very cost effective. The prevalence of AIDS puts particular emphasis on diffuse adenopathy and lymphocytosis. In some instances, a measurement of lymphocyte cell subsets in peripheral blood or bone marrow may be helpful. Individualization of treatment is necessary and must be related to diagnosis.

Lymphocytopenia.-This category is of current interest in relation to HIV infection, particularly in the later stages, when the CD4 count is reduced to less than 2OO/pl. Iatrogenic lymphopenia may result from the use of glucocorticoids or psoriasis therapy with psoralens (methoxsalen) and ultraviolet radiation, antilymphocyte globulin, and antineoplastic chemotherapy. Particularly among elderly patients in institutions, malnutrition and zinc deficiency may result in lymphopenia, which can be resolved with great benefit to the pa- tient. Congenital lymphopenias are discovered within the first year of life (Tables 37, 38).

A search for the cause of lymphocytosis leads to direct, specific therapy. Some episodes of lymphocytosis are transient, with no ap-

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TABLE 39. Diagnosis of multiple myeloma Major Criteria Marrow plasmacytosis z 30% Plasmacytoma identified at biopsy Monoclonal globulin spike at serum electrophoresis (13.5 gm/dl IgG, >2.0 gm/dl IgA, 1.0 gm/24hr IgM of 1c light chain excretion on urine electrophoresis in absence of amyloidosis)

Minor Criteria Marrow plasmacytosis > 10% to 30% Monoclonal spike less than for major criteria Lvtic bone lesions Normal values are IgM <OS0 gm/dl, IgA ~0.10 gmidm, and IgG ~6.0 gm/dl. Diagnosis confirmed with one major and one minor or three minor criteria.

parent cause. Lymphopenias likewise require special management that ranges from removal of an offending agent through chemo- therapy for a neoplastic disorder to bone marrow transplantation for severe, combined immune deficiency.

Plasma Cells Plasma cells are normally not seen in a peripheral smear. When

they are seen, it is most unusual and may be diagnostic of plasma cell leukemia. In such instances, the associated multiple myeloma is of advanced stage and has a poor prognosis.

Evaluation of multiple myeloma requires a bone marrow examina- tion (aspiration and biopsy) in addition to a complete blood cell count (CBC). Electrophoresis of plasma and urine should be performed out to identify a monoclonal spike. In addition, immunofixation analysis should be performed and calcium and albumin should be measured. Radiographs of the axial skeleton should be obtained to help identify lytic lesions or diffuse osteoporosis (Table 39).

Benign monoclonal gammopathy must be ruled out. Management of multiple myeloma with chemotherapy differs according to the age of the patient and the rate of progression of the disease. Therapy is toxic and prolongs life only somewhat.

THE LEUKEMLAS

For historical and practical reasons, the leukemias are viewed as acute or chronic, myelogenous, or lymphocytic. Diagnosis ultimately depends on the characteristics related to leukocyte morphology, stain- ing characteristics, and surface antigens. Therapy for the acute leu- kemias is often complex and is undergoing constant evaluation.

Acute Lymphocytic Leukemia Acute lymphocytic leukemia is the most common malignant dis-

ease among persons younger than 15 years of age. It occurs most

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TABLE 40. Morphologic features of acute lymphocytic leukemias FAB” type Morphologic features

Ll Small blasts, uniform size, scanty cytoplasm, round nucleus, small nucleolus

L, Large blasts, irregular size, more cytoplasm, irregular nucleus, prominent nucleolus

L:3 Large blasts, uniform size, abundant cytoplasm, vacuoles, round nucleus, prominent nucleolus

FAB, French-American-British classification.

TABLE 41. Immunologic markers in acute lymphocytic leukemia Phenotype Markers

Pre B CDlO+(CALLAJ,sIg- B cell CD104, sIg+ T cell CDlO-, sIg-, CD7+, CD5+, CD6+, CD3+

frequently between the ages of 3 and 7 years and has a lesser peak incidence among persons older than 50 years. The onset is abrupt; signs and symptoms are related to anemia, thrombocythemia, and granulocytopenia. Bone pain, sternal tenderness, and minimal adenopathy are common. Chemotherapy is highly successful in the treatment of patients with this leukemia.

Acute lymphocytic leukemia is classified according to morphologic and immunologic markers (Tables 40,41). Laboratory studies include peripheral blood smear, bone marrow study with special stains, and immunologic markers. Consultation with a hematologist is essential to differentiate the subtypes of acute lymphocytic leukemia and to establish the appropriate chemotherapeutic regimen.

Acute Myelogenous Leukemias These leukemias are often acute fulminating illnesses or may be

transformations from the more indolent myeloproliferative diseases. Although acute myelogenous leukemias are monoclonal malignant diseases of primitive precursor cells, differentiation frequently is suggestive of somewhat more mature forms.

Morphology is the anchor of classification; however, staining char- acteristics and immunologic markers are also of great value. The modified French-American-British (FAB) classification is shown in Table 42. Myeloid blast forms have fine chromatin and multiple nucleoli.

Immunologic markers (CD13 to CD45) are helpful in the classifica- tion of cells as myelogenous or lymphocytic and may be helpful in further differentiation of cell cytometry in the key list for marker dif- ferentiation. Cytogenetic studies may also be helpful. Promyelocytic

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TABLE 42. ModifiedFrench-American-British (FAB) classification of acute myelog- enous leukemia FAB Cell type Description Incidence (%)

Ml Undifferentiated Blasts 20 M2 Myeloblasts Blasts with early granulocytic 30

differentiation M3 Promyelocytic Clear promyelocytic characteristics 10 M4 Myelomonocytic Both granulocytic and monocytic 25

features M5 Monocytic Monocytic features 10 M6 Erythroleukemic Blasts with erythroid features 4 M7 Megakaryocytic Blasts with megakaryocytic features 1

leukemia that responds to retinoic acid is differentiated with a trans- location between chromosomes 15 and 1i’t (15;17)(q22;q21).

The keys to the diagnosis of acute myelogenous leukemia are pe- ripheral blood smear, bone marrow morphology, and special stains. Cell cytometry to demonstrate markers is often helpful. In addition, chromosome studies are of interest and may be helpful. Consulta- tion is necessary for diagnosis and for the management of complex chemotherapy or bone marrow transplantation, particularly in pa- tients who experience relapses.

Chronic Lymphocytic Leukemias Chronic lymphocytic leukemias are diseases of adults, generally

of persons of advanced age. As a group they are relatively common lymphoproliferative disorders. Lymphomas and chronic lymphocytic leukemias may at times be difficult to differentiate. Lym- phadenopathy and splenomegaly of varying degrees may be found with all of these disorders. Lymphocytic surface antigen markers are used to supplement morphology in making the diagnosis (Table 43).

The diagnosis of chronic lymphocytic leukemia depends on care- ful morphologic review of a peripheral blood smear, bone marrow with special stains, and cell cytometric evaluation of cell surface markers.

Most B-cell chronic lymphocytic leukemias are indolent. Therapy, when instituted, is palliative and therefore is instituted to reduce cell burden if the patient has symptomatic disease or if there are complica- tions with anemia, thrombocytopenia, or infections. A&dating agents such as chlorambucil continue to be the mainstay of therapy. Transfor- mation of chronic lymphocytic leukemia to a more aggressive lymphoma requires treatment with multiple-agent chemotheraphy. Other treat- ments include radiation therapy and second-line chemotherapy. Im- mune complications (anemia, thrombocytopenia) are treated with ste- roids. Infections with severe hypogammaglobulinemia may require treat- ment with intravenous gamma globulin.

176 DM, March 1996

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TABL

E 43

. Cl

assif

icatio

n of

chro

nic

lymph

ocyti

c leu

kemi

a

Comm

on

name

De

scrip

tion

Addit

ional

featur

es

Marke

rs

B-ce

ll typ

e Ma

ture

cell

(lymp

hocy

te nu

cleus

ma

y be

clea

ved)

Matur

e ce

ll (ly

mpho

cyte

nucle

us

may

be

cleav

ed)

Larg

e gr

anula

r lym

phoc

yte

Larg

e lym

phoc

yte

(long

cy

toplas

mic

proje

ction

s)

Auto

antib

odies

to

red

blood

ce

lls

and

platel

ets

may

or ma

y no

t be

pre

sent

Hypo

gamm

aglob

uline

mia

Retro

virus

as

socia

tion

may

or ma

y no

t be

pre

sent

HTLV

-I wi

th

or wi

thou

t sk

in inv

olvem

ent

More

ad

enop

athy

No

distin

guish

ing

clinic

al fea

tures

Prom

inent

inf

iltrat

ion

of sp

leen

and

liver

Po

sitive

ac

id tar

trate

resis

tant-a

cid

phos

phata

se

stain

Mildl

y ph

agoc

ytic

CD19

, CD

ZO,

t CD

5, L

sIg

T-ce

ll typ

e

T-ce

ll typ

e

Hairy

ce

ll

CD4T

CD4+

, CD

8, or

CD4

B ce

ll les

s fre

quen

t T

cell

marke

rs

Seza

ry ce

ll La

rge

lymph

ocyte

ce

rebr

iform

De

rmal

manif

estat

ions

includ

e nu

cleus

my

cosis

, fun

goide

s, er

ythro

derm

ia

‘f’ ,

incre

ased

; L,

dec

reas

ed;

HTLV

-I,

hum

an

T-ce

ll lym

phot

ropi

c vir

us

type

I.

CD4T

Page 51: Hematology for primary care physicians

T-cell chronic lymphocytic leukemias, being less indolent than B- cell leukemias, require more active therapy to cause cell reduction and to reduce adenopathy and skin lesions. Skin therapies may in- clude topical steroids or alkylating agents and ultraviolet ray phototherapy. Leukapheresis may be useful in cell reduction. Alky- lating agents or bischloroethylnitrosurea (BCNU) may be useful, and steroids are used judiciously. Doxorubicin hydrochloride (Adria- mycin), methotrexate, and bleomycin are more aggressive alterna- tive systemic therapies. Other therapies include 2-chloro- deoxyadenosine and monoclonal CD5, CD5 antibodies.

Hairy cell leukemia varies in its rate of progression and may re- quire alkylating agents or combination therapy. Defensive treatment with interferon, pentostatin, and %chlorodeoxyadenosine will pro- duce remission in 80% of cases. Splenectomy or careful splenic ra- diation may be required for hypersplenism.

Chronic Myelocytic Leukemias Chronic myelocytic leukemias are discussed in the section on

myeloproliferative disease.

MYELOPROLIFERATIVE DISEASE

The myeloproliferative disorders represent a loose assemblage of clonal neoplasias characterized by a protracted course and abnor- malities of more than one hematopoietic cell line. Myeloproliferative diseases are usually designated on the basis of the cell line most predominantly involvedW4 In this section, are considered’poly- cythemia rubra vera (PCRV), agnogenic myeloid metaplasia- myelofibrosis (AMM-MF), thrombocythemia, and paroxysmal noc- turnal hemoglobinuria (PNH).

POLYCYTHEMIA RUBRA VERA

Polycythemia rubra vera is a disorder of the later decades of life. It results from clonal expansion of a transformed stem cell. The disor- der is predominantly associated with expansion of the red blood cell mass and, to a lesser degree, of granulocytes and platelets. Usu- ally diagnosed in the early phase of red cell mass increase, PCRV initially may be asymptomatic. It must be differentiated from other disorders, non-neoplastic in nature, that produce increases in eryth- rocyte mass in response to increased erythropoietin or erythropoi- etin-like substances. Such disorders may also be caused by the pres- ence of erythrocyte precursors with altered affinity to substances such as interleukin-6 [IL-6). Over time, PCRV may transform to a cytopenic state, myelofibrosis, or any of a variety of leukemias.

The patient may have no symptoms, and the diagnosis may be

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TABLE 44. Diagnostic criteria for polvcvthemia rubra vera Category Criteria

A. 1. Increased red blood cell mass:d >36 ml/kg,?>32 ml/kg 2. Normal arterial 0, saturation: ~92% 3. Splenomegaly

B 1. Thrombocytosis: >400,000 x 103/mm3 (400 x log/L) 2. Leukocytosis >12,000/mm3 (12 x 106/L) (no infection) 3. Leukocyte alkaline phosphatase (r 100) 4. Serum vitamin B,, >900 pg/ml or unbound vitamin B,,-

finding capacity >220 pg/ml Positive if category Al, AZ, and A3 are all present, or if Al, AZ, and two from category B are present. Pelycythemia r&a vera in the absence of hemorrhage is characterized by a markedly decreased eryth- ropoietin level.

suggested by an increased hemoglobin level. Physical findings may include plethora, dusky cyanosis, venous engorgement of the retina and conjunctiva, and splenomegaly (75% of patients). Patients are predisposed to gout. Hyperuricemia is secondary to increased cell turnover. These patients have a high incidence of symptomatic duodenal ulcers, and headache, weakness, and pruritus are present in more than 40% of patients.

Increased hemoglobin and hematocrit are the characte ings in erythrocytosis of any cause. Tables 44 through 4

7

/$ istic find- are useful

in differentiating the various forms of erythrocytosis. PCRV can be differentiated from other causes of increased hemo-

globin or hematocrit. Erythrocytosis may be secondary to a broad range of disorders, the most frequent of which are th,e following: 1. Rare cases of familial or sporadic erythremia 1 2. Response to decreased tissue oxygenation, as in ‘high altitudes,

chronic lung disease, alveolar hypoventilation, cardiac right to left shunts, carbonmonoxyhemoglobinemia, or increased 2,3- diphosphoglycerate (2,3-DPG) level

3. Tumors producing erythropoietin or other stimuli: renal cell, hepa- tocellular, ovarian, or other tumors: carcinomas, pheochromo- cytoma, or cerebellar hemangioblastoma

4. Renal diseases 5. Excess of androgens, erythropoietin, or cobalt 6. Stress polycythemia (Gaisbock’s syndrome)

Erythrocytosis may be relative to a decrease in plasma volume. In this instance, the red blood cell mass will be normal. States that induce decreased plasma volume include diaphoresis, diabetes mellitus, diabetes insipidus, use of diuretics, salt and water depri- vation, and anaphylactic states with third spacing of fluids. In the secondary, or relative, conditions, erythropoietin level is not as re- duced as much as it is with PCRV History and physical examination coupled with selective testing frequently reveal the underlying cause.

Reduction of erythrocyte mass to normal without marked eleva-

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TABLE 45. Comparison of forms of polycythemia rubra vera (PCRV) Observation PCRV Secondary PCRV Relative PCRV

Splenomegaly + 0 0 Leukocytosis + 0 0 Thrombocytosis + 0 0 Increased red blood + 0 0

cell mass Arterial 0, N I orN N Serum vitamin B,,

T N N

Leukocyte alkaline N N phosphatase

Marrow biopsy General Erythroid N hyperplasia hyperplasia

Fwthronoietin J 1‘ 0 +, present; 0, not present; N, normal; f , increased; 1, decreased.

tion of platelets is the main objective of therapy. In the short term, this may be accomplished with phlebotomy of 250 to 500 ml every other day. As a chronic strategy, chemotherapeutic cytoreduction with hydroxyurea can be accomplished with minimal side effects. Hemoglobin values should be maintained in the low normal range. Until normal values are attained, gout should be prevented with administration of allopurinol. Therapy must be tailored to the pa- tient.

AGNOGENIC MYELOID METAPLASLA, MYELOFIBROSIS

This chronic monoclonal myeloproliferative disorder is identified by the presence of the following: 1. Immature granulocytes in the blood 2. Teardrop-shaped red blood cells 3. Marrow fibrosis with no apparent cause 4. Splenomegaly 5. Extramedullary hematopoiesis

Extramedullary hematopoiesis is found in the spleen, liver, and other sites. Fibrosis is likely related to an abnormal release of a vari- ety of growth factors from the a granules of platelets and megakaryo- cytes. Although splenomegaly is a distinguishing feature, the labo- ratory studies shown in Table 47 may at times be the first clue to the disorder.

History and physical examination, as always, are the keys to diag- nosis. Hepatosplenomegaly becomes a predominant feature at some time in the history of most patients. Peripheral smear abnormalities often provide the first clue to the disorder, and confirmation with bone biopsy is essential. Caution is essential in making the diagno- sis because tuberculosis and metastatic carcinoma may generate myelophthisic features with bone marrow fibrosis. Other

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TABLE 46. Laboratory features of myelofibrosis

Leukoerythroblastic peripheral blood smear is characterized by the following: 1. Nucleated erythrocytes 2. Basophilic stippling of erythrocytes 3. Teardrop-shaped erythrocytes 4. Pseudo-Pelger-Huet cells 5. Immature myeloid (granulocyte-monocyte) forms 6. Large platelets 7. Megakaryocyte fragments 8. Anemia: Degree varies, usually perichromic-normocytic

Leukocytosis: Modest, 15% of patients may have absolute neutropenia 9. Basophil increase common lO.Leukocyte alkaline phosphatase level usually elevated (low level tends to correlate

with neutropenia) ll.Vitamin B,, and vitamin B,, binding capacity elevated lZ.Bone marrow often a “dry tap” aspiration; bone biopsy is viable but demonstrates

fibrosis when stained with silver

TABLE 47. Laboratory diagnosis of paroxysmal nocturnal hemoglobinuria

Essential for diagnosis Variably present

Hemolysis Positive sucrose hemolysis, acid hemolysis tests Positive sucrose hemolysis test (Ham test) or positive acid hemolysis test Anemia

Iron deficiency Leukopenia Low alkaline phosphatase level Low platelet count (80% of patients) Marrow, erythroid hyperplasia aplasia; late manifes-

tations include stainable iron, but this sign is often absent

Constant hemosiderinuria; hemoglobinuria may be variable

myeloproliferative disorders such as partial early CML may strongly resemble myelofibrosis. Less difficult to differentiate are disorders such as osteopetrosis, lymphoma, lipid storage disease, and leukemoid reactions.

Treatment must be highly individualized and is often directed at complications and causes. Although anemia is generally attributed to decreased production and ineffective erythropoiesis, it may be secondary to vitamin B,, or folate deficiency, iron deficiency, hypersplenism, or even PNH. Each of these disorders requires spe- cific management. Thrombocytopenia may be related to ineffective megakaryopoiesis, autoimmune disorder, or hypersplenism. Paradoxi- cally, thrombocytosis may be associated with increased thrombosis or hemorrhage.

Symptomatic splenomegaly may, in rare instances, require care- ful low-dose radiation therapy or splenectomy. At times it may re-

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spond to chemotherapy. Severe myelofibrosis and pancytopenia are often refractory end-stage manifestations. As with other myelo- proliferative disorders, leukemic transformation to a secondary acute leukemia $9~ CPX.W .& with .atJw qyelsprolifesa~iw leukemias, these respond poorIy to therapy. In the nonleukemic phase, chemo- therapy alkylating agents, radioactive phosphorus, and other mo- dalities demonstrate variable effect. Hydroxyurea has received ini- tial positive evaluation. Therapy for fibrotic complications is un- dergoing intense evaluation.

7HROMBOCyTHEMLA Thrombocythemia results in a marked elevation in platelet count

(more than 600 x 103/mm3 with no apparent cause, deriving from an alteration in stem cell growth. As with other myeloproliferative disorders, all myeloid cell lines may be affected; however, platelet abnormalities predominate. Qualitative platelet defects include ab- normally large platelet fragments with occasional megakaryocyte fragments. Splenomegaly may be reversed in the presence of a high platelet count secondary to splenic infarction. The qualitative and quantitative platelet abnormalities may lead to hemorrhage, hy- percoagulability states, and a panoply of venous and thrombolytic events.

Thrombocythemia is often first found when abnormal laboratory findings occur. The strict criteria adopted by the Polycythemia Vera Study group include the following: 1. Thrombocytosis greater than 600 x 103/mm3 2. Bone marrow megakaryocytic hyperplasia 3. Hemoglobin level less than 13 gm/dl 4. Stainable bone marrow iron 5. Red blood cell mass in normal range if polycythemia vera is a

possibility 6. Philadelphia chromosome negative 7. Absence of clinically significant marrow fibrosis (less than one-

third the area of biopsy specimen) 8. Absence of other myeloproliferative disorders and reactive

thrombocytosis (carcinoma, splenectomy infection, and splenectomy)

Untreated thrombocythemia is not a benign disorder. Therapy with platelet pheresis may be used, although the effect is of short dura- tion. Anti-aggregating agents such as aspirin may be reserved for patients with thrombotic phenomena and should not be used in those with hemophagic phenomena. Although many agents may effectively lower platelet count, hydroxyurea has a high therapeutic index. Despite its toxicity, interferon a may be used effectively in younger patients. Anagrelide, which interferes with megakaryocyte matura- tion, is effective; however, its toxicity is under evaluation. 184 DM, March 1998

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PAROXY3UAL NOCTURNAL HEMOGLOBINURLA

Paroxysmal nocturnal hemoglobinuria is a hematopoietic stem cell disorder that results in the formation of abnormal erythrocytes, granu- locytes, and platelets. The most apparent clinical abnormality is the increased sensitivity of erythrocytes to the hemolytic action of complement. The resultant complement-induced lysis presents a variable degree of hemolysis, which may be constant or, less fre- quently, paroxysmal. In the latter instance, the patient may experi- ence a dramatic production of cola-colored urine caused by hemoglobinuria.

The apparent biochemical lesion is a failure to produce an effec- tive glycosyl phosphatidylinositol (GPI) cell surface anchor for sub- stances, some of which are complement-regulatory proteins (for ex- ample, CD55, CD59 acetylcholinesterase, leukocyte alkaline phos- phatase). The erythrocytes and other hematopoietic cells are ren- dered abnormally sensitive to complement lysis and other effects by the direct or alternative complement pathways.

The clinical features of PNH include hemoglobinuria (periodic or constant); chronic anemia related to chronic hemolysis and iron loss; hemorrhage secondary to thrombocytopenia; pregnancy, complicated frequently by abortion and venous thrombosis; renal dysfunction, progressive with renal infection, hypertension, and proteinuria; and neurologic manifestations, including cerebral infarction. The abnor- mal pattern of complement lysis is pathognomonic. Secondary diag- nostic features must support the diagnosis, although they may be variably present (Table 47). Therapy is complicated and is based on clinical features, Only transplantation of marrow is curative.

HEMOSTASIS

Evaluation of hemostatic mechanisms in patients can often be confusing. Therefore, a simple approach for these patients is de- scribed. Every effort has been made to consider costs in describing evaluation plans.

MEDICAL HISTORY

The most important screening device for the evaluation of a patient’s hemostatic competence is a detailed medical history. De- tailed information must be obtained with regard to the following: 1. Age at onset. Determining the age at onset of hemorrhagic events

will help define whether the disorder is inherited or acquired. In- herited disorders tend to occur in childhood; however, mild forms of inherited disorders may not be manifest until later years, when surgical intervention or trauma uncovers a bleeding tendency.

2. Family history. A positive family history is helpful in guiding the investigation of bleeding disorders. It is important to recall that

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the absence of a family history does not preclude the diagnosis of an inherited bleeding disorder.

3. Medication use. A detailed history of all medications used, par- ticularly over-the-counter drugs, must be obtained. Often medi- cations contain agents such as aspirin, and patients may be un- aware of this fact.

4. Other disorders. Patients with disorders such as renal failure or liver dysfunction may have disruptions of their hemostatic mechanisms.

5. Response to previous hemostatic stressors. The patient’s past re- sponse to hemostatic stress such as an operation or trauma is im- portant information.

PHYSICAL, ,!BAMINATION

The physical examination of patients with suspected bleeding dis- orders may be normal or may show manifestations of multisite bleed- ing. Bleeding from a single site in a patient who is recovering from an operation is most often due to a surgical defect, but may be made more severe by defective hemostasis. Bleeding that is superficial (that is, skin or mucous membrane bleeding) most often is the result of platelet-associated bleeding or vascular fragility (for example, senile purpura). Bleeding that is more deeply seated (that is, in muscles or joints) is more likely associated with coagulation factor deficiencies.

The following laboratory tests can provide an evaluation of the he- mostatic pathways to assure adequate hemostasis in most patients: bleeding time, platelet count, partial thromboplastin time, coagula- tion profile, prothrombin time (PT), and thrombin time or fibrinogen level. Together, these tests provide a coagulation profile. If the results of these tests are normal, adequate hemostasis should be present, and only an occasional patient will continue to have an increased risk of excessive bleeding associated with trauma or surgical therapy.

COAGULAlTON PROFILE

Activated partial Thrombopl@n Time The activated partial thromboplastin time (APTT) is a modern

version of the whole blood clotting time. The advantages of APTT over clotting time include the following: 1. Results are available more rapidly 2. Processing can be managed with automatic instrumentation 3. Evaluations can be performed on samples of frozen plasma, al-

lowing transport of specimens 4. New substrates have made the APTT more sensitive to clotting

factor levels in the intrinsic and common pathways 5. With selection of appropriate substrates, the APTT can be sensi-

tive to the presence of a lupus anticoagulant

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APTT may be prolonged in patients who have no symptoms, who are bleeding or have a bleeding history, or who have a history of thromboembolism. APTT also may be prolonged by a deficiency of any of the following clotting factors: XI, IX, VIII, X, V, prothrombin, or fibrinogen.

Patients with isolated factor XII deficiency do not have bleeding due to this factor deficiency. A patient with thromboembolism who has a prolonged APTT may have factor XII deficiency or lupus anti- coagulant. Factor XII is intimately concerned with the activation of plasminogen to plasmin. Plasmin is the enzyme responsible for fi- brinolysis. Thus, if factor XII is present in reduced amounts, the fibrinolytic process is slowed and clot dissolution may be inadequate. Very small amounts of factor XII are required to support the intrin- sic coagulation pathway.

The lupus anticoagulant phenomenon is poorly named. At least one-half of these patients do not suffer from systemic lupus erythematosus, and they may experience a thromboembolic disor- der as opposed to a bleeding disorder. The importance of the lupus anticoagulant when discovered in patients with no history of thromboembolism is yet to be clarified. For patients with documented thromboembolism, the presence of the lupus anticoagulant supports the need for prolonged anticoagulant therapy. We believe antico- agulant therapy should be continued for as long as the lupus antico- agulant phenomenon can be demonstrated in a patient who has suf- fered from a thrombotic event.

Prothrombin Time Prolongation of PT occurs if there is deficiency of any of the fol-

lowing coagulation factors: VII, X, V, prothrombin, or fibrinogen.

Mixing Studies or the Inhibitor Screening Test When a patient has a prolonged PT or APTT or both and there is

uncertainty about whether the patient has a coagulation factor defi- ciency or an antibody [inhibitor) directed against one or more clot- ting factors, mixing studies or an inhibitor screening test may be requested. The principle of the mixing study is to mix normal plasma with the patient’s plasma and evaluate the effect on PT and APTT

50% normal plasma Correction of PT or APTT \1

Factor deficiency 50% patient plasma

> Failure to correct PT or APTT

J Inhibitor present

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By determining whether the disorder is a clotting factor deficiency or an inhibitor is present, the physician can more accurately direct additional evaluation steps for the patient.

Reminder: Prolongation of APTT may be a clue to the presence of a lupus anticoagulant.

Thombin Time The thrombin time is a one-step test that evaluates the amount

and function of fibrinogen. Prolongation of thrombin time may be due to low levels of fibrinogen or the presence of dysfunctional fi- brinogen.

Fibrinogen Assay The fibrinogen assays used in most laboratories define only the

amount of fibrinogen present and not its functional capability.

“Non-patient” Reasons for Prolongation of APTT, P?; or Thornbin Time and Abnormal Fibrinogen Assay 1. Inadequate amounts of blood are placed in the specimen tube.

The anticoagulant in the specimen tube is present in a ounts that will anticoagulate the plasma of the correct amount o blood. When less than the required amount of blood is place I in the tube, there is excess of anticoagulant in the plasma, prolonging clot-based laboratory tests.

2. Hemolysis of specimens may alter the results of coagulation tests; either prolongation or shortening of the time may occur.

3. Heparin contamination of samples obtained from indwelling cath- eters can cause prolongation of clot-based laboratory tests.

4. Specimens obtained after difficult venipuncture may be contami- nated with tissue fluids, which can activate clotting. The data obtained with these samples may be incorrect and may not cor- rectly reflect the patient’s hemostatic status.

5. Improper handling of specimens can prolong times to clot (for example, specimens kept at room temperature for periods longer than 1 hour or improper centrifugation of blood to obtain plasma).

Bleeding Time In years gone by, the bleeding time was not a particularly reliable

test. The main difficulties related to difficulty with reproducing the test in a reliable manner, In recent years, new methods of perform- ing the bleeding time with a single-use, disposable device to make l-mm deep incisions on the forearm of the patient have led to a much more reliable bleeding-time test. A correctly conducted bleed- ing time test can provide reliable information concerning a patient’s hemostatic competence.

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The bleeding time will show varying degrees of prolongation de- pending on the degree of thrombocytopenia and the cause of the thrombocytopenia. Usually, prolongation of bleeding time will be seen if the platelet count falls to 40 to 50 x 103/mm3 (40 to 50 X log/ L) or less.

The causes of thrombocytopenia may be divided into four groups: 1. Decreased production of platelets by the bone marrow, as in pa-

tients receiving chemotherapy and radiation therapy and those with bone marrow failure, as in aplastic anemia, bone marrow infiltration,leukemia, or tumor invasion of the bone marrow

2. Increased peripheral destruction of platelets, as in idiopathic thrombocytopenic purpura or DIC

3. Splenic pooling or sequestration of platelets, which can be associ- ated with any disorder that causes splenomegaly

4. Combined causes. Thrombocytopenia of multiple disorders may occur from time to time

Platelet dysfunction causing a prolongation of the bleeding time may have the following causes:

1. Most commonly, the effect of medication consumed by the pa- tient, particularly nonsteroidal anti-inflammatory drugs (NSAIDs) (Note: The effect of aspirin on platelets is irreversible, whereas other NSAIDS appear to have reversible effects.)

2. Nonhematologic disorders associated with platelet dysfunction, such as uremia and liver dysfunction

3. Hematologic disorders associated with platelet dysfunction, such as myelodysplastic syndromes, myeloproliferative syndromes, multiple myeloma, von Willebrand’s disease, and severe hypofibrinogenemia

4. Intrinsic platelet defects that occur with prolonged bleeding times, including dense granule deficiency, Bernard-Soulier syndrome, and a number of other less common disorders Elderly patients, patients undergoing long-term corticosteroid

therapy, and patients with some congenital disorders may have frag- ile skin, which may cause prolongation of bleeding time. Before requesting a bleeding time, one should carefully examine the skin on the patient’s forearms to be sure that it is normal.

PROBLEM-SOLVING STRATEGIES IN HEMOSTASIS

For patients with a history of bleeding or bruising or patients undergoing an evaluation for bleeding risk, the coagulation labo- ratory can use simple tests to provide clues to the cause of the potential or actual bleeding. Results of the APTT, PT, and throm- bin time or fibrinogen assay can narrow the diagnostic possibili- ties:

DM, March 1996 189

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I. APTT PT Normal Prolonged

Thrombin time and fibrinogen normal 1

Factor VII deficiency II. APTT PT Prolonged Normal Thrombin time and fibrinogen normal

L von Willebrand’s disease

Factor VIII deficiency Factor IX deficiency Factor XI deficiency (Although Factor XII deficiency will give the same test re-

sults, it is not associated with bleeding) III. APTT PT Prolonged Prolonged

L A. Multiple factor deficiencies 1. Vitamin K deficiency 2. Liver disease 3. Warfarin therapy B. Heparin therapy

MOL?XULAR MARKERS OF HEMOSTAslS

D-Dimer and Fibrin-Fibrinogen Split Products The D-dimer and fibrin-fibrinogen split products (FSP) tests pro-

vide information about fibrinolysis, a process that is usually present, to a greater or lesser degree, in patients with DIC. Fibrinolysis is a normal response to clot formation and is an essential protective mechanism to maintain vascular patency. In patients with DIC, clini- cally significant secondary fibrinolysis often develops. Primary fi- brinolysis is a much less common phenomenon, occurring when excessive amounts of plasminogen activator are released into the blood, overwhelming the inhibitors of fibrinolysis. The most com- mon settings in which primary fibrinolysis occurs include excessive exercise, shock, severe trauma, heat stroke, carcinoma of the pros- tate, and thrombolytic therapy.

The D-dimer test is positive when fragments of cross-linked fibrin are present in the blood, requiring fibrin formation followed by diges- tion of the fibrin by plasmin. The FSP test is, positive in the presence of plasmin digestion products of both fibrinogen and fibrin. Thus the specificity of the test is somewhat less than that of the D-dimer test.

Both the D-dimer and FSP tests are rapidly performed, providing results within 60 to 90 minutes of collection of the sample of blood.

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The D-dimer test can be conducted on a sample of titrated plasma- the same sample one would use for obtaining a PT, APTT, or fibrino- gen assay. The FSP test requires a separate sample of blood and can- not be performed on a routine titrated plasma sample.

DISSEMINATED INTRAVASCULAR COAGULATION

Disseminated intravascular coagulation may become apparent as active multisite bleeding in a patient who has many different disor- ders. The most common settings in which DIC should be suspected include shock of any cause, sepsis, disseminated malignant disease, brain injury, obstetric complications, incompatible blood transfu- sions, heat stroke, or liver dysfunction. For a patient with an at-risk diagnosis, DIC may be considered even if there is no evidence of bleeding if laboratory results are abnormal.

One should consider the following cost-effective group of labora- tory studies when making the diagnosis of DIC8-11: l APTT l PT l Platelet count l Fibrinogen D-dimer or FSP l Euglobulin lysis time

These tests are chosen to evaluate DIC because of their 24-hour availability. Many physicians considered the D-dimer to be the most useful test because elevated levels are seen during the clotting phase of DIC, at which other tests may still show only minor changes.

The degree of abnormality shown with any or all of these tests will assist the physician in making the choice of hematologic sup- port, The most difficult decision one often faces is when or if to use heparin. The following questions are helpful: 1. Has the patient been resuscitated from shock? 2. Is there evidence of ongoing end-organ failure, such as hypoxia or

renal failure? 3. Does the patient have a treatable underlying cause of DIC?

If the answer to each of these questions is yes, heparin therapy should be considered. When the use of heparin is being considered for patients with DIC, transfusion support should be carefully planned. The following options are available for transfusion sup- port in patients with DIC: 1. Packed red blood cells may be used to maintain an adequate he-

moglobin level. 2. Cryoprecipitate is used as a source of fibrinogen. A rough guide is

one bag of cryoprecipitate for each 5 to 10 kg of body weight. After the infusion is complete, fibrinogen level is assessed to en- sure that it is greater than 100 mg/dl. (Cryoprecipitate may be contaminated by the hepatitis viruses and HIV)

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3. Fresh frozen plasma (FFP) may be used to provide coagulation factor support. The disadvantage of the use of FFP is the large volume of fluid given to achieve only small increases in clotting factor levels. (FFP may be contaminated by the hepatitis viruses and HIV)

4. Platelet transfusions should be considered when the platelet count falls to 20 x 103/mm3 or less. If the platelet count is between 20 and 50 x 103/mm3 but it is believed that this degree of thrombocytopenia is contributing to bleeding, platelet transfusions are justified. Platelet transfusions may be given in two ways. Platelets may be

given as random-donor platelets, in which the platelets from a single unit of blood are separated from the blood for transfusion. The usual dose is one bag of random donor platelets for each 10 kg of the recipient’s body weight. Platelets also may be given as single-donor platelets, which are obtained by platelet pheresis from a donor. One bag of single-donor platelets is equivalent to 6 to 8 units of random- donor platelets.

The most important advantage of single-donor platelets is reduced exposure of the patient to transfusion-related complications because there is one rather than multiple donors. The most important disad- vantage of the use of single-donor platelets is availability. Most large hospitals have random-donor platelets available in the blood bank for urgent use, but few hospitals stock single-donor platelets for ur- gent use.

There are non-transfusion methods that may assist in the control of bleeding associated with DIC. Epsilon aminocaproic acid and tranexamic acid are both agents that block the fibrinolytic pathway. These agents are particularly valuable in the treatment of patients with clinically significant secondary fibrinolysis that may be associ- ated with DIC. However, care must be exercised in the use of such agents. It is advisable to use heparin to block the thrombus forma- tion that occurs in DIC before introducing agents that will block the fibrinolytic pathway.

LABORATORYEV3LUATION OF INCREASED CLOTTING RZSK [HYPERCOAGULABL5lTYSTATES)

Laboratory assessment for patients believed to have an increased risk for thromboembolism has advanced rapidly in the past 10 years. Patients at risk for thromboembolic events fall into two groups- those with congenital disorders and those. with acquired disorders.

Congenital Disorders The more common causes of congenital hypercoagulability disor-

ders include antithrombin III deficiency, deficiency of proteins C

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and S, and activated protein C resistance. Antithrombin III and pro- teins C and S are naturally occurring anticoagulants.5-7

Acquired Disorders Acquired hypercoagulability states may occur as a result of a num-

ber of mechanisms, including the following: . Decreased production of naturally occurring anticoagulants (as

in liver failure) l Ineffective fibrinolysis due to a reduced level of or defective plas-

minogen l Very high levels of clotting factors, particularly fibrinogen, as in

response to the stress of severe illness or trauma l Platelet counts greater than 1000 X 103/mm3 (1000 X log/L) l The lupus anticoagulant phenomenon . Some cases of chronic DIC

Medical His tory The patient’s medical history will give useful information to al-

low one to decide whether studies should be performed to deter- mine the presence of a hypercoagulability state. The key points in the patient’s history are the occurrence of thromboembolic events before the age of 40 years, particularly if there has not been any period of immobility, and unexplained death of family members at less than 40 years of age.

Laboratory Studies The laboratory evaluation for patients who may have a hyperco-

agulability disorder should be undertaken with care, because the studies can be expensive. The following appear to be the most ap- propriate and cost-effective studies: 1. APTT. Prolongation may occur in patients with the presence of a

lupus anticoagulant or with factor XII deficiency. A search for a lupus anticoagulant is appropriate because the phenomenon may be quite common.

2. Platelet count 3. Fibrinogen level 4. Proteins C and S 5. Antithrombin III 6. Activated protein C resistance 7. Anticardiolipin and antiphospholipid antibodies 8. Plasminogen levels

If none of these studies provides an answer, further studies are less likely to yield a hematologic cause of the hypercoagulability state.

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REFERENCES 1. Lee GR, Bithell TC, Foerster J, et al. Wintrobe’s clinical hematology. 9th ed.

Philadelphia: Lea & Febiger, 1993. 2. Williams WJ, Buetler E, Lichtman MA, et al. Williams’ hematology. 5th ed.

New York: McGraw-Hill, 1995. 3. Hillman RS, Ault KA. Hematology in clinical practice: A guide to diagnosis

and management. New York: McGraw-Hill, 1995. 4. Wasserman LR, Berk PD, Berlin NI. Polycythemia Vera and the myeloproliferative

disorders. Philadelphia: WI3 Saunders, 1995. 5. Hellgren M, Svensson PJ, Dahlback B. Resistance to activated protein C as

a basis for venous thromboembolism associated with pregnancy and oral contraceptives. Am J Obstet Gynecol 1995;173:210-13,

6. Dahlback B. Factor V gene mutation causing inherited resistance protein C as a basis for venous thromboembolism. J Intern Med 1995;237;221-7.

7. Dahlback B. Inherited thrombophilia: resistance to activated protein C as a pathogenic factor of venous thromboembolism. Blood 1995;85:607-14.

8. Fareed J, Bick RL, Hoppensteadt DA, Bermes EW. Molecular markers of hemostatic activation: applications in the diagnosis of thrombosis and vascular and thrombotic disorders. Clin Appl Thromb Hemost 1995;1:87- 102.

9. Bick RL. Disseminated intravascular coagulation: objective criteria for clinical and laboratory diagnosis and assessment of therapeutic response. Clin Appl Thromb Hemost 1995;1:3-23.

10. Bick RL. Antiphospholipid syndromes. Semin Thromb Hemost 1994;20:3- 15.

11. Cines DB. Disseminated intravascular coagulation: is there a role for antithrombin III? Semin Hematol 1994;31(suppl 1).

194 DM, March 1996