1. This item describes two deficiencies. First, the hematology laboratory scientist should have washed his/her hands after removing the gloves and before leaving the laboratory. Second, the hematology laboratory scientist should have removed his/her laboratory coat before going to the meeting.
2. This item describes a deficiency. Storage of food in a specimen refrigerator is prohibited.
3. This may or may not be a deficiency. The laboratory employees may have had on a personal laboratory coat. A second laboratory coat could have been obtained by the employees to wear in public areas. Some laboratories require different colored lab coats for public areas.
4. No deficiency is indicated. Fire extinguishers should be placed every 75 feet.
5. This item describes a deficiency. Fire extinguishers should be inspected monthly and maintained annually.
6. This item represents a deficiency. All chemicals should be labeled.
7. This item represents a deficiency. The 1:10 bleach solution should be made fresh daily.
8. No deficiency is indicated. Gloves should be worn by all personnel handling specimens.
9. No deficiency is indicated. Safety data sheets can be received by fax.
10. This item describes a deficiency. Chemicals should not be stored alphabetically, but according to storage requirements specified in the safety data sheets.
1. d; 2. b; 3. c; 4. c; 5. a; 6. b; 7. c; 8. b; 9. b; 10. b; 11. d
The proper procedure is to ask the patient to state his/her full name and then confirm by asking his/her birth date and/or address. The phlebotomist should not prelabel tubes; tubes should be labeled after the blood is drawn and before leaving the patient.
Test results that can be affected by this selection of tubes and order of draw include the prothrombin time (PT), potassium, and type and screen.
• The light blue stopper tube for the PT should not have been collected after the serum separator tube (which contains an inert gel and clot activator). The clot activator could contaminate the blue stopper tube, activate coagulation factors, and cause an error in the PT results.
• The green stopper tube for potassium should not have been collected after a lavender stopper tube (which contains EDTA, usually as a potassium salt). The potassium-EDTA could be carried over into the green stopper tube and falsely elevate the potassium level. The potassium could, however, be assayed in the serum separator tube.
• The type and screen cannot be done on blood from the serum separator tube because the gel interferes with blood bank procedures; the lavender stopper tube, however, could be used for the type and screen.Box 3-2 contains the correct order of draw for evacuated tubes.
1. a; 2. b; 3. b; 4. c; 5. b; 6. b; 7. b; 8. d; 9. b; 10. c; 11. c; 12. a
The following things should be checked:
Is the slide right side up? This is the most common cause of inability to focus a slide under oil when it has been focused under 10× and 40× objectives.
If the slide is right side up, continue by checking the following:
• Is there sufficient oil on the slide? If not, clean off all the residual oil first and then apply another drop of oil.
• Is the objective screwed in tightly? If not, tighten the objective.
• If the slide has a coverslip, is there more than one coverslip on the slide? If so, gently remove the top coverslip.
• Has oil seeped into the seal on the oil objective? Examine by removing the objective and use an inverted eyepiece as a magnifier to check the seal. If the seal is broken, the objective must be replaced.
1. b; 2. c; 3. a; 4. b; 5. c; 6. c; 7. d; 8. a; 9. c; 10. d; 11. b
1. This is a systematic error because the magnitude of error remains constant at three ranges of test results.
2. It is not acceptable to continue using the instrument or to simply subtract the systematic error from test sample results. All the samples in a two out-of-control test run must be re-assayed after the error is corrected.
3. Determine from the quality control charts at what moment the error occurred. Investigate potential changes in instrument settings, calibration, reagent changes, or instrument malfunction that may have occurred at the time the error was recorded.
1. d; 2. c; 3. b; 4. b; 5. b; 6. a; 7. d; 8. d; 9. c; 10. c; 11. c; 12. a; 13. b; 14. c; 15. a; 16. b
1. b; 2. b; 3. a; 4. b; 5. c; 6. a; 7. d; 8. d; 9. c; 10. b; 11. a; 12. d; 13. b; 14. a
1. a; 2. d; 3. c; 4. a; 5. b; 6. c; 7. c; 8. b; 9. a; 10. d; 11. a; 12. b
1. When the blood is not well oxygenated, the bone marrow responds by producing more red blood cells to carry more oxygen.
2. The hormone that stimulates RBC production is erythropoietin (EPO). The peritubular cells of the kidney detect hypoxia. A hypoxia-sensitive transcription factor is produced that moves to the peritubular cell nucleus and upregulates transcription of the EPO gene. EPO acts by preventing apoptosis of the erythroid colony-forming unit. In RBC precursors, it also shortens the cell cycle time between mitoses and reduces the number of mitotic divisions; and it promotes early release of reticulocytes from the bone marrow.
3. Once the patient was receiving oxygen therapy, hypoxia diminished and EPO production also declined. Thus, production of new RBCs slowed. At the same time, RBCs reaching 120 days of age were removed from the circulation. Thus the total number of circulating RBCs decreased.
1. c; 2. a; 3. d; 4. b; 5. b; 6. d; 7. a; 8. d; 9. a; 10. c; 11. b; 12. b
1. A reducing agent is able to donate an electron to an oxidized compound so that the oxidized compound has one fewer unpaired proton. The compound receiving the electron becomes reduced and the donating compound becomes oxidized.
2. When heme iron is oxidized, the molecule cannot carry oxygen and patients become cyanotic. Because vitamin C eliminated the cyanosis, it must be able to reduce methemoglobin and restore the oxygen-carrying capacity of the blood.
3. Because this condition affected brothers, a hereditary condition was suggested in which hemoglobin became oxidized more than is usual. (The condition affecting these brothers was later identified as a hereditary deficiency of methemoglobin reductase.)
1. a; 2. c; 3. b; 4. d; 5. b; 6. b; 7. a; 8. c; 9. a; 10. a; 11. a; 12. a
1. The mother’s and infant’s hemoglobin results were within the reference intervals. (Reference intervals: adult women, 12.0 to 15.0 g/dL; newborns, 16.5 to 21.5 g/dL.)
2. The major hemoglobin at birth is Hb F. It has a high oxygen affinity because it weakly binds 2, 3-BPG resulting in decreased delivery of oxygen to the tissues. The hypoxia triggers an increase in secretion of erythropoietin by the fetal kidney, which results in an increase in the production and release of red blood cells from the fetal bone marrow. The resultant increase in red blood cell count, hemoglobin concentration, and hematocrit compensates for the high Hb F oxygen affinity and reduced oxygen transfer to tissues. The Hb F concentration gradually decreases to adult physiologic levels by 1 to 2 years of age as most of the Hb F is replaced by Hb A.
3. The hemoglobin assay measures concentration; high performance liquid chromatography (and hemoglobin electrophoresis) identifies and quantifies hemoglobin types.
4. These are the expected results for hemoglobin fractions for a healthy mother and infant. In the second and third trimesters of fetal life, the α- and γ-globin genes are activated producing α and γ globin chains that combine to form Hb F. In late fetal life, γ-β switching begins in which transcription of the β-globin gene begins to be activated and the γ-globin gene begins to be repressed. With the activation of the β-globin gene, the β chains combine with the α chains to form Hb A. The Hb F level decreases from 60% to 90% at birth to 1% to 2% by 1 to 2 years of age, while the Hb A increases from 10% to 40% at birth to greater than 95% at 1 to 2 years of age and throughout life. The synthesis of Hb A2 begins shortly before birth and remains at less than 3.5% throughout life.
1. d; 2. a; 3. a; 4. a; 5. a; 6. d; 7. c; 8. b; 9. d; 10. b; 11. c
1. Iron loss via blood donations and normal physiologic loss was not compensated by diet or supplementation.
2. Adaptation to the low iron levels. Iron stores of ferritin were mobilized first. But when storage iron declined, hepcidin levels declined, and as a result, duodenal iron absorption increased.
4. Transferrin saturation reflects the proportion of transferrin binding sites for iron that are actually filled with iron during transit in the plasma. Transferrin level is an indirect indicator of the iron storage compartment while serum iron is the transport compartment, so transferrin saturation effectively reflects both compartments.
1. c; 2. b; 3. c; 4. c; 5. c; 6. b; 7. d; 8. b; 9. d; 10. c; 11. b; 12. d
1. The patient had an asthmatic attack. Eosinophils play an important role in the initiation and maintenance of symptoms. Eosinophils release basic proteins, lipid mediators, and reactive oxygen species that cause inflammation and damage to the mucosal cells lining the airway.
2. Eosinophils are typically elevated in the peripheral blood and also in the sputum of asthmatic patients. The number of eosinophils in the blood correlates with the severity of the case.
3. IL-5 plays an important role in the differentiation and proliferation of eosinophils. Monoclonal antibodies to IL-5 block eosinophil development. Since eosinophils are reduced, the symptoms of asthma are controlled.
1. b; 2. d; 3. a; 4. c; 5. b; 6. c; 7. c; 8. a; 9. b; 10. d
1. Bleeding characterized by petechiae, purpura, and ecchymoses is known as mucocutaneous bleeding, also called systemic bleeding. By contrast, anatomic bleeding is bleeding into soft tissue, muscles, joints, or body cavities.
2. Thrombocytopenia, or low platelet count, is a common cause of mucocutaneous bleeding. Another is diseases that weaken vascular collagen such as scurvy.
3. No, the bone marrow megakaryocyte estimate is high, indicating an increase in platelet production.
4. Thrombopoietin and interleukin-11 have the greatest effect on recruitment and proliferation of megakaryocytes and their progenitors. Also involved in early progenitor recruitment are interleukin-3 and interleukin-6. Other cytokines and hormones that participate synergistically with thrombopoietin and the interleukins are KIT ligand, also called stem cell factor or mast cell growth factor; granulocyte-macrophage colony-stimulating factor; granulocyte colony-stimulating factor; and erythropoietin.
1. d; 2. d; 3. c; 4. b; 5. d; 6. d; 7. a; 8. a; 9. c; 10. d
1. HGB × 3 = HCT ± 3
15 × 3 = 45 ± 3 (42−48)
2. Hemoglobin can be falsely elevated by lipemia, increased WBC count, or presence of Hb S or Hb C. Hematocrit can be falsely decreased by a short draw in an EDTA-anticoagulated tube causing RBC shrinkage, or contamination of the specimen with intravenous fluids. In the microhematocrit method, false decreases can be caused by improper sealing of the capillary tube, errors in reading the microhematocrit reader, excessive centrifugation, and improper mixing of the specimen.
3. For lipemia, replace lipemic plasma with an equal amount of saline and retest; or use a plasma blank. For increased WBC count, centrifuge the hemoglobin/reagent solution and read the % T of the supernatant (manual procedure). For specimens with Hb S or Hb C, make a 1 : 2 dilution of blood with distilled water and multiply the result by 2. For the microhematocrit, check if the specimen tube was filled to the proper level, and ensure the procedure is performed correctly.
1. MCV = 59 fL; MCH = 18.1 pg; MCHC = 30.7 g/dL.
2. Microcytic, hypochromic red blood cells
3. Examine the patient’s peripheral blood film
1. The sodium concentration could affect the hematocrit. The sample electrolyte concentration is used to correct the measured conductivity prior to reporting hematocrit results. Factors that affect sodium concentration will therefore also affect the hematocrit.
2. A high sodium concentration would falsely decrease the hematocrit.
3. Factors that decrease the hematocrit by this method are low total protein, settling of red blood cells in the collection device, presence of cold agglutinins, and specimen contamination by intravenous solutions.
1. b; 2. c; 3. c; 4. d; 5. d; 6. b; 7. c; 8. c; 9. a; 10. d
1. d; 2. a; 3. d; 4. c; 5. c; 6. Impedance - c; RF - b; optical scatter - a; 7. b; 8. b; 9. c; 10. Abbott CELL-DYN Sapphire - b; Siemens ADVIA 2120i - c; Sysmex XN-1000 - d; Beckman Coulter UniCel DxH 800 - a.
1. The patient’s hemoglobin shows neither anemia or polycythemia; hence it is normal. Red blood cells are normocytic and normochromic with no anisocytosis. The blood picture shows leukocytosis and thrombocytopenia. The mean platelet volume is slightly low, which suggests small average platelet volume. There is no white blood cell (WBC) differential.
2. The platelet count and WBC count should be questioned because of platelet clumping. EDTA-induced pseudothrombocytopenia and pseudoleukocytosis most likely occurred.
3. The specimen should be redrawn in sodium citrate and processed through the automated analyzer. The new WBC and platelet counts should then be adjusted for the sodium citrate dilution by multiplying the results by the dilution factor 10/9 or 1.1. The following are the new results:
a. WBCs for specimen drawn in sodium citrate: (8.4 × 109/L) × 1.1 = 9.2 × 109/L (the corrected WBC count)
b. Platelets for specimen drawn in sodium citrate: (231 × 109/L) × 1.1 = 254 × 109/L (the corrected platelet count)
1. d; 2. c; 3. a; 4. c; 5. b; 6. c; 7. a; 8. b; 9. b; 10. a
1. Bone marrow cellularity, estimated from the core biopsy specimen, or the aspirate if a biopsy specimen is unavailable, provides information on blood cell production.
2. The ratio is 9:1, which indicates myeloid hyperplasia.
3. When a bone marrow aspirate or core biopsy specimen is reviewed, the normal megakaryocyte distribution is 2 to 10 per low-power field. Counts outside these limits are characterized as decreased or increased megakaryocytes. Megakaryocyte morphology is also reviewed for diameter, granularity, and nuclear lobularity.
1. c; 2. b; 3. c; 4. a; 5. d; 6. c; 7. d; 8. b; 9. b; 10. b; 11. b
1. Tube 3 or the least bloody tube.
2. A 1:53 dilution with saline is necessary for a satisfactory cytocentrifuge slide.
4. The most likely diagnosis is bacterial meningitis.
1. b; 2. a; 3. c; 4. b; 5. a; 6. b; 7. c; 8. d; 9. c; 10. a
1. Anemia is not a disease or diagnosis in itself but is the symptom of an underlying disorder. A complete history and physical examination are necessary to help identify the cause(s) of the anemia. If the underlying cause is not determined and corrected, the patient will continue to be anemic. Questions regarding lifestyle, medications, and bleeding history are only some of the questions that should be asked.
2. The reticulocyte count differentiates anemias into those involving impaired production (decreased reticulocyte count) and increased destruction (increased reticulocyte count). Anemia can also be classified on the basis of mean cell volume into normocytic, microcytic, or macrocytic. With that knowledge, appropriate laboratory testing can be ordered to determine the cause.
3. The peripheral blood film yields valuable information about the volume and hemoglobin content of the erythrocytes as well as any abnormal shapes, which may be correlated with specific causes. Some anemias are also associated with white blood cell and/or platelet abnormalities, which may be noted on the blood film.
1. c; 2. b; 3. d; 4. c; 5. c; 6. b; 7. c; 8. d; 9. b; 10. c; 11. d
1. The patient’s results demonstrate a severe hypochromic, microcytic anemia with anisocytosis. There is no evidence of a bone marrow response as there is no polychromasia mentioned in the morphology which does note unspecified poikilocytosis, anisocytosis, hypochromia, and microcytosis, all consistent with the numerical values. The white blood cells are unremarkable in number, distribution, and morphology as are the platelets.
2. Hypochromic, microcytic anemias to be considered include iron deficiency anemia, thalassemia, hemoglobin E disease, sideroblastic anemias, and possibly, anemia of chronic inflammation.
3. Thalassemia and hemoglobin E disease can be eliminated because they are not conditions that would be acquired late in life.
4. Anemia of chronic inflammation could be eliminated in this case because the woman is otherwise healthy. Although iron deficiency anemia is not as common in women after menopause, it is probably the most likely of the remaining possibilities for an anemia that is this severe.
5. Iron studies, including ferritin, would be useful in clarifying the patient’s diagnosis. Assuming that she is iron deficient, the ferritin, total serum iron, and percent saturation should all be decreased, whereas total iron-binding capacity (TIBC) would be expected to be increased. Upon hospitalization, the patient was immediately placed on oxygen while laboratory tests were ordered. With the confirmation by the hospital laboratory of a dangerously low hemoglobin, transfusions were ordered, and the patient received 3 units of packed cells over the first 2 days of hospitalization. The transfusions were administered very slowly so as not to stress her cardiovascular system with added volume. Noting the hypochromic, microcytic blood picture, the physician ordered iron studies on blood specimens drawn before the transfusions. The results were as follows: serum iron decreased, TIBC increased, percent saturation decreased, and ferritin decreased. The possibility of gastrointestinal bleeding as a cause for iron deficiency was investigated. Results of tests for occult blood in the stool were negative. The hospital dietitian assessed the patient’s usual diet of tea, toast, canned soup, and crackers and determined that it was quite inadequate not only in iron, but also in other important nutrients. The physician concluded that the patient’s dietary iron deficiency had developed slowly, which had allowed her to adapt to the exceedingly low hemoglobin level. Furthermore, her low level of activity meant that she rarely experienced the effects of the anemia. She was started on a course of oral iron supplementation and arrangements were made for her to receive one balanced meal daily from the Meals on Wheels program sponsored through a community service organization for senior citizens. She was quite responsible about taking her iron supplements, and her hemoglobin was within the reference interval within 3 months.
1. b; 2. a; 3. d; 4. a; 5. c; 6. a; 7. d; 8. c; 9. b; 10. d; 11. b; 12. d
1. The complete blood count findings for this patient (notably macrocytic, normochromic anemia; pancytopenia; hypersegmentation of neutrophils; and oval macrocytes) were consistent with the physician’s suspicion of megaloblastic anemia as suggested by the clinical findings.
2. Although the relative reticulocyte count was within the reference interval of 0.5% to 2.5%, and the calculated absolute reticulocyte count (approximately 40 × 109/L) was within the reference interval of 20 to 115 × 109/L, the calculated reticulocyte production index was 0.5, which was clearly inadequate to compensate for a substantial anemia (Chapter 14).
3. The patient’s vitamin assays point to a deficiency of vitamin B12, substantiated by an increase in serum methylmalonic acid.
4. Based on these results, a test for intrinsic factor blocking antibodies would be appropriate. However, the physician also inquired further about the patient’s dietary habits and learned that he enjoyed dishes of raw fish obtained from the surrounding lakes. Therefore, the physician ordered a stool analysis for ova and parasites. The study indicated the presence in the stool of both eggs and proglottids of the fish tapeworm Diphyllobothrium latum. The patient was treated with a suitable purgative, and the scolex of the tapeworm was discovered in a stool sample after a single treatment. The patient was counseled on the proper preparation of fresh fish to avoid reinfection. He received injections of cyanocobalamin to replenish his vitamin B12 stores. His hemoglobin returned to normal over the next month, and his neurologic symptoms subsided.
1. d; 2. c; 3. c; 4. b; 5. a; 6. b; 7. c; 8. d; 9. a; 10. c
1. The term used to describe a decrease in all cell lines is pancytopenia.
2. Acquired aplastic anemia should be considered due to the pancytopenia, reticulocytopenia, bone marrow hypocellularity, normal vitamin B12 and folate levels, absence of blasts and abnormal cells in the bone marrow and peripheral blood, normal myelopoiesis and megakaryopoiesis, and history of autoimmune hepatitis.
3. An increase in blasts or reticulin in the bone marrow suggests a diagnosis of myelodysplasia or leukemia.
4. The extent of the patient’s bone marrow hypocellularity, her hemoglobin concentration, and neutrophil and platelet counts place her disorder in the severe aplastic anemia category.
5. Because of her age and the severity of her aplastic anemia, hematopoietic stem cell transplant is the treatment of choice if she has an HLA-identical sibling. If an HLA-identical sibling is not available, an HLA-matched unrelated donor or immunosuppressive therapy (anthymocyte globulin and cyclosporine) may be considered. Blood product replacement should be given judiciously to avoid alloimmunization. In general, red blood cells would be transfused if the patient had symptoms of anemia, whereas platelet transfusions would be given if her platelet count fell below 10 × 109/L.
1. c; 2. d; 3. b; 4. d; 5. b; 6. d; 7. c; 8. d; 9. c; 10. d; 11. a
1. Intravascular hemolysis is suspected in the patient because the color of the urine suggests oxidized hemoglobin.
2. Tests for serum haptoglobin, serum unconjugated (indirect) bilirubin, serum lactate dehydrogenase, plasma hemoglobin, and urine hemoglobin and examination of a peripheral blood film can differentiate the mechanism of hemolysis as fragmentation or macrophage-mediated.
3. Due to the likelihood that the patient had hemoglobinuria, fragmentation hemolysis was suspected. Therefore, the serum haptoglobin would be markedly decreased, while the serum lactate dehydrogenase and plasma hemoglobin levels would be increased, if measured. Routine urinalysis should yield positive results for blood on the test strip with no intact red blood cells in the urine sediment. The serum indirect bilirubin does not increase immediately after an episode of intravascular hemolysis, but should begin to increase within several days. The peripheral blood film may demonstrate schistocytes immediately, but reticulocytosis several days later.
1. a; 2. b; 3. d; 4. b; 5. c; 6. a; 7. d; 8. b; 9. c; 10. c
1. On the basis of the patient’s jaundice and splenomegaly, history of gallstones, family history of anemia, low hemoglobin, increased mean cell hemoglobin concentration and red cell distribution width, and spherocytes and polychromasia on the peripheral blood film, hereditary spherocytosis (HS) is suspected.
2. Additional laboratory tests to confirm HS should demonstrate increased hemolysis (increased serum indirect bilirubin level and lactate dehydrogenase activity, decreased serum haptoglobin level), increased erythropoiesis to compensate for the premature hemolysis (increased reticulocyte count), and the nonimmune nature of the hemolysis (negative result on the direct antiglobulin test). Testing family members to establish a mode of inheritance is desirable. The osmotic fragility test is expected to show increased fragility and the eosin-5’-maleimide (EMA) binding test is expected to show low mean fluorescence intensity of the red blood cells when measured in a flow cytometer. However, special tests are not required for diagnosis of HS in a patient with a familial inheritance pattern and the typical clinical and laboratory findings.
3. HS is an inherited intrinsic hemolytic anemia caused by a mutation that disrupts the vertical protein interactions in the red blood cell (RBC) membrane. Various mutations in five known genes can result in the HS phenotype. The defective membrane protein causes the RBCs to lose unsupported lipid membrane over time due to a local disconnection between transmembrane proteins and the cytoskeleton. The loss of membrane with minimal loss of cell volume results in a decreased surface area-to-volume ratio and the formation of spherocytes. Spherocytes do not have the deformability of normal biconcave discoid RBCs. As the cells repeatedly go through the spleen, they lose more membrane due to splenic conditioning and eventually become trapped in the spleen and removed by the splenic macrophages. The RBC membrane also has abnormal permeability to cations, particularly sodium and potassium, likely due to the disruption of the cytoskeleton by the mutated protein.
1. b; 2. a; 3. a; 4. b; 5. a; 6. d; 7. a; 8. b; 9. b; 10. a; 11. d
1. Many malarial ring forms, with multiple ring forms in individual red blood cells (RBCs), are present in the thin peripheral blood film. Many ring forms and a crescent-shaped gametocyte are also present in the thick peripheral blood film.
2. The high parasitemia, the presence of multiple ring forms in individual RBCs, the crescent-shaped gametocyte on the thick film, and the absence of other parasite stages in the thin and thick peripheral blood films suggest a diagnosis of malaria due to Plasmodium falciparum.
3. The patient had typical symptoms of malaria after a recent 3-week trip to Ghana in West Africa. Malaria is endemic in Ghana, and according to the Centers for Disease Control and Prevention, 52 most of the malaria cases in Ghana are due to P. falciparum.
4. The only forms of P. falciparum that are seen on a peripheral blood film are ring forms and gametocytes, and the latter are characteristically crescent-shaped.
5. Anemia in malaria is due to direct lysis of infected RBCs during schizogony; immune destruction of infected and noninfected RBCs by macrophages in the spleen; and inhibition of erythropoiesis and ineffective erythropoiesis.
1. c; 2. a; 3. b; 4. b; 5. c; 6. b; 7. c; 8. c; 9. c; 10. d; 11. c
1. The WBC can be elevated due to an underlying infection or the autoimmune response itself (inflammation). The MCV is elevated due to the reticulocytosis; the RDW is slightly elevated due to the anisocytosis and occasional schistocytes. The reticulocyte count is increased due to a surge in RBC production in the bone marrow in response to the anemia.
2. In this immune process, spherocytes develop from IgG-sensitized RBCs that have had the immune complex (and a part of the cell membrane) removed by macrophages. The membranes seal and the cells become spherocytic. The red pulp of the spleen eventually entraps the spherocytes, which are less deformable, and macrophages engulf and digest them, thus shortening their life span.
3. The direct antiglobulin test detected an IgG autoantibody which attached to the patient’s RBCs in vivo, which is a hallmark of WAIHA. The IgG autoantibody was also detected in the serum with the antibody screen using the indirect antiglobulin test. The patient’s RBCs, sensitized with IgG autoantibody, were prematurely ingested and destroyed by macrophages (extravascular hemolysis); within the macrophages hemoglobin is degraded to polypeptide chains, iron, and the protoporphyrin ring. The protoporphyrin is converted to unconjugated bilirubin and is transported to the liver where it is conjugated with glucuronic acid to form conjugated bilirubin. When there is excessive hemolysis, the liver cannot process all the excess unconjugated bilirubin that is being formed, so it accumulates in the serum. The excess conjugated bilirubin formed in the liver is excreted through the bile duct to the intestines where it is converted to urobilinogen. Because of the increased urobilinogen produced in the intestines, an increased amount is reabsorbed into the blood, and an increased amount is excreted in the urine. There is also an increase in intravascular hemolysis which liberates lactate dehydrogenase and elevates the level in serum. Free hemoglobin is also liberated and is bound by haptoglobin. The hemoglobin-haptoglobin complex is taken up and degraded by macrophages, resulting in a decrease in serum haptoglobin. When the serum haptoglobin is depleted, the excess hemoglobin accumulates in the plasma. Some is salvaged by hemopexin, but the excess is filtered by the kidney. Some hemoglobin is absorbed by the proximal tubular cells; the iron is removed and converted to hemosiderin. When the tubular cells slough off into the urine, the hemosiderin can be detected. The excess hemoglobin that is not absorbed by the tubular cells flows into the urine resulting in hemoglobinuria.
4. Prednisone is a glucocorticosteroid with immunosuppressive properties, such as reducing WBC response to inflammation and production of inflammatory cytokines. When a patient with an autoimmune disorder is given prednisone, most of these inflammatory mechanisms are switched off or slowed down, which in turn reduces the body’s autoimmune response. The patient probably had an acute form of WAIHA because the symptoms and severe anemia developed suddenly and there was no evidence of an underlying condition.
1. b; 2. a; 3. a; 4. d; 5. d; 6. d; 7. a; 8. c; 9. c; 10. c; 11. c
1. The confirmatory test that should be performed is citrate agar electrophoresis at a pH between 6.0 and 6.2. In the citrate agar test, Hb C is separated from Hb A2, Hb O, and Hb E, and Hb S is separated from Hb D and Hb G (see Figure 27-7).
2. The characteristic morphologic feature on the peripheral blood film is a Hb SC crystal. They appear as fingerlike or quartzlike crystals of dense hemoglobin protruding from the RBC membrane.
3. On the basis of the electrophoretic pattern and RBC morphology, Hb SC disease is likely.
4. With parents of the genotypes SC and AS, 25% of the offspring would have each of the following genotypes: AS, SS, AC, and SC.
1. d; 2. b; 3. c; 4. b; 5. d; 6. b; 7. a; 8. b; 9. c; 10. a; 11. b; 12. d; 13. d; 14. b; 15. c; 16. d
1. The family history revealed a Mediterranean ethnic background; both α- and β-thalassemia are common in the Mediterranean population. The student’s mother had always been anemic, and her gallbladder “attacks” were probably caused by pigmented gallstones (calcium bilirubinate), which resulted from the mild hemolytic anemia of heterozygous β-thalassemia. A cousin on the mother’s side had children with thalassemia major. Because of the family history, it is quite likely that the student has β-thalassemia minor. Note that his mother was periodically given iron therapy. It is a common mistake to treat a thalassemic individual for iron deficiency anemia, especially in areas in which thalassemia is not common in the general population, because both iron deficiency anemia and thalassemia are microcytic, hypochromic anemias.
2. The student had a mild hypochromic (decreased mean cell hemoglobin concentration) and microcytic (decreased mean cell volume) anemia with target cells and basophilic stippling on his peripheral blood film. He had an elevated level of hemoglobin A2, which is a marker for β-thalassemia minor. His serum ferritin level was within the reference interval, which ruled out a diagnosis of iron deficiency anemia.
3. A microcytic, hypochromic anemia could be due to α- or β-thalassemia, Hb E disease or trait, iron deficiency anemia, or, more rarely, sideroblastic anemia (including lead poisoning) or anemia of chronic inflammation (see Figure 19-2). Iron deficiency anemia is the most common of these. Iron studies can differentiate these conditions. An incorrect presumption that a patient has iron deficiency may lead to inappropriate iron therapy or to unnecessary diagnostic procedures.
4. The potential mother should be screened for β-thalassemia trait, and if she is heterozygous for a β-thalassemia gene mutation, the couple should be advised that there is a 25% chance of having a baby with β-thalassemia major (homozygous or compound heterozygous for a β-thalassemia mutation). In addition, there is a 25% chance of having a baby who is homozygous for normal β-globin genes, and a 50% chance of having a baby heterozygous for a β-thalassemia mutation (β-thalassemia trait). Molecular genetic testing of the HBB gene is performed for carrier detection in couples seeking preconception counseling.
1. b; 2. c; 3. a; 4. d; 5. a; 6. c; 7. a; 8. c; 9. d; 10. a; 11. d; 12. b; 13. c; 14. d; 15. c
1. Chronic granulomatous disease.
2. Patient neutrophils are unable to form reactive oxygen species such as hydrogen peroxide.
3. Aggressive treatment of infections and use of antifungal agents have greatly increased survival rates so that the majority of patients survive into adulthood.
4. The majority of cases are X-linked.
1. Because of the reactive monocytosis, the blood film should be examined for possible circulating macrophages.
2. On the edges of the blood film, because macrophages are very large cells.
3. Circulating macrophages indicate sepsis.
4. A buffy coat preparation, which concentrates nucleated cells.
1. a; 2. d; 3. c; 4. b; 5. d; 6. c; 7. d; 8. c; 9. b; 10. c
1. G banding utilizes Giemsa staining to differentiate chromosomes into bands for identification of specific chromosomes. The chromosomes must be pretreated with the proteolytic enzyme trypsin.
2. The mutation is an example of a structural rearrangement between chromosomes 9 and 22, called the Philadelphia chromosome. The Philadelphia chromosome represents a balanced translocation between the long arms of chromosomes 9 and 22. At the molecular level, the gene for ABL1, an oncogene, joins a gene on chromosome 22 named BCR. The result of the fusion of these two genes is a new fusion protein.
3. Fluorescence in situ hybridization (FISH) is a molecular technique that uses DNA or RNA probes labeled directly with a fluorescent nucleotide or with a hapten (e.g., dinitrophenyl, digoxigenin, or biotin). Both the probe and either metaphase or interphase cells are made single-stranded (denatured) and then hybridized together. Cells hybridized with a direct-label probe are viewed with a fluorescence microscope. If the probe was labeled with a hapten, antibodies to the hapten, carrying a fluorescent tag, are applied to the cells. Once the antibodies bind to the RNA or DNA probe, the cells can be viewed using a fluorescence microscope. FISH complements standard chromosome analysis by confirming the G-band analysis and by improving resolution, which allows for analysis at the molecular level.
1. c; 2. d; 3. a; 4. d; 5. a; 6. c; 7. d; 8. c; 9. c; 10. b
1. DNA isolation for the detection of inherited mutations requires whole blood collected in a lavender stopper tube containing EDTA to preserve white blood cells.
2. The correct controls are present and include a positive control (Lane B), a negative control (Lane D), and a “no-DNA” control (Lane E). The no-DNA control is essential when any polymerase chain reaction (PCR) test is performed in the clinical laboratory. This control will demonstrate whether cross-contamination occurred during the setup of the PCR procedure. The no-DNA control region of the gel should lack a banding pattern, as seen in Figure 31-1. If a banding pattern is present in the no-DNA control region or this control is missing, the test must be repeated before reporting patient results.
3. Bands in the patient’s sample (Lane C) appear at 141, 104, and 82 bp.
4. The following band sizes are expected in factor V Leiden DNA analysis:
• Normal: 104 and 82 bp (37 bp is sometimes barely visible, as well)
• Heterozygous: 141, 104, and 82 bp (37 bp is sometimes barely visible, as well)
• Homozygous: 141 and 82 bp
5. The three bands in the patient sample indicate that this patient is heterozygous for the factor V Leiden mutation.
1. d; 2. b; 3. d; 4. b; 5. b; 6. c; 7. a; 8. a; 9. b; 10. b
1. The lymphoid population is the most prominent. Forward scatter demonstrates small to medium-sized cells. These cells are characterized by low side scatter indicative of sparse agranular cytoplasm.
2. The majority of cells express CD19, CD10, and κ light chain. There is also a small population of T cells positive for CD5 and negative for CD19 antigen.
3. Prominent κ light chain expression indicates a monoclonal B-cell population that is characteristic of lymphoma.
1. The low density of CD45 antigen coupled with relatively low side scatter is characteristic of a blast population. Such a prominent blast population can only be seen in acute leukemias.
2. The expression of immature markers (CD34 and HLA-DR) coupled with positivity for myeloid and megakaryoblastic antigens (CD33, CD41, and CD61) is seen in acute megakaryoblastic leukemias.
1. c; 2. b; 3. a; 4. d; 5. b; 6. a; 7. a; 8. a; 9. c; 10. a; 11. b
1. An elevated white blood cell (WBC) count with a left shift suggests a myeloproliferative neoplasm or a leukemoid reaction (reactive neutrophilia). However, in this patient the WBC count was extremely elevated, the left shift was rather deep (presence of promyelocytes and blasts), and basophilia was present, which suggests that a myeloproliferative neoplasm is likely present. Chronic myelogenous leukemia (CML) is the most likely cause of these laboratory findings.
2. The leukocyte alkaline phosphatase (LAP) score is low in CML due to inappropriate LAP synthesis in the secondary granules, whereas LAP is elevated in bacterial infections due to activation of enzyme synthesis.
3. The BCR/ABL1 fusion gene must be identified to confirm the diagnosis of CML. BCR/ABL1 can be demonstrated from a karyotype analysis showing the t(9;22) reciprocal translocation known as the Philadelphia chromosome (Chapter 30), by demonstration of the BCR/ABL1 fusion gene using fluorescence in situ hybridization (Chapter 30), or by demonstration of the BCR/ABL1 fusion mRNA by qualitative reverse transcriptase polymerase chain reaction (Chapter 31). Patients who have complete blood count and differential results that resemble those in CML but test negative for BCR/ABL1 are considered to have atypical CML, and the disorder is classified as myelodysplastic syndrome/myeloproliferative neoplasm (Chapter 34).
4. Cytogenetic studies are likely to show the t(9;22) mutation.
5. The t(9;22) translocation produces the BCR/ABL1 chimeric gene, which is observed in four primary molecular forms that produce three versions of the BCR/ABL chimeric protein: p190, p210, and p230.
6. First-line therapy for CML is the tyrosine kinase inhibitor imatinib mesylate (Gleevec). Allogeneic stem cell transplantation should be considered for all CML patients, because it is the only potentially curative treatment for CML. However, few CML patients qualify for allogeneic stem cell transplantation, because most do not meet the criteria for low risk: age younger than 40 years, disease in the chronic phase, transplantation within 1 year of diagnosis, and availability of an HLA-matched donor. For those patients who qualify for allogeneic stem cell transplantation, imatinib is used to induce remission prior to transplant, to treat minimum residual disease, and to provide rescue therapy if the transplant fails. Imatinib is continued as lifelong therapy until drug resistance is detected.
7. The majority of cases of imatinib resistance result from two primary causes: acquisition of additional BCR/ABL1 mutations and expression of point mutations in the adenosine triphosphate (ATP) binding site. Additional BCR/ABL1 mutations can occur through the usual translocation of the remaining unaffected chromosomes 9 and 22, which converts the hematopoietic stem cell from heterozygous to homozygous for the BCR/ABL1 mutation. A double dose of BCR/ABL1 can also be acquired from gene duplication during mitosis and accounts for 10% of secondary mutations. An additional BCR/ABL1 mutation will double the tyrosine kinase activity, which makes the imatinib dosage inadequate. In these cases higher dosages of imatinib will restore remission in most patients. Over 60 mutations have been identified in the ATP binding site, and these account for the remaining 50% to 90% of secondary mutations. Mutations in the ATP binding site reduce the binding affinity of imatinib, producing some level of resistance.
1. b; 2. c; 3. d; 4. c; 5. c; 6. c; 7. b; 8. d; 9. a; 10. c
1. The differential diagnosis of patients with pancytopenia should include megaloblastic anemia (vitamin B12 or folate deficiency), aplastic anemia, liver disease, alcoholism, and myelodysplastic syndrome (MDS).
2. The probable diagnosis is MDS.
3. This patient’s MDS should be classified as refractory anemia with ringed sideroblasts (RARS).
1. d; 2. a; 3. b; 4. b; 5. c; 6. c; 7. a; 8. d; 9. c; 10. c
1. Due to the presence of blasts on the peripheral blood film, the most likely diagnosis is acute leukemia. The thrombocytopenia and anemia support that diagnosis. According to the WHO classification, ≥ 20% blasts in the bone marrow is required for diagnosis of acute leukemia; an exception to this criterion are those cases that have specific genetic abnormalities (delineated in the WHO classification) that are diagnostic, regardless of blast count. Acute lymphoblastic leukemia (ALL) is more common in children. Immunophenotyping by flow cytometry determines the lineage and maturation stage of the blasts. Testing for genetic abnormalities is required for diagnosis and prognosis.
2. This child has clinical and laboratory features indicative of a favorable prognosis: young age, a white blood cell count less than 20 x 109/L (i.e., low tumor burden), and hyperdiploidy. The strongest predictor of patient outcome is the presence of certain genetic abnormalities; the immunophenotype also contributes to the prognosis.
3. Hyperdiploidy carries a favorable prognosis in B-cell ALL in children.
1. b; 2. b; 3. a; 4. d; 5. b; 6. d; 7. c; 8. c; 9. b; 10. b; 11. b; 12. b
1. Diffuse large B-cell lymphoma (DLBCL).
2. This lesion is expected to show exclusive (clonal) κ or λ light chain expression. Flow cytometry is particularly sensitive in detecting surface and cytoplasmic immunoglobulin light chains and is commonly used to confirm clonality of lymphoproliferative disorders. In addition, other pan–B-cell markers can be studied by flow cytometry (e.g., CD19, CD22, and FMC7 antigens) to demonstrate the B-cell origin of this lymphoma.
3. Most often DLBCL presents as a localized disease involving a group of lymph nodes. Bone marrow involvement is rare at presentation; however, it can occur later in the course of the disease.
1. d; 2. c; 3. d; 4. d; 5. b; 6. b; 7. b; 8. a; 9. c; 10. a
1. Given the family history, this may be an inherited condition, although pregnancy is an independent risk factor for thrombosis.
2. Thrombosis is probably caused by the deficiency of a coagulation inhibitor such as protein C, protein S, or antithrombin. It may be caused by a procoagulant gain-of-function mutation such as the factor V Leiden mutation or the prothrombin G20210A mutation.
1. b; 2. c; 3. b; 4. d; 5. b; 6. d; 7. b; 8. c; 9. a; 10. a
1. The combination of thrombocytopenia and prolonged prothrombin time (PT) and partial thromboplastin time (PTT) indicate probable liver disease. In the absence of a full medical history, the patient’s hemarthroses and description of himself as a “bleeder” lead to the presumption of hemophilia, possibly hemophilia A. It is possible that he contracted hepatitis C from an untreated blood product. Treatment of factor concentrates for viral disease began in 1984. Prior to 1984 most hemophilia patients eventually developed hepatitis B or C from factor concentrates. Hepatitis A is also a possibility. Liver disease may be confirmed using bilirubin and liver enzyme assays.
In advanced liver disease, poor liver circulation causes pressure in the portal circulation. This enlarges the spleen (splenomegaly). The enlarged spleen sequesters and clears platelets more rapidly than normal, a condition called hypersplenism, which causes thrombocytopenia. In most cases, platelet function is reduced. This reduced platelet function can be demonstrated in the laboratory using platelet aggregometry and is the reason for the patient’s epistaxis.
Vitamin K deficiency is also a possibility. To differentiate vitamin K deficiency from liver disease, a factor V and VII activity assay is performed. In vitamin K deficiency factor VII activity is reduced but factor V activity is normal. In liver disease, both are reduced.
2. In early liver disease the vitamin K–dependent factors II (prothrombin), VII, IX, and X are produced with diminished function. This can be corrected with a trial dose of oral or intravenous vitamin K. In people with true vitamin K deficiency secondary to an altered diet, the vitamin K therapy corrects bleeding and normalizes the PT and PTT, but in liver disease vitamin K may not have a lasting effect. This is because the liver cannot process the vitamin K normally.
In addition to vitamin K therapy, thawed frozen plasma (FP) transfusion at 1 to 2 units/day is effective in supplementing the liver’s production of all the coagulation factors. Cryoprecipitate may also be used to raise the fibrinogen concentration, and platelet concentrate may be used if the platelet count drops to below 50, 000/µL and there is continued evidence of mucocutaneous bleeding.
Administration of vitamin K, FP, cryoprecipitate, and platelets does not cure liver disease; these therapies only treat the bleeding symptoms. Additional treatment may include antibiotics, anti-virals, and anti-inflammatory drugs.
1. c; 2. d; 3. b; 4. c; 5. b; 6. b; 7. d; 8. a; 9. a; 10. c; 11. c
1. The following tests for congenital and acquired risk factors are included in a thrombophilia profile. Results for the items with asterisks are valid only when the test is performed 10 to 14 days after termination of antithrombotic therapy or resolution of a thrombotic event.
• Lupus anticoagulant profile*
• Prothrombin G20210A mutation
• Activated protein C resistance*
• Factor V Leiden mutation (confirmatory for activated protein C resistance)
• Anticardiolipin antibodies by immunoassay
• Protein C functional assay and follow-up immunoassay*
• Protein S functional assay and follow-up immunoassay*
• Antithrombin functional assay and follow-up immunoassay*
2. The most common acquired thrombotic risk factors are antiphospholipid antibodies and lupus anticoagulant, and these are most often implicated in a thrombotic event.
3. Patients with thrombotic risk factors may be instructed to avoid situations and practices that may trigger thrombosis, such as immobilization, smoking, and use of oral contraceptives or hormone replacement therapy. They may be provided with prophylactic antithrombotic therapy at times when circumstances increasing thrombotic risk cannot be avoided, such as when undergoing orthopedic surgery.
1. b; 2. a; 3. d; 4. c; 5. a; 6. b; 7. a; 8. a; 9. c; 10. d; 11. d; 12. d; 13. d; 14. c
1. Yes, the heparin is significant.
2. Heparin-induced platelet aggregation assay, serotonin release assay, or enzyme-linked immunosorbent assay (ELISA) should be ordered.
An ELISA was performed to look for the presence of heparin-induced antibodies. Results gave an optical density of 0.650, with a reference interval of less than 0.400 optical density. The patient was found to have clinically significant levels of heparin-induced antibodies.
The patient underwent an above-knee amputation of her right leg. No heparin or low molecular weight heparin was used during or after the procedure. The grafting surgeries were successful, and the patient recovered.
1. b; 2. d; 3. b; 4. c; 5. b; 6. b; 7. b; 8. c; 9. d; 10. a
1. Storage pool disease, aspirin-like defects, and use of antiplatelet agents such as aspirin are possibilities.
2. Storage pool disease or aspirin-like defects seem most likely.
3. Based on the results of the quantitative test for adenosine triphosphate release, the likely cause is dense granule storage pool disease.
These results were confirmed by the findings of electron microscopy of the patient’s platelets, which revealed the absence of detectable dense granules. Because the patient’s bleeding problems are due to an inherited abnormality that typically results in only mild bleeding problems, the patient was counseled to avoid antiplatelet agents, particularly aspirin, since they are known to exacerbate the bleeding problems encountered by patients with dense granule deficiency.
1. d; 2. a; 3. a; 4. b; 5. c; 6. c; 7. d; 8. b; 9. d; 10. a
1. The laboratory director questioned the phlebotomist about the problem. The phlebotomist admitted that he had erroneously collected blood in a red- and gray-stoppered “tiger-top” tube and, responding to the patient’s remark, had immediately poured the blood into a blue-stoppered tube for analysis. He thought the specimen would be okay because it had not clotted yet.
2. The red and gray marbleized stopper designates a serum separator tube. The phlebotomist poured the blood into the blue-stoppered tube before it had begun to clot; however, the activator from the tiger-top tube shortened the clotting time on the prothrombin time (PT) test, thus causing an erroneously short PT and low international normalized ratio (INR).
3. Unexpectedly short PTs during oral anticoagulant therapy are generally indicators of patient non-compliance to the drug regimen. The second most common circumstance that affects the PT is dietary changes, most often an increased intake of vitamin K–rich foods such as green leafy vegetables, liver, or avocado. In this instance the patient had been fully compliant, carefully following the prescribed dosage and timing, and her diet had not changed. These facts led the laboratory director to consider a specimen collection error.
Specimens collected in 3.2% sodium citrate may be stored for up to 24 hours at room temperature without a change in the PT. However, specimens stored at higher than 24° C deteriorate rapidly, which causes prolongation of the PT and increase in the INR. Prolonged storage at 2° to 4° C may activate factor VII, which slightly shortens the PT and slightly decreases the INR.
Many serum separator tubes contain particulate materials that hasten in vitro clotting. Core laboratory managers select these tubes to improve test result turnaround time when the required sample is serum. When blood is collected into a series of tubes that includes a blue-stoppered tube for hemostasis testing, the blue-stoppered tube should be filled first or should be filled after a tube without additives. It should not be filled immediately after filling a serum separator tube with clot activators, because the activators may carry over to the hemostasis specimen and affect test results.
In this case, an observant patient provided clues that led to identification of the pre-analytical error. The phlebotomist was carefully counseled about the effects of tube additives on hemostasis tests.
1. c; 2. b; 3. a; 4. b; 5. a; 6. a; 7. b; 8. b; 9. b; 10. d; 11. b; 12. d; 13. d; 14. c
1. The increase in anticoagulation could be caused by a change in diet, dietary supplements, or drugs. Any new drug that interferes with the cytochrome oxidase P-450 enzyme 2C9 pathway could reduce warfarin breakdown and excretion, and increase its effectiveness.
2. Determine what has caused the change in warfarin efficacy and eliminate it if possible, adjust the warfarin dosage, or give vitamin K orally or intravenously to stop bleeding if necessary.
3. The chromogenic factor X assay.
1. c; 2. c; 3. c; 4. a; 5. d; 6. b; 7. d; 8. c; 9. b; 10. b; 11. a; 12. a; 13. a; 14. d; 15. d
1. No. The description of the sample and the instrument flags indicating lipemia should alert the operator to a potentially invalid test result because lipemia is known to cause erroneous results on some photo-optical coagulation analyzers.
2. Two options are available to negate the effect of the lipemia and obtain valid test results:
a. Remove the lipids from the plasma by high-speed centrifugation or ultracentrifugation.
b. Perform testing using an endpoint detection method that is not susceptible to lipemia in the sample, such as mechanical clot detection.
3. Because the patient history includes previous surgical procedures without bleeding symptoms and there is no other indication of abnormal bleeding tendencies for this patient, it is probably safe to consider that the prolonged prothrombin time and activated partial thromboplastin time results are due to the lipemic nature of the sample. The patient would most likely not be at risk for bleeding during the surgery, and it would be anticipated that repeat testing using one of the options listed previously would yield test results within the reference interval.
1. b; 2. d; 3. a; 4. c; 5. b; 6. c; 7. c; 8. a; 9. d; 10. a
1. Yes, the newborn reference interval for hemoglobin is 16.5 to 21.5 g/dL and for the hematocrit is 48% to 68%.
2. These values are normal for newborns. Erythrocytes of a newborn are markedly macrocytic. There may be 2 to 24 nucleated red blood cells on the first postnatal day, but they are not present by day 5. The polychromasia reflects the reticulocytosis that persists for about 4 days.
3. These values are within the reference intervals for newborns. The white blood cell count of a newborn fluctuates a great deal with a reference interval of 9.0 to 37.0 × 109/L, and leukocytosis without evidence of infection is common. The differential may show an increase in neutrophils rather than the lymphocyte predominance seen after 2 weeks. In this case the neutrophils and lymphocytes were present in equal amounts, but no immature neutrophils were seen.
1. d; 2. b; 3. a; 4. b; 5. c; 6. c; 7. d; 8. b; 9. a; 10. c