Secrets – Pediatric: Hematology

Secrets – Pediatric: Hematology

BONE MARROW FAILURE
1. What are the types of bone marrow failure?
Bone marrow failure is manifested by pancytopenia or, at times, by cytopenia of a single cell type.
It can be acquired (acquired aplastic anemia) or inherited/genetic (e.g., Fanconi anemia, Kostmann syndrome, Diamond-Blackfan anemia, amegakaryocytic thrombocytopenia, thrombocytopenia-absent radius).

Chirnomas SD, Kupfer GM: The inherited bone marrow failure syndromes, Pediatr Clin North Am
60:1291–1310, 2013.
Hartung HD, Olson TS, Bessler M: Acquired aplastic anemia in children, Pediatr Clin North Am 60: 1311–1336, 2013.

2. What are the causes of acquired aplastic anemia?
After careful exclusion of the known causes listed below, 80% of cases remain classified as idiopathic. A variety of associated conditions include the following:
Radiation Immune diseases
• Eosinophilic fasciitis
• Hypogammaglobulinemia
Drugs and chemicals
• Regular: Cytotoxic (as in treatment for malignancy), benzene
• Idiosyncratic: Chloramphenicol, anti-inflammatory drugs, antiepileptics, gold, nifedipine
Viruses
• Epstein-Barr virus (EBV)
• Hepatitis (primarily B)
• Parvovirus (in immunocompromised hosts)
• Human immunodeficiency virus (HIV)
Thymoma Pregnancy
Paroxysmal nocturnal hemoglobinuria Preleukemia

Shimamura A, Guinana EC: Acquired aplastic anemia. In Nathan DG, Orkin SD, Ginsburg D, Look AT, editors: Nathan and Oski’s Hematology of Infancy and Childhood, ed 6. Philadelphia, 2003, WB Saunders, p 257.

3. What is the definition of severe aplastic anemia?
SEVERE disease includes a hypocellular bone marrow biopsy (<30% of the normal hematopoietic cell density for age) and decreases in at least 2 out of 3 peripheral blood counts: neutrophil count <500 cells/mm3, platelet count <20,000 cells/mm3, or reticulocyte count <1% after correction for the hematocrit. Categorization has important prognostic and therapeutic
implications.
4. What are the treatments and prognosis for children with aplastic anemia?
In the absence of definitive treatment, <20% of children with severe acquired aplastic anemia
survive for >2 years. When bone marrow transplantation is performed using a human leukocyte antigen (HLA)-identical sibling donor, the 2-year survival rate exceeds 85%. The usual approach to the newly
diagnosed child with severe acquired aplastic anemia is to perform bone marrow transplantation if there is an HLA-identical sibling to serve as the donor.

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About 80% of children with severe aplastic anemia do not have a sibling donor for bone marrow transplantation. These children receive medical therapy, usually the combination of antithymocyte globulin, cyclosporine, and hematopoietic growth factors, such as granulocyte-macrophage
colony-stimulating factor or granulocyte colony-stimulating factor. Two-year response and survival rates for combination medical therapy now exceed 80% in children.

Scheinberg P, Wu CO, Nunez O, et al: Long-term outcome of pediatric patients with severe aplastic anemia treated with antithymocyte globulin and cyclosporine, J Pediatr 153:814, 2008.

5. What is the probable diagnosis of a 6-year-old child with pancytopenia, short stature, abnormal thumbs, and areas of hyperpigmentation?
Fanconi anemia, or constitutional aplastic anemia, is a genetic disorder in which numerous physical abnormalities are often present at birth, and aplastic anemia occurs around the age of 5 years.
The more common physical abnormalities include hyperpigmentation, anomalies of the thumb and radius, small size, microcephaly, and renal anomalies (e.g., absent, duplicated, or pelvic horseshoe kidneys). Patients with Fanconi anemia are also susceptible to leukemia and epithelial carcinomas.
6. How is the diagnosis of Fanconi anemia made?
Chromosomal breakage analysis, for example with diepoxybutane (DEB), can be used to make the diagnosis, and molecular diagnosis can confirm the diagnosis and be used to test relatives. In studies of peripheral blood lymphocytes, a high percentage of patients with Fanconi anemia will have chromosomal breaks, gaps, or rearrangements. Many genes causing the Fanconi anemia syndrome have now been identified, and molecular diagnosis has assumed increasing importance as studies linking genotype and phenotypes such as aplastic anemia and leukemia can be analyzed.

De Rocco D, Bottega R, Cappelli E, et al: Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology, Haematol 99:1022–1031, 2014.

7. A 1-year-old child presents with pallor and lethargy and is found to have a normocytic anemia (hemoglobin 3.5 g/dL). The white blood cell (WBC) and platelet count are normal, and the exam is otherwise unremarkable. The reticulocyte count is 0.2%. What are two possible causes of this clinical scenario?
Transient erythroblastopenia of childhood (TEC) and Diamond-Blackfan anemia. Both are disorders of red-cell production that occur during early childhood. Both disorders are characterized by a low hemoglobin level and an inappropriately low reticulocyte count. The bone marrows of patients with these conditions may be indistinguishable, showing reduced or absent erythroid activity in both cases.
8. Why is distinguishing between the two conditions extremely important?
TEC is a self-limited disorder, whereas Diamond-Blackfan syndrome usually requires lifelong treatment.
9. How are the two conditions diagnosed?
Age of presentation: Although there is an overlap in the age of presentation, Diamond-Blackfan syndrome commonly causes anemia during the first 6 months of life, whereas TEC occurs more frequently after the age of 1 year.
Red cells: The red cells in patients with Diamond-Blackfan syndrome have fetal characteristics that are useful for distinguishing this disorder from TEC, including increased mean cell volume, elevated level of hemoglobin F, and presence of i antigen.
Adenosine deaminase: The level of adenosine deaminase may be elevated in patients with Diamond- Blackfan syndrome but normal in children with TEC.
Mutations: Twenty-five percent of white patients with Diamond-Blackfan anemia have been found to have mutations in the gene for ribosomal protein S19, and molecular diagnosis for these mutations is very helpful when positive. Recently additional gene mutations have been identified in Diamond- Blackfan anemia. These also affect ribosomal proteins. In total, about three-fourths of Diamond- Blackfan patients can be identified by mutational analysis.

Viachos A, Ball S, Dahl N, et al: Diagnosing and treating Diamond Blackfan anemia: results of an international clinical conference, Br J Hematol 142:859–876, 2008.

10. What is Kostmann syndrome?
Kostmann syndrome is severe congenital neutropenia. At birth, or shortly thereafter, very severe neutropenia (absolute neutrophil count of 0 to 200/mm3) is noted, often at the time of significant bacterial infection (e.g., deep skin abscess, pneumonia, sepsis). Even with antibiotic treatment, there is a high mortality during infancy unless granulocyte colony-stimulating factor (G-CSF) therapy is used to elevate the neutrophil count. Some recipients of G-CSF have survived the infection risk but have developed myelodysplastic syndrome or acute myeloid leukemia.
Therefore, individualized judgment and monitoring are essential in G-CSF treatment of severe congenital neutropenia. An alternative treatment is bone marrow transplantation from an
HLA-identical sibling donor. Patients with Kostmann syndrome may have mutations in ELANE
or HAX1 genes.

Boztug K, Klein C: Genetic etiologies of severe congenital neutropenia, Curr Opin Pediatr 23:21–26, 2011.

11. You are asked to evaluate a 9-month-old male with eczema and recurrent respiratory infections who was found to be thrombocytopenic. What is the most likely diagnosis?
Wiskott-Aldrich syndrome is an X-linked disease characterized by eczema, microthrombocytopenia, and combined B-cell and T-cell immunodeficiency. It is caused by mutations in the WAS gene.

Nurden P, Nurden A: Congenital disorders associated with platelet dysfunctions, Thromb Haemost 99:253–263, 2008.

12. A 4-year-old with failure to thrive and chronic diarrhea has a normal sweat test but is noted to have neutropenia on a routine complete blood count (CBC). What is the most likely diagnosis?
Shwachman-Diamond syndrome is characterized by exocrine pancreatic dysfunction (causing steatorrhea), skeletal abnormalities, growth retardation, and bone marrow insufficiency leading to neutropenia. It may initially be misdiagnosed as cystic fibrosis because of overlapping symptoms. Genetic testing for mutations in the SBDS gene is diagnostic.

Ganapathi K, Shimamura A: Ribosomal dysfunction and inherited marrow failure, Br J Hem 141:376–387, 2008.

CLINICAL ISSUES
13. What is the hemoglobin value below which children are considered to be anemic (lower limit of normal)?
• Newborn (full term): 13.0 g/dL
• 3 months: 9.5 g/dL
• 1 to 3 years: 11.0 g/dL
• 4 to 8 years: 11.5 g/dL
• 8 to 12 years: 11.5 g/dL
• 12 to 16 years: 12.0 g/dL

Dallman P, Siimes MA: Percentile curves for hemoglobin and red-cell volume in infancy and childhood, J Pediatr
94:26–31, 1979.

14. When does the physiologic anemia of infancy occur?
Physiologic anemia occurs at 8 to 12 weeks in full-term infants and at 6 to 8 weeks in premature infants. Full-term infants may exhibit hemoglobin levels as low as 9 g/dL at this time, and very premature infants may have levels as low as 7 g/dL.
15. Why does the physiologic anemia of infancy occur?
The mechanisms responsible for physiologic anemia are not completely understood. Red blood cell (RBC) survival time is decreased in both premature and full-term infants. Furthermore, the ability to increase erythropoietin production in response to ongoing tissue hypoxia is somewhat blunted, although the response to exogenous erythropoietin is normal.

16. In what settings of shortened RBC survival can the reticulocyte count be normal or decreased?
As a rule, the reticulocyte count is elevated in conditions of shortened RBC survival (e.g., hemoglobinopathies, membrane disorders, immune hemolysis) and decreased in anemias that are characterized by impaired RBC production (e.g., iron deficiency, aplastic anemia). The reticulocyte count may be unexpectedly low in a setting of shortened RBC survival in the following conditions:
• Aplastic or hypoplastic crisis is occurring at the same time, as is seen in patients with human parvovirus B19 infection.
• An autoantibody in immune-mediated hemolysis reacting with antigens that are present on reticulocytes leads to increased clearance of these cells.
• In patients in chronic states of hemolysis, the marrow may become unresponsive as a result of micronutrient deficiency (e.g., iron, folate) or because of a reduction in erythropoietin production, as is seen in patients with chronic renal failure.
17. How does the pathophysiology of anemia differ in chronic and acute infection? Chronic infection and other inflammatory states impair the release of iron from reticuloendothelial cells, thereby decreasing the amount of this necessary ingredient that is available for RBC production. The lack of mobilizable iron may be the result of the action of proinflammatory cytokines (e.g., interleukin -1 [IL-1], tumor necrosis factor [TNF]-alpha). Giving additional iron under these circumstances further increases reticuloendothelial iron stores and does little to help the anemia.
Acute infection may cause anemia through a variety of mechanisms, including bone marrow suppression, shortened RBC lifespan, red-cell fragmentation, and immune-mediated RBC destruction.
18. Describe the differential diagnosis for children with splenomegaly and anemia. Key question: Is the anemia the cause of the splenomegaly or is the splenomegaly the cause of the anemia?
Anemia-causing splenomegaly
• Membrane disorders
• Hemoglobinopathies
• Enzyme abnormalities
• Immune hemolytic anemia
Splenomegaly-causing anemia
• Cirrhotic liver disease
• Cavernous transformation of portal vessels
• Storage diseases
• Persistent viral infections
19. What is the significance of a leukemoid reaction?
A leukemoid reaction usually refers to a WBC count of >50,000/mm3 and an accompanying shift to the left (i.e., the differential count shows an increase in immature cells). Causes include bacterial sepsis, tuberculosis, congenital syphilis, congenital or acquired toxoplasmosis, and
erythroblastosis fetalis. Infants with Down syndrome may also have a leukemoid reaction that is often confused with acute leukemia during the first year of life.
20. Name the three most common causes of eosinophilia in children in the United States.
Eosinophilia, which is usually defined as more than 10% eosinophils or an absolute eosinophil count of 1000/mm3 or greater, is most commonly seen in three atopic conditions: atopic dermatitis, allergic rhinitis, and asthma.
21. What conditions are associated with extreme elevations of eosinophils in children?
• Visceral larval migrans (toxocariasis)
• Other parasitic disease (trichinosis, hookworm, ascariasis, strongyloidiasis)
• Eosinophilic leukemia
• Hodgkin disease
• Drug hypersensitivity
• Idiopathic hypereosinophilic syndrome

22. A 14-month-old child presents symptoms including marked cyanosis, lethargy, and normal oxygen saturation by pulse oximetry after drinking from a neighbor’s well. What is the likely diagnosis?
Methemoglobinemia should always be considered when a patient presents symptoms of cyanosis without demonstrable respiratory or cardiac disease. Methemoglobin is produced by the oxidation of ferrous iron in hemoglobin into ferric iron. Methemoglobin cannot transport oxygen. Normally, it constitutes <2% of circulating hemoglobin. Oxidant toxins (e.g., antimalarial drugs, nitrates in food or well water) can dramatically increase the concentration. Patients with cyanosis as a result of
methemoglobinemia can have normal oxygen saturation as measured by pulse oximetry because the oximeter operates by measuring only hemoglobin that is available for saturation.
23. What is the treatment for methemoglobinemia?
In an acute situation in which levels of methemoglobin are >30%, treatment consists of 1 to 2 mg/kg of 1% methylene blue administered intravenously over 5 minutes and repeated in 1 hour if levels have not fallen to normal. Failure to respond to therapy should raise the possibility of glucose-6-phosphate-
dehydrogenase (G6PD) deficiency, which prevents the conversion of methylene blue to the metabolite that is active in the treatment of methemoglobinemia. In these cases, hyperbaric oxygen therapy or exchange transfusion may be necessary.
24. Why are infants at greater risk for the development of methemoglobinemia?
• Antioxidant defense mechanisms (e.g., soluble cytochrome b5 and NADH-dependent cytochrome b5 reductase) are 40% lower in infants than teenagers.
• An infant’s intestinal pH is relatively alkaline as compared with older children’s. If nitrates are ingested (e.g., from fertilizer-contaminated well water), this higher pH more readily allows bacterial conversion of nitrate to nitrite, which is a potent oxidant.
• Infants are more susceptible to various oxidant exposures: nitrate reductase from foods such as undercooked spinach, menadione (vitamin K3) for the prevention of neonatal hemorrhage, over-the- counter teething preparations with benzocaine, and metoclopramide for gastroesophageal reflux.

Bunn HF: Human hemoglobins: Normal and abnormal. In Nathan DG, Orkin SH, editors: Nathan and Oski’s Hematology of Infancy and Childhood, ed 5. Philadelphia, 1998, WB Saunders, p 729.

25. What are the critical steps in planning for a teenager with a chronic hematologic condition to transition to adult-oriented health care?
All teenagers with chronic conditions face challenges when transitioning to adult-oriented health care. There are several steps recommended to ease the transition for these potentially complex patients.
• Begin planning early! Collaborate with the patient and family to create a written health care transition plan by age 14 that includes what services will be needed, who will provide them, and how they will be financed. This should be updated annually until the patient successfully transitions.
• Encourage pediatric patients to begin to assume developmentally appropriate responsibilities for their care (scheduling appointments, calling for refills, etc.).
• Identify a health-care professional who assumes responsibility for care coordination and future planning and can partner with the patient and family through the transition to ensure care is uninterrupted.
• Maintain an up-to-date health-care summary to communicate the pertinent medical history of the patient to their new providers.

American Academy of Pediatrics, American Academy of Family Physicians, American College of Physicians-American Society of Internal Medicine: A consensus statement on health care transitions for young adults with special health care needs, Pediatrics 110:1304–1306, 2002.

COAGULATION DISORDERS
26. What features on history or physical examination help pinpoint the cause of a bleeding problem?
• Platelet problems: Although there can be considerable overlap, in general, platelet problems result in petechiae, especially on dependent parts of the body and mucosal surfaces. Additional

manifestations of platelet disorders include epistaxis, hematuria, menorrhagia, and gastrointestinal (GI) hemorrhages.
• Coagulation factor deficiencies or platelet problems: Ecchymoses are suspicious for coagulation factor deficiencies or platelet problems when they occur in unusual areas, are out of proportion with the extent of described trauma (also seen in child abuse), or are present in different stages of healing. Delayed bleeding from old wounds and extensive hemorrhage (particularly into joint spaces or after immunizations) are also suggestive of coagulation protein disorders.
• Disseminated intravascular coagulation (DIC): Bleeding from multiple sites in an ill patient
is worrisome for DIC. If a patient has tolerated tonsillectomy and/or adenoidectomy or extraction of multiple wisdom teeth without major hemorrhage, a significant inherited bleeding disorder
is unlikely.
27. What do the activated partial thromboplastin time (aPTT) and the prothrombin time (PT) measure in the basic clotting cascade?
See Figure 9-1.

Anticoagulants

TFPI

P-C/S

AT-III P-C/S AT-III

Figure 9-1. Simplified pathways of blood coagulation. The area inside the dotted line is the intrinsic pathway measured by the activated partial thromboplastin time (aPTT). The area inside the solid line is the extrinsic pathway, measured
by the prothrombin time (PT). The area encompassed by both lines is the common pathway. AT-III, Antithrombin III; F, factor; HMWK, high-molecular-weight kininogen; P-C/S, protein C/S; PL, phospholipid; TFPI, tissue factor pathway inhibitor. (Adapted from Montgomery RR, Scott JP: Hemostasis. In Behrman RE, Kliegman RM, Jenson HB, editors: Nelson Textbook of Pediatrics, ed 16. Philadelphia, 2000, WB Saunders, 2000.)

28. What are the possible causes of a prolonged aPTT and PT?
See Table 9-1.
29. What is the INR?
The international normalized ratio (INR), introduced in an attempt to standardize the PT, results from
a calculation in which an individual patient’s PT test value is divided by the laboratory’s pooled normal plasma standard PT, then raised to an exponent applicable to each individual PT-initiating reagent available. Its utility is in monitoring Coumadin use in that the reported value has clinical utility regardless of which laboratory performed the PT test. The INR for individuals with normal coagulation proteins not receiving Coumadin therapy is 1.0 (+/ 0.1 to 0.2 based on that lab’s upper and lower range). For those receiving Coumadin therapy, the desired INR varies with the condition being treated, but it is often 2.0 to 3.0.

Table 9-1. Common Causes of Prolonged Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT)

SCENARIO COMMON AND IMPORTANT CAUSES
COMMENTS
Prolonged PT Vitamin K deficiency Liver disease Warfarin
Factor VII deficiency Disseminated intravascular
coagulation (DIC) Isolated PT elevation is sensitive marker early in DIC development
Prolonged aPTT Von Willebrand disease Hemophilia (factor VIII, IX, or XI
deficiency) Heparin
Antiphospholipid antibodies (associated with minor infections or, rarely, autoimmune or thromboembolic disease) Rare deficiencies of factor XII, congenital abnormalities of the receptor for vitamin B12-intrinsic factor complex
Gastric mucosal defects that interfere with the secretion of intrinsic factor or phosphokinase may also elevate aPTT but are not clinically significant
Half of children with prolonged aPTT do not have a bleeding disorder
Prolonged PT and aPTT Heparin Warfarin Liver disease DIC Fibrinogen measurement can help distinguish among liver disease and DIC (decrease in fibrinogen) and vitamin K (no decrease in fibrinogen)
From SAVAGE W, Takemoto C: Bleeding and bruising, Contemp Pediatr 26:66, 2009.

30. What are the frequency and the inheritance patterns of common bleeding disorders?
• von Willebrand disease: This is the most common coagulopathy and it is autosomal dominant in the majority of cases. Frequency is estimated to be between 1 in 100 to 1 in 500.
• Factor VIII deficiency (hemophilia A) and factor IX deficiency (hemophilia B): These conditions are inherited in an X-linked pattern so that females are carriers and males are affected. Inquiry about affected maternal male first cousins or uncles is appropriate. In general, heterozygotes
for clotting factor deficiencies are not clinically affected. Factor VIII deficiency is more common (1 in 5000) than factor IX deficiency, affecting 80% to 85% of all patients with clinically diagnosed factor deficiency.

Journeycake JM, Buchanan GR: Coagulation disorders, Pediatr REV 24:83–91, 2003.

31. Why is the lack of a family history of bleeding problems only moderate evidence against the likelihood of hemophilia A in a patient?
The abnormal factor VIII gene responsible for hemophilia A exhibits marked heterogeneity, and up to a third of cases (either the immediate-carrier mother or the son himself) may have developed a spontaneous mutation. Molecular diagnosis of the most common mutation in severe factor VIII deficiency—a gene inversion in the distal portion of the gene in the affected male, the mother, and maternal relatives—may help the physician with understanding the family history.
32. What are the clinical classifications for hemophilia A and B?
• Severe: <1% factor VIII or IX activity; spontaneous bleeding common; bleeding often involves joints, soft tissue, brain (intracranial hemorrhages in neonates), postcircumcision; most common type (50% to 70% of cases).
• Moderate: 1% to 5% factor VIII or IX activity; bleeding after minor trauma, but not usually spontaneous; may involve joints and soft tissue, but less commonly central nervous system (CNS) or postcircumcision; least common type (10% of cases)

• Mild: 6% to 30% factor VIII or IX activity; bleeding only after major trauma or surgery; joint and soft tissue involvement, but uncommon after circumcision; more common than moderate type (30% to 40% of cases)

Sharathkumar AA, Pipe SW: Bleeding disorders, Pediatr REV 29:121–129, 2008. National Hemophilia Foundation: www.hemophilia.org. Accessed on Jan. 9, 2015.

33. What are the primary measures for achieving hemostasis in individuals with bleeding disorders?
Never forget anatomic or surgical technical causes and corrections for hemorrhage. As a result, primary measures are local measures (“push on it, put a stitch or staple in it”), supplemented occasionally with licensed topical prothrombotic agents. Replacement of the deficient blood component
(s) is also important, but pharmacologic measures such as desmopressin acetate (DDAVP, which increases von Willebrand factor), antifibrinolytics such as epsilon aminocaproic acid (which stabilize clots), and topical hemostatic preparations such as fibrin glue can be useful.
34. To what degree should factor levels be raised for patients with hemophilia with or without life-threatening hemorrhage?
The following guidelines are applicable to patients with moderate (1% to 5% of normal factor levels) to severe (<1% of normal) hemophilia:
For minor hemorrhages (e.g., small muscle or oral), factor levels should be increased to 20% to 30% of normal.
For major bleeding episodes (e.g., hip bleeds, intracranial hemorrhage, bleeding around the airway), factor levels should be raised 70% to 100%, and repeat dosing should be strongly considered under close medical supervision.
35. How are doses of replacement factors calculated?
Recombinant factor VIII or factor IX concentrates are the treatments of choice. Each unit of factor
VIII or factor IX is equivalent to the activity of 1 mL of normal plasma. With the recombinant products, a dose of 1 unit/kg should increase the factor VIII level by 1.5% to 2% and the factor IX level by
1%. Calculations can be made as follows:
Factor VIII dose ðunitsÞ ¼ ðGoal % IncreaseÞ × ðkgÞ × 0:5 Factor IX dose ðunitsÞ ¼ ðGoal % IncreaseÞ × ðkgÞ
For example: If you have a 28-kg patient with a head bleed and severe factor VIII deficiency that you wish to correct 100%, your goal dose is 100 28 0.5 1400 units.
Another example: If you have a 50-kg patient with a minor bleed and severe factor IX deficiency that you wish to correct 30%, your goal dose is 30 50 1500 units.
Always round up to the nearest vial size so that there is no wastage of recombinant factor.
Of note, if there is an antibody inhibitor of the replacement factor, correction will not be achieved. Under these circumstances, alternate therapies are needed, such as porcine factor VIII, factor VIII inhibitor bypassing activity complexes, or recombinant factor VIIa.

Josephson N: The hemophilias and their clinical management, Hematology 2013:261–267, 2013.

36. In patients with severe hemophilia, can prophylaxis with factor replacement prevent severe hemorrhage?
In a study of boys with severe hemophilia A who were given regular recombinant factor VIII infusions up to 6 years of age, prophylaxis prevented joint damage and decreased the frequency of joint and other hemorrhages. Prophylaxis works. However, the cost was nearly $300,000 annually. How to reconcile the benefits and costs of effective expensive therapies remains a challenge for the health care system.

Manco-Johnson MJ, Abshire TC, Shapiro AD, et al: Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia, N Engl J Med 357:535–544, 2007.
Roosendaal G, Lafeber F: Prophylactic treatment for prevention of joint disease in hemophilia—cost versus benefit, N Engl J Med 357:603–605, 2007.

37. What are the half-lives of exogenously administered factors VIII and IX?
The half-lives for the first doses of factors VIII and IX are 6 to 8 hours and 4 to 6 hours, respectively. With subsequent doses, factor VIII has a half-life of 8 to 12 hours, whereas factor IX has a half-life of 18 to 24 hours. Thus, for serious bleeding, the second dose of factor VIII should be given 6 to 8 hours after the first, whereas the second dose of factor IX should be given 4 to 6 hours after the first. Subsequent doses are usually given every 12 hours for factor VIII replacement and every 24 hours for factor
IX replacement, but the measurement of actual factor levels may be necessary to guide therapy in life-threatening situations.

Gill JC: Transfusion principles for congenital coagulation disorders. In Hoffman R, Benz EJ, Shattil SJ, et al, editors:
Hematology: Basic Principles and Practice, ed 3. New York, 2000, Churchill Livingstone, p 2282.

38. Are longer-acting factors VIII and IX available?
Both long-acting recombinant Factor IX and long-acting recombinant factor VIII were recently approved in the United States. They significantly affect the frequency of dosing for factor, especially because each is used for prophylaxis. The half-lives are extended by fusion with the Fc moiety of immunoglobulin G (IgG), which prevents lysosomal degradation of the factor. Other mechanisms to prevent degradation and prolong the half-life of factors VIII and IX, including PEGylation (the process of covalent attachment of polyethylene glycol (PEG) polymer chains to the recombinant factors) are being evaluated. Gene therapy also holds future promise for long-term cure.

Shapiro A: Long-lasting recombinant factor VIII proteins for hemophilia A, ASH Education Program 1:37–43, 2013.

39. What can cause an elevation of the PT when other coagulation testing is normal? Factor VII deficiency. PT measures the function of the common pathway factors (including X, V, II, and fibrinogen) and the extrinsic pathway (tissue factor and factor VII). The aPTT measures the common pathway plus the function of the intrinsic pathway (including factors XII, XI, IX, and VIII). Isolated factor VII deficiency selectively elevates the PT. Other causes of elevated PT (e.g., liver disease, vitamin K deficiency, Coumadin toxicity) are not selective for lowering factor VII activity.
40. Who gets hemophilia C?
More commonly called factor XI deficiency, this is an uncommon type of hemophilia (<5% of total hemophilia patients). Unlike the X-linked nature of hemophilias A and B, it is an autosomal recessive disease that occurs most frequently in Ashkenazi Jews.

Asakai R, Chung DW, Davie EW, et al: Factor XI deficiency in Ashkenazi Jews in Israel, N Engl J Med 325:153–158, 1991.

41. Why is factor IX deficiency also called “Christmas disease”?
In 1952, investigators in England noted that, when blood from one group of hemophiliacs was added to the blood of another group of hemophiliacs, the clotting time was shortened. This provided
the basis for the discovery of plasma substances in addition to what was then called “antihemophilic globulin” (and now called factor VIII), which is responsible for normal clotting. The name was
derived because the first patient examined in detail with the unusual clotting deficiency (later designated as factor IX) was a boy named Christmas. The publication of the landmark article in fact occurred during the last week of December in 1952.

Biggs R, Douglas AS, Macfarlane RG, et al: Christmas disease: A condition previously mistaken for haemophilia, Br Med J 262:1378–1382, 1952.

KEY POINTS: HEMOPHILIA
1. X-linked recessive disorder
2. Hemophilia A: Factor VIII abnormalities (80% to 85% of total cases)
3. Hemophilia B: Factor IX abnormalities
4. Severity based on factor levels: Severe (<1%), moderate (1% to 5%), mild (5% to 30%)
5. Common initial presentation: Bleeding after circumcision

42. What is the von Willebrand factor (vWF)?
Synthesized in megakaryocytes and endothelial cells, vWF is a large multimeric protein that
binds to collagen at points of endothelial injury. It serves as a bridge between damaged endothelium and adhering platelets, and it facilitates platelet attachment. It also serves as a carrier protein for factor VIII in circulation; it minimizes the clearance of factor VIII from plasma and accelerates its cellular synthesis.
43. What are the coagulation abnormalities in von Willebrand disease?
von Willebrand disease is actually a group of disorders caused by qualitative or quantitative abnormalities in vWF. Coagulation abnormalities in children with severe disease can include a prolonged bleeding time, prolonged PTT, decreased factor VIII coagulant activity, decreased factor VIII antigen, and decreased ability of patient plasma to induce aggregation of normal platelets in the presence of ristocetin (the so-called “ristocetin cofactor activity”).
44. What are initial diagnostic tests for suspected von Willebrand disease?
• Quantification of vWF antigen
• Measurement of vWF function (either ristocetin-based platelet aggregation test, known as ristocetin cofactor assay) or vWF collagen-binding assay
• Factor VIII clotting activity
Screening tests for bleeding disorders (such as aPTT and bleeding time) can be normal in mild disease. Stress, pregnancy, or medications (e.g., oral contraceptives) can cause falsely elevated vWF levels in a patient. Once a diagnosis of vWF deficiency is suspected, vWF multimer analysis or genetic testing may assist in defining the subtype of vWF deficiency.

Cooper S, Takemoto C: Von Willebrand disease. Pediatr REV 35:136–137, 2014.

45. What does the ristocetin cofactor assay measure?
vWF activity. vWF will bind to the glycoprotein IB receptor on platelets in the presence of the antibiotic ristocetin. A patient’s plasma is serially diluted and mixed with platelets. The presence of vWF allows for platelet agglutination, which can then be quantified on the basis of the dilutions.
46. How is von Willebrand disease treated?
Treatment depends on the variant of von Willebrand disease that is identified:
• If protein is normal but diminished in quantity, desmopressin acetate (DDAVP) is given to stimulate endogenous release. DDAVP is now available for intravenous use and for intranasal use (Stimate). It is important to test von Willebrand disease patients for the safety and efficacy of either form of DDAVP before clinical use. It is also important to distinguish the form of intranasal DDAVP used for vWF therapy from that used for enuresis management.
• If protein is abnormal but bleeding is mild, desmopressin may also be of value.
• If protein is abnormal but bleeding is SEVERE, licensed vWF concentrates may be administered. A plasma-derived but highly purified product (trade name Humate P) provides both vWF and Factor VIII. The ristocetin cofactor activity is quantitated for each vial, which allows for more precise use.

Mannucci PM: Treatment of von Willebrand’s disease, N Engl J Med 351:683–694, 2004.

47. In an adolescent with menorrhagia, how likely is a bleeding disorder?
Up to 20% may have a bleeding disorder, particularly von Willebrand disease. The American College of Obstetrics and Gynecology recommends screening for any patient under age 18 with menorrhagia.

Kulp JL, Mwangi CN, Loveless M: Screening for coagulation disorders in adolescents with abnormal uterine bleeding, J Pediatr Adolesc Gynecol 21:27, 2008.

48. How does DDAVP work in the treatment of von Willebrand disease?
DDAVP is a synthetic analog of vasopressin, the antidiuretic hormone. Within 1 to 2 hours of its administration (either intravenous, subcutaneous, or intranasal), plasma vWF levels increase by 2-fold to 8-fold. DDAVP appears to act by causing the release of vWF from the endothelial cells. Factor VIII levels also increase in part because of the increased stabilization of vWF/factor VIII complex by

DDAVP, which lessens proteolytic degradation. As a caution, DDAVP administration in the setting of von Willebrand disease type IIB may cause a dangerous drop in platelet count due to increased vWF binding and platelet clearance.

Robertson J, Lilicrap D, James PD: von Willebrand disease, Pediatr Clin North Am 55:377–392, 2008.

49. Should children awaiting surgery undergo routine preoperative screening for potential abnormal bleeding?
This is controversial. A study from Philadelphia of 1600 pediatric patients scheduled for tonsillectomy who had a PT, aPTT, and bleeding time found only 2% with abnormal results, of which most were an isolated elevated aPTT. Of these patients, most had an antiphospholipid antibody, which was transient. A study of patients referred for isolated aPTT found that in the absence of symptoms and a negative family history, the diagnosis of a bleeding disorder was unlikely. Others argue that screening should be used despite the small yield to avoid missing an undiagnosed bleeding disorder.

Shah MD, O’Riordan MA, Alexander SW: Evaluation of prolonged aPTT values in the pediatric population, Clin Pediatr
45:347–353, 2006.
Burk CD, Miller L, Handler SD, et al: Preoperative history and coagulation screening in children undergoing tonsillectomy,
Pediatrics 89:691, 1992.

50. What is the role of vitamin K in coagulation?
Vitamin K is essential for the gamma-carboxylation of both procoagulants (including factors II, VII, IX, and X) and anticoagulants (proteins C and S). Gamma-carboxylation occurs in the liver and converts the proteins to their functional forms. Vitamin K is obtained in three ways: (1) as dietary fat-soluble K1 (phytonadione) from leafy vegetables and fruits; (2) as K2 (menaquinone) from synthesis by intestinal bacteria, and (3) as water-soluble K3 (menadione) from commercial synthesis.

51. In what settings outside the newborn period can vitamin K abnormalities contribute to a bleeding diathesis?
• Malabsorptive intestinal disorders (e.g., cystic fibrosis, Crohn disease, short-bowel syndrome)
• Prolonged antibiotic therapy (this diminishes intestinal bacteria)
• Prolonged hyperalimentation without supplementation
• Malnutrition
• Chronic hepatic disorders (hepatitis, alpha1-antitrypsin deficiency) can diminish both the absorption of fat-soluble vitamin K (as a result of diminished bile salt production) and the use of vitamin K in factor conversion
• Drugs that can disrupt vitamin K include phenobarbital, phenytoin, rifampin, and Coumadin

52. What is the best test for distinguishing coagulation disturbances that result from hepatic disease, DIC, and vitamin K deficiency?
Factors II, V, VII, IX, and X are made in the liver, and all of these factors (except factor V) are vitamin K dependent. Therefore, the measurement of factor V is a useful test to distinguish liver disease from vitamin K deficiency because this factor is reduced in the former and normal in the latter disorder. Factor VIII is reduced in patients with DIC because of the consumptive process, but this factor is normal or increased in patients with liver disease and vitamin K deficiency. Therefore, the factor VIII level
is a good test to distinguish DIC from the other two disorders (Table 9-2).

Table 9-2. Coagulation Abnormalities in Liver Disease, Vitamin K Deficiency, and Disseminated Intravascular Coagulation
FACTOR V FACTOR VII FACTOR VIII
Liver disease Low Low Normal or increased
Vitamin K deficiency Normal Low Normal
Disseminated intravascular coagulation Low Low Low

53. What is DIC?
DIC is an acquired syndrome that is precipitated by a variety of diseases and characterized by
diffuse fibrin deposition in the microvasculature, consumption of coagulation factors, and endogenous generation of thrombin and plasmin. The process is uncontrolled, and the result can be significant microthrombus formation with ischemic injury to multiple organ systems.
54. What tests are valuable for the diagnosis of suspected DIC?
See Table 9-3.

Table 9-3. Tests for Diagnosis of Disseminated Intravascular Coagulation
TEST USUAL RESULTS
Prothrombin time; activated partial thromboplastin time Prolonged
Fibrinogen <100 mg/dL*

Platelet count Low
D-Dimer >2 μg/mL
Factors II, V, and VIII Usually low*

*These results may be normal, however, especially in patients with mild disseminated intravascular coagulation because synthesis increases with accelerated consumption.
Data from Nathan DG, Orkin SH, Ginsburg D, Look AT, editors: Nathan and Oski’s Hematology of Infancy and Childhood, ed 6. Philadelphia, 2003, WB Saunders, p 1524.

55. What is the treatment of choice for DIC?
DIC occurs most commonly in the context of bacterial sepsis and hypotension. The best treatment is reversal of the underlying cause through treatment of the infection and appropriate fluid and pressor management. If bleeding is severe or if hemorrhage is occurring in a life-threatening location, platelets and fresh frozen plasma (FFP) should be given to make up for the loss of these elements, which is occurring from consumption. Heparin has not been proven to be effective for increasing survival in patients with sepsis and DIC. The replenishment of depleted antithrombin III levels with antithrombin III concentrate may decrease the risk of new thromboses.

Morley SL: Management of acquired coagulopathy in acute paediatrics, Arch Dis Child Educ Pract Ed 96:49–60, 2011.

56. What are the common hereditary disorders that predispose a child to thrombosis?
• Factor V Leiden: This is an abnormal factor V protein that is resistant to the normal antithrombotic effect of activated protein C.
• Protein C deficiency: Protein C inactivates factors V and VIII and stimulates fibrinolysis.
• Protein S deficiency: Protein S serves as a cofactor for the activity of protein C.
• Antithrombin III deficiency: Antithrombin III is involved in the inhibition of thrombin; factor X; and, to a lesser extent, factor IX.
• Prothrombin variation: Mutation at gene position 20210 increases prothrombin levels possibly through decreased mRNA degradation.
• Hyperhomocysteinemia: Often the result of a mutation of the MTHFR gene. Those with predisposition to hyperhomocystenemia due to thermolabile MTHFR variants benefit from folate supplementation, sometimes with vitamins B6 and B12 in addition.
• Antiphospholipid antibodies: These are passed from mother to infant prenatally. They can also be acquired, often in adolescence in the presence of systemic autoimmune diseases such as SLE.

Yang JY, Chan AK: Pediatric thrombophilia, Pediatr Clin North Am 60:1443–1462, 2013.

57. What are the inheritance patterns of the hypercoagulable states?
Factor V Leiden, protein C deficiency, and antithrombin III deficiency are all inherited in an autosomal dominant pattern. Factor V Leiden is transmitted with incomplete penetrance. Factor V mutation is present in 3% to 6% of white children, and evidence indicates that some of these

heterozygous individuals may have problems related to hypercoagulation (e.g., venous thrombosis). Nearly 200 pathogenic mutations have been described for protein C deficiency. Mutations in the SERPINC1 gene are responsible for antithrombin III abnormalities.
58. In an adolescent with an unprovoked deep vein thrombosis (DVT), what risk factors need to be assessed?
In young patients with a spontaneous DVT (not line-associated), one main concern is an inherited thrombophilia. Adolescents with unprovoked DVTs may have an inherited condition; however, they may also have additional modifiable risk factors that predispose them to DVTs, such as the use of estrogen-containing birth control pills, smoking, driving/sitting for prolonged periods of time, excessive repetitive motions, and pregnancy. Autoimmune phenomena, including antiphospholipid antibody syndrome, also are increased in frequency in adolescents and should be evaluated.
59. What anatomic variants will predispose individuals to venous thromboses?
• May-Thurner syndrome is an anatomic variant where the left common iliac vein is compressed by the right common iliac artery causing venous outflow tract obstruction predisposing patients to DVTs in the left lower extremity.
• Paget-Schroetter disease is a form of upper extremity DVT in the axillary or subclavian veins due to extrinsic compression or repetitive injury as the subclavian vein passes by the junction of the first rib and the clavicle. This is also called “effort thrombosis” as athletes (particularly pitchers and violin players) are susceptible.
60. What are the mechanisms for low molecular weight heparin and pentasaccharide as antithrombotic agents?
Low molecular weight heparin (LMWH) is the sulfated oligosaccharide heparin, derived from natural sources such as beef lung and pig intestine, that has been subjected to heparinase treatment to reduce the average molecular weight. Dosing and bioavailability are standardized, with less frequent or no monitoring of the anti-Factor Xa activity, depending on clinical circumstances. LMWH still works by binding antithrombin to enhance its anti-Factor IIa and anti-Factor Xa activities.
Pentasaccharide (Fondaparinux) is a synthetic five sugar agent that binds antithrombin and primarily inhibits Factor Xa. It has a longer half-life and reduced monitoring advantages over heparin, but currently no antidote is available clinically.
61. What are the direct thrombin inhibitors?
Direct thrombin inhibitors (DTIs) are anticoagulant drugs that block the enzymatic activity of thrombin without binding to antithrombin. There are two classes of DTIs. The first class includes natural or synthetic derivatives of leech hirudin, usually cleared renally. The second class includes synthetic small molecule drugs such as dabigatran, which are usually cleared hepatically. Use in children is reserved for conditions in which heparin is contraindicated, such as heparin-induced thrombocytopenia (HIT).

DEVELOPMENTAL PHYSIOLOGY
62. How do immunoglobulin (Ig) levels change during the first years of life?
• IgG levels in a full-term baby are equal or higher (5% to 10%) than maternal levels as a result of active placental transport. With an IgG half-life of 21 days, this transported maternal IgG reaches a nadir after 3 to 5 months. As the infant begins to make IgG, the level begins to rise slowly; it is 60% of adult level at 1 year of age, and it achieves the adult level by 6 to 10 years of age.
• IgM concentrations are normally very low at birth, and 75% of normal adult concentrations are usually achieved by about 1 year of age.
• IgA is the last immunoglobulin produced and approaches 20% of adult value by 1 year; however, full adult levels are not reached until adolescence. Because delays in the production of IgA are not unusual, the diagnosis of IgA deficiency is difficult to make with certainty in a child who is younger than 2 years.
• IgD and IgE, both of which are present in low concentrations in the newborn, reach 10% to 40% of adult concentrations by 1 year of age.
63. Why are antibodies not produced by the fetus in appreciable quantities?
• The fetus is in a sterile environment and is not exposed to foreign antigens.
• The active transport of maternal IgG across the placenta may suppress fetal antibody synthesis.
• Fetal and neonatal monocyte-macrophages may not process foreign antigens normally.

64. What is the role of the thymus?
The thymus is the primary lymphoid organ for the production and generation of T cells bearing the α/β T-cell antigen receptor. The thymus is responsible for the central selection of the T-cell repertoire, which allows for the establishment of tolerance toward self-antigens and responsiveness to nonself (i.e., foreign) antigens.
65. At what age does thymic function cease?
At birth, the thymus is at two-thirds of its mature weight, and it reaches its peak mass at about 10 years of age. Subsequently, thymic size declines, but substantial function (as measured by the output of new T cells) persists into very late adulthood (70 to 80 years of age).

Douek DC, McFarland RD, Keiser PH, et al: Changes in thymic function with age and during the treatment of HIV infection, Nature 396:690–695, 1998.

66. How does neutrophil function in the neonate compare with that of adults?
There is a diminished neutrophil storage in the neonate, and the cells display a reduced adhesion and migration capacity in response to chemotactic stimuli. By contrast, the efficiency for the ingestion and killing of bacteria is normal for these cells. Under suboptimal conditions, however, these effector functions may be diminished, and neutrophils from sick and stressed neonates can display a decreased microbicidal activity.

HEMATOLOGY LABORATORY
67. Of the seven red-cell parameters given by a Coulter counter, which are measured and which are calculated?
The Coulter counter, which is the most commonly used automated electronic cell counter, uses the impedance principle. A precise volume of blood passes through a narrow aperture and impedes an electrically charged field, and each “blip” is counted as a cell. The larger the red cell, the greater the electric displacement. In a separate chamber, the same volume is hemolyzed and colorimetrically analyzed to determine the hemoglobin concentration.
Measured values
• RBC count
• Mean corpuscular volume (MCV)
• Hemoglobin (Hb)
Calculated values
• Mean corpuscular hemoglobin (MCH, measured in pg/cell)¼(10 ×[Hb/RBC])
• Mean corpuscular hemoglobin concentration (MCHC, measured in g/dL)¼(100 ×[Hb/Hct])
• Hematocrit (Hct, given as a percentage)¼(RBC × [MCV/10])
• Red-cell distribution width (RDW)¼ coefficient of variation in RBC size
68. How does the mean corpuscular volume help provide a quick screen of the possible causes of anemia?
• Microcytic: Iron deficiency, thalassemias, sideroblastic anemia
• Normocytic: Autoimmune hemolytic anemia, hemoglobinopathies, enzyme deficiencies, membrane disorders, anemia of chronic inflammation
• Macrocytic: Disorders of B12 and folic acid metabolism, bone marrow failure
69. What is a quick rule of thumb for approximating MCV?
70 +(age in years). This number (in mm3) approximates the lower limit of MCV in children
<12 years old, below which microcytosis is present. After the age of 12 years, the lower limit for normal MCV is 82 fL.
70. In addition to an elevated reticulocyte count, what laboratory studies suggest increased destruction (rather than decreased production) of RBCs as a cause of anemia?
• Increased serum erythrocyte lactate dehydrogenase: More commonly seen in patients with hemolytic diseases, it can be greatly elevated in patients with ineffective erythropoiesis (e.g., megaloblastic anemia).

• Decreased serum haptoglobin: When RBCs lyse, serum haptoglobin binds the released hemoglobin and is excreted. However, up to 2% of the population has congenitally absent haptoglobin.
• Hyperbilirubinemia (indirect): This is usually increased with RBC lysis. However, it may also be elevated in patients with ineffective erythropoiesis (e.g., megaloblastic anemia). Additionally, 2% of the population has Gilbert disease. In these patients, acute infection can cause a transient elevation of bilirubin as a result of liver enzymatic dysfunction rather than hemolysis.
71. Why must the reticulocyte count sometimes be corrected?
Because the reticulocyte count is expressed as a percentage of total RBCs, it must be corrected according to the extent of anemia with the following formula: reticulocyte % (patient Hct/normal Hct) corrected reticulocyte count. For example, a very anemic 10-year-old patient with a hematocrit level of 7% (in contrast with an expected normal hematocrit of 36%) and a reticulocyte count of 5% has a corrected reticulocyte count of 1.0%: 5% (7%/36%) 1%. This is not appropriately elevated, as might be seen in patients with severe iron deficiency. The key concept is the appropriateness of the reticulocyte response to anemia. The corrected “retic count” should be elevated if the bone marrow is working properly and has all the right nutrients for making RBCs, including iron, folate, and vitamin B12.
72. What is the significance of targeting on an RBC smear?
Red-cell targets on a peripheral smear are caused by excessive membrane relative to the amount of hemoglobin. Therefore, target cells are found when the membrane is increased (e.g., in patients with liver disease) or when the intracellular hemoglobin is diminished (e.g., in patients with iron deficiency or thalassemia trait). Target cells may also be found in patients with certain hemoglobinopathies
(e.g., hemoglobins C and SC). In these instances, the target cells are caused by aggregation of the abnormal hemoglobin.
73. In what conditions are Howell-Jolly bodies found?
Howell-Jolly bodies are nuclear remnants that are found in the red cells of patients with reduced or absent splenic function (e.g., sickle cell disease, heterotaxy) and in patients with megaloblastic anemias. They are occasionally present in the red cells of premature infants. These remnants are part of the process of normal red cell maturation but are typically removed by a normal spleen. Howell-Jolly bodies are dense, dark, and perfectly round, and their characteristic appearance makes them easily distinguishable from other red-cell inclusions and from platelets overlying red cells (Fig. 9-2).

Figure 9-2. Red blood cells with Howell-Jolly bodies in a patient with hyposplenism. The cytoplasmic inclusions are nuclear remnants. (From Hoffman R, Benz EJ Jr, Silberstein LE, et al, editors: Hematology: Basic Principles and Practice, ed 6.
Philadelphia, 2013, ELSEVIER, p. 2259.)

74. What is the cause of Heinz bodies?
Heinz bodies represent precipitated denatured hemoglobin in the red cell. Heinz bodies occur when the hemoglobin is intrinsically unstable (e.g., hemoglobin Koln) or when the enzymes that normally protect hemoglobin from oxidative denaturation are abnormal or deficient (e.g., G6PD deficiency). These inclusions are not visible with a routine Wright-Giemsa stain but can be readily seen with methyl violet or brilliant cresyl blue stains.

75. What makes an “atypical lymphocyte” atypical?
Atypical lymphocytes (Fig. 9-3) are young lymphocytes (not lymphoblasts) that are characterized by an irregular plasma membrane with a large nucleus. Cytoplasm is typically basophilic. On a blood smear, where an atypical lymphocyte abuts an RBC, the shape of the lymphocyte will deform around it. Atypical lymphocytes are seen in a variety of illnesses, most commonly infectious mononucleosis.

Figure 9-3. Atypical lymphocyte. Note the deformation of the lymphocyte by the adjacent red cells. (From Zitelli BJ, DAVIS HW: Atlas of Pediatric Physical Diagnosis, 5th ed. Philadelphia, Mosby, 2007, p 421.)

76. A patient with oculocutaneous albinism has repeated Staphylococcus
aureus infections and the peripheral smear shown in Fig. 9-4. What is the likely diagnosis?
Chédiak-Higashi syndrome. This is an autosomal recessive disease with a defect in phagocytosis due to a mutation of a lysosomal trafficking regulator protein. Microtubules do not form normally and neutrophils do not respond to chemotactic stimuli. Giant lysosomal granules, which fail to function properly, are evident in a peripheral smear. Associated features include partial albinism, peripheral neuropathy, and a susceptibility to recurrent pyogenic infections.

Figure 9-4. Chédiak—Higashi syndrome, microscopic peripheral blood smear. (From Klatt EC: Robbins and Cotran Atlas of Pathology, ed 2, Philadelphia, 2010, Saunders ELSEVIER, p 67.)

HEMOLYTIC ANEMIA
77. What clinical features are suspicious for hemolytic anemia?
• Discolored urine (dark, brown, red)
• Jaundice
• Pallor
• Tachycardia
• Splenic and/or liver enlargement
• If very severe, hypovolemic shock or congestive heart failure
78. What two types of RBC forms are commonly seen on the peripheral smear in patients with hemolytic anemia?
• Spherocytes or microspherocytes: These forms can be seen in any hemolytic anemia that results from a loss of RBC membrane surface area (e.g., Coombs-positive hemolytic anemia, DIC, or hereditary spherocytosis).
• Schistocytes: These various forms of fragmented RBCs can be seen in patients with microangiopathic hemolytic anemia, which is a form of intravascular hemolysis caused by mechanical disruption (e.g., prosthetic heart valves, hemolytic-uremic syndrome, thrombotic thrombocytopenic purpura, cavernous hemangioma).
79. Name the two most common inherited disorders of red-cell membranes Hereditary spherocytosis is characterized by hemolysis (anemia, reticulocytosis, jaundice, splenomegaly); spherocytosis; and, in most cases, a family history of hemolytic anemia, early gallstones, or splenectomy. The diagnosis can be made by establishing the presence of the clinical findings and by the finding of increased osmotic fragility of the RBCs. Hereditary spherocytosis is inherited as an autosomal dominant disorder about 75% of the time.
Hereditary elliptocytosis is characterized by variable hemolysis, with a predominance of elliptocytes on the blood smear. It is usually inherited in an autosomal dominant pattern.
80. Which disorder is most commonly associated with an elevated MCHC?
Hereditary spherocytosis. The hyperchromic appearance of spherocytes and microspherocytes is the result of the loss of surface membrane, an excess of hemoglobin, and mild cellular dehydration. In other hemolytic anemias that are associated with spherocytosis, the percentage of spherocytes is usually insufficient to raise the MCHC.
81. What is the osmotic fragility test?
This is a test to confirm the diagnosis of hereditary spherocytosis. A normal RBC is discoid in shape as a result of its relative excess of surface area per cell volume from the redundancy of its cell membrane. In increasingly hypotonic solutions, more and more red cells will swell and burst at a standard rate. In spherocytosis, because there is less surface area to cell volume, more cells burst as compared with normal in these hypotonic solutions, particularly after incubating at 37 °C
for 24 hours. This tendency toward earlier lysis makes them osmotically fragile (Fig. 9-5).

1

.8 .7 .6

.5 .4 .3

Figure 9-5. Osmotic fragility curves in hereditary spherocytosis (HS). (From Nathan DG, Orkin SD, Ginsburg D, Look AT, editors: Nathan and Oski’s

% Saline concentration

Hematology of Infancy and Childhood, ed 6, Philadelphia, 2003, WB Saunders, p 610.)

Novel tests utilizing flow cytometric techniques and the eosin 5-maleimide (EMA) binding assay have also proven useful in the diagnosis of HS.

Bolton-Maggs PHB, Langer JC, Iolascon A, et al: Guidelines for the diagnosis and management of hereditary spherocytosis—2011 update, Br J Hem 156:37–49, 2012.

82. What is the difference between alloimmune and autoimmune hemolytic anemia?
• Alloimmune hemolytic anemia: Antibodies responsible for hemolysis are directed against another’s RBCs; it may cause acute or delayed hemolytic reactions.
• Autoimmune hemolytic anemia (AIHA): Antibodies are directed against the host’s red cells.
83. In which settings do alloimmune and AIHA most commonly appear?
Alloimmune: Red-cell antigen incompatibility between mother and fetus, transfusion of incompatible blood
Autoimmune:
• Primary: AIHA
• Secondary:
• Infections (e.g., Mycoplasma pneumoniae, EBV, varicella, viral hepatitis)
• Drugs (e.g., antimalarials, penicillin, tetracycline)
• Systemic autoimmune disorders (e.g., systemic lupus erythematosus, dermatomyositis)
84. How does the cause of AIHA vary by age?
AIHA in children <10 years old is more likely to be primary. In children >10 years old, AIHA is more likely to be secondary to an underlying disease.
85. What is the most important test to establish the diagnosis of AIHA?
The Coombs test or direct antiglobulin test (DAT). The diagnosis of AIHA requires the presence of autoantibodies that bind to erythrocytes and signs or symptoms of hemolysis. However, approximately 10% of patients with AIHA are Coombs negative. Thus, patients should be treated for AIHA if the disease is strongly suspected, even if the direct Coombs test is negative.
86. What are the differences between autoimmune hemolytic anemias caused by “warm” and “cold” erythrocyte autoantibodies?
• Warm (usually IgG antibodies with maximum activity at 37 °C): These are most commonly directed against the Rh antigens and generally do not require complement for in vivo hemolysis. Hemolysis is predominantly EXTRAVASCULAR—consumption occurs primarily in the spleen. Warm antibody-mediated hemolytic anemia is more likely to be associated with underlying disease (especially systemic lupus erythematosus in females) and to become chronic. Splenectomy and/or immunosuppression (e.g., with steroids) are often effective therapies.
• Cold (IgM antibodies with maximum activity between 0 to 30 °C): These are most commonly directed against I or i antigen. Hemolysis is most commonly INTRAVASCUlar via complement activation. Extravascular hemolysis that does occur primarily involves hepatic consumption. Cold antibody- mediated hemolytic anemia is more commonly associated with acute infection (e.g., Mycoplasma pneumoniae, EBV, cytomegalovirus). Patients are less likely to develop chronic hemolysis, and therapy (e.g., splenectomy, immunosuppression) is often ineffective.
87. A 6-year-old presents with acute anemia, fatigue, jaundice, and dark urine after an early spring swim in a local quarry. What is the likely diagnosis?
Paroxysmal cold hemoglobinuria is a transient autoimmune hemolysis due to a Donath-Landsteiner (D-L) antibody. This may be challenging to diagnose because the D-L antibody is a biphasic hemolysin that attaches to the RBC membrane at cold temperatures and initiates the complement cascade.
Once the RBCs are warmed by the body, the D-L antibody falls off, but the cells continue to lyse. The Coombs test is often negative. Significant hemolysis occurs after exposure to cold (such as swimming in an unheated body of water). The antibody is often triggered by a preceding infection. Treatment consists of warming the patient and providing any blood products as needed.
88. An 8-year-old black male developed jaundice and very dark urine 24 to 48 hours after beginning nitrofurantoin for a urinary tract infection. What is the likely diagnosis? G6PD deficiency is the most common hemolytic anemia caused by an RBC enzymatic defect. The enzyme G6PD is a key component of the pentose phosphate pathway, which ordinarily generates

sufficient nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) to maintain glutathione in a reduced state (and to make it available for combating oxidant stresses). The deficiency is inherited in an X-linked recessive fashion. In patients who are deficient (most commonly those of African,
Mediterranean, or Asian ancestry), oxidant stresses (particularly certain drugs) can result in hemolysis.
89. Why are “bite cells” seen in patients with G6PD deficiency”?
Bite cells (Fig. 9-6) are abnormally shaped RBCs with semicircular portions removed from the cell margin that give the appearance of a “bite” having been taken from the cell. These cells are seen in hemolytic anemias and anemias involving an altered, denatured hemoglobin (Heinz bodies), such as G6PD deficiency. These are cleaved by macrophages in the spleen which results in the abnormal appearance.

Figure 9-6. Bite cells in a patient with G6PD deficiency. (From Naeim F, Rao PN, Song SX, Grody WW, editors: Atlas of Hematopathology, London, 2013, Academic PRESS/ELSEVIER, p 698.)

90. In a patient with G6PD deficiency, why is the initial diagnosis often difficult in the acute setting?
The amount of G6PD enzymatic activity depends on the age of the RBC. Older RBCs have the least, and reticulocytes have the most. In an acute hemolytic episode, the older cells are destroyed first; younger ones may remain, and reticulocytes may increase. If erythrocytic G6PD levels are measured at this point, the result may be misleadingly near or above the normal range. If clinical suspicions remain, repeating the test when the reticulocyte count is reduced will give a more accurate measurement.
91. What is favism? FAVISM refers to the clinical syndrome of acute hemolytic anemia from the ingestion of fava beans as an oxidative challenge in patients with G6PD deficiency. This is particularly common in portions of the Mediterranean and Asia, where fava beans are a dietary staple.

IMMUNODEFICIENCY
92. How is neutropenia defined?
Neutropenia is arbitrarily defined as an absolute neutrophil count (ANC) of <1500/mm3. The ANC is determined by multiplying the percentage of bands and neutrophils by the total WBC count. An ANC of
<500/mm3 is severe neutropenia. Agranulocytosis is defined as an ANC of <100/mm3. As a rule, the lower the ANC, the greater the risk for infectious complications.

KEY POINTS: INFECTIONS IN IMMUNODEFICIENCIES
1. Increased frequency
2. Increased and prolonged severity
3. Unusual organisms (frequently opportunistic microorganisms)
4. Unexpected or severe complications of infection
5. Repeated infections without a symptom-free interval

93. How do children with neutrophil disorders present?
Neutrophil disorders include those that affect quantity (e.g., various neutropenias) and those that affect function (e.g., chemotaxis, phagocytosis, bactericidal activity). These defects should be considered part of the differential diagnosis in patients with delayed separation of the umbilical cord, recurrent infections with bacteria or fungi of low virulence (but minimal problems with recurrent
viral or protozoal infections), poor wound healing, and specific locales of infection (e.g., recurrent furunculosis, perirectal abscesses, gingivitis).
94. What is the most common cause of transient neutropenia in children?
Viral infections, including influenza, adenovirus, Coxsackie virus, respiratory syncytial virus, hepatitis A and B, measles, rubella, EBV, cytomegalovirus, and varicella. The neutropenia usually develops during the first 2 days of illness and may persist for up to a week. Multiple factors likely contribute to the neutropenia, including a redistribution of neutrophils (increased margination rather than circulation), sequestration in reticuloendothelial tissue, increased use in injured tissues, and marrow suppression. In general, otherwise healthy children with transient neutropenia as a result of viral infections are at low risk for serious infectious complications.

95. Excluding intrinsic defects in myeloid stem cells, what conditions are associated with neutropenia in children?
• Infection: Viral marrow suppression, bacterial sepsis-endotoxin suppression
• Bone marrow infiltration: Leukemia, myelofibrosis
• Drugs
• Immunologic factors: Neonatal alloimmune (secondary to maternal IgG directed against
fetal neutrophils) and autoimmune (e.g., autoimmune neutropenia of childhood, systemic lupus erythematosus, Evans syndrome)
• Metabolic factors: Hyperglycinemia, isovaleric acidemia, propionic acidemia, methylmalonic acidemia, glycogen storage disease type IB
• Nutritional deficiencies: Anorexia nervosa, marasmus, B12/folate deficiency, copper deficiency
• Sequestration: Hypersplenism

Segel GB, Halterman S: Neutropenia in pediatric practice, Pediatr REV 29:12–23, 2008.

96. Which is the most common form of chronic childhood neutropenia? Autoimmune neutropenia of infancy (ANI). This disorder displays a 3:2 female predominance and is caused by a chronic depletion of mature neutrophils. About 90% of all cases are detected within the first 14 months of life. The median duration of neutropenia is 20 months, and 95% of
patients with this condition have fully recovered by the time they are 4 years old. The ANC of infants with ANI is usually below 500/mm3, and the bone marrow displays normal cellularity despite an arrest at late stages of metamyelocytes or at the band stage. Antineutrophil antibodies are occasionally detected, but their presence is not necessary for the diagnosis of ANI.

97. How common are primary immunodeficiencies?
• Primary immune deficiencies: 1:10,000 (excluding asymptomatic IgA deficiency)
• B-cell defects: 65%
• Combined cellular and antibody deficiencies: 15% (severe combined immunodeficiency: 1 in 100,000 newborns)
• Phagocytic disorders: 10%
• T-cell–restricted deficiencies: 5%
• Complement component disorders: 5%

In a survey study of 10,000 American households, the calculated prevalence of a diagnosed immunodeficiency was 1 in 2000 in children, 1 in 1200 in people of all ages, and 1 in 600 households.

Boyle JM, Buckley RH: Population prevalence of diagnosed primary immunodeficiency diseases in the United States, J Clin Immunol 27:497–502, 2007.
Immune Deficiency Foundation: www.primaryimmune.org. Accessed on Jan. 9, 2015.
International Patient Organization for Primary Immunodeficiencies: www.ipopi.org. Accessed on Jan. 9, 2015.

98. What are the typical clinical findings of the various primary immunodeficiencies?
See Table 9-4.

Table 9-4. Clinical Findings of Primary Immunodeficiencies
PREDOMINANT B-CELL DEFICIENCY PREDOMINANT T-CELL DEFICIENCY
PHAGOCYTIC DEFECTS
COMPLEMENT DEFECTS
Age at onset After maternal Early infancy Early infancy Any age
antibodies have
disappeared
(usually >6 mo)
Type of
infection Gram-positive or gram-negative (encapsulated) bacteria, Mycoplasma, Giardia, Cryptosporidium, Campylobacter; enteroviruses Viruses, particularly CMV-1 and CBV;
systemic BCG after vaccination; fungal; Pneumocystis carinii Gram-positive or gram-negative bacteria; catalase- positive organisms in CGD, especially Aspergillus Streptococcus, Neisseria
Clinical
findings Recurrent respiratory tract infections, diarrhea, malabsorption, ileitis, colitis, cholangitis, arthritis, dermatomyositis, meningoencephalitis Poor growth and failure Poor wound
to thrive, oral healing, skin
candidiasis, skin diseases (e.g., rashes, sparse hair, seborrheic opportunistic dermatitis,
infections, impetigo,
graft-versus-host abscess),
disease, bony cellulitis
abnormalities, without pus, hepatosplenomegaly suppurative
adenitis, periodontitis, liver abscess, Crohn disease, osteomyelitis, bladder outlet obstruction Rheumatoid disorders, angioedema, increased susceptibility to infection
BCG Bacille Calmette-Guérin; CBV coxsackie B virus; CGD chronic granulomatous disease; CMV-1 cytomegalovirus type 1.

99. What is the single most important laboratory test if SCID is suspected?
A full blood count to document lymphopenia (2000/mm3) is the single most important laboratory test during the initial evaluation of a patient for suspected SCID. However, a minority of patients with SCID (about 20%) may have a normal absolute lymphocyte count.

100. Why are male children more likely to suffer from a primary immunodeficiency? Several primary immunodeficiency disorders are linked to the X-chromosome: agammaglobulinemia, hyper-IgM syndrome, severe combined immunodeficiency (the common cytokine receptor δ-chain deficiency), lymphoproliferative syndrome, Wiskott-Aldrich syndrome, one form of chronic granulomatous disease, and properidine deficiency. This fact accounts for the observation that the male- to-female ratio is 4:1 among patients with a primary immunodeficiency who are younger than 16 years.
101. Which is the most common type of primary immunodeficiency? Selective IgA deficiency is the most common primary immunodeficiency. The prevalence of selective IgA deficiency has been calculated to range from 1 in 220 to 1 in 3000, depending on the population studied. However, most IgA-deficient subjects remain healthy, which has been attributed to a compensatory increase of IgM in bodily secretions. A minority of these patients demonstrate normal levels of secretory IgA and normal numbers of IgA-bearing mucosal plasma cells. Although IgA represents less than 15% of total immunoglobulin, it is predominant on mucosal surfaces. Therefore, most patients with symptoms have recurrent diseases involving mucosal surfaces, including otitis media, sinopulmonary infections, and chronic diarrhea. Systemic infections are rare.
102. What are the diagnostic criteria for IgA deficiency? Serum concentrations of IgA lower than 0.05 g/L are diagnostic and almost invariably associated with a concomitant lack of secretory IgA. Serum levels for IgM are normal, and concentrations for IgG (particularly IgG1 and IgG3) may be increased in one-third of all IgA-deficient patients.
103. What is the association of autoimmune disorders and IgA deficiency? Autoimmune disorders have been described in up to 40% of patients with selective IgA deficiency. These include systemic lupus erythematosus, rheumatoid arthritis, thyroiditis, celiac disease, pernicious anemia, Addison disease, idiopathic thrombocytopenic purpura, and AIHA.
104. Why is immunoglobulin therapy not used as a treatment for selective IgA deficiency? Unless a patient has a concurrent IgG subclass deficiency (even in this setting, therapy is controversial), γ-globulin therapy is not indicated and is in fact relatively contraindicated because of the following:
• The short half-life of IgA makes frequent replacement therapy impractical.
• γ-Globulin preparations have insufficient IgA quantities to restore mucosal surfaces.
• Patients can develop anti-IgA antibodies with the potential for hypersensitivity complications, including anaphylaxis.

The Jeffrey Modell Foundation: www.info4pi.org. Accessed on Mar 20, 2015.

105. In an infant with panhypogammaglobulinemia, how can the quantitation of B and T lymphocytes in peripheral blood help distinguish the diagnostic possibilities?
• Normal numbers of T lymphocytes, no detectable B lymphocytes: X-linked agammaglobulinemia (Bruton disease)
• Normal numbers of T and B lymphocytes: Transient hypogammaglobulinemia of infancy, common variable immunodeficiency
• Decreased numbers of T lymphocytes, normal or decreased numbers of B lymphocytes: Severe combined immunodeficiency
• Decreased CD4 lymphocytes: HIV infection

KEY POINTS: WARNING SIGNS OF IMMUNODEFICIENCY
1. Eight or more new ear infections within 1 year
2. Two or more serious sinus infections within 1 year
3. Two or more months on antibiotics with little effect
4. Two or more severe pneumonia infections within 1 year
5. Failure of an infant to gain weight and grow normally
6. Recurrent deep skin or organ abscesses
7. Persistent thrush in mouth or elsewhere on skin after 1 year of age
8. Need for intravenous antibiotics to clear infections
9. Two or more deep-seated infections such as meningitis, osteomyelitis, cellulitis, or sepsis
10. A family history of primary immunodeficiency

106. What is the underlying disorder in an 8-year-old girl with atypical eczema, pneumatoceles, and bouts of severe furunculosis?
Hyper-IgE syndrome is the most likely diagnosis. This disease is clinically characterized by the following:
• Recurrent infections (almost invariably caused by S. aureus) of the skin, lungs (causing frequently persistent pneumatoceles), ears, sinuses, eyes, joints, and viscera
• Atypical eczema with lichenified skin
• Coarse facial features, especially the nose
• Osteopenia of unknown cause
• Delayed tooth exfoliation (i.e., prolonged retention of primary teeth)
The laboratory evaluation of the hyper-IgE syndrome reveals massively elevated IgE levels associated with IgG subclass and specific antibody deficiencies; variable dysfunctions of neutrophils; and an imbalance of cytokine production as a result of a Th2 predominance
(IL-4, IL-5).

Grimbacher B, Holland SM, Gallin JI, et al: Hyper-IgE syndrome with recurrent infections—an autosomal dominant multisystem disorder, N Engl J Med 340:697–702, 1999.

107. What are the proven indications for intravenous immunoglobulin (IVIG) therapy?
More than 75% of IVIG used in the United States is for the treatment of autoimmune or inflammatory conditions. Dosing in those conditions is typically 4 to 5 times greater than replacement therapy in immunodeficiency disease. Among the FDA-approved indications for IVIG are the following:
• Primary immunodeficiency disease
• Chronic lymphocytic leukemia
• Pediatric HIV disease
• Kawasaki disease
• Allogeneic bone marrow transplantation
• AIHA
• Idiopathic thrombocytopenic purpura
• Guillain-Barré syndrome (acute inflammatory demyelinating polyradiculopathy)
• Chronic inflammatory demyelinating polyradiculoneuropathy
• Cytomegalovirus-induced pneumonia in solid organ transplant recipients
• Various dermatologic conditions (including toxic epidermal necrolysis)

Gelfand EW: Intravenous immune globulin in autoimmune and inflammatory diseases, N Engl J Med 367:2015– 2025, 2012.

108. What are the pharmacologic characteristics of IVIG?
After the infusion, 100% of the IgG stays in the intravascular compartment. Over the course of the next 3 to 4 days, IgG equilibrates with the extracellular space, with 85% of the infused IgG still situated in the circulation. By the end of the first week, half of the IgG given has left the circulation, and by 4 weeks after the infusion, the serum levels have returned to baseline. However, these data apply to healthy individuals with a regular catabolism, and they have to be adjusted for both patients with a higher metabolic rate and for individuals transfused with increased IgG concentrations.

109. What are the adverse reactions to IVIG?
The common, infusion rate–related adverse events are chills, headache, fatigue and malaise, nausea and vomiting, myalgia, arthralgia, and back pain. Less frequent reactions are abdominal and chest pains, tachycardia, dyspnea, and changes in blood pressure. Serious but rare side effects include aseptic meningitis, thrombosis, DIC, renal and pulmonary insufficiency, and anaphylaxis in complete IgA-deficient individuals due to IgE antibodies specific for IgA. Subcutaneous therapy can reduce the occurrence of systemic adverse events in selected patients.

Orange JS, Hossny EM, Weiler CR, et al: Use of intravenous immunoglobulin in human disease, J Allergy Clin Immunol 117: S525–S553, 2006.

110. Which viral infections can result in hypogammaglobulinemia in the immunocompetent individual?
EBV, HIV, and congenital rubella. Single cases of hypogammaglobulinemia have also been described among children infected with cytomegalovirus and parvovirus B19.
111. What is the classic triad of Wiskott-Aldrich syndrome?
Thrombocytopenia with small platelets volume, eczema, and immunodeficiency. This syndrome is an X-linked disorder, and the initial manifestations are often present at birth and consist of petechiae, bruises, and bloody diarrhea as a result of thrombocytopenia. The eczema is similar in presentation to classical atopic eczema (antecubital and popliteal fossa). Infections are common and include
(in decreasing frequency): otitis media, pneumonia, sinusitis, sepsis, and meningitis. The severity of immunodeficiency may vary but usually affects both T-cell and B-cell functions. It is important to
note that this immunodeficiency is progressive and associated with a high risk for developing cancer; a teenager with this condition has a 10% to 20% statistical risk for developing a lymphoid neoplasm. Only about one-third of patients with Wiskott-Aldrich syndrome present with the classic triad.

Puck J, Candotti F: Lessons from the Wiskott-Aldrich syndrome, N Engl J Med 355:1759–1761, 2006.

112. What is the likely diagnosis of a patient presenting with a progressive ataxia, conjunctival abnormalities, and recurrent bacterial sinopulmonary infections? Ataxia-telangiectasia. In patients with ataxia-telangiectasia, primarily progressive cerebella ataxia develops during infancy and is typically associated with other neurologic symptoms (e.g., the loss or decrease of deep tendon reflexes, choreoathetosis, apraxia of eye movements). The signs of telangiectasia occur usually after the onset of ataxia, generally between 2 and 8 years of age. The telangiectasias are primarily at the bulbar conjunctivae (Fig. 9-7). Recurrent infections (as a consequence of a humoral and cellular immunodeficiency) are observed in 80% of patients with ataxia- telangiectasia and are typically localized to the middle ear and the upper airways.

Figure 9-7. Telangiectasia of the conjunctiva. (From Orth KAHM, Leung H, Andrews I, SachDEV R: Ataxia telangiectasia in a three-year-old girl, Pediatr Neurol 50:279, 2014.)

KEY POINTS: SUSPECT IMMUNODEFICIENCY IN INFANTS WITH THESE CONDITIONS
1. Failure to thrive
2. Persistent cough
3. Persistent candidiasis
4. Absolute lymphocyte count <2000/mm3

113. What disease did the “bubble boy” have?
Adenosine deaminase (ADA) deficiency. In this form of SCID, the lack of ADA results in abnormalities of B- and T-cell function and increased susceptibility to infection. The bubble served as a means of minimizing contagion but also promoted social isolation. Although bone marrow transplantation has been curative as a treatment for this condition, ADA deficiency is the first disease to be treated by gene therapy (i.e., insertion of functional ADA genes into the patient’s autologous cells and followed by infusion).

Aiuti A, Cattaneo F, Galimberti S, et al: Gene therapy for immunodeficiency due to adenosine deaminase deficiency,
N Engl J Med 360:447–458, 2009.

114. Describe the molecular defect of chronic granulomatous disease (CGD)
CGD is characterized by a profound defect in the oxygen metabolic burst in myeloid cells following the phagocytosis of microbes. The molecular mechanisms responsible for this disease are heterogenous because any defect of the four subunits that constitute the nicotinamide adenine dinucleotide phosphate hydrogen-oxidase can cause CGD. As a consequence, superoxide, oxygen radicals, and peroxide production are lacking, and patients with CGD cannot kill catalase-positive pathogenic bacteria and fungi (e.g., S. aureus; Nocardia, Serratia, and Aspergillus species).
115. Which laboratory tests are used for the diagnosis of CGD?
Patients suspected to have CGD can be diagnosed as a result of their failure to generate reactive oxygen species during the respiratory burst or, alternatively, as a result of their inability to kill catalase-positive bacteria (S. aureus, Escherichia coli) in vitro with their phagocytes. The screening tests for the production of superoxide are the slide nitroblue tetrazolium reduction test and the flow cytometric 20,70-dichlorofluorescein test.
116. What types of infections are commonly seen in children with CGD?
Superficial staphylococcal skin infections, particularly around the nose, eyes, and anus, are common. Severe adenitis, recurrent pneumonia, indolent osteomyelitis, and chronic diarrhea are frequent. A male child with a liver abscess should be considered to have chronic granulomatous disease until it is proved otherwise.
117. Which disorder has to be considered in a newborn patient with delayed separation of the umbilical cord?
Separation of the umbilical cord occurs normally on average by 10 days of life with a range of 3 to 45 days. Delayed separation can occur in patients with leukocyte adhesion deficiency type 1 (LAD1), who suffer from a profound impairment of leukocyte mobilization into extravascular sites. The hallmark of this disorder is the complete absence of neutrophils at the site of infection and inflammation (e.g., wound healing).
118. Which potential life-threatening disorder of the complement system is associated with nonpruritic swelling and occasional recurrent abdominal pain? Hereditary C1 inhibitor deficiency. Angioedema of any part of the body—including the airway and the intestine—can occur as a consequence of failure to inactivate the complement and kinin systems. The condition has also been called hereditary angioneurotic edema. Infections, oral contraceptives, pregnancy, minor trauma, stress, and other variables have been noted to precipitate this autosomal dominant disease. Diagnosis is confirmed by direct assay of the inhibitor level. Clinical presentations include the following:
• Recurrent facial and extremity swelling: Acute, circumscribed edema that is not painful, red, or pruritic, thereby clearly distinguished from urticaria; usually self-resolves in 72 hours
• Abdominal pain: Recurrent and often severe, colicky pain as a result of interstitial wall edema with vomiting and/or diarrhea; may be misdiagnosed as an acute abdomen
• Hoarseness, stridor: A true emergency because death by asphyxiation may occur as a result of laryngeal edema; epinephrine, hydrocortisone, and antihistamines are often of only limited benefit; and tracheostomy is needed if there is progression of symptoms

Bork K: An evidence-based therapeutic approach to hereditary and acquired angioedema, Curr Opin Allergy Clin Immunol 14:354-362, 2014.
Zuraw BL: Hereditary angioedema, N Engl J Med 359:1027–1036, 2008.

IMMUNOLOGY LABORATORY
119. Which are the initial screening tests for a suspected immunodeficiency?
The basic screening tests should include CBC (including hemoglobin, morphology, and absolute cellularity); quantification of immunoglobulin levels (IgM, IgG, IgE, and IgA); antibody responses to previous antigen exposures (e.g., vaccines, pathogen-defined infections); determination of isohemagglutinin titers; assessment of the classic complement pathway by determining the CH50; and workup of infections, including determination of C-reactive protein, blood cultures, and appropriate radiography. The choice of the laboratory tests is generally dependent on the clinical findings and the immunodeficiency suspected, and the results have to be compared with age-matched controls. It is important to note that there is no justification for a blanket screening; tests should only be ordered if their results will affect either the diagnosis or management of the patient.
120. Which laboratory tests allow for a broad evaluation of the humoral immune system?
Serum immunoglobulin levels, quantitative: IgM, IgG, IgA, and IgE. A combined IgG, IgA, and IgM level of <400 mg/dL suggests immunoglobulin deficiency; >5000 IU/mL for IgE suggests hyper-IgE syndrome.
IgG subclasses: These immunoglobulins should generally be measured primarily in patients
>6 years old, in certain circumstances (e.g., in patients with selective IgA deficiency and normal to low IgG concentrations but demonstrated functional antibody deficiency), and in patients with recurrent sinopulmonary infections.
• Specific antibody titers: In response to documented infections and vaccinations
• Isohemagglutinin titer (anti-A, anti-B): 1:4 or less after the age of 1 year suggests specific IgM deficiency
• Tetanus, diphtheria (IgG1)
• Pneumococcal polysaccharide antigens (IgG2)
• Viral respiratory agents (IgG3)
Determination of B-cell numbers: In the peripheral blood with the use of flow cytometry (CD19, CD20)
B-cell proliferation and immunoglobulin production: With the use of in vitro assays
121. Which diagnostic tests allow for the specific evaluation of T-cell functions?
• Total lymphocyte count: Although most T-cell immunodeficiencies are not associated with a decreased lymphocyte count, a total count of <1500/mm3 suggests a deficiency.
• T-cell subpopulations: Total T cells with <60% mononuclear cells, helper (CD4) cells
<200/μL, or CD4/CD8 < 1.0 suggest T-cell deficiency.
• Delayed-type hypersensitivity skin testing
• Proliferative responses to mitogens, antigens, and allogeneic cells
• Acquisition of activation markers on T cells (using flow cytometry)
• Cytotoxic assay
• Cytokine synthesis
• Adenosine deaminase and purine nucleoside phosphorylase determination in RBCs
• Molecular biologic studies (including karyotyping and fluorescent in situ hybridizations)
• Histology of thymic and lymph-node biopsies
122. What is the value of skin testing for the diagnosis of T-cell deficiencies?
Skin tests for the assessment of delayed-type hypersensitivity are difficult to evaluate. A positive test is useful for eliminating the diagnosis of severe T-cell deficiency, whereas a negative test may reflect a T-cell defect, or it may result from the lack of an anamnestic response to the antigens used. Seventy-five percent of normal children between the ages of 12 and 36 months will respond to Candida skin testing at 1:10 dilution, and, by 18 months, about 90% of normal children will respond to one of a panel of recall antigens (tetanus toxoid, trichophyton, and Candida); the younger the child, the less likely the reactivity. The cell-mediated reaction may
be obscured by a humoral (Arthus) reaction as a result of previous priming.
123. What is the importance of the CD4/CD8 ratio?
The CD4/CD8 ratio is an index of helper to suppressor and cytotoxic cells and may be significantly altered in patients with a variety of immunodeficiencies. In normal individuals, the ratio ranges from 1.4:1.0 to 1.8:1.0. In patients with viral infections (particularly HIV), the ratio can be reduced; in patients with bacterial infections, it can be increased.

124. Which laboratory tests appropriately evaluate the phagocytic system?
Absolute granulocyte count
Antineutrophil antibodies (however, antineutrophil antibodies are found in only one-half of the cases of autoimmune neutropenia of infancy)
Bone marrow biopsy (to differentiate increased consumption from decreased production)

Specific in VITRO and in VIVO assays:
• Determination of chemotaxis: in vivo (skin wounds) or in vitro (Boyden chambers): Measurements are not routinely used for diagnostic purposes
• Quantification of neutrophil adherence: Measurement of cell surface expression of leukocyte function antigen-1 (CD11/CD18) by flow cytometry; adherence to inert surfaces such as nylon, wool, or plastic
• Determination of the respiratory burst: (1) Nitroblue tetrazolium test (NBT) measures the ability of phagocytic cells to ingest and reduce a yellow dye to an intercellular blue crystal; (2) Dihydrorhodamine (DHR)—in activated granulocytes reactive oxygen intermediates reduce DHR 123 to rhodamine 123, which results in an increase in fluorescence that can be quantified by flow cytometry
• Enzyme assays (myeloperoxidase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, NADPH-oxidase)
• Test treatment with rHu granulocyte colony-stimulating factor. Autoimmune forms of neutropenia in small children respond to minor doses (1 mcg/kg) within a couple of days, whereas congenital forms require larger doses with responses after 2 to 3 weeks of treatment
• Mutational analysis
125. How is the classic complement cascade evaluated?
The primary screening test is the CH50. This test assesses the ability of an individual’s serum
(in varying dilutions) to lyse sheep RBCs after those cells are sensitized with rabbit IgM antisheep antibody. The CH50 is an arbitrary unit that indicates the quantity of complement necessary for 50% lysis of the RBCs in a standardized setting. Test results are usually expressed as a derived reciprocal of the test dilution needed for 50% lysis. The test is relatively insensitive because major reductions in individual complement components are necessary before the CH50 is altered. Therefore, determination C3 and C4 levels are often included in the initial screening of a child with a suspected complement deficiency.

IRON-DEFICIENCY ANEMIA
126. What is the world’s most common single-nutrient deficiency?
According to the World Health Organization, it is iron. It’s estimated that 2 billion people, or over 30% of the world’s population, are anemic, many due to iron deficiency. In developing countries, about 40% of preschool children are estimated to be anemic.

World Health Organization: www.who.int/nutrition. Accessed on Mar 18, 2015.

127. At what age do exclusively breast-fed infants become at risk for iron deficiency?
Healthy term infants who are exclusively breast-fed are at risk for iron deficiency after they are
4 to 6 months old. The AAP Committee on Nutrition has recommended that exclusively breast-fed infants be supplemented with iron (1 mg/kg per day) starting at 4 months of age and continued until appropriate iron-containing complementary foods have been introduced. The age of risk for exclusively breast-fed premature infants can be more complicated, particularly for the smaller and sicker infants. The lower iron stores of premature infants are more rapidly depleted as compared with term babies. The AAP Committee on Nutrition recommends that all preterm breast-fed infants receive an iron supplement (2 mg/kg per day) by 1 month of age and that it be continued until sufficient iron-containing foods or formula are being consumed.

Baker RD, Greer FR; Committee on Nutrition American Academy of Pediatrics: Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0-3 years of age), Pediatrics 126:1040–1050, 2010.

128. Why are infants who begin consuming cow milk at an early age susceptible to
iron-deficiency anemia?
Lower bioavailability. Although breast milk and cow milk contain about the same amount of iron (0.5 to 1.0 mg/L), nonheme iron is absorbed at 50% efficiency from breast milk but at only 10% from cow milk. In addition, cow milk may cause microscopic GI bleeding in younger infants
as a result of mucosal injury, possibly from sensitivity to bovine albumin. In older infants, cow milk may interfere with iron absorption from other sources.

Thorsdottir I, Thorsdottir AV: Whole cow’s milk in early life, Nestle Nutr Workshop Ser Pediatr Program 67: 29–40, 2011.
Sullivan P: Cow’s milk-induced intestinal bleeding in infancy, Arch Dis Child 68:240–245, 1993.

129. As iron becomes depleted from the body, what is the progression at which laboratory tests change?
The left end of the line for each test indicates the point at which the result deviates from its baseline. As shown in Fig. 9-8, in general, the depletion of marrow, liver, and spleen reserves (as represented by ferritin) occurs first. This is followed by a decrease in transport iron
(as represented by transferrin saturation) and finally a fall in hemoglobin and MCV. The figure illustrates that the absence of anemia does not exclude the possibility of iron deficiency and that iron depletion is relatively advanced before anemia develops. Tests of soluble transferrin receptor have become of interest in patients with iron-deficiency anemia because the elevated levels are very sensitive indicators.

Depleted iron stores

Iron deficiency without anemia

Iron deficiency anemia

Figure 9-8. Progression of laboratory test changes with iron depletion. MCV, Mean corpuscular volume. (From Dallman PR, Yip R,Oski FA: Iron deficiency and related nutritional anemias. In Nathan DG, Oski FA, editors: Nathan and Oski’s Hematology of Infancy and Childhood, ed 4, Philadelphia, 1993, WB Saunders, p 427.)

130. How might the reticulocyte hemoglobin content be helpful for the diagnosis of iron deficiency?
Because the reticulocyte is the most recently produced RBC in circulation, the earliest sign of iron deficiency may be a fall in the concentration of hemoglobin in reticulocytes. This number can be calculated from automated counting equipment and may be a reliable and inexpensive alternative to ferritin. Studies have indicated that patients with a concentration of 30 pg per cell have virtually no chance of iron deficiency.

Brugnara C, Zurakowski D, DiCanzio J, et al: Reticulocyte hemoglobin content to diagnose iron deficiency in children,
JAMA 281:2225–2230, 1999.
Cohen AR: Choosing the best strategy to prevent childhood iron deficiency, JAMA 281:2247–2248, 1999.

131. Why are tests for iron stores more difficult to interpret during acute inflammatory states?
The ferritin level, which is used to monitor body iron stores, is exquisitely sensitive to inflammation, increasing even with mild upper respiratory infections. Elevations of ferritin may persist for some time. By contrast, serum iron, transferrin LEVEL, and percent transferrin saturation may decrease with infection or inflammation. Free erythrocyte protoporphyrin should not be affected by acute inflammation but may increase in chronic inflammatory states.
132. What is the role of hepcidin in iron metabolism?
Hepcidin is part of the system of iron regulatory proteins that have undergone an explosive increase in our understanding. The iron regulatory system controls intestinal iron absorption,
blood transport, tissue deposition, and mobilization of stores for utilization. Hepcidin is synthesized in the liver and participates in the orchestration of uptake and utilization.

Collard KJ: Iron homeostasis in the neonate, Pediatrics 123:1208–1216, 2009.

133. What are the common causes of microcytic anemia in children?
• More common: Iron deficiency (from nutritional insufficiency and/or blood loss), thalassemia (α- or β-; major, minor, or trait)
• Less common: Lead toxicity, hemoglobinopathy (with or without thalassemia), chronic inflammation, copper deficiency, sideroblastic anemia
134. How is the RDW useful for distinguishing causes of microcytic anemia? The red blood cell distribution width (RDW) is a quantification of anisocytosis (variation in red-cell size). It is derived from the RBC size histogram that is measured by automated cell counters, and it is reported as a percentage. In children, normal values range from about 11.5% to 14.5% but can vary among instruments. Statistically, it is the coefficient of variation of red-cell volume distribution. When elevated in a patient with microcytosis, it suggests that iron deficiency is a more likely cause of anemia than the thalassemia trait. Children with the thalassemia trait tend to have values that overlap with normal RDW values. The combination of an RDW above the normal range with a free erythrocyte
protoporphyrin level of >35 μg/dL is more sensitive and specific for iron-deficiency anemia.
135. What is the Mentzer index?
MCV/RBC. This is one of the formulas used to distinguish the hypochromic, microcytic anemias of the thalassemia trait from iron deficiency. As a general rule, iron deficiency causes alterations in RBCs that tend to be variable, whereas thalassemia generally results in more uniformly smaller cells. In patients with the beta-thalassemia trait, the Mentzer index is usually <13; in patients
with iron deficiency, it is usually >13.
136. In a child with suspected iron-deficiency anemia, is a therapeutic trial with iron an acceptable diagnostic approach?
Yes. If an infant or child is otherwise well, a therapeutic trial of 4 to 6 mg/kg/day of elemental iron can substitute for additional diagnostic testing (e.g., ferritin, transferrin saturation, free erythrocyte protoporphyrin), because dietary iron deficiency is the most likely cause of microcytic anemia. If the child is iron deficient, compliant with therapy, and there is not ongoing undetected blood loss, the hemoglobin should rise by >1 g/dL in about 2 weeks. If the hemoglobin does rise, therapy should
be continued for an additional 2 months to replenish iron stores.
137. After iron therapy is initiated, how early can a response be detected?
2 to 5 days: Increase in reticulocyte count
7 to 10 days: Increase in hemoglobin level

For patients with mild iron-deficiency anemia, the hemoglobin level should be checked after several weeks of therapy. For patients with more severe anemia, it may be useful to check the hemoglobin and reticulocyte levels after several days to make certain that the hemoglobin has not declined to dangerous levels and that the reticulocyte response is beginning.
138. What foods affect the bioavailability of nonheme iron? It is decreased by phosphates, tannates, polyphenols, and oxalates found in cereal, eggs, milk, cheese, tea, and complex carbohydrates. It is increased by fructose; citrate; and especially, ascorbic acid found

in red kidney beans, cauliflower, and bananas. In children with iron deficiency, the administration of replacement iron with a vitamin-C-fortified fruit juice 30 minutes before a meal makes
physiologic sense.
139. What are the options for use of parenteral iron therapy?
When oral iron therapy has failed or cannot be used, there are several formulations of iron for intravenous use with generally good tolerance. These include formulations such as iron sucrose, sodium ferric gluconate, and ferric carboxymaltose. Usually repletion of iron stores requires multiple treatments over time; however, novel parenteral iron supplements (iron isomaltoside) may only require single dose administration. Care must be exercised in administration to avoid untoward side effects. Monitoring is required to ensure that anemia is reversed and that iron stores are restored.
140. What are the differences between pica, geophagia, and pagophagia?
All are clinical markers that suggest the diagnosis of iron deficiency. Pica is a more general term that indicates a hunger for material that is not normally consumed as food. Geophagia refers to the consumption of dirt or clay, and pagophagia refers to the excessive consumption of ice.
These are distinguished from cissa, which is the physiologic craving during pregnancy for unusual food items or combinations.
141. What is the derivation of the term pica?
The condition comes from the Latin term for the magpie, Pica hudsonia. This bird is believed to eat almost anything, hence the term pica for the tendency to eat nonnutritional substances.

Borgna-Pignatti C, Marsella M: Iron deficiency in infancy and childhood, Pediatr Ann 37:332–333, 2008.

142. Discuss the relationship between iron deficiency and development in infants and toddlers.
Multiple studies have shown an association between iron deficiency in infants between 9 and 24 months old and lower motor and cognitive scores and increased behavioral problems as compared with nonanemic controls. Some longer-term studies suggest that the developmental impairments may be long lasting. Debate remains about whether this relationship is causal and, if so, whether the correction of anemia leads to a reversal of the problems.

Baker RD, Greer FR; Committee on Nutrition American Academy of Pediatrics: Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0-3 years of age), Pediatrics 126:1040–1050, 2010. Buchanan GR: The tragedy of iron deficiency during infancy and childhood, J Pediatr 135:413–415, 1999.

143. What are the risk factors for iron deficiency or iron-deficiency anemia in a 1-year-old?
• Low socioeconomic status (especially children of Mexican-American descent)
• Exposure to lead
• History of prematurity or low birth weight
• Exclusive breastfeeding beyond 4 months of age without supplemental iron
• Introduction of whole milk before 1 year of age
• Feeding problems
• Poor growth
• Inadequate nutrition (particularly seen in infants with special care needs)

Baker RD, Greer FR; Committee on Nutrition American Academy of Pediatrics: Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0-3 years of age), Pediatrics 126:1040–1050, 2010.

144. Why are iron-deficient children at increased risk for lead poisoning?
• Pica associated with iron deficiency increases the likelihood of ingestion of lead- contaminated items.
• GI absorption of lead may be increased in patients who consume less iron-containing nutrients.

Watson WS, Morrison J, Bethel MI, et al: Food iron and lead absorption in humans, Am J Clin Nutr 44:248–256, 1986.

145. How and when should younger children be screened for iron deficiency?
This is controversial. AAP recommendations, which previously had advised selective screening only, began in 2010 to advocate universal screening at approximately 12 months of age with a hemoglobin measurement and an assessment of risk factors for iron deficiency/iron-deficiency anemia. Critics have argued that this type of screening process does not identify early enough those with iron problems, including by definition those with iron deficiency alone before the development of anemia. Other screening tests that have been suggested as better biomarkers of iron status include reticulocyte hemoglobin concentration, transferrin saturation, serum transferrin receptor 1 (TfR1) concentration, and zinc protoporphyrin. Zinc protoporphyrin is a red cell–specific intermediary metabolite required for the biosynthesis of hemoglobin.

Baker RD: Zinc protoporphyrin to prevent iron deficiency, JAMA Pediatr 167:393–394, 2013.
Baker RD, Greer FR; Committee on Nutrition American Academy of Pediatrics: Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0-3 years of age), Pediatrics 126:1040–1050, 2010.

KEY POINTS: IRON- DEFICIENCY ANEMIA
1. The introduction of whole cow milk before the age of 1 year increases risk as a result of occult gastrointestinal bleeding.
2. Red-cell distribution width is increased because deficiency results in uneven red-cell size (anisocytosis).
3. Low levels of ferritin indicate diminished tissue iron stores.
4. This condition impairs cognitive development in infants.
5. Absence of anemia does not exclude the possibility of iron deficiency. Iron depletion is relatively advanced before anemia occurs.

MEGALOBLASTIC ANEMIA
146. What is megaloblastic anemia?
Megaloblastic anemia is a macrocytic anemia that is characterized by large red-cell precursors (megaloblasts) in the bone marrow and that is usually caused by nutritional deficiencies of either folic acid (folate) or vitamin B12 (cobalamin).
147. Is megaloblastic anemia the most common cause of macrocytic anemia?
No. Macrocytic anemia can be found in conditions associated with a high reticulocyte count
(e.g., hemolytic anemia, hemorrhage), bone marrow failure (e.g., Fanconi anemia, aplastic anemia, Diamond-Blackfan anemia), liver disease, Down syndrome, and hypothyroidism.
148. What findings on a CBC are suggestive of megaloblastic anemia?
• RBCs: Elevated MCH and mean cell volume (often 106 fL or more), with normal MCHC; marked variability in cell size (anisocytosis) and shape (poikilocytosis)
• Neutrophils: Hypersegmentation (>5% of neutrophils with five lobes or a single neutrophil with six lobes)
• Platelets: Usually normal; thrombocytopenia in more severe anemia
149. What are the causes of vitamin B12 (cobalamin) deficiency in children?
Decreased intake
• May occur in vegetarians who consume no animal products
• Seen in exclusively breast-fed infants of B12-deficient mothers
• General malnutrition
Decreased absorption
• Ileal mucosal abnormalities (e.g., Crohn disease)
• Surgical resection of terminal ileum (e.g., infant with history of surgical necrotizing enterocolitis [NEC])
• Competition for cobalamin in bacterial overgrowth syndromes or infection with the fish tapeworm Diphyllobothrium latum
• Congenital abnormalities of the receptor for vitamin B12–intrinsic factor complex
• Gastric mucosal defects that interfere with the secretion of intrinsic factor

150. What are the best dietary sources of folate and B12?
• Folate: Folate-rich foods include liver, kidney, and yeast. Good sources also include green vegetables (particularly spinach) and nuts. Moderate sources include fruits, bread, cereals, fish, eggs, and cheese. Pasteurization or boiling destroys folate.
• Vitamin B12: Humans do not manufacture B12; bacteria and fungi do. Animals require it, whereas plants do not. Consequently, our major dietary source of vitamin B12 is the consumption of animal tissue, milk, or eggs. Seafood, which live on bacterial diets, are also a good dietary source. Of note is that B12 is required for normal folate metabolism.
151. What is pernicious anemia? Pernicious anemia is a megaloblastic anemia that is caused by a lack of intrinsic factor. Intrinsic factor is a glycoprotein that is released from the gastric parietal cells that binds to vitamin B12 to form a complex that is ultimately absorbed in the terminal ileum.
152. A 10-month-old child who was exclusively fed goat milk is likely to develop what type of anemia?
Megaloblastic anemia as a result of folic acid deficiency. Goat milk contains very little folic acid compared with cow milk. Infants who are consuming large amounts of goat milk— especially if they are not receiving significant supplemental solid foods—are susceptible to this type of anemia. In addition, the diagnosis can be complicated by the higher risk of coexistent iron-deficiency anemia in this
age group.

PLATELET DISORDERS
153. How can a platelet count be estimated from a peripheral smear?
As a rule, each platelet that is visible on a high-power microscopic field (100 objective) represents 15,000 to 20,000 platelets/mm3. If platelet clumps are observed, the count is usually > 100,000/mm3.
154. What are the main pathophysiologic processes that can result in thrombocytopenia?
• Peripheral destruction
• Consumptive coagulopathy
• Splenic sequestration
• Bone marrow failure
155. A previously healthy 3-year-old child develops mucosal petechiae, multiple ecchymoses, and a platelet count of 20,000/mm3 2 weeks after a bout of chicken pox. What is the most likely diagnosis?
Acute immune thrombocytopenic purpura (ITP). ITP is one of the most common bleeding disorders of childhood, and the presentation of symptoms occurs after infection in about 50% of cases.
156. What microscopic features would suggest a diagnosis other than ITP in a patient with a platelet count of 20,000/mm3?
• Platelet clumps (in vitro phenomenon caused by ethylenediaminetetraacetic acid (EDTA) that results in artifactually low platelet counts)
• Leukemic blasts
• RBC fragments (suggest a microangiopathic etiology such as hemolytic-uremic syndrome or Kasabach-Merritt syndrome)
• Large platelets (seen in inherited platelet disorders such as Bernard-Soulier syndrome, MYH9 syndromes, DiGeorge syndrome)
• Atypical lymphocytes (thrombocytopenia rarely occurs as part of infectious mononucleosis)
• Uniformly small platelets (a feature of Wiskott-Aldrich syndrome)

Thachil J, Hall GW: Is this immune thrombocytopenic purpura? Arch Dis Child 93:76–81, 2008
Drachman JG: Inherited thrombocytopenia: when a low platelet count does not mean ITP, Blood 103:390–398, 2004.

157. What is the natural history of acute childhood ITP?
With or without medical treatment, 50% to 60% of patients with acute ITP will have normal platelet counts within 1 to 3 months of diagnosis, and 75% are well after 6 months. By 1 year, only 10% of

children with ITP remain thrombocytopenic, and some of the children with chronic ITP still improve as long as 5 to 10 years after diagnosis. About 5% of patients have recurrent ITP. Because of this predominantly benign natural course of ITP, careful consideration is necessary before instituting treatment that is hazardous or irreversible.
158. In a toddler with suspected ITP, what is the significance of a palpable spleen on examination?
Although patients with ITP may rarely have a palpable spleen tip, the presence of splenomegaly
in a patient with thrombocytopenia warrants more aggressive evaluation for an associated problem (e.g., collagen-vascular disease, hypersplenism, leukemia, glycogen storage disorder).
159. In patients with suspected ITP, should a bone marrow evaluation be done?
Recent guidelines suggest that with classic ITP, there is no need for a bone marrow examination even in patients who have failed IVIG and may require steroids. However, if a patient has features that are potentially consistent with an alternative diagnosis, such as other cytopenias, organomegaly or an atypical history and physical exam, a bone marrow evaluation should be considered.

Neunert C, Lim W, Crowther M, et al: The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia, Blood 117:4190–4207, 2011.

160. When should medical treatment be given for acute ITP without active bleeding? Because the long-term prognosis of ITP does not appear to be influenced by medical treatment, the management of a newly diagnosed child with ITP and no serious bleeding is observation and specific instructions for thrombocytopenic precautions. Historically, the concern at very low platelet counts
(<10,000/mm3) was the risk of intracranial hemorrhage which was rare (<1% of affected patients), but had mortality rates that ranged from 30% to 50%. However, current data suggest that the risks
of up-front therapy in a child with minimal or no bleeding outweighs the potential benefits. Up-front therapy should be considered for those patients who may fail to follow thrombocytopenic precautions (such as active toddlers) or those who already have significant bleeding.

Cooper N: A review of the management of childhood immune thrombocytopenia: how can we provide an evidence-based approach? Br J Haematol 165:756–767, 2014.

161. What are thrombocytopenic precautions in children?
The goal of thrombocytopenic precautions is to prevent significant trauma in children who may be at risk for bleeding as a result of their ITP. An easy rule of thumb for families is for the child to keep one foot on the ground at all times (no climbing, swinging, diving, etc.) as this limits the height of fall a child could take.
162. How do treatments for ITP compare?
• IVIG: 0.8 to 1.0 g/kg/day, raises the platelet count in approximately 85% of patients. The response usually occurs within 48 hours and persists for 3 to 4 weeks. Up to 75% of patients will have some degree of limited adverse reaction (e.g., nausea, vomiting, headaches, fever). IVIG is more expensive than steroids.
• Corticosteroids: Corticosteroids are similarly effective, but oral steroids take about twice as long (4 days) to raise the platelet count significantly. The steroid effect may be multifactorial because signs of hemorrhage tend to decrease before the increase in platelets occurs. This may include microvascular endothelial stability. Side effects of long-term frequent steroid use are multiple.
• Anti-D immunoglobulin: Anti-D immunoglobulin (immunoglobulin with antibody Rh [D]) should be given intravenously to individuals with adequate hemoglobin count, (Rh)D-positive RBCs, and intact splenic function. It is more rapidly given than IVIG, with a slightly smaller proportion of responders.
• Splenectomy: When done laparoscopically, splenectomy successfully restores the platelet count to safe (>50 k/μL) or normal (>150 k/μL) in 75% to 80% of patients who fail drug therapy. Preoperative immunization against encapsulated bacteria is necessary to minimize the risk of postsplenectomy sepsis; many also advocate oral antibiotic prophylaxis postoperatively.
• Anti-CD20 (rituximab): In refractory severe cases, antibody therapy directed at B-lymphocyte CD20 has achieved some partial and complete responses that are sustained.

163. Which children with ITP are candidates for splenectomy?
Splenectomy improves the platelet count in up to 90% of patients. Because spontaneous remission is common in acute ITP, splenectomy is usually limited to bleeding that is life threatening and unresponsive to medical therapies. Patients with ITP lasting >1 year with continued bleeding, severe thrombocytopenia, or unacceptable restrictions may be reasonable candidates for splenectomy.
164. What evaluations should be considered in a patient with persistent refractory thrombocytopenia?
• Antinuclear antibody, double-stranded DNA, C3, C4, p-ANCA (to rule out systemic lupus erythematosus and other collagen vascular diseases)
• Quantitative immunoglobulin levels, pneumococcal titers (to rule out common variable immune deficiency)
• Bone marrow aspiration or biopsy (to evaluate for possible myelodysplastic syndrome or marrow failure)
• Viral studies (including polymerase chain reaction for HIV, hepatitis C, EBV, cytomegalovirus, parvovirus, and human herpesvirus 6 and 8)

Kalpatthi R, Bussel JB: Diagnosis, pathophysiology and management of children with refractory immune thrombocytopenic purpura, Curr Opin Pediatr 20:8–16, 2008.

165. How is neonatal alloimmune thrombocytopenia diagnosed and treated?
Neonatal alloimmune thrombocytopenia may occur when a fetus expresses platelet antigens inherited from the father that the mother lacks. Some mothers, especially those with “permissive” HLA types, form IgG antibodies that cross the placenta and cause moderate to severe thrombocytopenia in the fetus. Infants of mothers with first pregnancies can be affected, and there is a high recurrence risk. Both mother and father should have the common platelet alloantigens typed for incompatibility, and the mother should be tested for IgG antiplatelet antibodies recognizing that difference. In second and subsequent pregnancies at risk, especially for intracranial hemorrhage, maternal IVIG has been demonstrated to be of benefit. Affected infants should receive washed maternal platelets; antigen- matched platelets or in exceptionally dire circumstances, untyped platelets with or without concomitant treatment with IVIG and steroids. Under investigation is whether prenatal platelet typing is of benefit in prevention of the substantial proportion of cases in first pregnancies.

Bussel, JB, Sola-Visner, M: Current approaches to the evaluation and management of the fetus and neonate with immune thrombocytopenia, Semin Perinatol 33:35–42, 2009.

166. In what conditions of children is thrombocytosis most commonly seen?
• Acute infections (e.g., upper and lower respiratory tract infections)
• Chronic infections (e.g., tuberculosis)
• Iron-deficiency anemia
• Hemolytic anemia
• Medications (e.g., vinca alkaloids, epinephrine, corticosteroids)
• Inflammatory disease (e.g., Kawasaki disease)
• Malignancy (e.g., chronic myelogenous or megakaryocytic leukemia)

Chiarello P, Magnolia M, Rubino M, et al: Thrombocytosis in children, MINERVA Pediatr 63:507–513, 2011.
Yohannan MD, Higgy KE, al-Mashhadani SA, Santhosh-Kumar CR: Thrombocytosis. Etiologic analysis of 663 patients,
Clin Pediatr 33:340–343, 1994.

167. What level of thrombocytosis requires treatment? A high platelet count in most children does not appear to be a cause of significant morbidity because it is often transient. In some centers, aspirin in doses of 81 mg daily are administered when the platelet count exceeds 1.5 106/mm3. The early introduction of aspirin therapy may be more important if the patient has other problems that might contribute to hyperviscosity, such as a high WBC count or hemoglobin level.

Denton A, Davis P: Extreme thrombocytosis in admissions to paediatric intensive care: no requirement for treatment, Arch Dis Child 92:515–516, 2007.

SICKLE CELL DISEASE
168. What is the mutation that results in sickle cell disease?
On the β-globin gene on chromosome 11, the seventeenth nucleotide is changed from thymine
to adenine and thus the sixth amino acid in the β-globin chain becomes valine instead of glutamic acid. Thus, only a single nucleotide substitution is required (GTG for GAG), but the result is sickle hemoglobin (HbS), which polymerizes on deoxygenation, makes the RBC more rigid, and causes structural damage to the RBC membrane. This change leads to hemolytic anemia and contributes to vasoocclusion. The α-chain is normal.

NHLBI Evidence Based Management of Sickle Cell Disease: Expert Panel Report 2014. http://www.nhlbi.nih.gov/health- pro/guidelines/sickle-cell-disease-guidelines. Accessed on Mar. 18, 2015.
NHLBI Comprehensive Sickle Cell Centers: www.everythingsicklecell.com. Accessed on Mar. 20, 2015. Sickle Cell Disease Association of America: www.sicklecelldisease.org. Accessed on Jan. 9, 2015.
169. Why is sickle cell disease often asymptomatic during the first months of life? During the neonatal period, the presence of large amounts of fetal hemoglobin reduces the rate of polymerization of HbS and the sickling of RBCs that contain this abnormal hemoglobin. As the amount of fetal hemoglobin decreases after age 3 to 6 months, patients with sickle cell disease are increasingly likely to experience their first clinical manifestations.
170. What are the various genotypes that can cause the clinical syndrome of sickle cell disease?
Genotypes depend on which two genes make up the β chain component. In general, severity varies from SS> Sβ0— thalassemia > SC> Sβ+ thalassemia> S-hereditary persistence of fetal hemoglobin (HPFH). Hemoglobin concentrations increase from an average of 6 to 8 g/dL with HbSS to 11 to 14 g/dL for HbS-HPFH, which contributes to the variation in clinical severity.
Only Sβ+ thalassemia has any hemoglobin A on electrophoresis (5% to 30%).
171. What are the two major pathophysiologic mechanisms in sickle cell anemia that cause the morbidities associated with the disease?
• Hemolysis: Sickled RBCs undergo both intravascular and extravascular hemolysis, which leads to anemia, reticulocytosis, jaundice, gallstones, and occasional aplastic crisis. It now
appears that chronic hemolysis impacts on the utilization and bioavailability of NO (nitric oxide), a potent vasoactive agent. Long-term hemolysis has been associated with pulmonary hypertension and right-sided heart failure.
• Vasoocclusion: Intermittent and chronic vasoocclusion result in both acute exacerbations (e.g., painful crisis, stroke) and chronic disease manifestations (e.g., retinopathy, renal disease). The adhesion of sickled erythrocytes to inflamed vascular endothelium is a principal pathologic component. Activation of leukocytes and platelets, as well as components of the coagulation protein cascade, is also prominent.
172. A 6-month-old black male has painful swelling of both hands. What is the most likely diagnosis?
Hand-foot syndrome, or dactylitis. This common early manifestation of sickling disorders in infants and young children is characterized by painful swelling of the hands, feet, and proximal fingers and toes caused by symmetric infarction in the metacarpals, metatarsals, and phalanges (Fig. 9-9).

Figure 9-9. Swelling of the fingers from dactylitis. (From Lissauer T, Clayden G: Illustrated Textbook of Pediatrics. London, 1997, Mosby,
p 238.)

A lack of systemic signs, the presence of symmetric involvement, and young patient age help distinguish hand-foot syndrome from the much less common osteomyelitis, which may also complicate sickle cell disease.
173. When does functional asplenia occur in children with sickle cell disease?
It may begin as early as 5 or 6 months of age, and it may precede the presence of Howell-Jolly bodies in the peripheral smear. Most children with HbSS who are >5 years old have functional asplenia, with a small, atrophied spleen. Clinical experience indicates that the period of increased risk for serious bacterial infection parallels the development of functional asplenia. Consequently, in addition to routine vaccinations, antibiotic prophylaxis with penicillin is recommended beginning
at 2 months of age. Loss of splenic function usually occurs later in patients with HbSC or HbSβ+ thalassemia or those receiving chronic transfusion therapy.
174. What is the most common cause of death in children with sickle cell disease?
Infection. Splenic dysfunction causes increased susceptibility to meningitis and sepsis (particularly pneumococcal). The incidence of infection can be reduced by 84% with daily penicillin taken orally and initiated early in infancy (before 4 months of age) and continued into childhood. Though studies demonstrated no further benefit after 5 years of age, many providers continue penicillin prophylaxis into the teenage years. Pneumococcal and meningococcal vaccines may provide further protection.
175. What are the four main categories of acute events requiring intervention in patients with sickle cell disease?
• Aplastic crisis: Hemoglobin may fall as much as 10% to 15% per day without reticulocytosis
• Acute hemolytic crisis: Acute hemolysis may be precipitated by infection or febrile illness. Hemoglobin may fall while total and indirect bilirubin levels are elevated. Reticulocyte count may be preserved or elevated. Patients present with jaundice and dark urine.
• Vasoocclusive events: Includes painful crises (most common), acute chest syndrome, acute central nervous system events (stroke), and priapism
• Acute splenic sequestration: May occur rapidly, with profound hypotension and cardiac decompensation

Dover GJ, Platt OS: Sickle cell disease. In Nathan DG, Orkin SD, Ginsburg D, Look AT, editors: Nathan and Oski’s Hematology of Infancy and Childhood, ed 6, Philadelphia, 2003, WB Saunders, p 802.

176. How should a child with a vasoocclusive (painful) event be managed? For outpatients with an acute painful crisis, ibuprofen or acetaminophen and codeine are reasonable choices. Patients with intensely painful crises require day unit or inpatient
hospitalization for opioid (including morphine) analgesia, ideally given intravenously. Use of meperidine in this situation is no longer recommended unless the patient has a specific preference for the medication or allergy to other morphine derivatives. Patient-controlled analgesia offers the dual benefit of a constant infusion and intermittent boluses of an analgesic. Other supplementary agents, including nonsteroidal analgesics (e.g., ketorolac), and vasodilators/membrane active agents (e.g., arginine) are under study. For severe crises, blood transfusions to reduce the
percentage of sickle cells to <30% may be beneficial as part of a multimodal pain approach.
177. A 15-month-old with sickle cell disease presents with pallor and fatigue, but no jaundice. On exam, his spleen is palpable to his umbilicus. What is the diagnosis and how should it be managed?
Acute splenic sequestration represents a true emergency and is the second leading cause of death in young children with sickle cell disease. The clinical problem is primarily one of hypovolemic shock as a result of the pooling of blood in the acutely enlarged spleen.
The hemoglobin level may drop to as low as 1 to 2 g/dL. The major therapeutic effort should be directed toward volume replacement with whatever fluid is handy. In most instances, normal saline or colloid solutions will be adequate until properly cross-matched blood is available.
Patients may experience “auto-transfusion” where, after a bolus of fluid, the spleen begins to shrink and formerly trapped RBCs reenter circulation, raising the hemoglobin beyond what

one would expect with transfusion or fluids alone. Close monitoring of hemoglobin and repeated assessment of spleen size are critical to ensure the patient does not become polycythemic.
Splenectomy may be considered for patients with recurrent splenic sequestration.

Yawn BP, Buchanan GR, Afenyi-Annan AN, et al: Management of sickle cell disease: summary of the 2014 evidence-based report by expert panel members, JAMA 312:1033–1048, 2014.

178. What is “acute chest syndrome” in sickle cell patients?
Acute chest syndrome refers to the constellation of findings (e.g., fever, cough, chest pain, pulmonary infiltrates) that can resemble pneumonia or pulmonary infarction. The exact mechanism is unknown, and the cause is likely multifactorial. Various infections (e.g., viral, chlamydial, mycoplasmal) may initiate respiratory inflammation, which ultimately causes localized hypoxia; increased pulmonary sickling may then result. Rib and other bone infarcts can also occur, and hypoventilation may
result from chest splinting. Pulmonary fat embolism has been seen to occur, particularly in the setting of a preceding bony painful crisis (e.g., the thigh).

Zar HJ: Etiology of sickle cell chest, Pediatr Pulmon 26:S188–S190, 2004.

179. How should the acute chest syndrome in sickle cell patients be treated?
• Aggressively because rapid progression to respiratory failure is possible.
• Optimization of ventilation is vital, including supplemental oxygen, analgesics adequate to minimize splinting, incentive spirometry, and other possible measures (e.g., bronchodilators, nitrous oxide).
• Judicious hydration: Overly vigorous hydration can lead to pulmonary edema.
• Antibiotics: These should typically be given to cover Chlamydia, Mycoplasma, and
Streptococcus pneumoniae.
• Blood transfusion, including erythrocytapheresis (automated RBC exchange transfusion), has been shown to improve the status of patients with acute chest syndrome; this should be considered for patients with severe or worsening disease.

Rees DC, Williams TN, Gladwin MT: Sickle-cell disease, Lancet 376:2018–2031, 2010.
Graham LM: Sickle cell disease: Pulmonary management options, Pediatr Pulmonol 26:S191–S193, 2004.

180. How often is priapism a problem in children with sickle cell disease?
Priapism is an unwanted, painful erection that is usually unrelated to sexual activity. It is an underappreciated morbidity in adolescents with sickle cell disease, usually occurring at least once by the age of 20 years and typically by the age of 12 years. Most patients are unaware of the term and the consequences; early urologic intervention may prevent irreversible penile fibrosis and impotence.

Rachid-Filho D, Cavalcanti AG, Favorito LA, et al: Treatment of recurrent priapism in sickle cell anemia with finasteride: a new approach, Urology 74:1054–1057, 2009.
Maples BL, Hagemann TM: Treatment of priapism in pediatric patients with sickle cell disease, Am J Health Sys Pharm 61:355–363, 2004.

181. What are some long-term morbidities associated with sickle cell disease?
• Stroke
• Chronic lung disease
• Renal failure
• Congestive heart failure
• Retinal damage
• Leg ulcers
• Aseptic necrosis of the hip or shoulder
• Poor growth
182. How can stroke be prevented in children with sickle cell disease?
Children with sickle cell disease are at increased risk of stroke. The risk of stroke increases and peaks around 2 to 5 years of age then declines, only to increase again in the late teens and

throughout adulthood. To prevent initial strokes, children (age 2 to 16 years old) should
undergo annual screening with transcranial Doppler ultrasounds (TCDs) to assess flow velocities of the intracranial vessels. Elevated velocities are predictive of increased stroke risk. Regular blood transfusions are an effective primary prevention strategy to prevent strokes. The goal
of transfusion therapy is to keep the HbS percent below 30%. Children who have already suffered an overt stroke also benefit from regular transfusions as a secondary prevention method.

Yawn BP, Buchanan GR, Afenyi-Annan AN, et al: Management of sickle cell disease: summary of the 2014 evidence-based report by expert panel members, JAMA 312:1033–1048, 2014.
Armstrong-Wells J. Grimes B. Sidney S, et al: Utilization of TCD screening for primary stroke prevention in children with sickle cell disease, Neurology 72:1316–1321, 2009.

183. If initiated for either primary or secondary stroke prevention, when should blood transfusions be discontinued?
Currently, there is no clear recommendation as to when it is safe to discontinue routine transfusions for either primary or secondary stroke prevention. Patients may have normalization of their TCDs while on chronic transfusions; however, once a routine transfusion program has been stopped, they may quickly revert to their high-risk velocities. One large study tried to transition patients with a history of stroke from transfusions to hydroxyurea combined with phlebotomy in an effort to maintain the same level of stroke protection while also alleviating the transfusional iron burden. However, the study was closed early because investigators saw an increase in stroke events without a decrease in iron burden.

Ware RE and Helms RW: Stroke with transfusions changing to hydroxyurea, Blood 119:3925–3932, 2012. Adams RJ and Brambilla D: Discontinuing prophylactic transfusions used to prevent stroke in sickle cell disease, N Engl J Med 353:2769–2778, 2005.

184. What is the primary mechanism by which hydroxyurea is beneficial for sickle cell disease?
Hydroxyurea is a cytotoxic drug that has been used primarily to treat chronic myelogenous leukemia and polycythemia vera. However, its use was shown to increase hemoglobin F (HbF) totals. It is unclear if this is due to direct effects on γ chain transcription sites or due to preferential γ chain production during erythroid regeneration following cytotoxic insult.
Increased concentrations of HbF (particularly >20%) are associated with decreased sickling of the RBCs and decreased hemolysis, which results in increased hemoglobin. Clinically,
patients experience fewer vasoocclusive painful events, episodes of acute chest syndrome, transfusion requirements, and hospitalizations. It is unclear if hydroxyurea can prevent or reverse organ damage.

Platt OS: Hydroxyurea for the treatment of sickle cell anemia, N Engl J Med 358:1362–1369, 2008.
Strouse JJ, Lanzkron S, Beach MC, et al: Hydroxyurea for sickle cell disease: a systematic review for efficacy and toxicity in children, Pediatrics 122:1332–1342, 2008.

185. Hydroxyurea treatment in young children: how early and how beneficial? Infants (9 months to 18 months) have been started on hydroxyurea (maximum dose 20 mg/kg/ day) with minimal toxicity and no increased rate of infection. In a recent Phase 3 randomized controlled trial comparing hydroxyurea to placebo, infants started on hydroxyurea had lower rates of recurrent vasoocclusive events, dactylitis, acute chest syndrome, transfusion, and hospitalization. TCD velocity rates were also lower, although it is unclear if this translates into decreased risk of stroke. There were no significant differences between the groups in their splenic or renal function.

Thornburg CD, Files BA, Luo Z, et al: Impact of hydroxyurea on clinical events in the BABY HUG trial, Blood 120:4304– 4310, 2012.
Wang WC, Ware RE, Miller ST, et al: Hydroxycarbamide in very young children with sickle-cell anaemia: a multicenter, randomized, controlled trial (BABY HUG), Lancet 377:1663–1672, 2011.

186. How common is the sickle cell trait in the United States?
Heterozygosity for the sickle gene occurs in about 8% of blacks in the United States; 3% of Hispanics in the eastern United States; and a much smaller percentage of individuals of Italian, Greek, Arabic, and Veddah Indian heritage. Of note is that 2% of blacks in the United States have the hemoglobin C trait.
187. Does sickle cell trait have any significant morbidity?
Under normal physiologic conditions, no significant morbidity is associated with sickle cell trait. RBCs in individuals with sickle cell trait contain only 30% to 40% sickle hemoglobin, which is insufficient to cause sickling. However, in hypoxic settings, sickling may occur. Portions of the kidney may have physiologically low oxygen concentrations that can interfere with function and lead to an inability to concentrate urine (hyposthenuria) and hematuria (usually microscopic and asymptomatic). At high altitudes (e.g., when mountain climbing or in an unpressurized aircraft), splenic infarction is possible.

KEY POINTS: SICKLE CELL DISEASE
1. A genetic mutation leads to abnormal beta-globin chain that promotes polymerization of hemoglobin and sickling in the setting of hypoxia.
2. Eight percent of blacks have the sickle cell trait.
3. Acute events include aplastic, hemolytic, vasoocclusive, and sequestration.
4. The risk of serious bacterial infection is increased among these patients as a result of functional asplenia.
5. Dactylitis (painful hand/foot swelling) is often the earliest manifestation.

188. What is the second most common worldwide hemoglobin variant?
Hemoglobin E. This variant is particularly high in the Southeast Asian population (especially those of Laotian, Thai, and Cambodian heritage). Heterozygotes are asymptomatic; homozygotes can have a mild microcytic anemia. The most common abnormal findings on a peripheral smear are microcytosis and target cells.

THALASSEMIA
189. What are the thalassemias? The thalassemias are a heterogeneous group of disorders of hereditary anemia due to diminished or absent normal globin chain production. Normally, four alpha-globin genes and two beta-globin genes are expressed to make the tetrameric globin protein, which then combines with a heme moiety to make the predominant hemoglobin that is found in red cells, HbA (sub-units α2β2). Depending on the number of genes that are deleted, the production of polypeptide chains is diminished. In patients with
alpha-thalassemia, alpha-globin production is lowered; in patients with beta-thalassemia, beta-globin production is lowered. When one class of polypeptide chains is diminished, this leads to a relative excess of the other chain. The result is ineffective erythropoiesis, precipitation of unstable hemoglobins, and hemolysis as a result of intramedullary RBC destruction.
190. Where was β-thalassemia first described?
Despite its incidence being highest in the Mediterranean region, β-thalassemia was first described by a hematologist, Dr. Denton Cooley, in 1925 in Detroit. Why Detroit and not Europe for the first recognition? Speculation is that the condition was thought to be malaria, endemic to that region and with similar clinical features of hemolysis, anemia, and splenomegaly.

Weatherall DJ, Clegg JB: Historical perspectives: the many and diverse routes to our current understanding of the thalassemias. In Weatherall DJ Clegg JB, editors: The Thalassemia Syndromes, ed 4. Oxford, 2001, Blackwell Science, p 3.

191. What accounts for the variability in the clinical expression of the thalassemias? Clinical heterogeneity results from variability in the number of gene deletions (particularly in alpha- thalassemia). As a rule, the greater the number of deletions, the more severe the symptoms. A large number of point mutations have been identified in various populations; this can contribute to the phenotypic diversity. In addition, the inheritance of other thalassemia genes (e.g., delta-thalassemia) or the persistence of fetal hemoglobin can modify the clinical course.

192. How is the diagnosis of thalassemia made in most clinical laboratories? Homozygous beta-thalassemia is detected by the absence (β0) or reduction (β+) of the amount of HbA (α2β2) relative to HbF (α2γ2 or fetal hemoglobin) on hemoglobin electrophoresis. The carrier state for beta-thalassemia is characterized by a low mean cell volume and, in most instances, an increased level of HbA2 (α2δ2) or HbF. The levels of these two hemoglobins are most accurately measured by column chromatography. Estimation or quantitation from electrophoretic patterns is frequently misleading. The alpha-thalassemia trait remains a diagnosis of exclusion (low mean cell volume in the absence of an identifiable cause) in the clinical laboratory, although the enumeration of missing alpha genes for the most common deletions in specific ethnic populations is accomplished by molecular techniques. Newer polymerase chain reaction-based DNA tests for the common variants have become very useful.
193. Describe the clinical features of the alpha-thalassemia syndromes
When all four alpha-globin genes are missing or nonfunctional, this results in severe intrauterine anemia and hydrops fetalis. Extraordinary therapy such as in utero transfusion may result in survival. Absence of three functional alpha-globin genes results in HbH disease, which is a chronic moderate to severe anemia with jaundice and splenomegaly that may necessitate RBC transfusion therapy. Absence of two alpha-globin genes is associated with mild microcytic anemia. Absence of one alpha-globin gene is clinically silent (Table 9-5).

Table 9-5. Clinical Features of α-Thalassemia

SYNDROME USUAL GENOTYPE α GENE NUMBER
CLINICAL FEATURES
Normal αα/αα 4 Normal
Silent carrier α-/αα 3 Normal
α-Thalassemia trait α-/α- 2 Mild microcytic anemia
HbH disease – -/αα 1 Moderate microcytic anemia Splenomegaly
Jaundice

194. What is hemoglobin Barts?
Hemoglobin Barts is a tetramer of γ-chains often noted on the newborn screen due to α-chain deletions. It can be present, in varying degrees, in the setting of alpha thalassemia trait, HbH disease, or fetal hydrops.
195. What are the clinical features of the beta-thalassemia syndromes?
• Thalassemia minor: Minimal or no anemia (hemoglobin 9 to 12 g/dL); microcytosis; elevated RBC count; no need for transfusion
• Thalassemia intermedia: Microcytic anemia with hemoglobin usually >7 g/dL; growth failure; hepatosplenomegaly; hyperbilirubinemia; thalassemic facies (i.e., frontal bossing, mandibular malocclusion, prominent malar eminences due to extramedullary hematopoiesis) develop
between the ages of 2 and 5 years; intermittent or variable transfusion requirements
• Thalassemia major (Cooley’s anemia): Severe anemia (hemoglobin 1 to 6 g/dl) usually during the first year of life; hepatosplenomegaly; growth failure; transfusion dependent

Olivieri NF: The beta-thalassemias, N Engl J Med 341:99–109, 1999.

196. How can coexistent iron deficiency increase the difficulty of diagnosing beta- thalassemia?
The beta-thalassemia trait is usually diagnosed by hemoglobin electrophoresis, with quantitative hemoglobins revealing elevated HbA2 and/or HbF levels. Iron deficiency can cause a lowering of HbA2, thereby masking the diagnosis. With iron replacement, the hemoglobin A2 will rise to the expected elevated levels seen in patients with the beta-thalassemia trait.

197. What are the adverse effects of chronic transfusional iron overload in children with thalassemia?
• Cardiac effects include congestive heart failure; dysrhythmias; and less frequently, pericarditis. Cardiac T2* MRI imaging is both diagnostic and prognostic. Significant cardiac iron deposition predicts rates of heart failure and arrhythmia over the subsequent year.
• Endocrine effects include delays in growth and sexual development, hypoparathyroidism, and hypothyroidism. Diabetes as a result of iron overload is irreversible, even with intensive chelation
• Hepatic effects include progressive liver fibrosis and cirrhosis. Monitoring with dedicated MRI imaging is recommended.

Kirk P, Roughton M, Porter JB, et al: Cardiac T2* magnetic resonance for prediction of cardiac complications in thalassemia major, Circulation 120:1961–1968, 2009.

198. What are the two most common diseases that are associated with transfusion- related iron overload?
Thalassemia major and sickle cell disease.
199. How do you reduce iron accumulation in children who require repeated transfusions?
• Chelation therapy: Subcutaneous or intravenous deferoxamine has been the standard therapy for transfusional overload; however, new oral iron chelators including daily deferasirox and three times daily deferiprone have demonstrated efficacy as single agents. Studies of combination chelation therapy demonstrate acceptable toxicity profiles with improved iron status.
• Splenectomy: This is used primarily in patients with thalassemia (and a small subgroup of sickle cell patients) who have hypersplenism, which results in the premature destruction of RBCs and increased transfusion requirements.
• Diet: Drinking tea with meals reduces dietary iron absorption and may be most helpful in patients with diseases such as thalassemia intermedia, in which the bulk of excessive iron is dietary in origin.
• Erythrocytapheresis: Automated erythrocytapheresis (red cell exchange) rather than repeated simple transfusions may markedly reduce transfusional iron loading in patients with sickle cell disease.

Lo L, Singer ST: Thalassemia: Current approach to an old disease, Pediatr Clin North Am 49:1165–1192, 2002.

200. In addition to iron loading, what are additional risks of chronic transfusion therapy? Patients on chronic transfusion therapy are at risk of alloimmunization to RBC antigens, which may make transfusions a challenge going forward, or HLA antigens, which may make bone marrow transplantation (BMT) a challenge. With every transfusion, patients experience an ongoing risk of transfusion-related infection and a risk of experiencing a transfusion reaction.

KEY POINTS: THALASSEMIA
1. Normal hemoglobin (HbA): Tetramer of two alpha and two beta chains
2. Associated with quantitative reduction in globin synthesis
3. Homozygous beta-thalassemia is most severe form, with pallor, jaundice, hepatosplenomegaly, growth retardation
4. Expansion of facial bones resulting from extramedullary hematopoiesis
5. Severity of alpha-thalassemia depends on number of genes deleted (1 to 4)
6. Alpha-thalassemia: More common among people of Southeast Asian ethnicity
7. Beta-thalassemia: More common in people of Mediterranean ethnicity

TRANSFUSION ISSUES
201. What is the difference between the direct and indirect Coombs tests?
• Direct test: Coombs serum (antihuman globulin) is added directly to a patient’s washed RBCs. The occurrence of agglutination means that the patient’s RBCs have been coated in vivo by an antibody. Direct Coombs testing is vital for diagnosing AIHAs.

• Indirect test: This involves incubating a patient’s serum with RBCs of a known type and adding Coombs serum. If in vitro sensitization occurs, agglutination will result, which indicates that antibodies in the serum are binding to the antigens on the RBCs. Indirect testing is key for blood cross-matching.
202. What is the difference between forward and reverse blood typing?
A forward type determines antigens on patient or donor RBCs. It uses reagent monoclonal antibodies against A or B or Rh(D) and tests for agglutination. A REVERSE type determines antibodies in patient or donor serum or plasma. It tests for agglutination with RBCs of a known phenotype, ensuring the patient has appropriate naturally forming antibodies (anti-A or anti-B isoagglutinins). Outside of the neonatal period (age> 4 months), both a forward and a reverse type must be
performed for a patient to have a valid ABO type.
203. What can cause a patient to be ABO indeterminate?
Inconsistencies with either the forward or reverse type may cause a patient to be ABO indeterminate. A compatible, but out of group, transfusion may cause the patient to be ABO indeterminate in both the forward or reverse direction. Often, these will result in “mixed field” results where the
laboratory notes two populations of cells. Infants <4 months old may not have developed the naturally forming isoagglutinins, so the reverse type may be invalid, and therefore, is not required
to result their specimens. Usually the reverse type resolves by 6 months to 1 year of age. Leukemia or a history of bone marrow transplantation may cause a patient to be ABO indeterminate.
Hypogammaglobulinemia may cause a patient to lack their appropriate serum isoagglutinins, causing a discrepancy in the reverse type. In contrast, IVIG infusion, a cold autoantibody or a cold alloantibody may cause excessive reactivity in the reverse type, thus making the patient ABO indeterminate.
204. What is a naturally forming RBC antibody?
RBC alloantibodies are antibodies against RBC antigens that the patient lacks. These are usually only formed after exposure to those antigens through transfusion or pregnancy. However, there are some alloantibodies (such as anti-A and anti-B) that do not require such exposure. This is because similar antigens are widely expressed in nature (e.g., aeroallergens, gut flora) and thus the individual is exposed “naturally.”
205. What is the difference between a type and screen and a type and cross? When a type and screen is performed, both a forward and reverse type are performed on the patient sample. In addition, patient serum or plasma is then incubated with RBCs of a known
phenotype to assess for the presence of any alloantibodies. When a type and cross is performed, the patient sample has all of the elements of a type and screen performed, but then the plasma or serum of the patient is tested with RBCs of potentially compatible units of blood. If compatible, those units are reserved for the patient.
206. What are the indications for the use of leukoreduced RBCs?
When packed red cells are prepared from whole blood and then filtered, most of the remaining white cells are removed from the product. Because febrile transfusion reactions are usually the result of leukocytes, filtered products should be used for patients who have experienced such reactions to previous blood transfusions. Filtered red cells are also effective for reducing the transmission of cytomegalovirus in at-risk individuals. In addition, the use of filtered blood components reduces the risk of HLA alloimmunization, which is desirable for patients who have undergone repeated transfusions and for those who may need stem cell or solid organ transplants.
207. What is the estimated total blood volume of children?
Estimation of blood volume is dependent on both age and weight. Older children have a lower proportion of their weight as blood compared with younger children. As a rule of thumb, blood volume is estimated as follows:
Children >3 months of age: 70 mL/kg Premature infants: 90 to 100 mL/kg Term infants: 80 to 90 mL/kg

Morley SL: Red blood cell transfusions in acute paediatrics, Arch Dis Child Pract Ed 94:65–73, 2009.

208. What is the RBC transfusion threshold for infants <4 months of age? Transfusion thresholds vary significantly by gestational age, postnatal age, and clinical status because of the complex physiology of neonates and young infants. In an effort to limit the risks
associated with transfusions, restrictive transfusion practices are being examined to determine if reduced transfusion requirements might be used without increased morbidity and mortality. One set of proposed guidelines is as follows:
• Hct <20% with low reticulocyte count and symptomatic anemia
• Hct <30% requiring oxygen support or significant symptoms including apnea, bradycardia, tachycardia, or tachypnea.
• Hct <35% on >35% oxygen hood or escalated ventilatory support.
• Hct <45% on extracorporeal membrane oxygenation (ECMO) or with congenital cyanotic heart disease.

Kirpalani H, Whyte RK, Andersen C, et al: The Premature Infants in Need of Transfusion (PINT) study: a randomized, controlled trial of a restrictive (low) versus liberal (high) transfusion threshold for extremely low birth weight infants, J Pediatr 149:301–307, 2006.
Roseff SD, Luban NLC, Manno CS: Guidelines for assessing appropriateness of pediatric transfusion, Transfusion
42:1398–1413, 2002.

209. What is the packed red blood cell (PRBC) transfusion threshold for children
>4 months of age?
Transfusion thresholds vary significantly by age and clinical status in older children as
well. Restrictive practices are also being evaluated in older children. One study involving 637 children in a pediatric ICU found no difference in outcome between using hemoglobin thresholds of 7 g/dL versus 9.5 g/dL as the threshold for transfusion. One set of proposed guidelines is
as follows:
• Hct <24% with symptoms of anemia
• Acute blood loss (>15%) unresponsive to other interventions
• Hct <40% on ECMO or severe cardiopulmonary disease
• Sickle cell disease with stroke, acute chest, symptomatic anemia, splenic sequestration,
preoperatively for general anesthesia with goal for Hb of 10 g/dL
• Chronic transfusion for patients with failure of RBC production (beta thalassemia, Diamond- Blackfan anemia, etc.) with goal for Hb of 10 to 12 g/dL

Lacroix J, Hebert PC, Hutchison JS, et al: Transfusion strategies for patients in pediatric intensive care units,
N Engl J Med 356:1609–1619, 2007.
Roseff SD, Luban NLC, Manno CS: Guidelines for assessing appropriateness of pediatric transfusion, Transfusion
42:1398–1413, 2002.

210. In patients with severe chronic anemia, how rapidly can transfusions be given? When anemia is chronic, there has been cardiovascular adaptation and a relatively normal blood volume. Excessively rapid transfusions can lead to congestive heart failure. For patients with a hemoglobin level of <5 g/dL who exhibit no signs of cardiac failure, a safe regimen is to transfuse PRBCs at a rate of 1 to 2 mL/kg per hour by continuous infusion until the desired
target is reached. In most patients, 1 mL/kg will raise the hematocrit level by 1%. Judicious use of a diuretic such as furosemide (or automated erythrocytapheresis, in larger children) can be considered.

Jayabose S, Tugal O, Ruddy R, et al: Transfusion therapy for severe anemia, Am J Pediatr Hematol Oncol
15:324–327, 1993.

211. At typical doses, what are the expected increases and likely average survival of packed RBCs, platelets, and fresh frozen survival?
See Table 9-6.

Table 9-6. Quick Facts About Blood-Product Dosing
PRODUCT DOSE EXPECTED INCREASE SURVIVAL
PRBCs 10-15 mL/kg 10 mL/kg increases Hb by 2-3 g/dL May persist 60-90 days in circulation
Platelets 10 mL/kg or 0.1-0.2 unit/kg 40 k/μL Hours to days
FFP 10-15 mL/kg 1 mL/kg increases factor levels by 1% 4-6 hours
FFP ¼ Fresh frozen plasma; PRBCs ¼ packed red blood cells.

212. What are the components of cryoprecipitate?
Cryoprecipitate is a plasma product of concentrated factors VIII, XIII, vWF and fibrinogen which precipitates as FFP is thawed and is collected by centrifugation. Indications for use include situations when specific factor concentrates are not available (e.g., hemophilia), reversal of anticoagulation or DIC. Fibrinogen concentrate is also available for children with congenital fibrinogen deficiencies, including afibrinogenemia and hypofibrinogenemia.
213. What are the most common types of transfusion reactions?
Transfusion reactions occur infrequently, with approximately 2 to 7 events per 1000 units transfused. However, these reactions can be serious and even fatal. The most common transfusion reactions are febrile nonhemolytic transfusion reactions. Patients experience fever and chills, without evidence of hemolysis, while receiving a transfusion or several hours later. Patients may be treated with antipyretics and meperidine if the chills are significant. Allergic transfusion reactions are the second most common type. Often occurring with plasma containing platelets
or FFP, symptoms may range from mild urticaria and pruritus to significant anaphylaxis with hypotension and angioedema. Antihistamines, steroids, or epinephrine may be necessary depending upon the severity of the reaction.
214. What is the most common cause of transfusion-related death in the United States?
Transfusion-related acute lung injury (TRALI) is the most common cause of transfusion related death. TRALI is characterized as acute respiratory distress with bilateral lung infiltrates and hypoxia within 6 hours of transfusion. HLA and human neutrophil antigen (HNA) antibodies have been implicated in the pathogenesis of this syndrome.
215. What is the difference between an acute hemolytic transfusion reaction and a delayed hemolytic transfusion reaction? Acute hemolytic transfusion reactions result in rapid hemolysis during or within 24 hours of an infusion of incompatible blood products. Often, these are due to ABO incompatible transfusions. Patients may experience fever, chills, back pain, and a sense of “impending doom.” They may also have hemoglobinuria, which may result in renal failure. Fluids, mannitol, and other supportive care measures may be necessary to treat this type of reaction.
Delayed hemolytic transfusion reactions may occur from 24 hours up to 28 days after transfusion, though usually present 10 to 14 days after transfusion. These are often due to RBC alloantibody incompatibility. The symptoms may be similar to acute hemolytic reactions, although they are
often milder.
216. Why do some blood products require irradiation?
Irradiation with either x-rays or gamma irradiation prevents transfusion-associated graft versus host disease (TA-GVHD). TA-GVHD is caused by a proliferation of donor T-cells within the transfusion recipient that then attack the host. Symptoms include rash, hepatitis, and GI symptoms similar to classic GVHD; however, the hallmark of this disease is pancytopenia. It is greater than 90% fatal when it occurs. Patients at risk for TA-GVHD include patients with known or suspected cellular immunodeficiency;
significant immunosuppression due to chemotherapy or BMT; infants <1200 g at birth or those who received in utero transfusions; and any patient receiving HLA-matched components, granulocytes or
blood components from directed donors.

217. In what clinical settings is apheresis utilized? During apheresis, whole blood is removed from the patient and components are centrifugally separated by density: RBCs> WBCs> platelets> plasma. Components as desired are removed with the remaining components returned to the patient along with replacement fluid or replacement blood products.
• Erythrocytapheresis: Patient RBCs are removed and replaced with donor PRBCs; this is usually performed on patients with sickle cell disease who want to prevent iron accumulation or who require an acute reduction in their HbS percent such as in the setting of acute stroke or acute chest syndrome.
• Leukapheresis: WBCs are removed; patient’s RBCs, platelets, and plasma are returned along with some fluid; usually this is performed in the setting of acute leukemia with elevated WBC and signs or symptoms of leukostasis (>100, 000/μL in acute myelogenous leukemia [AML],
>200,000 to 250,000/μL in acute lymphoblastic leukemia [ALL], and >400,000/μL in chronic
myelogenous leukemia [CML]).
• Plasmapheresis: Plasma is removed; all cellular components are returned to the patient often with 5% albumin and saline as replacement fluid; replacement with FFP can be used if patients have a coagulopathy or when plasmapheresis is performed for certain conditions such as thrombotic thrombocytopenic purpura or hemolytic-uremic syndrome.
• Thrombocytapheresis: Platelets only are removed; usually only performed after platelet count is >1,500,000/μL; not usually performed in pediatrics.
218. How are transfusion-transmitted diseases prevented?
Direct testing and donor screening/deferral. Direct testing of blood products includes serologic testing for the presence of antibodies to known pathogenic antigens and nucleic acid amplification testing (NAAT), which detects viral DNA/RNA. All blood products or donors are tested using either or both of the above methods for HIV, hepatitis B, hepatitis C, HTLV-I/II, syphilis, West Nile Virus, and Trypanosoma cruzi. With implementation of NAAT testing, the window period for HIV detection has been
reduced to 9 days and the window period for hepatitis C to <8 days. Transfusion-transmitted diseases without an FDA-approved donor screening test, such as malaria and prion diseases, are prevented
through donor screening questions and donor deferrals. For example, individuals are excluded from donating blood for 1 year after traveling to malaria-endemic areas, and for 3 years after long-term residence (5 years or more) in a malaria-endemic area.

Galel S: Infectious disease screening. In Roback JD, Grossman BJ, Harris T, Hillyer CD, editors: Technical Manual, ed 17. Bethesda, MD, 2011, American Association of Blood Banks, p 239.

219. What role does molecular testing play in providing blood products to patients? Molecular phenotyping of RBC antigens, which currently uses PCR-based, microarray technology, is an exciting and expanding area of transfusion medicine. This technology is able to identify the expected phenotype of a patient’s or donor’s RBCs at multiple (>30) antigens from DNA. This
can be used to identify donors with rare RBC phenotypes to be added to the American Rare
Donor Program (ARDP). It can also be used in clinical practice to allow for better matching of RBC products and to clarify serologic ambiguities.
Acknowledgment
The editors gratefully acknowledge contributions from Dr. Anne F. Reilly, Dr. Greg A. Holländer, and Dr. Anders Fasth that were retained from previous editions of Pediatric Secrets.

BONUS QUESTIONS
220. Do socioeconomic factors affect the ability of pediatric patients to successfully transition health care?
Yes. Patients who live in a low-income household, live in a non–English-speaking household or are either Hispanic or black are more likely to experience discontinuities in care. Patients with sickle cell disease and other hematologic disorders may be disproportionately affected.
221. Which are the characteristics of immunoglobulin transport across the placenta? IgG is the only isotype that is transferred across the placenta. All IgG subclasses cross the placenta, and their relative concentrations in the cord serum are comparable with those of the maternal serum. Transfer of IgG can first be detected as early as 8 weeks of gestation, and levels rise steadily between 18 and 22 weeks. By 30 weeks, the serum concentrations of IgG are about 50% of those observed in neonates born at term. IgG concentrations comparable with those of the mother are achieved by 34 weeks of gestation, and values at term can be higher by about 10% compared with maternal serum levels as a result of the active transport across the placenta.
222. What are the allergies associated with IgA deficiency? There is a strong association between IgA deficiency and allergic disorders. The most common diseases are allergic conjunctivitis, rhinitis, urticaria, atopic eczema, food allergies, and asthma.

National Institute of Allergy and Infectious Disease: http://www.niaid.nih.gov. Accessed Jan. 9, 2015.

223. Is there a role for stimulating platelet production in ITP therapy?
In chronic, severe, refractory ITP, use of agonists of the thrombopoietin (TPO) receptor have shown efficacy in raising platelet counts to safe levels. The effect lasts only during the time period in which the drug is being administered. Romiplostim, a TPO peptide mimetic, is administered subcutaneously and activates the TPO receptor through binding at the hematopoietic receptor domain. Eltrombopag, a TPO nonpeptide mimetic, is administered orally and activates the TPO receptor by binding at the transmembrane domain.
224. In addition to stroke, what other sickle cell–related conditions benefit from chronic transfusion regimens?
• Pulmonary hypertension
• Anemia related to renal failure
• Heart failure
• Recurrent splenic sequestration
Controversial indications for chronic transfusion therapy include:
• Prevention of recurrent vasoocclusive (pain) events
• Prevention of recurrent acute chest syndrome
• Prevention of recurrent priapism
• Leg ulcers
225. What are the indications for platelet transfusion in neonates and children? Again, transfusion thresholds vary significantly by age and clinical status. One proposed general guideline is as follows:
• Active bleeding in the setting of qualitative platelet defect
• Patient receiving ECMO therapy and platelet count <100,000/μL or bleeding
• Platelet count <10,000/μL with failure of platelet production
• Platelet count <30,000/μL in asymptomatic neonate
• Platelet count <50,000/μL in premature infant with active bleeding or requiring invasive procedure
• Platelet count <100,000/μL in sick premature infant with active bleeding or requiring invasive procedure

Roseff SD, Luban NLC, Manno CS: Guidelines for assessing appropriateness of pediatric transfusion, Transfusion 42:1398– 1413, 2002.