1. Diagnosis of sickle cell disorders
Mohammad Faranoush , MD
Professor of pediatric hematologist & oncologist
Iran University of medical sciences
Rasool Akram Hospital

2. Introduction Sickle cell disease (SCD) :
Hemoglobin S (HbS)
Homozygosity for the sickle mutation in the beta globin chain of hemoglobin (HbSS)
Compound heterozygosity of a sickle beta globin mutation with another beta globin mutation (eg, sickle-beta thalassemia)
The hallmarks of SCD are vaso-occlusive phenomena and hemolytic anemia.
Sickle cell trait is a benign carrier condition characterized by heterozygosity for the sickle hemoglobin mutation.
11/13/2018
Faranoush , SCD,Qeshm

3. Background
Hemoglobin normally is soluble in the erythrocyte and does not polymerize.
Point mutation in the beta globin gene that causes the substitution of a valine for glutamic acid as the sixth amino acid of the beta globin chain.
The resulting hemoglobin tetramer (alpha2/beta S2) becomes poorly soluble when deoxygenated
11/13/2018
Faranoush , SCD,Qeshm

4. Pathophysiology
Polymerization alone does not account for the pathophysiology of SCD.
Subsequent changes in red cell membrane structure and function, disordered cell volume control, and increased adherence to vascular endothelium also play an important role
Hemolysis generates reactive oxygen species and free plasma hemoglobin (Hb).
This free plasma Hb is a powerful scavenger of nitric oxide, whose deficiency plays an important role in the vascular pathology of SCD
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Faranoush , SCD,Qeshm

5. Sickle cell disorders
If the other beta globin gene is normal, the individual has sickle cell trait, a benign carrier condition that is not a disease.
The combination of one sickle mutation and one alpha globin gene mutation results in two benign carrier conditions, sickle cell trait and alpha thalassemia trait (also called alpha thalassemia minima), and no clinical disease
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Faranoush , SCD,Qeshm

6. Sickle cell disorders
Sickle cell disease (SCD) is an umbrella term that includes all patients who have the sickle mutation plus a second beta globin gene mutation, the combination of which causes clinical sickling.
The other beta globin mutation could be the sickle mutation or a different mutation in the beta globin gene (eg, one associated with beta thalassemia or hemoglobin C disease, or others).
Patients with a sickle cell disease exhibit a clinical phenotype, anemia, and laboratory evidence of sickling.
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Faranoush , SCD,Qeshm

7. Overview of Diagnostic Testing
The goals and methods of diagnosis of sickle cell disease (SCD) vary with the age of the patient.
DNA-based testing is used for prenatal diagnosis
The diagnostic methods used after birth are those that separate hemoglobin species according to amino acid composition (hemoglobin electrophoresis or thin layer isoelectric focusing), solubility testing, and examination of the peripheral blood smear
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Faranoush , SCD,Qeshm

8. Prenatal Testing
The sickle hemoglobin (Hb) diseases (ie, sickle cell anemia, HbSC disease, sickle cell-beta thalassemia) are chronic, debilitating, and sometimes fatal.
The severe clinical nature of these diseases, particularly sickle cell anemia and sickle cell-beta0 thalassemia, and the absence of curative therapy are the primary stimulants for the development of fetal sampling and DNA-based diagnostic methodology.
Couples at risk should be offered hemoglobinopathy testing early in pregnancy and the opportunity for prenatal diagnosis where appropriate.
11/13/2018
Faranoush , SCD,Qeshm

9. Prenatal Testing
Currently, fetal DNA samples are obtained by chorionic villus sampling at 8 to 10 weeks gestation
Preimplantation diagnosis and testing fetal cells isolated from the maternal circulation are not yet in routine use.
Costly with limited availability
The many social and ethical issues that must be faced by the patient and physician in making decisions about prenatal testing for the hemoglobinopathies and the sampling methods that may be used
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Faranoush , SCD,Qeshm

10. New Methods
PCR
restriction analysis
Allele-specific hybridization
Reverse dot blotting
Allele-specific fluorescence PCR
PCR-based diagnosis for HbSC disease can be made using specific molecular methods for detecting the HbC gene
Diagnosis of sickle cell-beta thalassemia can be made using reverse dot blot methodology
11/13/2018
Faranoush , SCD,Qeshm

11. Newborn Screening
Infants with sickle cell disease (SCD) generally are healthy at birth and develop symptoms only when fetal hemoglobin levels decline later in infancy or early childhood.
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Faranoush , SCD,Qeshm

12. Goals of newborn screening (NBS)
Early recognition of affected infants
Early medical intervention to reduce morbidity and mortality, particularly from bacterial infections
Institution of regular and ongoing comprehensive care through a multidisciplinary sickle cell clinic, in collaboration with the primary care physician, whenever feasible
Access for families of children with SCD to accurate information about the diagnosis, clinical manifestations, treatment options, and age-appropriate anticipatory guidance toward the management of these emerging issues
11/13/2018
Faranoush , SCD,Qeshm

13. Example
The effectiveness of these programs, the use of prophylactic penicillin and the provision of comprehensive medical care have reduced the mortality of SCD during the first five years of life from approximately 25 percent to less than 3 percent
11/13/2018
Faranoush , SCD,Qeshm

14. Newborn screening (NBS)
Selective screening of infants of high-risk parents (20% missing)
Universal testing of newborns
Universal testing seems preferable due to its economy and superiority of detection
universal screening identifies more infants with disease and prevents more deaths
heel stick" filter paper screen is performed within 72 hours of life, with a second screen required at one to two weeks.
Testing is by isoelectric focusing, and abnormal specimens are confirmed by repeat testing or by DNA analysis
11/13/2018
Faranoush , SCD,Qeshm

15. Diagnostic in NBS
Solubility testing may not be valid, because of the large amount of HbF present in fetal blood.
Hb electrophoresis testing after transfusion of red cells can result in an incorrect diagnosis. In such cases, SCD diagnosis should utilize DNA testing or be postponed for at least four months after transfusion.
11/13/2018
Faranoush , SCD,Qeshm

16. Cont,
In very premature babies, HbA may not be detected, resulting in misdiagnosis.
Very premature babies with sickle cell trait may be found to have HbS levels greater than HbA, resulting in the incorrect diagnosis of hemoglobin S/beta+ thalassemia
11/13/2018
Faranoush , SCD,Qeshm

17. FS pattern HbSS sickle cell-beta0 thalassemia
sickle cell-hereditary persistence of fetal hemoglobin
sickle cell-hemoglobin D disease and sickle cell-hemoglobin G disease
11/13/2018
Faranoush , SCD,Qeshm

18. FAS and FSA patterns
FAS /sickle cell trait will have HbF, HbA, and HbS
HbA is greater than that of HbS
If the quantity of HbS exceeds that of HbA, the FSA pattern, the presumptive diagnosis is sickle cell-beta+ thalassemia.
It may not be possible to distinguish FAS and FSA patterns in newborns; thus, DNA-based testing or repeat hemoglobin testing at age three to six months is recommended.
11/13/2018
Faranoush , SCD,Qeshm

19. Future NBS
PCR-based diagnosis from blood spotted onto a filter paper may be used to detect sickle cell genes directly
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Faranoush , SCD,Qeshm

20. The following is appropriate while awaiting specialist referral:
Inform the family of the diagnosis of an SCD.
Initiate prophylactic penicillin (125 mg orally twice daily).
Inform the family that they should seek immediate medical attention if the infant has a fever of 101°F (38.3°C) and inform the treating clinician of the SCD diagnosis.
Provide information regarding the genetic inheritance of the disorder.
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Faranoush , SCD,Qeshm

21. Older Children & Adults
Clinical signs & symptoms
Laboratory methods
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Faranoush , SCD,Qeshm

22. Methods for hemoglobin analysis
High performance liquid chromatography (HPLC)
It is highly sensitive and specific and provides both quantitative and qualitative interpretation
Major disadvantages are the initial cost of the apparatus and reagents.
Thin-layer isoelectric focusing
Highly accurate and cost effective tool for the diagnosis of sickle or other hemoglobin variants
Cellulose acetate electrophoresis at pH 8.4
Standard method of separating hemoglobin S (HbS) from other Hb variants (SGD)
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Faranoush , SCD,Qeshm

23. Sickle cell prep
The sickle cell prep is a method for visualizing sickled cells that involves treating a blood sample with a reducing agent such as sodium metabisulfite or dithionite; these agents reduce the oxygen tension in the sample and promote sickling, which is then examined visually. This method is rarely used clinically and is generally of historic interest.
There are two exceptions:
The sickle cell solubility test is useful to detect sickling variants that are caused by a second mutation in addition to the sickle hemoglobin mutation (ie, compound heterozygosity) but show a different pattern from that produced by Hb S on electrophoresis and HPLC. Examples include Hb S-Antilles and Hb S-Oman.
Several point-of-care assays are under development that take advantage of the degree of sickle hemoglobin polymerization. These might be especially relevant for use in low resource settings.
11/13/2018
Faranoush , SCD,Qeshm

24. Hemoglobin electrophoresis (alkaline pH)
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Faranoush , SCD,Qeshm

25. Cont, Citrate agar electrophoresis at pH 6.2
Separates HbS from HbD and G, which co-migrate with HbA in this system
A solubility test such as the Sickledex also distinguishes HbD and G from HbS
only HbS precipitates using this test
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Faranoush , SCD,Qeshm

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34. DNA testing
DNA testing may be useful in selected cases (eg, where high concentrations of HbF raise the possibility of hereditary persistence of fetal hemoglobin)
11/13/2018
Faranoush , SCD,Qeshm

35. Point of care (POC) diagnostics
Point of care (POC) diagnostics are of potential use in low-resource countries with an at-risk population
Unlike the standard laboratory-based methods listed above, POC testing does not require expensive laboratory equipment and infrastructure.
11/13/2018
Faranoush , SCD,Qeshm

36. POC
Several promising techniques for simple, rapid, inexpensive diagnosis of SCD are being developed:
Paper-based tests that quantify sickle hemoglobin
Density-based rapid test using aqueous multiphase systems
Lateral flow immunoassays
These techniques appear to have excellent sensitivity and specificity to diagnose sickle cell anemia using dry blood and/or liquid samples.
11/13/2018
Faranoush , SCD,Qeshm

37. Hemoglobin A2 measurement
Valuable for the diagnosis of concomitant beta thalassemia
δβ thalassemia heterozygotes have a normal or decreased HbA2 level with a high HbF level
HbA2 levels are influenced by closely migrating hemoglobin variants such as HbE
11/13/2018
Faranoush , SCD,Qeshm

38. Findings in sickle cell anemia
Chronic hemolysis in those with sickle cell anemia (ie, HbSS) is usually associated with a mild to moderate anemia (hematocrit 20 to 30 percent), reticulocytosis of 3 to 15 percent (accounting for high or high-normal mean corpuscular volume [MCV]), unconjugated hyperbilirubinemia, and elevated serum lactate dehydrogenase (LDH) and low serum haptoglobin.
The peripheral blood smear reveals sickled red cells , polychromasia indicative of reticulocytosis, and Howell-Jolly bodies reflecting hyposplenia
The red cells are normochromic unless there is coexistent thalassemia or iron deficiency. If the age-adjusted MCV is not elevated, the possibility of sickle cell-beta thalassemia, coincident alpha thalassemia, or iron deficiency should be considered
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Faranoush , SCD,Qeshm

39. Cont,
The HbF level is usually slightly to moderately elevated and HbA is absent
The amount of HbF is a function of the number of reticulocytes that contain HbF, the extent of selective survival of HbF-containing reticulocytes to become mature HbF-containing erythrocytes, and the amount of HbF per red cell.
Each variable is separately regulated and the expression of each shows interpatient variability
In some patients with sickle cell anemia alone, values are as high (1 to 4 percent) as the modest elevations seen in heterocellular hereditary persistence of fetal hemoglobin
11/13/2018
Faranoush , SCD,Qeshm

40. CBC
In patients with sickle cell anemia, mean white blood cell (WBC) counts were higher than normal, particularly in those under the age of 10, and mean platelet counts were elevated, particularly in those under the age of 18.
In contrast, mean WBC and platelet counts were not elevated in HbSC disease or sickle cell-beta+ thalassemia.
11/13/2018
Faranoush , SCD,Qeshm

41. Bilirubin
Mean serum bilirubin concentrations were higher in those with sickle cell anemia than in those with HbSC disease or sickle cell-beta+ thalassemia due to the greater hemolytic rate.
The serum bilirubin rose after the first decade, possibly due to chronic hepatobiliary dysfunction.
Serum aminotransferase concentrations were often elevated, particularly in adults with sickle cell anemia, but mean levels were normal.
Serum alkaline phosphatase was elevated in all genotypes until puberty, suggesting an origin from bone rather than the liver.
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Faranoush , SCD,Qeshm

42. Creatinine
Serum creatinine concentrations were low in all genotypes until 18 years of age, when males experienced a rise apparently related to increasing muscle mass.
The low serum creatinine was in part due to an initial elevation in glomerular filtration rate.
Serum creatinine increased with age in all genotypes, presumably due to declining renal function
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Faranoush , SCD,Qeshm

43. Diagnostic patterns Other sickle cell disorders :
Several of the sickle cell disorders may have similar results with electrophoresis or isoelectric focusing.
Additional information from examination of the peripheral smear often helps to separate the sickle cell diseases
11/13/2018
Faranoush , SCD,Qeshm

44. SCD variants
Sickle cell-hemoglobin D-Punjab (D-Los Angeles) and sickle cell-hemoglobin O-Arab are moderate to severe diseases characterized by anemia, reticulocytosis, and often, macrocytosis.
Sickle cell-hemoglobin C-Harlem is slightly milder than sickle cell anemia with a similar blood film.
Sickle cell-hemoglobin Lepore, also a moderately severe disease, is more characterized by microcytosis and a blood smear comparable to sickle cell-beta thalassemia.
Sickle cell-hereditary persistence of fetal hemoglobin is usually asymptomatic or extremely mild.
Mild anemia and reticulocytosis may be noted. The electrophoresis may be misread as sickle cell anemia with an elevated HbF.
Definitive diagnosis requires family studies or DNA analysis
Sickle cell-hemoglobin E is a clinically mild disease.
The blood film shows targeting and variable microcytosis.
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Faranoush , SCD,Qeshm

45. Need SOCIETY GUIDELINE INFORMATION FOR PATIENTS Patient education
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Faranoush , SCD,Qeshm

46. SUMMARY AND RECOMMENDATIONS
The diagnosis of sickle cell disorders can take place in several settings: prenatal testing, newborn screening, diagnosis of symptomatic individuals, and counseling of those with sickle cell trait.
Diagnosis of one of the sickle cell disorders is generally made via high performance liquid chromatography (HPLC), isoelectric focusing (IEF), or gel electrophoresis techniques.
In prenatal diagnosis and in areas with high frequency of non-sickle-cell disorders, polymerase chain reaction (PCR) techniques, or direct DNA testing may be used.
11/13/2018
Faranoush , SCD,Qeshm

47. SUMMARY AND RECOMMENDATIONS
Only general patterns of hemoglobin production are available during the newborn period because beta globin production (which includes production of hemoglobins A and S [HbA and HbS]) is not fully developed
If questions arise as to interpretation, the tests should be repeated at age three to six months, when beta globin production is complete. DNA testing can be performed if clinically indicated
11/13/2018
Faranoush , SCD,Qeshm

48. SUMMARY AND RECOMMENDATIONS
Hemoglobin electrophoresis testing after transfusion of red cells can result in an incorrect diagnosis.
In such cases, sickle cell diagnosis should utilize DNA testing or be postponed for at least four months after transfusion.
In very premature babies, HbA may not be detected, resulting in misdiagnosis.
In addition, very premature babies with sickle cell trait may be found to have HbS levels greater than HbA, resulting in the incorrect diagnosis of hemoglobin S/beta+ thalassemia
11/13/2018
Faranoush , SCD,Qeshm

49. SUMMARY AND RECOMMENDATIONS
For children and adults, the combination of HPLC and IEF allows for a definitive diagnosis of one of the sickle cell disorders
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Faranoush , SCD,Qeshm

50. SUMMARY AND RECOMMENDATIONS
Sickle cell trait – In sickle cell trait, the usual pattern is to find >50 percent HbA, 35 to 45 percent HbS, and <2 percent HbF. The presence of <35 percent HbS suggests the presence of alpha thalassemia.
Sickle cell anemia – In sickle cell anemia (ie, HbSS), there is 0 percent HbA, <2 percent HbF, normal amounts of HbA2, and the remainder HbS.
Hemoglobin SC disease – In hemoglobin SC disease, HbS and HbC are both present.
Sickle cell-beta+ thalassemia – In sickle cell-beta+ thalassemia, there is 5 to 30 percent HbA, increased HbA2, with the remainder HbS. Target cells and hypochromic red cells are also present
Sickle cell-beta0 thalassemia – In sickle cell-beta0 thalassemia, there is 0 percent HbA, along with variable amounts of HbF, increased amounts of HbA2, with the remainder HbS. Target cells and hypochromic microcytic red cells are also present.
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Faranoush , SCD,Qeshm

51. SUMMARY AND RECOMMENDATIONS
Informing the parents of the results of newborn screening has value for the child and potentially other family members
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Faranoush , SCD,Qeshm

52. THANK YOU
Live as if you were to die tomorrow Learn as if you were to live forever
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Faranoush , SCD,Qeshm