Hereditary spherocytosis laboratory findings: Difference between revisions
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However, the following are appropriate in virtually all individuals: | However, the following are appropriate in virtually all individuals: | ||
●'''CBC and RBC indices''' – All individuals with suspected HS based on family history, neonatal jaundice, or other findings should have a complete blood count (CBC) with reticulocyte count and red blood cell (RBC) indices. The mean corpuscular hemoglobin concentration (MCHC) is often the most useful parameter for assessing spherocytosis; an MCHC ≥36 g/dL is consistent with spherocytes. A low mean corpuscular volume (MCV) is also helpful in some cases, especially in neonates, but variable degrees of reticulocytosis make the MCV less useful in older children and adults. | ●'''CBC and RBC indices''' – All individuals with suspected [[Hereditary spherocytosis|HS]] based on [[family history]], [[neonatal jaundice]], or other findings should have a [[Complete blood count|complete blood count (CBC)]] with [[reticulocyte count]] and [[Red blood cell|red blood cell (RBC)]] indices. The [[Mean corpuscular hemoglobin concentration|mean corpuscular hemoglobin concentration (MCHC)]] is often the most useful parameter for assessing [[spherocytosis]]; an MCHC ≥36 g/dL is consistent with [[Spherocyte|spherocytes]]. A low [[Mean corpuscular volume|mean corpuscular volume (MCV)]] is also helpful in some cases, especially in [[neonates]], but variable degrees of [[reticulocytosis]] make the [[MCV]] less useful in older children and adults. | ||
●'''Blood smear review''' – All individuals with suspected HS should have a blood smear reviewed by an experienced individual. RBC parameters to be assessed include the presence and abundance of spherocytes, other abnormal RBC shapes, and the degree of polychromatophilia, which reflects reticulocytosis. | ●'''[[Blood smear]] review''' – All individuals with suspected [[Hereditary spherocytosis|HS]] should have a [[blood smear]] reviewed by an experienced individual. [[Red blood cell|RBC]] parameters to be assessed include the presence and abundance of [[Spherocyte|spherocytes]], other abnormal [[RBC]] shapes, and the degree of polychromatophilia, which reflects [[reticulocytosis]]. | ||
●'''Hemolysis testing''' – Testing for hemolysis is also appropriate in all patients. This includes lactate dehydrogenase (LDH), indirect bilirubin, haptoglobin, and reticulocyte count. Findings consistent with hemolysis include increased LDH and indirect bilirubin, decreased or absent haptoglobin, and an elevated reticulocyte count. | ●'''[[Hemolysis]] testing''' – Testing for [[hemolysis]] is also appropriate in all patients. This includes [[Lactate dehydrogenase|lactate dehydrogenase (LDH)]], indirect bilirubin, [[haptoglobin]], and [[reticulocyte count]]. Findings consistent with [[hemolysis]] include increased [[Lactate dehydrogenase|LDH]] and indirect bilirubin, decreased or absent [[haptoglobin]], and an elevated [[reticulocyte count]]. | ||
●'''Coombs testing''' – If hemolysis is present, Coombs testing (also called direct antiglobulin testing [DAT]) is usually done to eliminate the possibility of immune-mediated hemolysis, which may be due to hemolytic disease of the fetus and newborn (HDFN) in neonates or autoimmune hemolytic anemia (AIHA) in older children and adults. The results of testing may also be useful to the transfusion service if transfusion is indicated. Coombs testing in HS is negative. | ●'''[[Coombs test|Coombs testing]]''' – If [[hemolysis]] is present, Coombs testing (also called direct antiglobulin testing [DAT]) is usually done to eliminate the possibility of immune-mediated hemolysis, which may be due to [[Hemolytic disease of newborn|hemolytic disease of the fetus and newborn (HDFN)]] in neonates or [[Autoimmune hemolytic anemia|autoimmune hemolytic anemia (AIHA)]] in older children and adults. The results of testing may also be useful to the [[transfusion]] service if [[transfusion]] is indicated. [[Coombs test|Coombs testing]] in [[Hereditary spherocytosis|HS]] is negative. | ||
'''Neonates''' — The evaluation of a neonate with suspected HS depends on whether a parent (or both parents) are known to have HS. | '''[[Neonates]]''' — The evaluation of a neonate with suspected [[Hereditary spherocytosis|HS]] depends on whether a parent (or both parents) are known to have HS. | ||
●If an infant with hyperbilirubinemia has a known family history of HS, then the likelihood of HS is high, and we rely heavily on the RBC indices. As noted above, an MCHC ≥36 g/dL is highly suggestive of HS. | ●If an infant with [[hyperbilirubinemia]] has a known family history of [[Hereditary spherocytosis|HS]], then the likelihood of [[Hereditary spherocytosis|HS]] is high, and we rely heavily on the RBC indices. As noted above, an [[Mean corpuscular hemoglobin concentration|MCHC]] ≥36 g/dL is highly suggestive of [[Hereditary spherocytosis|HS]]. | ||
●If an infant with hyperbilirubinemia or hemolytic anemia does not have a known family history of HS, then a number of other possible diagnoses must be considered. Appropriate therapy should not be delayed while determining the underlying cause; likewise, the importance of making the diagnosis of HS should be emphasized regardless of the management interventions needed. Hemolytic anemia with a negative Coombs test and a high MCHC (eg, >36 g/dL) is consistent with HS but must be considered in the context of the entire clinical picture. | ●If an infant with [[hyperbilirubinemia]] or [[hemolytic anemia]] does not have a known family history of [[Hereditary spherocytosis|HS]], then a number of other possible diagnoses must be considered. Appropriate therapy should not be delayed while determining the underlying cause; likewise, the importance of making the diagnosis of [[Hereditary spherocytosis|HS]] should be emphasized regardless of the management interventions needed. [[Hemolytic anemia]] with a negative [[Coombs test]] and a high [[Mean corpuscular hemoglobin concentration|MCHC]] (eg, >36 g/dL) is consistent with [[Hereditary spherocytosis|HS]] but must be considered in the context of the entire clinical picture. | ||
●[[Neonates]] with [[Hereditary spherocytosis|HS]] tend to have an elevated [[Mean corpuscular hemoglobin concentration|MCHC]] (typical range in HS, 35 to 38 g/dL) [83]. This is a useful discriminator between [[Hereditary spherocytosis|HS]] and [[Hemolytic disease of the newborn|hemolytic disease of the fetus and newborn (HDFN)]] because HDFN [[Red blood cell|RBCs]] tend to have [[Mean corpuscular hemoglobin concentration|MCHC]] in the range of 33 to 36 g/dL [88]. [[Spherocyte|Spherocytes]] on the [[blood smear]] are helpful if present, but up to one-third of neonates with HS do not have prominent [[Spherocyte|spherocytes]], and some neonates without [[Hereditary spherocytosis|HS]] have [[Spherocyte|spherocytes]] [83]. | |||
●It may be difficult to assess spherocytes on the peripheral blood smear in a neonate, either because neonates with HS may have fewer spherocytes or because spherocytic cells are often present after birth in neonates without HS [93]. If the infant is well, it is reasonable to postpone testing until approximately six months of age or older, at which time the RBC morphology will be easier to assess [80]. If there is greater urgency to establish a diagnosis, specialized testing may be used. | ●It may be difficult to assess [[Spherocyte|spherocytes]] on the [[peripheral blood smear]] in a neonate, either because neonates with [[Hereditary spherocytosis|HS]] may have fewer [[Spherocyte|spherocytes]] or because spherocytic cells are often present after birth in [[neonates]] without [[Hereditary spherocytosis|HS]] [93]. If the infant is well, it is reasonable to postpone testing until approximately six months of age or older, at which time the [[RBC]] morphology will be easier to assess [80]. If there is greater urgency to establish a diagnosis, specialized testing may be used. | ||
'''Older children and adults''' — HS may be suspected in a patient of any age who has evidence of hemolysis (eg, elevated serum LDH, elevated indirect bilirubin, reduced haptoglobin, increased reticulocyte count) or hemolytic anemia that is Coombs-negative and not explained by another condition. | '''Older children and adults''' — HS may be suspected in a patient of any age who has evidence of [[hemolysis]] (eg, elevated serum [[Lactate dehydrogenase|LDH]], elevated indirect bilirubin, reduced [[haptoglobin]], increased [[reticulocyte count]]) or [[hemolytic anemia]] that is Coombs-negative and not explained by another condition. | ||
HS may also be suspected in an individual who presents with a complication of hemolysis, such as splenomegaly, pigmented gallstones, or an abrupt drop in hemoglobin level when the bone marrow cannot compensate for hemolysis (eg, during a viral illness, pregnancy, or other condition). In such cases, a CBC will be obtained and RBC indices will be available; the reticulocyte count should also be measured if not done already. | [[Hereditary spherocytosis|HS]] may also be suspected in an individual who presents with a complication of [[hemolysis]], such as [[splenomegaly]], pigmented gallstones, or an abrupt drop in [[hemoglobin]] level when the bone marrow cannot compensate for [[hemolysis]] (eg, during a viral illness, pregnancy, or other condition). In such cases, a [[Complete blood count|CBC]] will be obtained and [[RBC]] indices will be available; the [[reticulocyte count]] should also be measured if not done already. | ||
Evidence consistent with HS as the likely diagnosis in an older child or adult include the following: | Evidence consistent with [[Hereditary spherocytosis|HS]] as the likely diagnosis in an older child or adult include the following: | ||
●Positive family history of HS, although this is not always present as some cases arise as new mutations and not all individuals will have a complete family history available. | ●Positive family history of [[Hereditary spherocytosis|HS]], although this is not always present as some cases arise as new mutations and not all individuals will have a complete family history available. | ||
●Chronic hemolytic anemia, although in mild cases, there may be chronic compensated hemolysis without anemia. | ●Chronic [[hemolytic anemia]], although in mild cases, there may be chronic compensated [[hemolysis]] without [[anemia]]. | ||
●Jaundice and/or splenomegaly, although these may be absent if the hemolysis is mild. | ●Jaundice and/or splenomegaly, although these may be absent if the hemolysis is mild. | ||
●[[Spherocyte|Spherocytes]] on the peripheral blood smear. The percentage of [[Spherocyte|spherocytes]] is variable. The typical [[reticulocyte count]] in older children and adults with HS is approximately 5 to 20 percent, but it may be as high as 20 to 30 percent in severe cases. Certain genetic defects have been associated with specific [[spherocyte]] morphologies, although the diagnostic value of these findings has not been rigorously tested [3,24,27,94,95]. | |||
* Pincered or notched spherocytes – Band 3 deficiency | * Pincered or notched [[Spherocyte|spherocytes]] – [[Band 3]] deficiency | ||
* Acanthocytic spherocytes – Spectrin deficiency | * Acanthocytic spherocytes – [[Spectrin]] deficiency | ||
* Dense and irregularly shaped cells – Spectrin/ankyrin deficiency | * Dense and irregularly shaped cells – Spectrin/ankyrin deficiency | ||
* Elliptocytic spherocytes – Spherocytic elliptocytosis | * Elliptocytic spherocytes – Spherocytic [[elliptocytosis]] | ||
●RBC indices consistent with spherocytosis (eg, MCHC >36 g/dL; normal to slightly low MCV). The MCV and red cell distribution width (RDW) may be increased by greater degrees of reticulocytosis in older children and adults; thus, the MCHC is the most useful of the RBC indices. The combination of increased MCHC and increased RDW further improves diagnostic performance [96]. If reticulocyte indices are available, a higher-than-average reticulocyte MCHC and a low reticulocyte MCV are also consistent with HS (table 2) [80]. | ●RBC indices consistent with [[spherocytosis]] (eg, MCHC >36 g/dL; normal to slightly low MCV). The [[MCV]] and red cell distribution width (RDW) may be increased by greater degrees of [[reticulocytosis]] in older children and adults; thus, the [[Mean corpuscular hemoglobin concentration|MCHC]] is the most useful of the RBC indices. The combination of increased [[Mean corpuscular hemoglobin concentration|MCHC]] and increased RDW further improves diagnostic performance [96]. If [[reticulocyte]] indices are available, a higher-than-average [[reticulocyte]] [[Mean corpuscular hemoglobin concentration|MCHC]] and a low [[reticulocyte]] [[Mean corpuscular volume|MCV]] are also consistent with [[Hereditary spherocytosis|HS]] (table 2) [80]. | ||
As noted below, in the appropriate clinical setting, this testing is sufficient to establish a diagnosis of HS. | As noted below, in the appropriate clinical setting, this testing is sufficient to establish a diagnosis of [[Hereditary spherocytosis|HS]]. | ||
In cases that are unclear or if additional diagnostic confirmation is needed, specialized testing can be pursued. | In cases that are unclear or if additional diagnostic confirmation is needed, specialized testing can be pursued. | ||
'''Confirmatory tests''' — A number of tests are available for confirming the diagnosis of HS. We perform confirmatory testing in all cases, although some experts may omit this testing, especially in resource-limited settings and/or if there are classic clinical findings in an individual with a known family history of HS. | '''Confirmatory tests''' — A number of tests are available for confirming the diagnosis of [[Hereditary spherocytosis|HS]]. We perform confirmatory testing in all cases, although some experts may omit this testing, especially in resource-limited settings and/or if there are classic clinical findings in an individual with a known family history of HS. | ||
Available tests include the following: | Available tests include the following: | ||
●'''EMA binding''' – If specialized testing is indicated, EMA binding is our preferred test. EMA (eosin-5-maleimide) is an eosin-based fluorescent dye that binds to RBC membrane proteins, especially band 3 and Rh-related proteins [97]. The mean fluorescence of EMA-labeled RBCs from individuals with HS is lower than controls, and this reduction in fluorescence can be detected in a flow cytometry-based assay. Two case series of individuals with HS have found the EMA fluorescence in individuals with HS to be approximately two-thirds that of controls [97,98]. Samples can be stored and tested; one of the studies also analyzed the effect of delayed testing and found that samples stored for 24 hours in the darkness gave similar results to those tested immediately [97]. | ●'''EMA binding''' – If specialized testing is indicated, EMA binding is our preferred test. EMA (eosin-5-maleimide) is an eosin-based fluorescent dye that binds to RBC membrane proteins, especially [[band 3]] and Rh-related proteins [97]. The mean fluorescence of EMA-labeled RBCs from individuals with [[Hereditary spherocytosis|HS]] is lower than controls, and this reduction in [[fluorescence]] can be detected in a [[flow cytometry]]-based assay. Two case series of individuals with [[Hereditary spherocytosis|HS]] have found the EMA [[fluorescence]] in individuals with [[Hereditary spherocytosis|HS]] to be approximately two-thirds that of controls [97,98]. Samples can be stored and tested; one of the studies also analyzed the effect of delayed testing and found that samples stored for 24 hours in the darkness gave similar results to those tested immediately [97]. | ||
Advantages of EMA binding include its high sensitivity and specificity; rapid turnaround time (approximately two hours); and need for only a minimal amount of blood (a few microliters), which is especially advantageous for testing neonates [99-101]. In addition, EMA testing can be used to identify HS in a patient who has recently received a transfusion [102]. In various studies, the sensitivity and specificity of the test appear to be in the ranges of 93 to 96 and 93 to 99 percent, respectively [99,103-105]. | Advantages of EMA binding include its high sensitivity and specificity; rapid turnaround time (approximately two hours); and need for only a minimal amount of blood (a few microliters), which is especially advantageous for testing neonates [99-101]. In addition, EMA testing can be used to identify HS in a patient who has recently received a transfusion [102]. In various studies, the sensitivity and specificity of the test appear to be in the ranges of 93 to 96 and 93 to 99 percent, respectively [99,103-105]. | ||
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EMA binding may also be positive in some forms of hereditary elliptocytosis (HE; eg, hereditary pyropoikilocytosis [HPP] and Southeast Asian ovalocytosis [SAO]), in individuals with congenital dyserythropoietic anemia (CDA) type II, and/or in autoimmune hemolytic anemia [98,103]. False negative results may be seen in mild cases of HS. | EMA binding may also be positive in some forms of hereditary elliptocytosis (HE; eg, hereditary pyropoikilocytosis [HPP] and Southeast Asian ovalocytosis [SAO]), in individuals with congenital dyserythropoietic anemia (CDA) type II, and/or in autoimmune hemolytic anemia [98,103]. False negative results may be seen in mild cases of HS. | ||
●'''Osmotic fragility''' – If EMA binding is not available, osmotic fragility testing (OFT) is another useful specialized test for HS. In this test, RBCs are incubated in hypotonic buffered salt solutions of various osmolarities, and the fraction of hemoglobin released (due to hemolysis) is measured. The test takes advantage of the increased sensitivity of spherocytes to hemolysis, which is due to their reduced surface area to volume (SA/V) ratio (figure 3). Incubation of patient samples for 24 hours prior to testing may accentuate osmotic fragility and improve diagnostic yield. | ●'''Osmotic fragility''' – If EMA binding is not available, osmotic fragility testing (OFT) is another useful specialized test for HS. In this test, RBCs are incubated in hypotonic buffered salt solutions of various osmolarities, and the fraction of [[hemoglobin]] released (due to hemolysis) is measured. The test takes advantage of the increased sensitivity of [[Spherocyte|spherocytes]] to [[hemolysis]], which is due to their reduced [[Surface area to volume ratio|surface area to volume (SA/V) ratio]] (figure 3). Incubation of patient samples for 24 hours prior to testing may accentuate osmotic fragility and improve diagnostic yield. | ||
The OFT has relatively low sensitivity and specificity. It fails to identify a significant number of individuals with HS, and, particularly in the newborn, it may be positive in other conditions including immune hemolytic anemia, hemolytic transfusion reactions, RBC enzyme defects such as glucose-6-phosphate dehydrogenase (G6PD) deficiency, and unstable hemoglobin variants [93]. In one series of 86 individuals with HS, only 57 (66 percent) had positive osmotic fragility testing [106]. | The OFT has relatively low sensitivity and specificity. It fails to identify a significant number of individuals with HS, and, particularly in the newborn, it may be positive in other conditions including [[Immune hemolytic anemias|immune hemolytic anemia]], hemolytic transfusion reactions, RBC enzyme defects such as [[Glucose-6-phosphate dehydrogenase deficiency|glucose-6-phosphate dehydrogenase (G6PD) deficiency]], and unstable hemoglobin variants [93]. In one series of 86 individuals with HS, only 57 (66 percent) had positive osmotic fragility testing [106]. | ||
●'''Glycerol lysis''' – The glycerol lysis test (GLT) and the acidified GLT (AGLT) are modifications of the OFT that add glycerol (in the GLT) or glycerol plus a sodium phosphate (to lower the pH to 6.85, in the AGLT) to the hypotonic buffered salt solutions in which the patient's RBCs are incubated [75,93]. Like the OFT, these tests may also be positive in acquired spherocytosis conditions such as AIHA. | ●'''Glycerol lysis''' – The glycerol lysis test (GLT) and the acidified GLT (AGLT) are modifications of the OFT that add glycerol (in the GLT) or glycerol plus a sodium phosphate (to lower the pH to 6.85, in the AGLT) to the hypotonic buffered salt solutions in which the patient's [[RBCs]] are incubated [75,93]. Like the OFT, these tests may also be positive in acquired spherocytosis conditions such as AIHA. | ||
The "pink test" is a modification of the GLT in which the final extent of hemolysis is measured in a blood sample incubated in the glycerol solution at pH 6.66 [107]. A further modification has been proposed (the direct pink test) in which the test sample is obtained from fingerprick (or heel puncture in newborns), rather than venipuncture, and incubated directly in the glycerol solution; this requires only a few microliters of blood [108]. | The "pink test" is a modification of the GLT in which the final extent of [[hemolysis]] is measured in a blood sample incubated in the glycerol solution at pH 6.66 [107]. A further modification has been proposed (the direct pink test) in which the test sample is obtained from fingerprick (or heel puncture in newborns), rather than venipuncture, and incubated directly in the glycerol solution; this requires only a few microliters of blood [108]. | ||
●'''Cryohemolysis''' – In the cryohemolysis test, RBCs are suspended in a hypertonic solution, briefly heated to 37°C, then cooled to 4°C for 10 minutes [109]. Ease of performance and the wide separation in degree of hemolysis between spherocytes and normal cells are two attractive features of this test [110]. This test has limited availability in the United States. | ●'''Cryohemolysis''' – In the cryohemolysis test, [[RBCs]] are suspended in a hypertonic solution, briefly heated to 37°C, then cooled to 4°C for 10 minutes [109]. Ease of performance and the wide separation in degree of [[hemolysis]] between [[Spherocyte|spherocytes]] and normal cells are two attractive features of this test [110]. This test has limited availability in the United States. | ||
===Red cell indices=== | ===Red cell indices=== | ||
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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]
Overview
Laboratory Findings
Initial testing
All patients — Some aspects of the initial evaluation differ in neonates versus older children and adults since affected neonates tend to have more severe disease and less useful laboratory parameters.
However, the following are appropriate in virtually all individuals:
●CBC and RBC indices – All individuals with suspected HS based on family history, neonatal jaundice, or other findings should have a complete blood count (CBC) with reticulocyte count and red blood cell (RBC) indices. The mean corpuscular hemoglobin concentration (MCHC) is often the most useful parameter for assessing spherocytosis; an MCHC ≥36 g/dL is consistent with spherocytes. A low mean corpuscular volume (MCV) is also helpful in some cases, especially in neonates, but variable degrees of reticulocytosis make the MCV less useful in older children and adults.
●Blood smear review – All individuals with suspected HS should have a blood smear reviewed by an experienced individual. RBC parameters to be assessed include the presence and abundance of spherocytes, other abnormal RBC shapes, and the degree of polychromatophilia, which reflects reticulocytosis.
●Hemolysis testing – Testing for hemolysis is also appropriate in all patients. This includes lactate dehydrogenase (LDH), indirect bilirubin, haptoglobin, and reticulocyte count. Findings consistent with hemolysis include increased LDH and indirect bilirubin, decreased or absent haptoglobin, and an elevated reticulocyte count.
●Coombs testing – If hemolysis is present, Coombs testing (also called direct antiglobulin testing [DAT]) is usually done to eliminate the possibility of immune-mediated hemolysis, which may be due to hemolytic disease of the fetus and newborn (HDFN) in neonates or autoimmune hemolytic anemia (AIHA) in older children and adults. The results of testing may also be useful to the transfusion service if transfusion is indicated. Coombs testing in HS is negative.
Neonates — The evaluation of a neonate with suspected HS depends on whether a parent (or both parents) are known to have HS.
●If an infant with hyperbilirubinemia has a known family history of HS, then the likelihood of HS is high, and we rely heavily on the RBC indices. As noted above, an MCHC ≥36 g/dL is highly suggestive of HS.
●If an infant with hyperbilirubinemia or hemolytic anemia does not have a known family history of HS, then a number of other possible diagnoses must be considered. Appropriate therapy should not be delayed while determining the underlying cause; likewise, the importance of making the diagnosis of HS should be emphasized regardless of the management interventions needed. Hemolytic anemia with a negative Coombs test and a high MCHC (eg, >36 g/dL) is consistent with HS but must be considered in the context of the entire clinical picture.
●Neonates with HS tend to have an elevated MCHC (typical range in HS, 35 to 38 g/dL) [83]. This is a useful discriminator between HS and hemolytic disease of the fetus and newborn (HDFN) because HDFN RBCs tend to have MCHC in the range of 33 to 36 g/dL [88]. Spherocytes on the blood smear are helpful if present, but up to one-third of neonates with HS do not have prominent spherocytes, and some neonates without HS have spherocytes [83].
●It may be difficult to assess spherocytes on the peripheral blood smear in a neonate, either because neonates with HS may have fewer spherocytes or because spherocytic cells are often present after birth in neonates without HS [93]. If the infant is well, it is reasonable to postpone testing until approximately six months of age or older, at which time the RBC morphology will be easier to assess [80]. If there is greater urgency to establish a diagnosis, specialized testing may be used.
Older children and adults — HS may be suspected in a patient of any age who has evidence of hemolysis (eg, elevated serum LDH, elevated indirect bilirubin, reduced haptoglobin, increased reticulocyte count) or hemolytic anemia that is Coombs-negative and not explained by another condition.
HS may also be suspected in an individual who presents with a complication of hemolysis, such as splenomegaly, pigmented gallstones, or an abrupt drop in hemoglobin level when the bone marrow cannot compensate for hemolysis (eg, during a viral illness, pregnancy, or other condition). In such cases, a CBC will be obtained and RBC indices will be available; the reticulocyte count should also be measured if not done already.
Evidence consistent with HS as the likely diagnosis in an older child or adult include the following:
●Positive family history of HS, although this is not always present as some cases arise as new mutations and not all individuals will have a complete family history available.
●Chronic hemolytic anemia, although in mild cases, there may be chronic compensated hemolysis without anemia.
●Jaundice and/or splenomegaly, although these may be absent if the hemolysis is mild.
●Spherocytes on the peripheral blood smear. The percentage of spherocytes is variable. The typical reticulocyte count in older children and adults with HS is approximately 5 to 20 percent, but it may be as high as 20 to 30 percent in severe cases. Certain genetic defects have been associated with specific spherocyte morphologies, although the diagnostic value of these findings has not been rigorously tested [3,24,27,94,95].
- Pincered or notched spherocytes – Band 3 deficiency
- Acanthocytic spherocytes – Spectrin deficiency
- Dense and irregularly shaped cells – Spectrin/ankyrin deficiency
- Elliptocytic spherocytes – Spherocytic elliptocytosis
●RBC indices consistent with spherocytosis (eg, MCHC >36 g/dL; normal to slightly low MCV). The MCV and red cell distribution width (RDW) may be increased by greater degrees of reticulocytosis in older children and adults; thus, the MCHC is the most useful of the RBC indices. The combination of increased MCHC and increased RDW further improves diagnostic performance [96]. If reticulocyte indices are available, a higher-than-average reticulocyte MCHC and a low reticulocyte MCV are also consistent with HS (table 2) [80].
As noted below, in the appropriate clinical setting, this testing is sufficient to establish a diagnosis of HS.
In cases that are unclear or if additional diagnostic confirmation is needed, specialized testing can be pursued.
Confirmatory tests — A number of tests are available for confirming the diagnosis of HS. We perform confirmatory testing in all cases, although some experts may omit this testing, especially in resource-limited settings and/or if there are classic clinical findings in an individual with a known family history of HS.
Available tests include the following:
●EMA binding – If specialized testing is indicated, EMA binding is our preferred test. EMA (eosin-5-maleimide) is an eosin-based fluorescent dye that binds to RBC membrane proteins, especially band 3 and Rh-related proteins [97]. The mean fluorescence of EMA-labeled RBCs from individuals with HS is lower than controls, and this reduction in fluorescence can be detected in a flow cytometry-based assay. Two case series of individuals with HS have found the EMA fluorescence in individuals with HS to be approximately two-thirds that of controls [97,98]. Samples can be stored and tested; one of the studies also analyzed the effect of delayed testing and found that samples stored for 24 hours in the darkness gave similar results to those tested immediately [97].
Advantages of EMA binding include its high sensitivity and specificity; rapid turnaround time (approximately two hours); and need for only a minimal amount of blood (a few microliters), which is especially advantageous for testing neonates [99-101]. In addition, EMA testing can be used to identify HS in a patient who has recently received a transfusion [102]. In various studies, the sensitivity and specificity of the test appear to be in the ranges of 93 to 96 and 93 to 99 percent, respectively [99,103-105].
EMA binding may also be positive in some forms of hereditary elliptocytosis (HE; eg, hereditary pyropoikilocytosis [HPP] and Southeast Asian ovalocytosis [SAO]), in individuals with congenital dyserythropoietic anemia (CDA) type II, and/or in autoimmune hemolytic anemia [98,103]. False negative results may be seen in mild cases of HS.
●Osmotic fragility – If EMA binding is not available, osmotic fragility testing (OFT) is another useful specialized test for HS. In this test, RBCs are incubated in hypotonic buffered salt solutions of various osmolarities, and the fraction of hemoglobin released (due to hemolysis) is measured. The test takes advantage of the increased sensitivity of spherocytes to hemolysis, which is due to their reduced surface area to volume (SA/V) ratio (figure 3). Incubation of patient samples for 24 hours prior to testing may accentuate osmotic fragility and improve diagnostic yield.
The OFT has relatively low sensitivity and specificity. It fails to identify a significant number of individuals with HS, and, particularly in the newborn, it may be positive in other conditions including immune hemolytic anemia, hemolytic transfusion reactions, RBC enzyme defects such as glucose-6-phosphate dehydrogenase (G6PD) deficiency, and unstable hemoglobin variants [93]. In one series of 86 individuals with HS, only 57 (66 percent) had positive osmotic fragility testing [106].
●Glycerol lysis – The glycerol lysis test (GLT) and the acidified GLT (AGLT) are modifications of the OFT that add glycerol (in the GLT) or glycerol plus a sodium phosphate (to lower the pH to 6.85, in the AGLT) to the hypotonic buffered salt solutions in which the patient's RBCs are incubated [75,93]. Like the OFT, these tests may also be positive in acquired spherocytosis conditions such as AIHA.
The "pink test" is a modification of the GLT in which the final extent of hemolysis is measured in a blood sample incubated in the glycerol solution at pH 6.66 [107]. A further modification has been proposed (the direct pink test) in which the test sample is obtained from fingerprick (or heel puncture in newborns), rather than venipuncture, and incubated directly in the glycerol solution; this requires only a few microliters of blood [108].
●Cryohemolysis – In the cryohemolysis test, RBCs are suspended in a hypertonic solution, briefly heated to 37°C, then cooled to 4°C for 10 minutes [109]. Ease of performance and the wide separation in degree of hemolysis between spherocytes and normal cells are two attractive features of this test [110]. This test has limited availability in the United States.
Red cell indices
- Reticulocytosis
- Decreased mean corpuscular volume
- Increased mean corpuscular hemoglobin concentration
- Increased red cell distribution width
Peripheral blood smear
- In a peripheral blood smear, the abnormally small red blood cells lacking the central pallor i.e. spherocytes are seen.