Hereditary spherocytosis laboratory findings: Difference between revisions

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==Overview==
==Overview==
The initial [[laboratory]] [[Test|testing]] for [[hereditary spherocytosis]] include; [[Complete blood count|complete blood count (CBC)]], [[Mean corpuscular hemoglobin concentration|mean corpuscular hemoglobin concentration (MCHC)]], [[Blood film|blood smear review]], [[hemolysis]] [[Test|testing]] and [[Coombs test|coombs testing]]. All individuals suspected of having [[hereditary spherocytosis]] based on [[family history]], [[neonatal]] [[jaundice]] or other findings should have a [[Complete blood count|complete blood count (CBC)]], [[reticulocyte count]] and [[Red blood cell|RBC]] indices done. Confirmatory [[Test|tests]] for [[hereditary spherocytosis]] includes EMA binding test, osmotic fragility test, glycerol lysis test, cryohemolysis and [[Cell membrane|plasma membrane]] [[electrophoresis]].
==Laboratory Findings==
==Laboratory Findings==
'''Initial testing'''
'''Initial testing'''
*<nowiki/>'''CBC and RBC indices''' - The mean cell hemoglobin concentration (MCHC) and red cell distribution width (RDW) are both raised in hereditary spherocytosis.<ref name="Farias2017">{{cite journal|last1=Farias|first1=Mariela Granero|title=Advances in laboratory diagnosis of hereditary spherocytosis|journal=Clinical Chemistry and Laboratory Medicine (CCLM)|volume=55|issue=7|year=2017|issn=1437-4331|doi=10.1515/cclm-2016-0738}}</ref>
 
* '''[[Blood smear]] review''' – shows the spherocytes and increased reticulocytes.
'''CBC and RBC indices'''  
* '''[[Coombs test|Coombs testing]]''' – used when hemolysis is present, to differentiate hereditary spherocytosis from autoimmune hemolytic anemia (AIHA). Coombs test is negative in hereditary spherocytosis.
* All individuals with suspected [[Hereditary spherocytosis|hereditary spherocytosis]] 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]]; a [[Mean corpuscular hemoglobin concentration|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 [[Child|children]] and [[Adult|adults]].<ref name="Farias2017">{{cite journal|last1=Farias|first1=Mariela Granero|title=Advances in laboratory diagnosis of hereditary spherocytosis|journal=Clinical Chemistry and Laboratory Medicine (CCLM)|volume=55|issue=7|year=2017|issn=1437-4331|doi=10.1515/cclm-2016-0738}}</ref>
 
'''Red cell indices'''
* [[Reticulocytosis]]
* Decreased [[mean corpuscular volume]]
* Increased [[mean corpuscular hemoglobin concentration]]
* Increased [[Red blood cell distribution width|red cell distribution width]]
 
'''[[Blood smear]] review'''  
* All individuals with suspected [[Hereditary spherocytosis|hereditary spherocytosis]] should have a [[blood smear]] reviewed by an experienced individual. In a [[peripheral blood smear]], the abnormally small [[Red blood cell|red blood cells]] lacking the central pallor i.e. [[Spherocyte|spherocytes]] are seen. Other abnormal [[RBC]] shapes, and the degree of polychromatophilia, which reflects [[reticulocytosis]].
 
'''[[Hemolysis]] testing'''
* [[Test|Testing]] for [[hemolysis]] is also appropriate in all [[Patient|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 test|Coombs testing]]'''
* If [[hemolysis]] is present, [[Coombs test|Coombs testing]] (also called [[Coombs test|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 [[Child|children]] and [[Adult|adults]]. The results of [[Test|testing]] may also be useful to the [[transfusion]] service if [[transfusion]] is indicated.
* [[Coombs test|Coombs testing]] in [[Hereditary spherocytosis|hereditary spherocytosis]] is negative.
 
'''[[Neonates]]'''
* The evaluation of a [[neonate]] with suspected [[Hereditary spherocytosis|hereditary spherocytosis]] depends on whether a parent (or both parents) are known to have [[hereditary spherocytosis]].
* If an infant with [[hyperbilirubinemia]] has a known family history of [[Hereditary spherocytosis|hereditary spherocytosis]], then the likelihood of [[Hereditary spherocytosis|hereditary spherocytosis]] is high, and we rely heavily on the [[Red blood cell|RBC]] indices. As noted above, an [[Mean corpuscular hemoglobin concentration|MCHC]] ≥36 g/dL is highly suggestive of [[Hereditary spherocytosis|hereditary spherocytosis]].
* If an [[infant]] with [[hyperbilirubinemia]] or [[hemolytic anemia]] does not have a known [[family history]] of [[Hereditary spherocytosis|hereditary spherocytosis]], then a number of other possible [[Diagnosis|diagnoses]] must be considered.  
* Appropriate [[therapy]] should not be delayed while determining the underlying [[Causality|cause]]; likewise, the importance of making the [[diagnosis]] of [[Hereditary spherocytosis|hereditary spherocytosis]] 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|hereditary spherocytosis]] but must be considered in the context of the entire [[clinical]] picture.
 
* [[Neonates]] with [[Hereditary spherocytosis|hereditary spherocytosis]] tend to have an elevated [[Mean corpuscular hemoglobin concentration|MCHC]] (typical range in hereditary spherocytosis, 35 to 38 g/dL).
* This is a useful discriminator between [[Hereditary spherocytosis|hereditary spherocytosis]] 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.
* [[Spherocyte|Spherocytes]] on the [[blood smear]] are helpful if present, but up to one-third of [[Neonate|neonates]] with [[hereditary spherocytosis]] do not have prominent [[Spherocyte|spherocytes]], and some [[neonates]] without [[Hereditary spherocytosis|hereditary spherocytosis]] have [[Spherocyte|spherocytes]]. <ref name="pmid26009624">{{cite journal| author=Christensen RD, Yaish HM, Gallagher PG| title=A pediatrician's practical guide to diagnosing and treating hereditary spherocytosis in neonates. | journal=Pediatrics | year= 2015 | volume= 135 | issue= 6 | pages= 1107-14 | pmid=26009624 | doi=10.1542/peds.2014-3516 | pmc=4444801 | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=26009624  }}</ref>
* It may be difficult to assess [[Spherocyte|spherocytes]] on the [[peripheral blood smear]] in a [[neonate]], either because [[neonates]] with [[Hereditary spherocytosis|hereditary spherocytosis]] may have fewer [[Spherocyte|spherocytes]] or because [[Spherocyte|spherocytic]] [[Cell (biology)|cells]] are often present after [[birth]] in [[neonates]] without [[Hereditary spherocytosis|hereditary spherocytosis]].<ref name="pmid25790109">{{cite journal| author=King MJ, Garçon L, Hoyer JD, Iolascon A, Picard V, Stewart G et al.| title=ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. | journal=Int J Lab Hematol | year= 2015 | volume= 37 | issue= 3 | pages= 304-25 | pmid=25790109 | doi=10.1111/ijlh.12335 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=25790109  }}</ref> If the [[infant]] is well, it is reasonable to postpone [[Test|testing]] until approximately six months of [[Ageing|age]] or older, at which time the [[RBC]] [[morphology]] will be easier to assess.<ref name="pmid22055020">{{cite journal| author=Bolton-Maggs PH, Langer JC, Iolascon A, Tittensor P, King MJ, General Haematology Task Force of the British Committee for Standards in Haematology| title=Guidelines for the diagnosis and management of hereditary spherocytosis--2011 update. | journal=Br J Haematol | year= 2012 | volume= 156 | issue= 1 | pages= 37-49 | pmid=22055020 | doi=10.1111/j.1365-2141.2011.08921.x | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=22055020  }}</ref>
 
'''Older children and adults'''
* [[Hereditary spherocytosis]] may be suspected in a [[patient]] of any [[Ageing|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 [[Disease|condition]].
 
* [[Hereditary spherocytosis|Hereditary spherocytosis]] may also be suspected in an individual who presents with a [[Complication (medicine)|complication]] of [[hemolysis]], such as [[splenomegaly]], [[Gallstones|pigmented gallstones]], or an abrupt drop in [[hemoglobin]] level when the [[bone marrow]] cannot compensate for [[hemolysis]] (eg, during a [[Virus|viral]] [[illness]], [[pregnancy]], or other [[Disease|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 [[Hereditary spherocytosis|hereditary spherocytosis]] as the likely [[diagnosis]] in an older [[child]] or [[adult]] include the following:
** Positive [[family history]] of [[Hereditary spherocytosis|hereditary spherocytosis]], although this is not always present as some cases arise as new [[Mutation|mutations]] and not all individuals will have a complete [[family history]] available.
** [[Chronic (medical)|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.
** [[Spherocyte|Spherocytes]] on the [[Blood film|peripheral blood smear]]. The percentage of [[Spherocyte|spherocytes]] is variable. The typical [[reticulocyte count]] in older [[Child|children]] and [[Adult|adults]] with [[hereditary spherocytosis]] is approximately 5 to 20 percent, but it may be as high as 20 to 30 percent in severe cases. Certain [[Genetic disorder|genetic defects]] have been associated with specific [[spherocyte]] [[Morphology (biology)|morphologies]], although the [[Diagnosis|diagnostic]] value of these findings has not been rigorously [[Test|tested]].<ref name="pmid8943874">{{cite journal| author=Jarolim P, Murray JL, Rubin HL, Taylor WM, Prchal JT, Ballas SK et al.| title=Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency. | journal=Blood | year= 1996 | volume= 88 | issue= 11 | pages= 4366-74 | pmid=8943874 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=8943874  }}</ref><ref name="pmid9207476">{{cite journal| author=Hassoun H, Vassiliadis JN, Murray J, Njolstad PR, Rogus JJ, Ballas SK et al.| title=Characterization of the underlying molecular defect in hereditary spherocytosis associated with spectrin deficiency. | journal=Blood | year= 1997 | volume= 90 | issue= 1 | pages= 398-406 | pmid=9207476 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=9207476  }}</ref><ref name="pmid8102379">{{cite journal| author=Becker PS, Tse WT, Lux SE, Forget BG| title=Beta spectrin kissimmee: a spectrin variant associated with autosomal dominant hereditary spherocytosis and defective binding to protein 4.1. | journal=J Clin Invest | year= 1993 | volume= 92 | issue= 2 | pages= 612-6 | pmid=8102379 | doi=10.1172/JCI116628 | pmc=294892 | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=8102379  }}</ref><ref name="pmid2961992">{{cite journal| author=Coetzer TL, Lawler J, Liu SC, Prchal JT, Gualtieri RJ, Brain MC et al.| title=Partial ankyrin and spectrin deficiency in severe, atypical hereditary spherocytosis. | journal=N Engl J Med | year= 1988 | volume= 318 | issue= 4 | pages= 230-4 | pmid=2961992 | doi=10.1056/NEJM198801283180407 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=2961992  }}</ref>
** Pincered or notched [[Spherocyte|spherocytes]] – [[Band 3]] [[deficiency]]
** [[Acanthocyte|Acanthocytic]] [[Spherocyte|spherocytes]] – [[Spectrin]] [[deficiency]]
** Dense and irregularly shaped [[Cell (biology)|cells]] – [[Spectrin]]/[[ankyrin]] [[deficiency]]
** [[Elliptocyte|Elliptocytic]] [[Spherocyte|spherocytes]] – [[Spherocyte|Spherocytic]] [[elliptocytosis]]
** [[Red blood cell|RBC]] indices consistent with [[spherocytosis]] (eg, [[Mean corpuscular hemoglobin concentration|MCHC]] >36 g/dL; normal to slightly low [[Mean corpuscular volume|MCV]]). The [[MCV]] and [[Red blood cell distribution width|red cell distribution width (RDW)]] may be increased by greater degrees of [[reticulocytosis]] in older [[Child|children]] and [[Adult|adults]]; thus, the [[Mean corpuscular hemoglobin concentration|MCHC]] is the most useful of the [[Red blood cell|RBC]] indices. The combination of increased [[Mean corpuscular hemoglobin concentration|MCHC]] and increased [[Red blood cell distribution width|RDW]] further improves [[Diagnosis|diagnostic]] performance.<ref name="pmid9202619">{{cite journal| author=Michaels LA, Cohen AR, Zhao H, Raphael RI, Manno CS| title=Screening for hereditary spherocytosis by use of automated erythrocyte indexes. | journal=J Pediatr | year= 1997 | volume= 130 | issue= 6 | pages= 957-60 | pmid=9202619 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=9202619  }}</ref> 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|hereditary spherocytosis]].<ref name="pmid220550202">{{cite journal| author=Bolton-Maggs PH, Langer JC, Iolascon A, Tittensor P, King MJ, General Haematology Task Force of the British Committee for Standards in Haematology| title=Guidelines for the diagnosis and management of hereditary spherocytosis--2011 update. | journal=Br J Haematol | year= 2012 | volume= 156 | issue= 1 | pages= 37-49 | pmid=22055020 | doi=10.1111/j.1365-2141.2011.08921.x | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=22055020  }}</ref>
* In cases that are unclear or if additional [[Diagnosis|diagnostic]] confirmation is needed, specialized [[Test|testing]] can be pursued.
 
'''Confirmatory tests'''
'''Confirmatory tests'''
** EMA (eosin 5 maleimide binding assay) test<ref>{{Cite journal
* A number of [[Test|tests]] are available for confirming the [[diagnosis]] of [[Hereditary spherocytosis|hereditary spherocytosis]]. We perform confirmatory [[Test|testing]] in all cases, although some experts may omit this [[Test|testing]], especially in resource-limited settings and/or if there are classic [[clinical]] findings in an individual with a known [[family history]] of [[hereditary spherocytosis]].
* Available [[Test|tests]] include the following:
 
'''EMA (eosin-5-maleimide) binding'''
* If specialized [[Test|testing]] is indicated, EMA (eosin-5-maleimide) binding is our preferred test. EMA is an eosin-based fluorescent dye that binds to [[Red blood cell|RBC]] [[Cell membrane|membrane]] [[Protein|proteins]], especially [[band 3]] and [[Rhesus|Rh-related]] [[Protein|proteins]].<ref name="pmid23023797">{{cite journal| author=Ciepiela O, Kotuła I, Górska E, Stelmaszczyk-Emmel A, Popko K, Szmydki-Baran A et al.| title=Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis. | journal=Clin Chem Lab Med | year= 2013 | volume= 51 | issue= 4 | pages= 817-23 | pmid=23023797 | doi=10.1515/cclm-2012-0240 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=23023797  }}</ref>
* The mean fluorescence of EMA-labeled [[Red blood cell|RBCs]] from individuals with [[Hereditary spherocytosis|hereditary spherocytosis]] 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|hereditary spherocytosis]] have found the EMA [[fluorescence]] in individuals with [[Hereditary spherocytosis|hereditary spherocytosis]] to be approximately two-thirds that of controls. Samples can be stored and tested; one of the studies also analyzed the effect of delayed [[Test|testing]] and found that samples stored for 24 hours in the darkness gave similar results to those tested immediately.<ref name="pmid230237972">{{cite journal| author=Ciepiela O, Kotuła I, Górska E, Stelmaszczyk-Emmel A, Popko K, Szmydki-Baran A et al.| title=Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis. | journal=Clin Chem Lab Med | year= 2013 | volume= 51 | issue= 4 | pages= 817-23 | pmid=23023797 | doi=10.1515/cclm-2012-0240 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=23023797  }}</ref>
* Advantages of EMA binding include its high [[Sensitivity (tests)|sensitivity]] and [[Specificity (tests)|specificity]]; rapid turnaround time (approximately two hours); and need for only a minimal amount of [[blood]] (a few microliters), which is especially advantageous for [[Test|testing]] [[Neonate|neonates]] [99-101].
* In addition, EMA [[Test|testing]] can be used to identify [[hereditary spherocytosis]] in a [[patient]] who has recently received a [[Blood transfusion|transfusion]]. In various studies, the [[Sensitivity (tests)|sensitivity]] and [[Specificity (tests)|specificity]] of the test appear to be in the ranges of 93 to 96 and 93 to 99 percent, respectively.<ref name="pmid11122157">{{cite journal| author=King MJ, Behrens J, Rogers C, Flynn C, Greenwood D, Chambers K| title=Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia. | journal=Br J Haematol | year= 2000 | volume= 111 | issue= 3 | pages= 924-33 | pmid=11122157 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=11122157  }}</ref><ref name="pmid18782334">{{cite journal| author=Kar R, Mishra P, Pati HP| title=Evaluation of eosin-5-maleimide flow cytometric test in diagnosis of hereditary spherocytosis. | journal=Int J Lab Hematol | year= 2010 | volume= 32 | issue= 1 Pt 2 | pages= 8-16 | pmid=18782334 | doi=10.1111/j.1751-553X.2008.01098.x | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=18782334  }}</ref>
* EMA binding may also be positive in some forms of [[hereditary elliptocytosis]] (HE; eg, [[Hereditary pyropoikilocytosis|hereditary pyropoikilocytosis [HPP]]] and Southeast Asian [[ovalocytosis]] [SAO]), in individuals with [[Congenital dyserythropoietic anemia|congenital dyserythropoietic anemia (CDA) type II]], and/or in [[autoimmune hemolytic anemia]]. [[False negative|False negative results]] may be seen in mild cases of [[hereditary spherocytosis]].
 
'''Osmotic fragility'''
* If EMA binding is not available, osmotic fragility testing (OFT) is another useful specialized test for [[hereditary spherocytosis]]. In this test, [[Red blood cell|RBCs]] are incubated in hypotonic [[Buffer solution|buffered]] salt solutions of various [[Osmolarity|osmolarities]], and the fraction of [[hemoglobin]] released (due to [[hemolysis]]) is measured.
* The test takes advantage of the increased [[Sensitivity (tests)|sensitivity]] of [[Spherocyte|spherocytes]] to [[hemolysis]], which is due to their reduced [[Surface area to volume ratio|surface area to volume (SA/V) ratio]]. Incubation of [[patient]] [[Sample (statistics)|samples]] for 24 hours prior to [[Test|testing]] may accentuate osmotic fragility and improve [[Diagnosis|diagnostic]] yield.
* The OFT has relatively low [[Sensitivity (tests)|sensitivity]] and [[Specificity (tests)|specificity]]. It fails to identify a significant number of individuals with [[hereditary spherocytosis]] and, particularly in the [[newborn]], it may be [[positive]] in other conditions including [[Immune hemolytic anemias|immune hemolytic anemia]], [[Transfusion reaction|hemolytic transfusion reactions]], [[Red blood cell|RBC]] [[enzyme]] [[Defect|defects]] such as [[Glucose-6-phosphate dehydrogenase deficiency|glucose-6-phosphate dehydrogenase (G6PD) deficiency]], and unstable [[hemoglobin]] variants.<ref name="pmid257901092">{{cite journal| author=King MJ, Garçon L, Hoyer JD, Iolascon A, Picard V, Stewart G et al.| title=ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. | journal=Int J Lab Hematol | year= 2015 | volume= 37 | issue= 3 | pages= 304-25 | pmid=25790109 | doi=10.1111/ijlh.12335 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=25790109  }}</ref> In one series of 86 individuals with [[hereditary spherocytosis]], only 57 (66 percent) had positive osmotic fragility [[Test|testing]].<ref name="pmid8783633">{{cite journal| author=Cynober T, Mohandas N, Tchernia G| title=Red cell abnormalities in hereditary spherocytosis: relevance to diagnosis and understanding of the variable expression of clinical severity. | journal=J Lab Clin Med | year= 1996 | volume= 128 | issue= 3 | pages= 259-69 | pmid=8783633 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=8783633  }}</ref>
 
'''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 phosphates|sodium phosphate]] (to lower the [[pH]] to 6.85, in the AGLT) to the [[hypotonic]] [[Buffer solution|buffered]] [[Saline (medicine)|salt solutions]] in which the [[Patient|patient's]] [[RBCs]] are incubated.<ref name="pmid1554800">{{cite journal| author=Eber SW, Pekrun A, Neufeldt A, Schröter W| title=Prevalence of increased osmotic fragility of erythrocytes in German blood donors: screening using a modified glycerol lysis test. | journal=Ann Hematol | year= 1992 | volume= 64 | issue= 2 | pages= 88-92 | pmid=1554800 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=1554800  }}</ref><ref name="pmid17016037">{{cite journal| author=Stoya G, Gruhn B, Vogelsang H, Baumann E, Linss W| title=Flow cytometry as a diagnostic tool for hereditary spherocytosis. | journal=Acta Haematol | year= 2006 | volume= 116 | issue= 3 | pages= 186-91 | pmid=17016037 | doi=10.1159/000094679 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=17016037  }}</ref>
* Like the OFT, these [[Test|tests]] may also be [[positive]] in acquired [[spherocytosis]] conditions such as [[Autoimmune hemolytic anemia|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|glycerol solution]] at [[pH]] 6.66. A further modification has been proposed (the direct pink test) in which the [[test]] [[Sample (statistics)|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]].<ref name="pmid3631064">{{cite journal| author=Judkiewicz L, Szczepanek A, Bugała I, Bartosz G| title=Modified end-point glycerol hemolysis assay as a screening test for hereditary spherocytosis that requires no venipuncture. | journal=Am J Hematol | year= 1987 | volume= 26 | issue= 1 | pages= 89-91 | pmid=3631064 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=3631064  }}</ref>
 
'''Cryohemolysis'''
* In the cryohemolysis [[test]], [[RBCs]] are suspended in a [[Hypertonic|hypertonic solution]], briefly [[Heat|heated]] to 37°C, then cooled to 4°C for 10 minutes.<ref name="pmid9662272">{{cite journal| author=Streichman S, Gescheidt Y| title=Cryohemolysis for the detection of hereditary spherocytosis: correlation studies with osmotic fragility and autohemolysis. | journal=Am J Hematol | year= 1998 | volume= 58 | issue= 3 | pages= 206-12 | pmid=9662272 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=9662272  }}</ref>
* The ease of performance and the wide separation in degree of [[hemolysis]] between [[Spherocyte|spherocytes]] and normal [[Cell (biology)|cells]] are two attractive features of this [[test]].<ref name="pmid9204617">{{cite journal| author=Romero RR, Poo JL, Robles JA, Uriostegui A, Vargas F, Majluf-Cruz A| title=Usefulness of cryohemolysis test in the diagnosis of hereditary spherocytosis. | journal=Arch Med Res | year= 1997 | volume= 28 | issue= 2 | pages= 247-51 | pmid=9204617 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=9204617  }}</ref>
* This [[test]] has limited availability in the [[United States]].
* '''[[Cell membrane|Plasma membrane]] [[electrophoresis]]'''<ref>{{Cite journal
  | author = [[Olga Ciepiela]]
  | author = [[Olga Ciepiela]]
  | title = Old and new insights into the diagnosis of hereditary spherocytosis
  | title = Old and new insights into the diagnosis of hereditary spherocytosis
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  | pmid = 22058213
  | pmid = 22058213
}}</ref>
}}</ref>
** Osmotic fragility test
** Acidified glycerol lysis test
** Cryohemolysis
** Plasma membrane protein electrophoresis
'''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 [[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.
==== Red cell indices: ====
* [[Reticulocytosis]]
* Decreased [[mean corpuscular volume]]
* Increased [[mean corpuscular hemoglobin concentration]]
* Increased red cell distribution width
●'''[[Blood smear]] review''' – All individuals with suspected [[Hereditary spherocytosis|HS]] should have a [[blood smear]] reviewed by an experienced individual. In a [[peripheral blood smear]], the abnormally small [[Red blood cell|red blood cells]] lacking the central pallor i.e. spherocytes are seen. 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|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 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 [[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 [[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 [[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]]. <ref name="pmid26009624">{{cite journal| author=Christensen RD, Yaish HM, Gallagher PG| title=A pediatrician's practical guide to diagnosing and treating hereditary spherocytosis in neonates. | journal=Pediatrics | year= 2015 | volume= 135 | issue= 6 | pages= 1107-14 | pmid=26009624 | doi=10.1542/peds.2014-3516 | pmc=4444801 | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=26009624  }}</ref>
●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]].<ref name="pmid25790109">{{cite journal| author=King MJ, Garçon L, Hoyer JD, Iolascon A, Picard V, Stewart G et al.| title=ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. | journal=Int J Lab Hematol | year= 2015 | volume= 37 | issue= 3 | pages= 304-25 | pmid=25790109 | doi=10.1111/ijlh.12335 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=25790109  }}</ref> 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.<ref name="pmid22055020">{{cite journal| author=Bolton-Maggs PH, Langer JC, Iolascon A, Tittensor P, King MJ, General Haematology Task Force of the British Committee for Standards in Haematology| title=Guidelines for the diagnosis and management of hereditary spherocytosis--2011 update. | journal=Br J Haematol | year= 2012 | volume= 156 | issue= 1 | pages= 37-49 | pmid=22055020 | doi=10.1111/j.1365-2141.2011.08921.x | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=22055020  }}</ref> 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 [[Lactate dehydrogenase|LDH]], elevated indirect bilirubin, reduced [[haptoglobin]], increased [[reticulocyte count]]) or [[hemolytic anemia]] that is Coombs-negative and not explained by another condition.
[[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 [[Hereditary spherocytosis|HS]] as the likely diagnosis in an older child or adult include the following:
●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]].
●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.<ref name="pmid8943874">{{cite journal| author=Jarolim P, Murray JL, Rubin HL, Taylor WM, Prchal JT, Ballas SK et al.| title=Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency. | journal=Blood | year= 1996 | volume= 88 | issue= 11 | pages= 4366-74 | pmid=8943874 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=8943874  }}</ref><ref name="pmid9207476">{{cite journal| author=Hassoun H, Vassiliadis JN, Murray J, Njolstad PR, Rogus JJ, Ballas SK et al.| title=Characterization of the underlying molecular defect in hereditary spherocytosis associated with spectrin deficiency. | journal=Blood | year= 1997 | volume= 90 | issue= 1 | pages= 398-406 | pmid=9207476 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=9207476  }}</ref><ref name="pmid8102379">{{cite journal| author=Becker PS, Tse WT, Lux SE, Forget BG| title=Beta spectrin kissimmee: a spectrin variant associated with autosomal dominant hereditary spherocytosis and defective binding to protein 4.1. | journal=J Clin Invest | year= 1993 | volume= 92 | issue= 2 | pages= 612-6 | pmid=8102379 | doi=10.1172/JCI116628 | pmc=294892 | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=8102379  }}</ref><ref name="pmid2961992">{{cite journal| author=Coetzer TL, Lawler J, Liu SC, Prchal JT, Gualtieri RJ, Brain MC et al.| title=Partial ankyrin and spectrin deficiency in severe, atypical hereditary spherocytosis. | journal=N Engl J Med | year= 1988 | volume= 318 | issue= 4 | pages= 230-4 | pmid=2961992 | doi=10.1056/NEJM198801283180407 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=2961992  }}</ref>
* Pincered or notched [[Spherocyte|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 [[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.<ref name="pmid9202619">{{cite journal| author=Michaels LA, Cohen AR, Zhao H, Raphael RI, Manno CS| title=Screening for hereditary spherocytosis by use of automated erythrocyte indexes. | journal=J Pediatr | year= 1997 | volume= 130 | issue= 6 | pages= 957-60 | pmid=9202619 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=9202619  }}</ref> 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]].<ref name="pmid220550202">{{cite journal| author=Bolton-Maggs PH, Langer JC, Iolascon A, Tittensor P, King MJ, General Haematology Task Force of the British Committee for Standards in Haematology| title=Guidelines for the diagnosis and management of hereditary spherocytosis--2011 update. | journal=Br J Haematol | year= 2012 | volume= 156 | issue= 1 | pages= 37-49 | pmid=22055020 | doi=10.1111/j.1365-2141.2011.08921.x | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=22055020  }}</ref>
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.
'''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:
●'''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.<ref name="pmid23023797">{{cite journal| author=Ciepiela O, Kotuła I, Górska E, Stelmaszczyk-Emmel A, Popko K, Szmydki-Baran A et al.| title=Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis. | journal=Clin Chem Lab Med | year= 2013 | volume= 51 | issue= 4 | pages= 817-23 | pmid=23023797 | doi=10.1515/cclm-2012-0240 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=23023797  }}</ref> 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.<ref name="pmid230237972">{{cite journal| author=Ciepiela O, Kotuła I, Górska E, Stelmaszczyk-Emmel A, Popko K, Szmydki-Baran A et al.| title=Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis. | journal=Clin Chem Lab Med | year= 2013 | volume= 51 | issue= 4 | pages= 817-23 | pmid=23023797 | doi=10.1515/cclm-2012-0240 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=23023797  }}</ref>
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.<ref name="pmid11122157">{{cite journal| author=King MJ, Behrens J, Rogers C, Flynn C, Greenwood D, Chambers K| title=Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia. | journal=Br J Haematol | year= 2000 | volume= 111 | issue= 3 | pages= 924-33 | pmid=11122157 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=11122157  }}</ref><ref name="pmid18782334">{{cite journal| author=Kar R, Mishra P, Pati HP| title=Evaluation of eosin-5-maleimide flow cytometric test in diagnosis of hereditary spherocytosis. | journal=Int J Lab Hematol | year= 2010 | volume= 32 | issue= 1 Pt 2 | pages= 8-16 | pmid=18782334 | doi=10.1111/j.1751-553X.2008.01098.x | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=18782334  }}</ref>
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. 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 [[Spherocyte|spherocytes]] to [[hemolysis]], which is due to their reduced [[Surface area to volume ratio|surface area to volume (SA/V) ratio]]. 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 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.<ref name="pmid257901092">{{cite journal| author=King MJ, Garçon L, Hoyer JD, Iolascon A, Picard V, Stewart G et al.| title=ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. | journal=Int J Lab Hematol | year= 2015 | volume= 37 | issue= 3 | pages= 304-25 | pmid=25790109 | doi=10.1111/ijlh.12335 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=25790109  }}</ref> In one series of 86 individuals with HS, only 57 (66 percent) had positive osmotic fragility testing.<ref name="pmid8783633">{{cite journal| author=Cynober T, Mohandas N, Tchernia G| title=Red cell abnormalities in hereditary spherocytosis: relevance to diagnosis and understanding of the variable expression of clinical severity. | journal=J Lab Clin Med | year= 1996 | volume= 128 | issue= 3 | pages= 259-69 | pmid=8783633 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=8783633  }}</ref>
●'''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.<ref name="pmid1554800">{{cite journal| author=Eber SW, Pekrun A, Neufeldt A, Schröter W| title=Prevalence of increased osmotic fragility of erythrocytes in German blood donors: screening using a modified glycerol lysis test. | journal=Ann Hematol | year= 1992 | volume= 64 | issue= 2 | pages= 88-92 | pmid=1554800 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=1554800  }}</ref><ref name="pmid17016037">{{cite journal| author=Stoya G, Gruhn B, Vogelsang H, Baumann E, Linss W| title=Flow cytometry as a diagnostic tool for hereditary spherocytosis. | journal=Acta Haematol | year= 2006 | volume= 116 | issue= 3 | pages= 186-91 | pmid=17016037 | doi=10.1159/000094679 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=17016037  }}</ref> 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. 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.<ref name="pmid3631064">{{cite journal| author=Judkiewicz L, Szczepanek A, Bugała I, Bartosz G| title=Modified end-point glycerol hemolysis assay as a screening test for hereditary spherocytosis that requires no venipuncture. | journal=Am J Hematol | year= 1987 | volume= 26 | issue= 1 | pages= 89-91 | pmid=3631064 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=3631064  }}</ref>
●'''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.<ref name="pmid9662272">{{cite journal| author=Streichman S, Gescheidt Y| title=Cryohemolysis for the detection of hereditary spherocytosis: correlation studies with osmotic fragility and autohemolysis. | journal=Am J Hematol | year= 1998 | volume= 58 | issue= 3 | pages= 206-12 | pmid=9662272 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=9662272  }}</ref> Ease of performance and the wide separation in degree of [[hemolysis]] between [[Spherocyte|spherocytes]] and normal cells are two attractive features of this test.<ref name="pmid9204617">{{cite journal| author=Romero RR, Poo JL, Robles JA, Uriostegui A, Vargas F, Majluf-Cruz A| title=Usefulness of cryohemolysis test in the diagnosis of hereditary spherocytosis. | journal=Arch Med Res | year= 1997 | volume= 28 | issue= 2 | pages= 247-51 | pmid=9204617 | doi= | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=9204617  }}</ref> 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 cell]]s lacking the central pallor i.e. spherocytes are seen.


==References==
==References==

Latest revision as of 02:28, 19 December 2018

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]

Overview

The initial laboratory testing for hereditary spherocytosis include; complete blood count (CBC), mean corpuscular hemoglobin concentration (MCHC), blood smear review, hemolysis testing and coombs testing. All individuals suspected of having hereditary spherocytosis based on family history, neonatal jaundice or other findings should have a complete blood count (CBC), reticulocyte count and RBC indices done. Confirmatory tests for hereditary spherocytosis includes EMA binding test, osmotic fragility test, glycerol lysis test, cryohemolysis and plasma membrane electrophoresis.

Laboratory Findings

Initial testing

CBC and RBC indices

Red cell indices

Blood smear review

Hemolysis testing

Coombs testing

Neonates

Older children and adults

Confirmatory tests

EMA (eosin-5-maleimide) binding

Osmotic fragility

Glycerol lysis

Cryohemolysis

References

  1. ↑ Farias, Mariela Granero (2017). "Advances in laboratory diagnosis of hereditary spherocytosis". Clinical Chemistry and Laboratory Medicine (CCLM). 55 (7). doi:10.1515/cclm-2016-0738. ISSN 1437-4331.
  2. ↑ Christensen RD, Yaish HM, Gallagher PG (2015). "A pediatrician's practical guide to diagnosing and treating hereditary spherocytosis in neonates". Pediatrics. 135 (6): 1107–14. doi:10.1542/peds.2014-3516. PMC 4444801. PMID 26009624.
  3. ↑ King MJ, Garçon L, Hoyer JD, Iolascon A, Picard V, Stewart G; et al. (2015). "ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders". Int J Lab Hematol. 37 (3): 304–25. doi:10.1111/ijlh.12335. PMID 25790109.
  4. ↑ Bolton-Maggs PH, Langer JC, Iolascon A, Tittensor P, King MJ, General Haematology Task Force of the British Committee for Standards in Haematology (2012). "Guidelines for the diagnosis and management of hereditary spherocytosis--2011 update". Br J Haematol. 156 (1): 37–49. doi:10.1111/j.1365-2141.2011.08921.x. PMID 22055020.
  5. ↑ Jarolim P, Murray JL, Rubin HL, Taylor WM, Prchal JT, Ballas SK; et al. (1996). "Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency". Blood. 88 (11): 4366–74. PMID 8943874.
  6. ↑ Hassoun H, Vassiliadis JN, Murray J, Njolstad PR, Rogus JJ, Ballas SK; et al. (1997). "Characterization of the underlying molecular defect in hereditary spherocytosis associated with spectrin deficiency". Blood. 90 (1): 398–406. PMID 9207476.
  7. ↑ Becker PS, Tse WT, Lux SE, Forget BG (1993). "Beta spectrin kissimmee: a spectrin variant associated with autosomal dominant hereditary spherocytosis and defective binding to protein 4.1". J Clin Invest. 92 (2): 612–6. doi:10.1172/JCI116628. PMC 294892. PMID 8102379.
  8. ↑ Coetzer TL, Lawler J, Liu SC, Prchal JT, Gualtieri RJ, Brain MC; et al. (1988). "Partial ankyrin and spectrin deficiency in severe, atypical hereditary spherocytosis". N Engl J Med. 318 (4): 230–4. doi:10.1056/NEJM198801283180407. PMID 2961992.
  9. ↑ Michaels LA, Cohen AR, Zhao H, Raphael RI, Manno CS (1997). "Screening for hereditary spherocytosis by use of automated erythrocyte indexes". J Pediatr. 130 (6): 957–60. PMID 9202619.
  10. ↑ Bolton-Maggs PH, Langer JC, Iolascon A, Tittensor P, King MJ, General Haematology Task Force of the British Committee for Standards in Haematology (2012). "Guidelines for the diagnosis and management of hereditary spherocytosis--2011 update". Br J Haematol. 156 (1): 37–49. doi:10.1111/j.1365-2141.2011.08921.x. PMID 22055020.
  11. ↑ Ciepiela O, Kotuła I, Górska E, Stelmaszczyk-Emmel A, Popko K, Szmydki-Baran A; et al. (2013). "Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis". Clin Chem Lab Med. 51 (4): 817–23. doi:10.1515/cclm-2012-0240. PMID 23023797.
  12. ↑ Ciepiela O, Kotuła I, Górska E, Stelmaszczyk-Emmel A, Popko K, Szmydki-Baran A; et al. (2013). "Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis". Clin Chem Lab Med. 51 (4): 817–23. doi:10.1515/cclm-2012-0240. PMID 23023797.
  13. ↑ King MJ, Behrens J, Rogers C, Flynn C, Greenwood D, Chambers K (2000). "Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia". Br J Haematol. 111 (3): 924–33. PMID 11122157.
  14. ↑ Kar R, Mishra P, Pati HP (2010). "Evaluation of eosin-5-maleimide flow cytometric test in diagnosis of hereditary spherocytosis". Int J Lab Hematol. 32 (1 Pt 2): 8–16. doi:10.1111/j.1751-553X.2008.01098.x. PMID 18782334.
  15. ↑ King MJ, Garçon L, Hoyer JD, Iolascon A, Picard V, Stewart G; et al. (2015). "ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders". Int J Lab Hematol. 37 (3): 304–25. doi:10.1111/ijlh.12335. PMID 25790109.
  16. ↑ Cynober T, Mohandas N, Tchernia G (1996). "Red cell abnormalities in hereditary spherocytosis: relevance to diagnosis and understanding of the variable expression of clinical severity". J Lab Clin Med. 128 (3): 259–69. PMID 8783633.
  17. ↑ Eber SW, Pekrun A, Neufeldt A, Schröter W (1992). "Prevalence of increased osmotic fragility of erythrocytes in German blood donors: screening using a modified glycerol lysis test". Ann Hematol. 64 (2): 88–92. PMID 1554800.
  18. ↑ Stoya G, Gruhn B, Vogelsang H, Baumann E, Linss W (2006). "Flow cytometry as a diagnostic tool for hereditary spherocytosis". Acta Haematol. 116 (3): 186–91. doi:10.1159/000094679. PMID 17016037.
  19. ↑ Judkiewicz L, Szczepanek A, Bugała I, Bartosz G (1987). "Modified end-point glycerol hemolysis assay as a screening test for hereditary spherocytosis that requires no venipuncture". Am J Hematol. 26 (1): 89–91. PMID 3631064.
  20. ↑ Streichman S, Gescheidt Y (1998). "Cryohemolysis for the detection of hereditary spherocytosis: correlation studies with osmotic fragility and autohemolysis". Am J Hematol. 58 (3): 206–12. PMID 9662272.
  21. ↑ Romero RR, Poo JL, Robles JA, Uriostegui A, Vargas F, Majluf-Cruz A (1997). "Usefulness of cryohemolysis test in the diagnosis of hereditary spherocytosis". Arch Med Res. 28 (2): 247–51. PMID 9204617.
  22. ↑ Olga Ciepiela (2018). "Old and new insights into the diagnosis of hereditary spherocytosis". Annals of translational medicine. 6 (17): 339. doi:10.21037/atm.2018.07.35. PMID 30306078. Unknown parameter |month= ignored (help)
  23. ↑ Paola Bianchi, Elisa Fermo, Cristina Vercellati, Anna P. Marcello, Laura Porretti, Agostino Cortelezzi, Wilma Barcellini & Alberto Zanella (2012). "Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics". Haematologica. 97 (4): 516–523. doi:10.3324/haematol.2011.052845. PMID 22058213. Unknown parameter |month= ignored (help)

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