Strategy for screening for hereditary spherocytosis
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2007年06月26日 13:49:24 Tuesday
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作者：D.Kutter 作者单位：Laboratoires réunis,L 6101-Junglinster （Grand Duchy of Luxembourg）
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【摘要】 Approximately 94,000 unselected,mostly adult routine patients have been screened for hereditary spherocytosis （HS） using the percentage of hyperchromic red blood cells delivered by a last generation haematological analyser.A specific strategy based on long term results of this parameter,on family data and on determination of membrane proteins is presented.The high incidence of HS already found in previous investigations,as well as the scarcity of serious clinical symptoms resulting from reduced erythrocyte life span,is confirmed.Iron overload is the major biochemical consequence,increasing with age in males and after menopause in women.A higher incidence of diabetes is statistically confirmed.
【关键词】 hereditary and secondary spherocytosis consequences screening haemolysis iron overload diabetes
INTRODUCTION
Hereditary spherocytosis （HS） is described a rare haemolytic disease with serious consequences such as aplasia,anaemia,icterus with free bilirubin,cholelithiasis,splenomegalia,iron overload and the presence of “microspherocytes” on stained blood films［1-3］.The disease is considered hereditary.Most of the cases actually mentioned in literature have been detected through one or several of these clinical consequences and by family investigation.The incidence is claimed to be approximately 1∶5,000.Modern haematological analysers of the ADVIA type,using the double beam laser technology allows to determine simultaneously volume and haemoglobin concentration of approximately 50,000 red blood cells （RBC）,editing not only mean values of these 2 parameters,but also precise percentages of abnormal cells.The percentage of hypochromic RBC （%Hypo） is precious for the diagnosis of iron deficiency.Hyperchromia equivalent with “overfill” of the RBC -percentage of spherocytes,making it a valuable tool for diagnosis of both hereditary and secondary,acquired spherocytosis.Impressed by the high incidence of markedly increased %Hyper in daily routine,we decided systematic screening for both types of spherocytosis.Statistical evaluation of 7,000 unselected %Hyper values disclosed 3 slightly overlapping subpopulations:the large normal group with values up to 3.4 %,mild increase 3.5%～8.9 % and marked increase ≥ 9.0%［4］.
Hereditary trans-mission of HS follows the dominant pathway in most of the cases.Recessive transmission is rarer.Even de novo mutations occur.HS in direct family members,such as parents,offspring and sibs represents a valuable argument for HS.
In the course of a large screening action we elaborated a systematic strategy to follow when confronted with increased %Hyper values.
MATERIALS AND METHODS
During the years 2001～ 2006 we recorded %Hyper from approximately 94,000 unselected patients presenting to the laboratory for the first time,with 54,600 females,37,600 males.They represent the multiethnic population of the Grand Duchy of Luxembourg,of mainly central European origin,with large Mediterranean and minor African immigration.
Blood was sampled in K-EDTA vacuum tubes and processed within 4 hours.Complete haematology-reticulocytes included-was performed on an ADVIAhaematological analyser.As there exists no sufficiently precise control material for %Hyper,we relayed on the daily mean value delivered by the instrument and situatedbetween 1.5% and 1.9 %.
Additional chemical parameters,mainly ferritin,serum iron,blood glucose,haptoglobin and liver enzymes,were determined by routine methods in use in the laboratory,under permanent intern and extern quality control.
In several cases HS was confirmed by determination of RBC membrane proteins by the biochemistry laboratory of Erasmus Hospital in Brussels （Prof.B.Gulbis）.
SCREENING PROCEDURE
The follow up of 18 patients with known HS is represented in Figure 1.It clearly shows that the %Hyper is not constant,eventually dropping into the area of mild increase.
Figure 1 Evolution of the percentage of hyperchromic red blood cells in 18 patients with confirmed hereditary spherocytosis
Permanently increased %Hyper is used as leading parameter.Normal values < 3.5 % are ignored but stored in the archives awaiting relevant further results.All values ≥ 3.5 % are checked for anterior data.In cases without anamnesis or with increased values over less than one year,family investigations are attempted.Even single results ≥ 9.0 % are stored in separate file and continuously updated.Single results generally allow no final conclusion unless they appear within a clear familiar setting.Data exceeding one year are examined for permanence.Permanent increase between 3.5% and 8.9 % is suspicious for HS,which may eventually be confirmed by determination of membrane proteins.This labour intensive procedure is restricted to highly interesting cases.Normally these data are just stored in the archives.HS in direct family members is serious additional argument for HS in the proband.We provisionally conclude HS from permanently increased %Hyper over one year with at least one value ≥ 9.0 %.Before issuing a final diagnosis of HS,the eventuality of the secondary origin must be considered［5］.While the increase of %Hyper is generally not permanent in diseases such as infectious mononucleosis,HELLP syndrome,ethanol abuse,in pregnancy and during chemotherapy,it is permanent in G-6-PDH deficiency，homozygous drepanocytosis （HbSS,HbSC）［6］,long lasting autoimmune,haemolytic anaemia （AIHA）.In other words increased %Hyper can be seen as indicator of haemolysis of origin［7］.
An interruption of a series of even marked hyperchromia by normal values may be due to anaemia and/or iron deficiency［8］.Figure 2 illustrates the screening procedure.
RESULTS
Our screening program selected 992 potential cases of HS.Five hundred and forty-seven of them were finally identified as such disease.Seventy-one cases were detected by family investigations.Figure 3 shows a typical pedigree of a family of Portuguese immigrants with multiple cases of HS.Even the cases with only one increased %Hyper were considered HS.
In 8 cases HS was at first sight veiled by anaemia and/or iron deficiency;final diagnosis was reached by the analysis of membrane proteins［8］.In the case shown in Figure 4,HS became evident only after correction of iron deficiency.Figure 5 illustrates a case of falsely diagnosed haemochromatosis treated by phlebotomy.The patient turned out to be double heterozygote for the HFE genotype C282Y and H63D,HS remaining as the cause of heavy iron overload.
Figure 2 Screening for hereditary spherocytosis（HS）
Figure 3 Dominantly transmitted hereditary spherocytosis
over 3 generations in a family of Portuguese immigrants,even the 3 members with mild inerease are considered HSFigure 4 Case D.-R.Y.Evolution of hematological indices and ferritin in a cases of hereditary spherocytosis and temporary iron deficiency caused by chronic blood loss
Figure 5 Case of HS falsely diagnosed as primary haemochromatosis and treated by pllebotomy.HFE genotype turned out to be double heterozygote for C282Y and H63D genes.HS was the cause of heavy iron overload Age and gender distribution of the entire program is shown in Table 1.The total of 618 cases among 94,000 individuals is the minimum.In 263 cases diagnosis was withheld for insufficient data （single values or observation period < 1 year）.In 66 cases we concluded secondary spherocytosis.The causes ranged from permanent haematological anomalies,HbSC,HbSS,G-6-PDH deficiency （1 case each） to acquired affections,such as extremely low plasma electrolytes （4 cases）,chemotherapy （8 cases）,prostate cancer under specific treatment （5 cases）,severe ethanol abuse （2 cases）,infectious mononucleosis （4 cases）,HELLP syndrome （1 case）,AIHA （2 cases）,terminal renal insufficiency （2 cases）.Ribavirin was found responsible for haemolysis and high %Hyper in a case of hepatitis C.In 34 cases %Hyper increased from normal values to values ≥ 9.0 % in the course of normal pregnancy.
In 45 cases marked hyperchromia occurred only occasionally.These events were registered as they may correspond to haemolytic episodes eventually related to specific medications.
Table 1 Screening for Hereditary Spherocytosis among 94,000 Routine Patients
We attempted to collect a maximum of clinical and laboratory data of all detected HS cases.As our laboratory performs analysis strictly according to medical prescriptions and as many physicians hesitate to reveal clinical data for reason of data protection,our files are often incomplete.Symptoms such as splenomegalia were often overlooked,as palpation of the spleen is not an obligate part of medical examination.
Clinical symptoms ranged from marked anaemia （5 cases）,permanent or intermittent jaundice （31 cases）,cholelithiasis （5 cases）,splenomegalia （5 cases） to macroscopic urobilinuria （2 cases）.Only 11 of these 48 cases have the symptoms leading to the diagnosis of HS.This explains the discrepancy between our results and the figures regularly reported in world literature.The latter are mainly restricted to clinical symptoms,while we base our diagnosis on an item obtained systematically and free of costs in every routine haematological analysis.
Laboratory data mainly concerned haematology,available in all 618 cases.Serious anaemia was found in only 5 cases.Marked reticulocytosis occurred in 21 of 105 cases.This means that haemolysis is well compensated and tolerated.Off hand prescription for determination of haptoglobin is rare.Significant decrease was observed in only 26 of 64 cases.This is explained by the fact that haemolysis in HS is mainly extravascular,decrease of haptoglobin resulting only from lysis of more fragile RBC in the blood stream.Increase of free plasma bilirubin occurred in 139 of 350 cases.The test for urinary urobilinogen is performed as part of complete urinalysis.It was positive in only 5 of 334 cases.
HS being a haemolytic affection,we expected more or less systematic iron overload［9］.Our results show manifest hyperferritinaemia in of the males,rare in childhood and adolescence,increasing with age.The same symptom appeared in women,less frequently in child bearing age,where it was probably kept veiled by menstrual blood loss［8］.The incidence clearly increases after menopause （Table 2）.The difference of these results and the ones of an age and gender matched control group with normal haematology is highly significant ［10］.
Table 2 Increase of Iron Overload in Hereditary Spherocytosis （HS）
In analogy of primary haemochromatosis the higher incidence of diabetes was expected.Close to 30 % diabetes and glucose intolerance among HS patients is therefore not astonishing.The comparison of these values to those of an age and gender matched control group is highly significant （Table 3）.In this context it must be stressed that diabetes management via HbA1c is not reliable in case of HS.Due to the shorter life span of the RBC,glycation of haemoglobin does not reach the expected level,simulating an overoptimistic compliance［11］.
Table 3 Incidence of Diabetes and Glucose Intolerance in Hereditary Spherocytosis
DISCUSSION
Even if only a minority of our detected cases of HS suffers from serious clinical consequences,we maintain the necessity for systematic screening for this anomaly.The diagnosis of HS may be way-leading for the explanation of many symptoms.
Iron overload,the most striking biochemical symptom,is deserved discussion.Whatever its origin,it is considered toxic for many organs of the human body,leading to damage of liver,heart,to diabetes,to hormonal abnormalities［12-14］.Several authors implicate it in neoplastic disease,viral and bacterial infection［15-18］.
A link between primary haemochromatosis and diabetes is generally admitted.Our results at least suggest a similar link between diabetes and HS.Systematic reduction of iron overload is controversial.American blood banks often accept donations from individuals with iron overload,while they are mostly refused in Europe.
Unfortunately,most physicians are unaware of the significance of the parameter “%Hyper” and do not systematically consider a possible diagnosis of HS.On the other hand,many laboratories do not even edit “%Hyper”.Even in the absence of marked symptoms,we attempt to attract physicians’ attention to possible HS,so that they keep follow up of eventual complications in mind.Information of the patient himself may be useful in some cases,but should be withheld from overanxious hypochondriacs.
CONCLUSION
HS may no longer be listed under rare haematological anomalies.With an overall smoothed incidence of 1∶200 routine patients （1∶100 men,1∶500 women） it must,without doubt,be considered as frequent.Even if it occurs without marked clinical consequences in most cases,its diagnosis may often be way-leading,accounting for hitherto unexplained symptoms.The use of phlebotomy,commonly used for treatment of iron overload in haemolytic disorders,should be considered as possible issue.
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（Editor LEE ）
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