False Positive Diagnosis of Lysosomal Storage Disease Based on Dried Blood Spot Sample; Leucocyte Number of a Challenging Factor

Aim:Recently dried blood spot (DBS) samples have been recommended as a screening test for Lysosomal Storage diseases. Although DBS samples have many advantages including non-invasiveness, cost and transportation, usage of these samples is limited by its high false positive rate. We aimed to investigate any possible effect of the leucocyte number on enzyme activity in dried blood samples in a retrospective study.Materials and Methods:Data was collected from subjects (n=263) for whom hematological parameters were available in the database of Ege University Hospital. The lysosomal enzyme activity results (alpha glycosidase, glycocerebrosidase, alpha galactosidase, sphingomyelinase and galactocerebrosidase) were re-evaluated with regard to the leucocyte number. Enzyme activities were measured using fluorometric and liquid chromatography-tandem mass spectrometry methods.Results:All enzyme activities closely correlated with the total number of leucocyte, since leucocytes are the main source of lysosomal enzymes. Glycocerebrosidase and galactocerebrosidase presented a positive correlation with the number of neutrophils and sphingomyelinase showed a positive correlation with the number of lymphocytes. When we recalculated the lysosomal enzyme activities with regard to the leucocyte number, the false positive rates for glycocerebrosidase, sphingomyelinase and alpha galactosidase decreased from 20%, 10.5% and 10.8% to 4.5%, 4.4% and 4.2%, respectively.Conclusions:Our data indicated that the enzyme activity in dried blood samples including low leucocyte number might be found lower than reference intervals resulting in false positive diagnosis. We concluded that the calculation of enzyme activity with regard to the number of leucocytes might produce more reliable results and might be helpful in decreasing the false positive rate

Lizozomal depo hastalıklarının lökositlerinde hastalık ile ilgili spesifik enzim aktivitelerinin ölçülmesi

Lizozomal depo hastalıkları (LDH) 40' tan fazla genetik hastalık grubunu kapsar. LDH'ta yıkılamayan moleküller, endozom/ lizozomlar içerisinde depolanmaya başlar. Depo edilen metabolitler genellikle mukopolisakkarid (MPS), sfingolipid, mukolipid, glikoprotein, oligosakkarid ve glikojendir. Lizozomlardaki bu birikim bir süre sonra hücrelerin şişmesine, fonksiyonlarının bozulmasına ve hücre ölümüne neden olur. LDH'ların tanısında kuru kan örneğinde ve lökositlerde enzim aktivitesi tayini en önemli ve en sık kullanılan yöntemdir. Ancak özellikle heterozigot hastalarda yalancı pozitiflik veya negatiflik görülebilir. Bu araştırmada bu hastaların T lenfositleri, B lenfositleri ve monosit-makrofajları flow sitometri ile CD8, CD19, CD14, CD33 reseptörleri kullanılarak izole edilerek enzim aktiviteleri tayin edilmiştir. Bu şekilde özellikle heterozigot hastaların tanısı için daha güvenli bir tanı yöntemi araştırılmıştır. Son yıllarda, Gaucher hastalarında multipl myelom ve B hücreli lenfoma insidansında artış gösterilmiştir. Bu nedenle, çalışmada Gaucher hastalarında lökosit infiltrasyonu ve lenfoid malignite gelişmesi arasındaki ilişkiyi araştırmak amacıyla Gaucher hastaları ve sağlıklı kontrollerde lökosit yüzey antijenlerinin oranı belirlenmiştir Lökosit hücre yüzeyi antijenlerinin miktarı % dağılım olarak hesaplanmıştır. Çalışmamızda GD hastalarında CD33, CD14 ve CD8 antijenleri kontrole göre hafif yüksek olarak bulunurken CD19+ lökositlerin oranı istatistiksel olarak anlamlı yüksek bulunmuştur (p0.05). CD33, CD19, CD14 ve CD8 pozitif lökositler akım sitometresinde toplanmış ve enzim aktiviteleri belirlenmiştir. Enzim aktiviteleri hasta ve kontrol grubu arasında anlamlı farklı bulunmuştur( p≤0.00) GD hastaları genetik özelliklerine göre kendi aralarında ve kontrol grubu arasında karşılaştırılmıştır.Hücre sayı oranları incelendiğinde Homozigot GD olanlarda CD33+ lökosit oranı anlamlı yüksek olduğu görüldü (p≤0.038). Çalışmamızda bulunan CD19 antijen ekspresyonu artışı B hücre kaynaklı malignitelerin gelişiminde bu antijenin rolü olabileceğini düşündürmüş ve Gaucher'de inflamatuvar aktivasyon hipotezini desteklemiştir. CD19+ lökosit miktarının belirlenmesinin bu hastalarda malignitenin ön göstergesi olarak kullanılabileceğini düşündürmüştür. Anahtar kelimeler: Gaucher; CD19; CD33.Lysosomal Storage Diseases (LSD) are a group of metabolic disorders that contain almost more than 40 inherited disorders and its approximate incidence in births is 1/40.000-1/100.000.000.Lysosomal storage diseases result defects in lysosomal functions because of mutations on genes that code lysosomal enzymes. If one of these enzymes is defective, the large molecules accumulate within the cell. Storeged molecules cause cell disfunction and death.These are usually mukopolisakkarid (MPS), sfingolipid, mucolipid, glycoprotein, oligosaccharide and glycogen. Measuring the enzyme activity in leukocyte or dried blood sample,is the most common and principal method.However, sometimes it can be wrong positive or wrong negative results in dried blood samples. So enzyme activity in leukocyte is offered for certain diagnosis. But in case of manuel leukocyte izolation, results are changeable for especially heterozygote patients. İn this study, patients's and healty people's T lymphocyte, B lymphocyte,monocyte- macrophage are izolated, using CD8, CD14, CD19, CD33 receptors, by flow cytometry and measured enzyme activities. Comparison of levels of each leukocyte cell surface antigen between GD patients and healthy controls were expressed as % percentage. Our results show that in GD patients CD33, CD14 and CD8 leukocyte cell surface antigens were insignificantly higher compared to controls. Only the percentage of CD19+ leukocytes in GD patients were significantly higher compared to the control group (p0.05). There was statistically significant difference in glucocerebrosidase activities of leucocytes (positive for CD33, CD19, CD14 and CD8) between Gaucher patients and healthy controls(p≤0.001). We also investigated the genetic properties of GD patients. Homozygote GD and Heterozygote GD are compared each other and with control groups. CD33+ percent of Homozygote GD are significanly higher than the heterozygote patients (p≤0.038). Treatment duration and L1-L4 scores of Homozygote GD patients are significantly lower than those of heterozygote GD patients (p≤0.029, p≤0.016). There are no significant differences in terms of other biochemical parametres. When comparisonsa were made regarding to hepatomegaly, Z score of GD patients without hepatomegaly, is significantly higher than the patients having hepatomegaly (p≤0.009). There are no significant differences in terms of other biochemical parametres. This study indicates that the increase in CD 19+ leukocytes may serve as a useful marker to predict the leokocyte infiltration in GD patients. Since experimental anti-CD19 drugs are in development for direct treatment specifically towards B-cell cancers, close monitorization of the CD19+ leukocyte levels may also serve as a marker to show the response to treatment. Key Worlds: Gaucher; CD19; CD3