adipose triglyceride lipase ( atgl , ec 3.1.1.3 ) deficiency is caused by mutations in atgl gene , also called pnpla2 
 , , .
it presents profound lipid accumulation mainly in skeletal and cardiac muscles , manifesting neutral lipid storage disease with myopathy ( nlsd - m)/triglyceride deposit cardiomyovasculopathy ( tgcv ) , , , , . only up to 40 patients
most of the reported cases were diagnosed in adulthood except for one case each in childhood and adolescence , . in adulthood
, the myopathy and cardiomyopathy can be severe and rapidly progressive , and refractory to various therapies .
affected patients with atgl deficiency exclusively exhibit persistent lipid droplets in the cytoplasm of circulatory neutrophils known as jordans ' anomaly ( fig . 
1a ) , , , , , , , , , , . in earlier life ,
clinical symptoms seem to be absent or minimal in most cases , however jordans ' anomaly has been documented in subclinical or preclinical adolescents with atgl deficiency , . blood smear examination with may - giemsa
this report concerns a simple , easy and feasible laboratory test using a routine automated hematological analyzer that detects leukocyte abnormalities in patients with myopathy or cardiomyovasculopathy and possibly leads to a diagnosis of homozygous atgl deficiency .
four homozygous atgl - deficient patients ( 3 males and 1 female , 4560  years of age ) ( table  1 ) and nine heterozygous family members ( 4 males and 5 females , 1783  years ) , lacking atgl deficiency - associated symptoms , were enrolled .
the diagnosis of atgl deficiency was based on gene analyses together with clinical manifestations of myopathy , including easy fatigability , reduced exercise capability and limb weakness , and cardiomyopathy .
forty - three healthy subjects ( 14 males and 29 females , 3284 years ) lacking the mutations in atgl gene and having no abnormality under the physical examination were also enrolled as controls .
blood specimens were analyzed by the xe-5000 automated hematology analyzer ( sysmex , kobe , japan ) and investigated all the parameters including wbc / baso channel of the xe-5000 to screen for jordans ' anomaly . in the wbc / baso channel ,
its hemolyzing reagent , stromatolyzer fb ( sysmex ) , lyses plasma membranes of cells other than basophils in the specimen and , as a result , the cytosolic components of non - basophils are released from the cells .
lipid droplets released from atgl - deficient leukocytes which retain their shape in aqueous environment due to their own lipid monolayer membranes can be detected as smaller particles .
thus , in the wbc / baso scattergram , almost intact basophils , nucleus of non - basophil leukocytes , and relatively large cytosolic components including lipid droplets or debris are detected as particles ( fig . 
parameters named baso - wx and baso - wy , which stand for the spread of particle distribution in side and forward scattered light , respectively , are calculated in the wbc / baso channel .
an analysis of welch 's t - test was performed to compare differences in baso - wx and baso - wy values between homozygous and heterozygous or controls .
four homozygous atgl - deficient patients ( 3 males and 1 female , 4560  years of age ) ( table  1 ) and nine heterozygous family members ( 4 males and 5 females , 1783  years ) , lacking atgl deficiency - associated symptoms , were enrolled .
the diagnosis of atgl deficiency was based on gene analyses together with clinical manifestations of myopathy , including easy fatigability , reduced exercise capability and limb weakness , and cardiomyopathy .
forty - three healthy subjects ( 14 males and 29 females , 3284 years ) lacking the mutations in atgl gene and having no abnormality under the physical examination were also enrolled as controls .
blood specimens were analyzed by the xe-5000 automated hematology analyzer ( sysmex , kobe , japan ) and investigated all the parameters including wbc / baso channel of the xe-5000 to screen for jordans ' anomaly . in the wbc / baso channel ,
its hemolyzing reagent , stromatolyzer fb ( sysmex ) , lyses plasma membranes of cells other than basophils in the specimen and , as a result , the cytosolic components of non - basophils are released from the cells .
lipid droplets released from atgl - deficient leukocytes which retain their shape in aqueous environment due to their own lipid monolayer membranes can be detected as smaller particles .
thus , in the wbc / baso scattergram , almost intact basophils , nucleus of non - basophil leukocytes , and relatively large cytosolic components including lipid droplets or debris are detected as particles ( fig . 
parameters named baso - wx and baso - wy , which stand for the spread of particle distribution in side and forward scattered light , respectively , are calculated in the wbc / baso channel .
an analysis of welch 's t - test was performed to compare differences in baso - wx and baso - wy values between homozygous and heterozygous or controls .
after confirming that all the specimens from the four atgl - deficient patients had jordans ' anomaly by examining their blood smears stained with may - giemsa ( fig . 
1a ) , we investigated all the parameters of the xe-5000 automated hematology analyzer to find any change corresponding to jordans ' anomaly .
the wbc / baso scattergram revealed an increased number of small particles in the homozygous patients , typically shown as the blue dots in fig . 
the observed small particles are supposed to be the lipid droplets released from the patients ' leukocytes , because lipid droplets can be expected to retain their spherical forms with neutral lipid core and phospholipid monolayer surface in this aqueous environment .
the baso - wx and baso - wy values obtained from the wbc / baso channel of xe-5000 were significantly higher in the atgl - deficient patients than those in the non - affected heterozygotes and the controls ( fig . 
1c ) : 251.8    100/132.0    7.7 ( baso - wx / baso - wy , mean    sd ) for the atgl - deficient patients , 80.2    3.8/74.8    8.2 for the heterozygous carriers , and 80.4    5.5/70.3    8.2 for the controls .
we , therefore , anticipate that detection of the leucocyte abnormality in a routine automated hematological analysis may be a first step toward the diagnosis of homozygous atgl deficiency .
we further suggest that detected positive subjects should undergo more detailed analyses , including atgl gene analyses , to establish the diagnosis in the clinical practice
. in homozygous atgl deficiency , once the clinical presentations of myopathy and cardiomyopathy occur , it has been difficult so far to regress or resolve symptoms through any conventional treatments , , , , , , , , , .
we recently provided data indicating that up - regulation of peroxisome proliferated activated receptor- and the related genes may promote triglyceride accumulation in the skeletal and cardiac muscles in atgl deficiency .
we believe that the development of an easy method to detect cellular triglyceride accumulation is desired .
it is quite likely that the change in the leukocyte emerges before the development of myopathy or cardiomyovasculopathy , as it does in other congenital lipid storage diseases such as gaucher 's and niemann  pick disease , which show distinct lipid storage in circulatory and bone marrow macrophages . in our settings ,
heterozygous carriers could not be differentiated from control subjects , even though jordans ' anomaly has been reported in some heterozygous atgl deficiency .
we speculate that one of the reasons for this may be that heterozygous leukocytes may have enough atgl enzymatic activity to reduce the number and/or size of intracellular lipid droplets , so that baso - wx and baso - wy parameters were not different between heterozygous and control subjects .
it has been known that jordans ' anomaly in leukocytes is present not only in atgl deficiency but also in chanarin 
dorfman syndrome also called nlsd with ichthyosis , which is caused by deficiency of the protein cgi-58 , an activator of the atgl enzyme , . further , in carnitine palmitoyltransferase deficiency type 1 , a fatty acid beta - oxidation disorder engendering hypoglycemia and acidosis , this anomaly can sometimes be found in blood smears .
it would be of interest to know whether the present system can detect leucocyte abnormalities in these disorders , even we did not have the chance to test the possibility because of the disease rarity .
the sensitivity and specificity of baso - wx and baso - wy for atgl deficiency remains to be investigated , however we believe that this automatic detection of changes in leukocytes with an automated hematology analyzer may provide an earlier diagnostic clue for atgl deficiency to clinicians , who encounter patients with neuromuscular and cardiovascular disorders , whose causes are unknown . in order to increase information on the natural history and pathophysiology in nlsd / tgcv patients , we have started an international registry system on the web ( http://www.tgcv.org/r/home.html ) .
the baso - wx and baso - wy values obtained from automated hematology analyzer xe-5000 could help to detect jordans ' anomaly .
a notification system using an automated hematology analyzer may prompt the earlier and easier diagnosis of homozygous atgl deficiency .
 adipose triglyceride lipase ( atgl ) deficiency manifesting neutral lipid storage disease with myopathy / triglyceride deposit cardiomyovasculopathy presents distinct fat - containing vacuoles known as jordans ' anomaly in peripheral leucocytes . to develop an automatic notification system for jordans ' anomaly in atgl - deficient patients 
 , we analyzed circulatory leukocyte scattergrams on automated hematology analyzer xe-5000 . 
 the baso - wx and baso - wy values were found to be significantly higher in patients than those in non - affected subjects . 
 the two parameters measured by automated hematology analyzer may be expected to provide an important diagnostic clue for homozygous atgl deficiency . 