We describe an improved cytochemical procedure for detecting the presence of cytoplasmic lipid droplets (Jordans' bodies) in blood cells from patients suffering from neutral lipid storage diseases (NLSDs). The method employs Oil red O (ORO), Nile red (NR) or, preferably, Bodipy (4,4‐difluoro‐1,3,5,7,8‐pentamethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene) staining, coupled with DAPI (2‐(4‐amidinophenyl)‐6‐indolecarbamidine) staining of nuclei, to visualise clearly by fluorescence microscopy the presence of abundant neutral lipids (triacylglycerols and cholesterol esters) in granulocytes and monocytes. Using these reagents, we easily identified Jordans' bodies in buffy coats from patients affected by Chanarin–Dorfman syndrome (NLSD with ichthyosis) and NLSD with progressive myopathy, a laboratory finding critical for the diagnosis of both inherited metabolic disorders. Due to their yellow‐gold and green fluorescence arising selectively from neutral lipid binding and their water solubility that makes alcohol unnecessary for staining, NR and particularly Bodipy are superior to ORO for the specific detection of Jordans' bodies in leucocytes.
Neutral lipid storage diseases (NLSDs) are a clinically heterogeneous group of non‐lysosomal inherited disorders characterized by a cytoplasmic accumulation of lipid droplets (LDs) in most tissues. Rather than being an inert lipid inclusion, an impression given prima facie by their simple morphological structure under the light microscope, LDs were recently shown to be distinct organelles consisting of a core of neutral lipids, predominantly triacylglycerols or cholesteryl esters, that are surrounded by a monolayer of phospholipids and associated proteins.1,2 LDs take active part in lipid metabolism as well as in membrane trafficking and other cell fuctions.3
Since the pioneering observation by Jordans of numerous LDs in the leucocytes of two Dutch brothers suffering from progressive muscular dystrophy,4 several reports have described the presence of abundant triacylglycerol deposits in the non‐adipose cells of patients manifesting a variety of defects. Clinical phenotypes include myopathy (skeletal and heart muscle), liver damage, ataxia, neurosensory hearing loss, ichthyosis, subcapsular cataracts, nystagmus, strabismus and, rarely, mental retardation. When non‐bullous congenital ichthyosiform erythroderma, presenting as fine scaling on an erythematous skin, is the dominant feature of NLSD since birth, the disorder is commonly referred to as Chanarin–Dorfman syndrome (CDS).5,6 Neutral lipid storage skeletal myopathy4,7 and cardiomyopathy8 are two clinical variants of NLSD in which no skin involvement is observed.
Since NLSD is a genetic and allelic heterogeneous autosomal recessive disorder, so far mutational analysis is of limited use in clinical practice for a correct diagnosis of the congenital metabolic syndrome. The presence of 2–6 May–Grünwald–Giemsa (MGG)‐negative round vacuoles (cytoplasmic LDs)—called Jordans' bodies (JBs)—in otherwise normal neutrophils and eosinophils from peripheral blood is the most common laboratory finding in NLSDs and, together with normal serum carnitine levels, is of clinical value for a rational diagnostic approach to the disorder. However, since MGG‐negative spots in granulocytes and monocytes may result either from cell inclusions not containing neutral lipids (NLs) or from staining artefacts, a more specific cytochemical procedure seems desirable for an unambiguous identification of JBs.
To obviate this problem and to facilitate accurate and quantitative detection of LDs in NLSD leucocytes, we suggest the use of lipophilic dyes coupled with chromatin staining for visualising and counting JBs in buffy coat cells under fluorescent light.
Materials and methods
Five patients for which a clinical and molecular (ABHD5 mutation detection) diagnosis of CDS had been unambiguously established and four patients affected by NLSD with skeletal myopathy and cardiomyopathy, together with their unaffected relatives, were included in this study. Informed consent was obtained from each patient and the control subjects.
Fresh EDTA‐treated peripheral blood samples (6 ml) from patients, carriers and control subjects were centrifuged at 3300 g for 10 min. Buffy coats were carefully collected by gentle pipette suction, immediately smeared onto slide glasses, dried completely, and fixed with Biofix (Bio‐Optica, Milan, Italy). Cells were sequentially washed with Dulbecco's phosphate‐buffered saline (DPBS; Gibco, Grand Island, NY, USA), stained at room temperature for 20 min with Oil red O (ORO), Nile red (NR) or 4,4‐difluoro‐1,3,5,7,8‐pentamethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene (Bodipy 493/503), and then with 2‐(4‐amidinophenyl)‐6‐indolecarbamidine (DAPI). ORO (Sigma‐Aldrich, St Louis, MO, USA) staining was performed by a 0.2% (w/v) solution in 60% (v/v) isopropanol–water, twice filtered immediately before use. NR (Sigma‐Aldrich) staining solution was freshly prepared in DPBS (1:100 v/v) from a saturated solution (1 mg/ml) in dimethylsulphoxide. Bodipy (Molecular Probes, Eugene, OR, USA), 1 mg/ml in absolute ethanol, was diluted 1:500 (v/v) in DPBS to a final concentration of 2 μg/ml. DAPI (Sigma‐Aldrich) staining was performed using a 5 μg/ml water solution. After rinses with distilled water, cells were mounted on a glass slide with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and observed by a Leica MB5000B microscope equipped with a 100× Fluortar oil‐immersion objective. Fluorescence images were captured using a Leica DFC480 R2 digital camera and a Leica Application Suite (LAS) software which allows the acquisition of digital images under defined set‐up conditions. Set‐up (exposure time, saturation, gamma and gain, respectively) was as follows: ORO, 4 s, 1.2, 1.43 and 1×; NR, 1 s, 1.1, 1.21 and 1.1×; Bodipy, 7 s, 1.1, 1.28 and 1.3×; and DAPI, 0.03–0.05 s, 1.1, 1.52 and 1×. LDs present in leucocytes were analysed for number and dimension using ImageJ 1.35 software (NIH, Bethesda, MD, USA). Traditional MGG staining was carried out according to the standard procedure.
Results and discussion
Using fluorescent dyes which specifically bind to the NL core of LDs, their presence and distribution have been investigated in many cells, both in culture and within tissue samples.9,10,11,12,13 However, these selective dyes have not yet been used to detect LDs in leucocytes of NLSD patients, where they are identified as JBs.
To investigate how LDs differ in number and size among NLSD patients, carriers and healthy controls, we performed ORO, NR and Bodipy, as well as MGG staining of buffy coats from blood samples. Representative microphotographs of leucocytes (100×) obtained by traditional and NL‐specific fluorescence staining are reported in fig 1. The presence of LDs is negligible in control subjects (A1–A4), whereas LDs of variable number and size are clearly detectable both in NLSD patients without ichthyosis (C1–C4) and in CDS patients (D1–D4). Interestingly, a few small LDs were also identified in the leucocytes of heterozygous (ABHD5 mutation) subjects (B1–B4), which is of diagnostic value for the carrier status.
Figure 1 Microphotographs of Bodipy‐, Nile red (NR)‐, Oil red O (ORO)‐, and May–Grünwald–Giemsa (MGG)‐stained buffy coats: A1–A4, control subjects; B1–B4, carriers (ABHD5 mutation); C1–C4, patients affected by neutral lipid storage disease without ichthyosis; and D1–D4, Chanarin–Dorfman syndrome patients. Filters were I3 (excitation: 450–490 nm) for NR and Bodipy, TX2 (excitation: 540–580 nm) for ORO, and A4 (excitation: 340–380 nm) for 2‐(4‐amidinophenyl)‐6‐indolecarbamidine (DAPI). Bodipy, NR and ORO fluorescence images were merged with DAPI images by Adobe Photoshop software (v. 7). Scale bar: 10 μm.
While no NL‐specific fluorescence could be detected in the stained leucocytes of control subjects, we were able to perform a quantitative analysis of the LD fluorescence signal from leucocytes of patients. Using an image analysis software (ImageJ), the mean number and dimension (perimeter; area) of LDs per cell were determined for each slide. Values reported in table 1 represent the means of 20 cells and were obtained from different microscopic fields chosen at random for image analysis. For each blood sample, the average number of LDs per cell is significantly different in Bodipy‐, NR‐ and ORO‐stained smears. Also, total LD perimeter and area per cell increase progressively from Bodipy to NR and ORO staining of buffy coat preparations. Earlier studies on LD staining with the same fluorescent dyes in cells other than leucocytes provide evidence for an explanation of these striking results. Fukumoto and Fujimoto14 demonstrated that the presence of isopropanol in ORO staining solutions causes a deformation of adjacent LDs, eventually leading to their fusion. NR or Bodipy staining does not affect LD morphology to such an extent since these dyes are applied in alcohol‐free aqueous solutions. Furthermore, while Bodipy binds only to the NL core of LDs, NR often causes diffuse background fluorescence. NR fluorescence arising from the dye–lipid interaction can be selectively measured using an excitation wavelength of 450–500 nm for NLs (yellow‐gold emission) and 515–560 nm for polar lipids (red emission).15,16 Since NR red fluorescence is very intense, there might be a possible red spill‐over into the yellow‐gold fluorescence as a consequence of leaking of stray red light through the filter barriers.17,18 Together with our results, these observations point to Bodipy as the best tracer of LDs in leucocytes as well as in other cell types.
Table 1 Average number, total perimeter and total area of LDs per cell as determined by Bodipy, Nile red and Oil red O staining of buffy coats from NLSD patients with and without ichthyosis. Values are given as the mean (SD).
| Bodipy (n = 98) | NR (n = 138) | ORO (n = 224) | |
|---|---|---|---|
| Average LD number per cell* | 4.9 (2.43) | 6.7 (2.56) | 11.2 (7.32) |
| Total LD perimeter (μ) per cell† | 21.45 (9.36) | 41.05 (14.73) | 53.6 (36.09) |
| Total LD area (μ2) per cell‡ | 8.4 (4.16) | 18.6 (8.16) | 23.25 (21.22) |
LD, lipid droplet; NR, Nile red; ORO, Oil red O; SD, standard deviation; n, number of LDs.
*pBodipy/NR<0.05; pBodipy/ORO<0.001; pNR/ORO<0.05.
†pBodipy/NR<0.001; pBodipy/ORO<0.001; pNR/ORO = 0.158.
‡pBodipy/NR<0.001; pBodipy/ORO<0.001; pNR/ORO = 0.366.
In conclusion, this study shows that a specific and quantitative detection of JBs in granulocytes and monocytes can be easily performed using lipophilic fluorescent dyes, leading to an improved cytochemical method for the laboratory diagnosis of NLSDs.
Take home messages
Neutral lipid storage diseases (NLSDs) are a clinically heterogeneous group of non‐lysosomal inherited disorders characterised by a cytoplasmic accumulation of lipid droplets in most tissues.
The presence of May–Grünwald–Giemsa‐negative lipid droplets (Jordans' bodies) in otherwise normal neutrophils and esosinophils from peripheral blood is the most common laboratory finding in NLSDs.
We describe an improved cytochemical method for detecting Jordans' bodies in buffy coats which employs lipophilic fluorescent dyes such as Oil Red O, Nile red and Bodipy, coupled with DAPI staining of nuclei.
Our results show that a specific and quantitative detection of Jordans' bodies can be easily performed in human leucocytes using Nile red or, preferably, Bodipy, allowing an unambiguous laboratory diagnosis of NLSDs.
Acknowledgements
The skillful assistance of Chiara Redaelli in analysing digital microphotographs and the generous technical collaboration of Enrico Italo are gratefully acknowledged. This study was supported by grants from the Italian Ministry of University and Scientific and Technological Research (MURST‐FIRB) and the Cariplo Foundation (Milan).
Footnotes
Competing interest. None declared.
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