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The Hospital for Special SurgeryMusculoskeletal Integrity Program

535 East 70th Street Adele Boskey, Ph.D., DirectorNew York, NY 10021 Phone: 212-606-1453

Fax: 212-774-7877

PATHOLOGY CONSULTATION REPORTCase Number: S2009-15395

Analysis performed by:Rhima Coleman, Ph.D., Postdoctoral FellowPhone: 212-774-2908Fax: 212-774-7877

IntroductionThe patient sample presented to our lab demonstrated evidence of intracellular

accumulation of a material that stained positive with acidic Alcian Blue (pH 2.5), indicating

that the substance could be a proteoglycan (PG). We were asked to confirm these resultsusing Fourier Transform-Infrared Microscopic Imaging (FTIRI). FTIRI combines an FTIRspectrometer and light microscopy, which allows the identification and spatial distribution oftissue matrix components, like classic histology. Quantitative determination of theseparameters reveals repeatable, measurable differences between diseased and healthytissues. Using this method, we confirmed that there is increased proteoglycan content inseveral regions throughout the patients synovium compared to control tissue.

MethodFour micron thick paraffin embedded sections were supplied on BaF2 window for

analysis. FTIRI analysis was performed using a Spectrum Spotlight 300 spectrometer

(Perkin-Elmer, Waltham, MA). Rectangularregions throughout the synovial section were

acquired at a spatial resolution of 6.25 mand a spectral resolution of 4cm-1. The scansproduce a spectrum like that shown in Figure1 for each pixel. The Amide I peak is due tothe stretching of C=O bonds in collagen. TheAmide II and III peaks are due to the CNstretch associated with the NH bend incollagen and the stretching of the CC and NH bonds of collagen, respectively. The PG

peak is due to COC and COH vibrations insugar groups within the tissue.To determine proteoglycan and collagen content, analysis was performed using ISys

software version 3.1 (Spectral Dimensions, Olney, MD). The integrated area of the Amide I(1594-1718 cm-1), Amide II (1490-1594 cm-1), and PG (998-1180 cm-1) peaks were calculated.The integrated PG was normalized by the integrated Amide I and the Amide I peaknormalized by the Amide II peak to account for variations in tissue thickness. Theseparameters are reported as PG/Amide I and Amide I/Amide II, respectively. Fifteen different

Figure 1. Sample spectra of synovial tissue

0

0.2

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0.6

0.8

1

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9001100130015001700

Wavenumber (cm -1)

Absorban

Amide I

Amide II

Amide III PG

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regions in the patients tissue and 3 different regions in the control sample were isolated andanalyzed as described above.

Results

The distribution of the relative PG content is presented as color coded image maps inFigure 2 (top left). Quantitatively, the distribution is presented as histograms (not shown) andthe average values and standard deviations of relative PG content is presented in the plotshown in the top right of Figure 2. These data show that there was a significant increase inrelative proteoglycan content compared to control tissue. The H+E stain of PG in sequentialhistological sections show the distribution of PG and is presented to correspond with theFTIRI image maps (Figure 2, bottom). Qualitatively, the color maps show that the amount ofproteoglycans is increased in the patients synovium over the control tissue and thedistribution of the proteoglycans is broader. This is demonstrated by the mostly blue (low)distribution through the majority of the control sample compared to the patients tissue, whichis mostly green to yellow, with localizations of high PG concentration as indicated with red

(high). It was also apparent that not all sites analyzed in the patients synovium (3 out of 15fields) showed this effect. The Amide I/Amide II ratio was the same in both patient and controltissue (not shown), indicating that the makeup of the protein (predominately collagen) iscomparable in both tissues.

Control Synovium Patients Synovium

FTIRIP

roteoglycan

Distribution

PG/Amide I

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0.1

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1Control Patient

Corresponding

Hema

toxylinandEosin

St

ainedSection

Figure 2. Top: FTIRI color map showing the distribution of proteoglycan throughout thesynovial tissues. The significant accrual of PGs in the patients tissue is demonstrated in theplot of average PG/Amide I on the right. Bottom: Hematoxylin and eosin staining of theseregions on sequential sections. Note the enlarged lysosomes in the cells of the patients tissue.

0.30

* p < 0.02

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ConclusionsThe substance accumulating in the cells of the patients tissue is most likely a

proteoglycan. The intracellular accumulation of the proteoglycan appears to have no effect onthe structure of the collagen within the patients synovium. Further studies with the tissue will

be necessary to discern the precise chemical composition of this material.