Light chain immunofluorescence in various nephropathies

1. Moderator: Dr Rashmi C
Presenter: Dr G Santhi Priya
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2.  Introduction
 Types of renal biopsy
 Fixation and processing of sections
 Special stains
 Immunofluoresence
 Light chain structure and production
 Metabolism of light chains
 Diseases associated with increase in light chains
 Light chain(myeloma) cast nephropathy
 Proximal tubulopathies- monoclonal light chain mediated
 Tubulointestitial nephritis- monoclonal light chain mediated
 Deposition disease inducing light/heavy/light-heavy chain related
 Amyloidosis inclusing AL,AH related
 Summary
 References
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3.  There are two types
 One is a needle biopsy specimen
 Percutaneous specimen
 Transjugular specimens
 wedge biopsy specimen
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4.  Different methods of fixation
 Simplest- 10% formalin
 Several advantages
1. All routine -work well
2. Many immunohistologic methods also work well
3. If necessary-electron microscopy
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6.  Hematoxylin and eosin or periodic acid schiff
 Periodic acid-methenamine silver-basement membranes
 Connective tissue stain-hematoxylin van Gieson or a
trichrome
 Congo red
 Gram stain
 Perls’ Prussian blue
 von Kossa’s
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7.  Many renal diseases are complications of disorders of
the immune system
 Immunofluorescence techniques on frozen sections
 Immunoenzyme techniques on paraffin sections
 Advantages and disadvantages
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9.  Unfixed cryostat sections are the best
 Unfixed tissue to be frozen within brief
period of time can be placed on normal
saline/ PBS and transported to the lab in a
sealed container
 Tissue that will not be frozen for several
hours is best preserved using a transport
medium (Michel’s Medium)
 Snap freezing in isopentane+ liquid
nitrogen(stored at -80 o c)
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10.  Transport medium
 N-ethylmaleimide: anti autolytic agent
 Ammonium sulfate: precipitate tissue bound
immunoglobulins without losing their antigenicity
 Multiple washing in PBS to reverse the precipitation
of immunoglobulins
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11. Remove from the transport medium and placed in biopsy wash
solution for a minimum of 30 minutes
Remove excess wash solution
Chuck with frozen OCT and biopsy tissue should be left at −25°C for at
least 30 minutes
Cut in 5 µm sections
Sections are checked at regular intervals for the presence of
glomeruli; toluidine blue and light microscope
Air dry for a minimum of 30 minutes
Slides are kept in aluminium foil and stored at −20°C until ready for
staining
OCT: optimum cutting temperaturecompound
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12. Section is circled on the slide
Washed in phosphate buffered saline (PBS) for 5 minutes
Removed from the wash tank and excess PBS removed
Flooded with working-strength antibody or conjugated antibody and
incubated for 30 minutes
Washed in PBS for 10 minutes
Removed from wash tank and excess PBS is removed
Second stage conjugated antibody and incubate for 30 minutes
Washed in PBS for 10 minutes
Mounted in buffered glycerol using coverslips
Store the slides at 4°C until review3/4/2019 seinar pesimsr 12

13. 3 micron sections
Fix at 37 ̊C overnight or at 60 ̊Cfor 15 mins
Sections down to water
Rinse in tris buffer having pH 7.6 at 37 ̊Cfor 30 min
Add 100 μl proteinase-k on the sections and keep at 37 ̊Cfor 20 min
Stop enzymatic digestion with tris buffer at 4 ̊Cfor 40 mins
Rinse in PBS for 10 mins
Add FITC conjugated antibodies
Incubate in a wet chamber at 4 ̊Cfor 30 mins
Mount either with vector shield aqueous mounting media or PBG3/4/2019 seinar pesimsr 13

14.  Light chains are polypeptides
 Synthesized by plasma cells
 Assembled with heavy chains to form the various
classes of immunoglobulins.
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16.  Normally, there is a 40% overproduction of light chain
compared with heavy chain.
 On proliferation of an aberrant clone of B-cell lineage,
there may be additional secretion of clonal FLC.
 The k-FLCs are monomeric and the l-FLCs dimeric,
although higher oligomeric and polymeric forms of both
FLCs occur.
 FLCs rapidly disperse and are present in roughly equal
concentrations throughout extracellular compartments;
almost 80% of FLC is extravascular.
 Two-thirds of light chain production is k and this is
reflected in a serum k/λ ratio of 1.8:1.
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18.  Three clinical entities -dysproteinemia:
 Multiple myeloma (often referred to as myeloma),
 Plasma cell dyscrasia (dysproteinemia)
 Monoclonal gammopathy of unknown significance
(MGUS).
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19.  Heterogenous
 70% tubulopathies
 Light chain cast nephropathy
 Proximal tubulopathy
 Tubulointerstitial nephritis
 30% glomerupathies
 Amyloidosis
 Deposition diseases
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20.  Most typical presentation: acute renal functional
deterioration
 Most common cause of acute renal failure in patients
with myeloma
 Precipitating factors:
 Dehydration,
 Hypercalcemia,
 Contrast media,
 NSAIDS,
 Hyperuricemia,
 Infections,
 Nephrotoxins
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21.  Gross Pathology: no specific gross features in
kidneys
 The kidneys may have subcapsular pathology,
including granularity and occasional petechiae, but
these are likely related to vascular disease
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23. PAS Positive casts3/4/2019 seinar pesimsr 23

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25. k staining
λ staining
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26.  Electron microscopy: the casts contain abundant
fibrillary material admixed with cellular debris.
 The diagnosis can be confirmed by using
ultrastructural immunogold labeling to demonstrate
monoclonality when immunofluorescence studies
fail to make the diagnosis
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27.  Differential diagnosis
 Nephropathies with cast formation
 Acute tubulointerstitial nephritis
 Antirejection drugs: tacrolimus and rapamycin
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28.  Direct tubular damage
 Fanconi syndrome associated with proximal
tubulopathy in patients with plasma cell dyscrasias
 “lysosomal indigestion with constipation syndrome”
 Rapidly or slowly progressive renal failure
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29.  Gross pathology: enlarged with pale cortical areas
 Light Microscopy
 Crystal formation: Needle-like intracytoplasmic tubular
inclusions: PAS and trichrome stains
 Noncrystalline
(a) tubular damage with features of acute tubular necrosis,
(b) basolateral deposition of the light chain with interstitial
inflammatory response, and
(c) lysosomal accumulation with enlargement and atypical
lysosomal forms (lysosomal indigestion/constipation
pattern)
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31. • Immunofluorescence: Monoclonal light chains detected in
the cytoplasm of the tubular cells
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34.  Frequent pattern of renal damage associated with
plasma cell dyscrasias
 Mimics acute tubulointerstitial nephritis
 Gross pathology: Not significant
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36.  Immunofluorescence :
 Linear monotypic light chain staining -tubular
basement membranes
 Association with the most intense interstitial
inflammation
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38.  Electron Microscopy
 punctate to powdery, electron-dense material along
the outer aspect of the tubular basement membranes
 Differential Diagnosis
 Acute allergic tubulointerstitial nephritis-
eosinophils
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39.  Systemic disorders
 Light Chain Deposition Disease
 Heavy chain deposition disease
 Light and heavy chain deposition diseases
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40.  Acute renal failure
 Proteinuria
 Hematuria
 Renal insufficiency
 Tubular dysfunction
 Renal transplantation has been successful in patients
with disease confined to the kidneys
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42. • No capsular drop and hyaline cap lesions
• Mesangial nodules- evenly distributed
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43.  Silver methenamine stained: lamellation of the
peripheral portions of the mesangial nodules
 Congo red negative
 Glomerular morphologic patterns including
mesangial, membranoproliferative, and crescentic,
that precede the nodular glomerulopathy
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47.  Glomerulonephritis Associated With Monoclonal
IgG Deposits
 Monoclonal Gammopathy of Unknown Significance
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48.  Deposits of abnormally folded proteins share unique
staining properties and a fibrillar ultrastructural
appearance
 Gross - enlarged kidneys with pale, waxy-appearing
cut surfaces
 Normal or small kidneys
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53.  Amyloid deposits can be found in any of the renal
compartments: in the glomeruli, the interstitium, or
the extraglomerular blood vessels.
 Glomerular amyloid formation begins in the
mesangium, eventually replacing the normal
mesangial matrix and extending into peripheral
capillary walls.
 Amyloid deposition may occur in segmental, diffuse
mesangial, nodular, and pure basement membrane
patterns
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55.  It is a lymphoproliferative disorder characterized by
a monoclonal proliferation of lymphoplasmacytic
cells producing IgM, which can be detected as an M
spike in SPEP
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57.  Minimal change disease
 Membranous nephropathy
 Immunotactoid
 Fibrillary glomerulopathy
 Crescentic glomerulonephritis
 MIDD
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58.  Characteristic fibrillar deposits in the mesangium
and glomerular capillary walls
 Extracellular deposition of nonbranching, randomly
arrayed fibrils approximately 20 nm in diameter
 Resemble amyloid fibrils superficially but differ
ultrastructurally
 Do not stain with Congo red
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59.  Light microscopy
 The glomerular lesions usually show
membranoproliferative or mesangioproliferative
 IF
 Polyclonal IgG-IgG4 subclass
 complement C3
 Igκ and Igλ light chains
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61.  Microtubular structures ranging from 30 to 50 nm in
width arranged in parallel or stacked arrays
 Mesangial hypercellularity, expansion with
amorphous PAS-positive material, and thickening of
the glomerular capillary walls.
 Resemble mesangial proliferative,
membranoproliferative, focal and diffuse
proliferative, and membranous glomerulonephritis
 Kappa and lambda light chains are detected
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63. Renal
manifestati
on
Site of
deposits
Composition
of deposits
Histological features
Cast
nephropathy
(myeloma
kidney)
PTEC,
intestitium
and distal
tubules
LC+ u modulin PTEC damage, intersitial inflammation and
fibrosis . Distal tubular casts with giant cell
reaction.Found in up to 70% of cases of
dialysis dependent ARF
Amyloidosis All
compartments
LC:K/L=1:3
HC
Deposits stain with congo red giving apple
green birefringence under polarized light. EM:
fibrile 7-12 nm wide and 30-1000 nm long
MIDD-
LCDD
Mesangium ,
peritubular
areas, vascular
and GBM
LC Prominent mesangial nodules and thickening of
periperal basement membrane
IF: LC in mesangial nodules, peritubular
regions, vessels, intersitium and GBM
EM: Fine granular deposits
Light chain
fanconi
syndrome
Lysosomes in
PTEC
K/L=2:1 Tubular atrophy and intersitial fibrosis, crystals
concentrated in PTEC lysosomes
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64. Renal
manifestation
Site of
deposits
Compositio
n of deposits
Histological features
Waldenstrom’s
macroglobuline
mia
glomerulonephri
tis
Glomeruli IgM-k or -l,
LC
Nodular glomerulosclerosis may be seen. Most
patients have interstitial infiltrates.
IF: IgM deposits within capillary lumina
Immunotactoid
glomerulopathy
(including
GOMMID)
Glomeruli IgG-k or -l Membranous nephropathy or
membranoproliferative GN.
Congo red-negative organized glomerular
deposits.
EM: subendothelial-mixed granular and organized
deposits with microtubular (10–60nm diameter)
Organization
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65.  Mw S, De S, Fc G. Renal Diseases Associated With Plasma Cell
Dyscrasias, Amyloidoses, and Waldenström Macroglobulinemia.
Heptinstall:78.
 Other tubulointerstitial diseases. In Kumar V, Abbas A K, Aster J C,
Robbins and cotran patholology basis of disease. 9th ed. Elsevier Inc:
2016. P.936-7.
 Basnayake K, Stringer SJ, Hutchison CA, Cockwell P. The biology of
immunoglobulin free light chains and kidney injury. Kidney Int. 2011
Jun;79(12):1289–301.
 Brebner JA, Stockley RA. Polyclonal free light chains: a biomarker of
inflammatory disease or treatment target? F1000 Med Rep [Internet].
2013 Feb 1 [cited 2019 Mar 3];5. Available from:
http://www.f1000.com/prime/reports/m/5/4/
 Uppin M, Prayaga A, Srinivas B, Rapur R, Desai M, Dakshina Murthy K.
Light chain immunofluorescence in various nephropathies. Indian J
Pathol Microbiol. 2011;54(1):55.
 Renal Biopsy Pathology. :8.
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66. Thank you!!!
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