Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_3099_Библиотеки_им_академика_М_И_Перельмана.pdf
Tissue Examination in a Diagnostic Procedure
87
invasive, realised under the local anaesthesia by a fine-needle aspiration with
basic medical equipment. The possible moderate complications are bruising
and soreness; patients should be questioned about bleeding history, because
coagulopathy may increase the risk of haematoma. The obtained fat tissue
sample can be further used for various types of analysis. Both methods –
Congo red and proteomic analysis – are simultaneously applied in an
increasing number of centres. Fat tissue aspirate is divided into two parts, one
for Congo red-dyeing of tissue smears on glass slides and the second one for
the proteomic MS-based analysis.
Smears are prepared by placing washed fat tissue fragments on one glass
slide, which is then compressed by second perpendicular glass slide while
obtaining two slides with the tissue smear. Congo-red staining follows.
Proteomic analysis of subcutaneous fat aspirates belongs among effective
tools with high sensitivity and specificity also suitable for screening analysis
[11, 31]. Electron and immuno-electron microscopic examination can also be
used in fat tissue aspirates examination as these approaches provide
encouraging results with a high success rate in difficult cases [29].
A risk of misdiagnosis emerges in cases of insulin-based amyloid deposits
arising rarely in the areas of repeated subcutaneous application of insulin in
patients with diabetes mellitus. In case of detection of amyloid in the
subcutaneous fat tissue aspirate the typisation of amyloid is necessary to avoid
confusion with AL amyloid, which may have similar clinical symptoms
(nephropathy, neuropathy) [11].
CONCLUSION
A diagnosis of amyloidosis is a multilevel process. The determination of
the subtype of amyloid deposits is crucial in management of patients and has
been based on tissue examination so far. Therefore, the close cooperation
between clinicians and pathologists is of great importance and it must be
emphasized that making the diagnosis requires an experienced team, ideally in
specialised regional centres. It is important to concentrate on early detection.
Pavla Flodrova, Tomas Pika and Patrik Flodr
88
ACKNOWLEDGMENT
Photographs were obtained with the support of the grant by Ministry of
Health of the Czech Republic, grant nr. 15-31156A.
REFERENCES
[1] Sipe, JD; Cohen, AS. Review: history of the amyloid fibril. J Struct
Biol., 2000 Jun, 130(2-3), 88-98.
[2] Pepys-Vered, ME; Pepys, MB. Targeted treatment for amyloidosis. Isr
Med Assoc J., 2014 May, 16(5), 277-80.
[3] Pepys, MB. Amyloidosis. Annu Rev Med., 2006, 57, 223-41.
[4] Chiti, F; Dobson, CM. Protein misfolding, functional amyloid, and
human disease. Annu Rev Biochem., 2006, 75, 333-66.
[5] Westermark, GT; Johnson, KH; Westermark, P. Staining methods for
identification of amyloid in tissue. Methods Enzymol., 1999, 309, 3-25.
[6] Elghetany, MT; Saleem, A; Barr, K. The congo red stain revisited. Ann
Clin Lab Sci., 1989 May-Jun, 19(3), 190-5.
[7] Puchtler, H; Sweat, F; Levine, M. On the binding of Congo red by
amyloid. J Histochem Cytochem, 1962, 10, 355-364.
[8] Howie, AJ; Brewer, DB; Howell, D; Jones, AP. Physical basis of colors
seen in Congo red-stained amyloid in polarized light. Lab Invest., 2008
Mar, 88(3), 232-42.
[9] Howie, AJ; Brewer, DB. Optical properties of amyloid stained by Congo
red: history and mechanisms. Micron., 2009 Apr, 40(3), 285-301.
[10] Vrana, JA; Gamez, JD; Madden, BJ; Theis, JD; Bergen, HR; 3rd,
Dogan, A. Classification of amyloidosis by laser microdissection and
mass spectrometry-based proteomic analysis in clinical biopsy
specimens. Blood., 2009 Dec 3, 114(24), 4957-9.
[11] Vrana, JA; Theis, JD; Dasari, S; Mereuta, OM; Dispenzieri, A;
Zeldenrust, SR; Gertz, MA; Kurtin, PJ; Grogg, KL; Dogan, A. Clinical
diagnosis and typing of systemic amyloidosis in subcutaneous fat
aspirates by mass spectrometry-based proteomics. Haematologica., 2014
Jul, 99(7), 1239-47.
[12] D’Souza, A; Theis, J; Quint, P; Kyle, R; Gertz, M; Zeldenrust, S; Vrana,
J; Dogan, A; Dispenzieri, A. Exploring the amyloid proteome in
immunoglobulin-derived lymf node amyloidosis using laser
Tissue Examination in a Diagnostic Procedure
89
microdissection/tandem mass spectrometry. Am J Hematol., 2013 Jul,
88(7), 577-80.
[13] Richards, DB; Cookson, LM; Berges, AC; Barton, SV; Lane, T; Ritter,
JM; Fontana, M; Moon, JC; Pinzani, M; Gillmore, JD; Hawkins, PN;
Pepys, MB. Therapeutic Clearance of Amyloid by Antibodies to Serum
Amyloid P Component. N Engl J Med., 2015 Sep17, 373(12), 1106-14.
[14] Kolstoe, SE; Jenvey, MC; Purvis, A; Light, ME; Thompson, D; Hughes,
P; Pepys, MB; Wood, SP. Interaction of serum amyloid P component
with hexanoyl bis(D-proline)(CPHPC). Acta Crystallogr D Biol
Crystallogr., 2014 Aug, 70(Pt 8), 2232-40.
[15] Schönland, SO; Hegenbart, U; Bochtler, T; Mangatter, A; Hansberg, M;
Ho, AD; Lohse, P; Röcken, C. Immunohistochemistry in the
classification of systemic forms of amyloidosis: a systematic
investigation of 117 patients. Blood., 2012 Jan12, 119(2), 488-93.
[16] Chee, CE; Lacy, MQ; Dogan, A; Zeldenrust, SR; Gertz, MA. Pitfalls in
the diagnosis of primary amyloidosis. Clin Lymphoma Myeloma Leuk.,
2010 Jun, 10(3), 177-80.
[17] Kebbel, A; Röcken, C. Immunohistochemical classification of amyloid
in surgical pathology revisited. Am J Surg Pathol., 2006 Jun, 30(6),
673-83.
[18] Leung, N; Nasr, SH; Sethi, S. How I treat amyloidosis: the importance
of accurate diagnosis and amyloid typing. Blood., 2012 Oct 18, 120(16),
3206-13.
[19] Satoskar, AA; Efebera, Y; Hasan, A; Brodsky, S; Nadasdy, G; Dogan,
A; Nadasdy, T. Strong transthyretin immunostaining: potential pitfall in
cardiac amyloid typing. Am J Surg Pathol., 2011 Nov, 35(11), 1685-90.
[20] Brambilla, F; Lavatelli, F; Merlini, G; Mauri, P. Clinical proteomics for
diagnosis and typing of systemic amyloidoses. Proteomics Clin Appl.,
2013 Jan, 7(1-2), 136-43.
[21] Marcus, A; Sadimin, E; Richardson, M; Goodell, L; Fyfe, B.
Fluorescence microscopy is superior to polarized microscopy for
detecting amyloid deposits in Congo red-stained trephine bone marrow
biopsy specimens. Am J Clin Pathol., 2012 Oct, 138(4), 590-3.
[22] Picken, MM; Herrera, GA. Thioflavin T Stain: An Easier and More
Sensitive method for Amyloid Detection. In: Picken MM, Dogan A,
Herrera G, editor. Amyloid and related disorders: Surgical patology and
clinical correlations. New York: Humana Press, Springer
Science+Business Media, 2012, 187-207.
Pavla Flodrova, Tomas Pika and Patrik Flodr
90
[23] Khurana, R; Coleman, C; Ionescu-Zanetti, C; Carter, SA; Krishna, V;
Grover, RK; Roy, R; Singh, S. Mechanism of thioflavin T binding to
amyloid fibrils. J Struct Biol., 2005 Sep, 151(3), 229-38.
[24] Biancalana, M; Koide, S. Molecular mechanism of Thioflavin-T binding
to amyloid fibrils. Biochim Biophys Acta., 2010 Jul, 1804(7), 1405-12.
[25] Fradley, MG; Thakuria, JV; Collins, AB; Moore, SA; Stone, JR. Direct
tissue evaluation via immunofluorescence: in the diagnosis of hereditary
transthyretin cardiac amyloidosis. Tex Heart Inst J., 2012, 39(1), 71-5.
[26] Collins, AB; Smith, RN; Stone, JR. Classification of amyloid deposits in
diagnostic cardiac specimens by immunofluorescence. Cardiovasc
Pathol., 2009 Jul-Aug, 18(4), 205-16.
[27] Sethi, S; Theis, JD; Leung, N; Dispenzieri, A; Nasr, SH; Fidler, ME;
Cornell, LD; Gamez, JD; Vrana, JA; Dogan, A. Mass spectrometry-
based proteomic diagnosis of renal immunoglobulin heavy chain
amyloidosis. Clin J Am Soc Nephrol., 2010 Dec, 5(12), 2180-7.
[28] Fernández de Larrea, C; Verga, L; Morbini, P; Klersy, C; Lavatelli, F;
Foli, A; Obici, L; Milani, P; Capello, GL; Paulli, M; Palladini, G;
Merlini, G. A practical approach to the diagnosis of systemic
amyloidoses. Blood., 2015 Apr 2, 125(14), 2239-44.
[29] Arbustini, E; Verga, L; Concardi, M; Palladini, G; Obici, L; Merlini, G.
Electron and immuno-electron microscopy of abdominal fat identifies
and characterizes amyloid fibrils in suspected cardiac amyloidosis.
Amyloid., 2002 Jun, 9(2), 108-14.
[30] Greenwald, J; Riek, R. Biology of amyloid: structure, function, and
regulation. Structure., 2010 Oct 13, 18(10), 1244-60.
[31] Brambilla, F; Lavatelli, F; Di Silvestre, D; Valentini, V; Rossi, R;
Palladini, G; Obici, L; Verga, L; Mauri, P; Merlini, G. Reliable typing of
systemic amyloidoses through proteomic analysis of subcutaneous
adipose tissue. Blood., 2012 Feb 23, 119(8), 1844-7.
[32] Casadonte, R; Caprioli, RM. Proteomic analysis of formalin-fixed
paraffin-embedded tissue by MALDI imaging mass spectrometry. Nat
Protoc., 2011 Oct13, 6(11), 1695-709.
[33] Casadonte, R; Kriegsmann, M; Deininger, SO; Amann, K; Paape, R;
Belau, E; Suckau, D; Fuchser, J; Beckmann, J; Becker, M; Kriegsmann,
J. Imaging mass spectrometry analysis of renal amyloidosis biopsies
reveals protein co-localization with amyloid deposits. Anal Bioanal
Chem., 2015 Jul, 407(18), 5323-31.
In: Amyloidosis ISBN: 978-1-63485-884-7
Editor: Raquel Watts © 2017 Nova Science Publishers, Inc.
Chapter 5
PROTEIN STUDIES IN
LIGHT-CHAIN AMYLOIDOSIS
Pavel Lochman
Faculty Hospital Olomouc, Department of Clinical Chemistry,
Olomouc, Czech Republic
ABSTRACT
Introduction. AL amyloidosis is a rare disease belonging to the group
of monoclonal gammopathies. The basis of the disease is the deposition
of fibrils formed by molecules of monoclonal immunoglobulin light
chains into the form of amyloid - amorphous proteinaceous material.
Detection and quantification of intact monoclonal immunoglobulin (MIg)
or immunoglobulin light chains is a key aspect in the laboratory diagnosis
and monitoring of patients with AL amyloidosis.
Aim. The content of this contribution is our own experience with the
determination of monoclonal immunoglobulin in patients with AL
amyloidosis together with other biochemical parameters applied to
diagnostics and monitoring of the disease. In addition to the usual
parameters such as urine analysis or the monoclonal immunoglobulin
level assays, the benefits of free light immunoglobulin chains (FLC)
serum level assay and its application to treatment response evaluation are
pointed out, as well as the benefits of heart function indicators such as
troponins and natriuretic peptides and other biochemical parameters for
the stratification of AL amyloidosis patients.
Patients and Methods. The analyzed group included 27 patients with
systemic AL amyloidosis. In all patients we performed detection and
Pavel Lochman
92
quantification of monoclonal immunoglobulins in serum and urine using
electrophoresis and immunofixation techniques, serum immunoglobulin
free light chains levels were determined by immunoturbidimetry
FreeLite
TM
system. In group of 13 patients determination of heavy/light
chain pairs of immunoglobulins (HLC), using immunoturbidimetry
HevyLite
TM
system was performed. In part of patients cardiac enzymes
and natriuretic peptides on Roche Cobas system were determined.
Results. The immunofixation and electrophoresis of samples revealed
the presence of monoclonal components in 17 of 27 (63%) patients and
the median concentration was 3.8 g/l (0.3 to 14.2 g/l). 5 findings of
complete molecules of the IgG isotype (1x IgG-κ, 4x IgG-λ), 4 findings
IgA-λ and in one finding of IgD-λ were found by immunofixation. In 7
patients only λ immunoglobulin light chains in serum were detected.
When analyzing the urine excretion, Bence - Jones protein was detected
in 15 (56%) patients. In all cases the quantity was over 200 mg/24 hours,
while in 3 patients we found the presence of complete molecules of
monoclonal immunoglobulin in urine (1x IgA λ, 2x IgG-λ). Analysis of
serum immunoglobulin free light chains showed abnormal values in all
patients with median of 310 mg/l (53.6-1562 mg/l), pathology of κ/λ ratio
was found in 25 patients (92.5%). HLC analysis was performed in 13
patients, where those with a complete immunoglobulin molecule (IgGλ,
IgAλ) showed pathological levels of HLC isotype and change in HLC
index. In 2 patients with electrophoretic quantitation of λ light chains
only and in 3 patients with electrophoretic negativity suppression of HLC
levels in IgG class was observed. The patient set monitored for cardiac
markers consisted of 22 patients with histologically confirmed AL
amyloidosis, of whom 18 met the criteria for cardiac involvement. Levels
of cardiac biomarkers troponin T (TnT) and N‑terminal pro‑brain
natriuretic peptide (NT‑ProBNP) were determined in all patients. Of the
4 patients without cardiac involvement, 1 has shown positive levels of
TnT and 2 positive levels of NT‑ProBNP. All cardiac involvement
patients exhibited abnormal levels of NT‑ProBNP (median 4,752 ng/ l;
415.7– 35,000) as well as positive levels of TnT (median 0.0815 μg/ l;
0.02– 0.986). The application of the Mayo stratification system to the set
had determined 2 patients at stage I, 5 patients at stage II and 15 patients
at stage III.
Conclusion. Determination of selected biochemical indicators plays
an irreplaceable role in diagnosis and monitoring of disease progress and
requires a close cooperation of hematologists and biochemical stuff.
Advanced laboratory facilities with modern methods are a necessity for
quality care for patients suffering from AL amyloidosis and other
monoclonal gammopathies.
Protein Studies in Light-Chain Amyloidosis
93
INTRODUCTION
AL amyloidosis (light chain amyloidosis) is a systemic or organ-limited
disease belonging to the group of monoclonal gammopathies or plasma cells
dyscrasias [1-3]. The incidence is approximately 5-12 patients per million
inhabitants per year and approximately 10-20% of cases are associated with
multiple myeloma or Waldenström’s macroglobulinemia [4].
The disease is characterized by extracellular deposition of insoluble fibrils
formed by fragments or complete molecules of monoclonal immunoglobulin
light chains produced by clonal plasma cells, which finally leads to tissue
disorganization and subsequent malfunction of the affected organs - mostly
kidneys, heart, liver and peripheral nervous system [5, 6].
Diagnosis of AL amyloidosis must always be confirmed histologically by
visualization of amyloid deposits. The basic method for the detection of
amyloid is a special staining with Congo red, which is now to be extended by
histochemical and immunohistochemical detection of amyloid type.
Immunohistochemical detection of AL amyloid deposition is most often
carried out by indirect immunohistochemistry using antibodies against an
epitopes of the kappa/lambda chains with a standard visualization of an avidin-
biotin technique of formalin-fixed, paraffin-embedded tissue blocks. At
present, many laboratories use advanced visualization techniques and typing of
amyloid masses, including electron microscopy and mass spectrometric
techniques [2, 6]. In addition to the most commonly used bone marrow
histobiopsy, enabling the necessary assessment of the presence of monoclonal
plasmocellular population, it is also used histological sampling of material
from the tongue, salivary glands, rectal mucosa, subcutaneous fat, and targeted
biopsy of the affected organs - mostly kidneys, liver or endomyocardial
biopsy.
An identification of plasmacytic clone and its protein product - a
monoclonal immunoglobulin, represents one of the key aspects in the
laboratory diagnostics and monitoring of patients with AL amyloidosis [2]. In
recent years, serum immunoglobulin free light chain test (FreeLite
TM
, The
Binding Site, UK) is already widely available and significantly extends
analytical possibilities of laboratory diagnostics of monoclonal gammopathies
[7]. The aim of the present chapter is our experience with the determination of
serum free light chains of immunoglobulins (FLC) and other laboratory
parameters utilized in patients with AL amyloidosis.
Heart disease is one of the dominant prognostic factors in patients with
AL amyloidosis. For a detailed evaluation of the presence and extent of
Pavel Lochman
94
cardiac disability a number of non-invasive methods are in use, including
echocardiography and magnetic resonance imaging. Laboratory parameters
represent troponins and natriuretic peptides. In addition to the detection of
heart affection, cardiac biomarkers are used as relatively strong stratification
and prognostic factor.
METHODS FOR AL AMYLOIDOSIS
LABORATORY DIAGNOSTICS
Free Light Chains (FLC) and Monoclonal Immunoglobulins
(MIg)
Detection of presence, identification and quantitation of monoclonal
immunoglobulin or its subunits (i.e., heavy and light chain) is an indispensable
and basic laboratory examination in the diagnostics of patients with AL
amyloidosis [2, 8]. Until FLC determination was set up as a part of laboratory
examination, a basic approach for monitoring of the quantity of MIg was a use
of an agarose or capillary electrophoresis of serum with subsequent
quantitation, immunofixation specifying of type of immunoglobulin and 24-
hour urine collection Bence-Jones protein determination only.
This combination allows detection MIg at approximately 94% of patients
with AL amyloidosis [8, 9, 10, 11]. It a large part of patients, however, the
results did not allow effectively assessing the level of treatment, i.e.,
hematological response, because of low levels MIg in serum (˂10 g/L) or
urine (˂200 mg/day). (The level of MIg does not exceed 10 g/L in any patient
in our cohort too). Monitoring of the disease and evaluation of the therapy
efficiency was therefore very difficult.
Recently, determination of serum free light chains (FLC) with κ/λ ratio
calculating is already commonly available. Κ/λ index provides information
about the amount of clonal production of FLC. The determination of FLC is
positive in 88-92% of patients with AL and in combination with the standard
techniques of MIg detection, positive detection reaches up to 98% of patients
with AL amyloidosis [8-11]. The main advantage of determining the FLC is a
measurable parameter for a large group of patients with oligosecretory disease,
enabling regular monitoring of patients and evaluation of the effect of therapy.
We used periodic determination of serum FLC in 84% of our patients. In
remaining patients regular electrophoretic examination of serum and
Protein Studies in Light-Chain Amyloidosis
95
determination of waste Bence-Jones protein in the urine are used to evaluate
the effect of therapy.
The currently revised IMWG (International Myeloma Working Group)
and ISA (International Society for Amyloidosis) criteria state, that measurable
disease is considered from the absolute value of the difference between
dominant and uninvolved FLC (dFLC) ˃50 mg/L, achieving of partial
remission matches ˃50% reduction of input values, a very good partial
response is defined as the absolute value dFLC < 40 mg/L and conversely
progression of the disease is defined as a growth of FLC ˃ 50% (at least 100
mg/L) (Table 1) [12-15]. Complete remission is defined as a negative
immunofixation and electrophoresis of serum and urine, absence of clonal
plasma cells in the bone marrow and normal κ/λ index. The assessment of
treatment response by determining of FLC is preferred, whilst evaluation by
electrophoresis of serum and/or urine should be used in patients without
pathological levels or very low levels of FLC only. For evaluating of the
therapy effect, FLC better reflect achieved hematological response than the
determination of complete molecules of MIg and it is known that reduction of
the input levels of FLC by ˃90% represents a favorable prognostic factor for
further development of the disease in patients, who cannot reach the complete
hematologic remission (e.g., by reason of expressive organ impairments or
comorbidities) [16]. FLC serum levels correlate with the severity and number
of affected organs, as well as correlate with levels of cardiac markers as
troponins and natriuretic peptides. They are used as a prognostic factor, even
in combination with other indicators, such as cardiac enzymes and proteins
[17, 18].
Table 1. Criteria for hematologic response evaluation in patients with
AL amyloidosis
Complete response
Negative immunofixation of serum and urine,
absence of clonal plasma cells in the bone
marrow and normal κ/λ index
Very good partial remission
Absolute value of the difference between
concentrations of the dominant and alternative
FLC (dFLC) < 40 mg/L
Partial remission
˃50% reduction of input FLC levels
Disease progression
˃50% increase of FLC level (at least 100 mg/L)
Pavel Lochman
96
Heavy/Light Chains Pairs (HLC)
The newest test used for the analysis of MIg is determination of
immunoglobulins by isotype heavy/light chain pairs of intact immunoglobulins
(HLC), using HevyLite
TM
system (The Binding Site, UK).
It is a promising method, which enriches the spectrum of examinations
used in multiple myeloma and monoclonal gammopathies of undetermined
significance [19].
Cardiac Enzymes: Troponin T (TnT), Troponin I (TnI)
Troponins are known to be highly sensitive indicators of myocardial
damage, and they are used not only in the diagnosis of acute coronary
syndrome and other forms of coronary artery disease presently, but also in the
evaluation of the severity of pulmonary embolism, or alternatively, to assess
the severity of ischemic vascular stroke. Myocardial involvement in AL
amyloidosis is the most important prognostic factor. Therefore the
determination of levels of cardiac enzymes in the diagnostic algorithm is
irreplaceable. It is known that absolute levels of troponins got at the time of
diagnosis of disease seem to be very important prognostic indicators.
Dispenzieri et al. published a study of 261 patients with AL amyloidosis
treated with conventional therapy and with established levels of troponin T
(≥0.01 mg/L) and troponin I (≥0.03 mg/L), with a median survival of patients
with detectable levels of TnT and TnI 6 and 8 months, while the survival of
patients with levels below the detection limit was 22 and 21 months [20].
Similar results were published in a number of other authors [21, 22]. In
addition to the prognostic significance, the levels of TnT are currently used
along with levels of NT-proBNP as discriminatory parameters in widely used
staging system published by a team from the Mayo Clinic, which allows not
only to stratify patients into three risk groups, but helps to determine the
appropriate therapy (Stage I: TnT ˂ 0.035 µg/mL and NT-proBNP ˂332 ng/L
(OS 26.4 months), Stage II: TnT ˃ 0.035 µg/mL or NT-proBNP ˃332 ng/L
(OS 10.5 months) stage III: TnT ˃ 0.035 µg/mL and NT-proBNP ˃332 ng/L
(OS 3.5 months) [23, 24]. It is known that patients in stage III with significant
heart disease are candidates for high-dose therapy with the support of
autologous graft and should be considered for a different treatment strategy. At
present, this stratification system has been further modified and enriched by