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M. Ciaccio
nostic application for liquid biopsy. In all those cases where
the traditional biopsy on a primary tumor is not feasible or
informative, the molecular analysis conducted on CTC or
ctDNA represents a valid and noninvasive alternative to characterize the tumor and select patients for targeted therapies.
Moreover, given the extreme dynamism with which the
tumor evolves, it may develop resistance to targeted therapies by acquiring mutations that make the molecular target
insensitive to the drug’s action. In these cases, the possibility
of longitudinal monitoring, i.e., repeating serial controls over
time, represents an exciting opportunity to identify the
appearance of resistance mutations early. Unlike traditional
biopsy, liquid biopsyallows this approach dueto the limited
invasiveness of the diagnostic procedure, which involves a
simple blood sample.
However, liquid biopsy may have the most signicant
impact inthe early cancerdetection and early identication
of itsprogression. For example, detection of minimal residual disease after potentially curative therapy may lead to
second-line therapies. In patients with localized tumors, evidence of CTC or ctDNA could help stratify the risk of recurrence to select patients who may benet from adjuvant
therapy effectively. This approach requires greater analytical
sensitivity than is required by mutational analysis for targeted therapy, and most technology platforms available
today cannot provide it. With the advent of technologies with
increasingly higher analytical performance, it is conceivable
that liquid biopsy could also be applied in screening highrisk subjects. Finally, the knowledge of CTCs and ctDNA
can contribute to understanding the molecular mechanisms
underlying tumor evolution and its molecular heterogeneity,
as well as identifying molecular targets for new drugs.
24h after complete resection of the tumor. The morphology
of CTCs is highly heterogeneous. They may be damaged,
have an apoptotic phenotype, or a morphology similar to
cells in the corresponding tissue biopsy. The proliferative
power of CTCs is extremely variable, even within the same
patient.
Technologies fortheIsolation ofCTCs
The main limitation in isolating CTCs is that they are
extremely rare compared to the total number of blood cells in
circulation.
In most patients with tumor, the concentration of CTCs is
around less than 10 cells/mL, and only in a few selected cases
is it possible to nd several hundred ofCTCs per mL of blood.
In recent years, several technologies have been developed
to isolate CTCs. Among the most important technical
aspects, in addition to the already mentioned need to select
cells at very low concentrations, we must also emphasize the
need to ensure the lowest possible degree of molecular or
morphological alterations for subsequent characterization.
Moreover, keeping the degree of contamination by blood
cells lowis desirable.
CTC isolation strategies can be classied generically
based on cell selection mode. In particular, we distinguish:
– Methods based on the physical properties of CTCs (e.g.,
cell volume)
– Methods based on immunophenotyping (expression of
specic immunological biomarkers)
– Methods based on removing leukocytes without specic
molecular tags.
Circulating Tumor Cells
CTCs represent a biological material of great relevance
because they offer, on the one hand, the possibility to understandthe biological mechanisms underlying the metastatic
spread of the primary tumor and, on the other hand, toevaluate the response to primary treatment and to early recognize
the progression.
CTCs are released into the circulation from both the primary tumor and metastases. How tumor cells are released
into the circulation is not well understood. Active mechanisms, in which cells with more signicant metastatic
potential, such as those that have undergone epithelial–mesenchymal transition, and passive diffusion mechanisms of
single cells or cell clustersmay be involved. In the circulation, CTCs can be found either as isolated cells or in large
clusters of more than 50 cells. Once in circulation, CTCs
have a short half- life. In patients with localized cancer
undergoing surgical therapy, CTCs disappear approximately
Methods Based ontheCTCsPhysical
Properties
Tumor cells are often larger than leukocytes (mean diameter
15μm vs. 10μm). Based on this observation, ltration-based
isolation methods have been developed. The platforms supporting these methods have the advantage of being easy to
use, but with a reduced quality of the isolated cells, since the
passage through a static lter generates hemodynamic stress
that can compromise the integrity of CTCs. This approach is
also limited by the extreme variability of tumor cell volume,
sometimes even smaller than that of leukocytes, and by the
fact that patients undergoing chemotherapy may present
bone marrow megakaryocytes in the circulation. Other isolation methods based on the physical properties of CTCs
exploit their different density, electrical charge, or photoacoustic resonance.
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Immuno-phenotyping Methods
These CTC isolation methods are based on antibodies targeted to antigens expressed explicitly by tumor cells. This
approach is among the most widespread and is exploited by
some instrumental platforms, among which the best known
is CellSearch®. The latter evaluates the contextual expression
of several markers: a membrane marker, the epithelial cell
adhesion molecule (EpCAM), and cytoplasmic markers, epithelial cytokeratins 8, 18, and 19 (CK). Based on this technology, CD45+ leukocytes are negatively selected and
excluded from the analysis, while CTC nuclei are evaluated
by DAPI staining. Briey, after immunomagnetic capture by
EpCAM recognition and enrichment, reagents are added to
identify and count CTCs. Ultimately, the system recognizes
as CTCs those cells whose characteristics correspond to
those of a tumor cell, i.e., cells with EpCAM+, CK+, DAPI+,
and CD45-phenotype.
Methods Based ontheLeukocytesRemoval
Among the new microuidic CTC isolation strategies, the
most promising is the removal of leukocytes from the whole
blood sample, leaving unlabeled CTCs for subsequent
molecular characterization. This technology’s biological
rationale is that leukocyte markers are well characterized
while tumor cells can express different markers, even in the
same patient. In addition, non-epithelial tumors, such as melanoma, do not express EpCAM, while others undergo epithelial–mesenchymal transition, which results in the loss of
EpCAMexpression and otherepithelial markers. In the socalled CTC-iChip, an integrated microuidic device, magnetophoresis rst separatesthe nucleated cells from the other
blood components (platelets, erythrocytes, plasma). In the
second step, the leukocytes are discarded by immunological
marking. With this technology, the yield, i.e., the recovery of
CTCs, is 97%, starting from a sample of 8 mL of whole
blood. Moreover, the methodology is compatible with the
standard cytopathological and molecular analyses foreseen
for CTCs.
Molecular Characterization ofCTCs
Once isolated, CTCs should be counted and characterized.
The classic characterization criterion is positivity for the
cytokeratin marker and negativity for the leukocyte antigen
CD45. Characterization of CTCs is performed by uorescence microscopy, although not all platforms are equipped
with a sufcient number of channels for staining with multiple markers.
Although early studies of CTCs have suggested that their
absolute value is associated with poorer survival, the kinetics
of CTCs assessed post-therapy are of great clinical signicance. Indeed, it has been shown that CTCs decrease rapidly
in patients with a good therapeutic response. Interestingly,
while CTC count in individual patients with advanced tumors
correlates with clinical response to treatment, it does not correlate with tumor mass as assessed both morphologically, by
imaging techniques, and biochemically by circulating tumor
markers.
Molecular characterization of CTCs can also be performed by evaluating the expression of specic RNAs.
Preliminary studies have demonstrated the efcacy of the
analysis of specic translocations (e.g., EML4-ALK in nonsmall- cell lung cancer and TMPRSS2-ERG in prostate cancer) on CTCs.
Finally, the recent introduction of NGS techniques has
allowed an increasing characterization of CTCs, opening
exciting scenarios on tumor biology and the diagnostic
potential of these methods.
Circulating Tumor DNA
The presence of cell-free DNA in circulation has been
widely documented. All living cells release discrete
amounts of free DNA into circulation. Following normal
cell turnover, cells in apoptosis release DNA fragments into
the circulation. Under physiological conditions, apoptotic
and necrotic cells are rapidly removed, and cfDNA levels
are relatively low. More than 90% of healthy individuals
have less than 25ng of cfDNA per mL.Under certain pathological conditions, including inammation, exercise, tissue injury, or surgery, cfDNA levels can increase
signicantly. Generally, cfDNA levels can increase up to
magnitude even in patients with tumors. More specically,
ctDNA refers to the proportion of free DNA released from
cancer cells. Thus, in patients with cancer, a fraction of
cfDNA derives from the tumor and is referred to as
ctDNA. The ctDNA can originate from primary tumors,
metastatic lesions, or CTCs. The fraction of ctDNA relative
to the total circulating free DNA can range from <0.1% to
>10%. The mechanisms of ctDNA release have not yet
been elucidated. Proposed hypotheses include passive
mechanisms, such as cell necrosis, and controlled release
mechanisms, such as apoptosis. The hypothesis that ctDNA
may result from cell necrosis is supported by the observation that more advanced stage tumors, characterized by a
higher degree of necrosis, are associated with higher levels
of ctDNA.The hypothesis that ctDNA is released following
apoptosis is supported by its high degree of fragmentation.
Indeed, ctDNA is typically fragmented into segments of the
same length, 160–180 bp, as the nucleosome-protected
DNA observed in apoptotic cells. CtDNA can also be
released within exosomes, although this mode appears
more appropriate for low-molecular-weight nucleic acids
such as miRNAs (Fig.34.1).
470
Pr
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imary cancer/
metastasis
Release of ctDNA
Secretion
Necrosis
Apoptosis
Release of CTC
CTC
M. Ciaccio
Point mutations
Rearrangements
Insertions/deletions
ctDNA
Fig. 34.1 Primary tumor and/or metastasis release ctDNA into the circulation by secretion, necrosis, and apoptosis. Circulating tumor cells
(CTCs), another source of ctDNA, are also released. The ctDNA has the
The clearance of ctDNA occurs in the spleen, kidney, and
liver. Its half-life is very short, 16min according to some
Authors, about 1h for the rapid phase, and 13h for the late
phase, according to others. In any case, the half-life ismuch
shorter than many of the protein markers.
same alterations as the tumor cell of origin (point mutations, large rearrangements, insertions, deletions) and can be subjected to molecular
analysis. (Copyright EDISES 2021. Reproduced with permission)
able methods is often insufcient to identify ctDNA molecules, which in some cases represent little more than 0.01%
of total cfDNA.
Isolation of ctDNA is far more feasible than that of CTCs;
however, two factors make manipulation difcult:
The ctDNA can provide quantitative information by measuring the number of copies in the circulation, and qualitative
information, by searching for tumor-specic mutations. In
the rst case, given the extreme interindividual variability of
ctDNA levels, it would seem to be the kinetics of ctDNA,
– The extreme interindividual variability in ctDNA
concentration;
– The “dilution” effect of circulating ctDNA because a
large amount of cfDNA from healthy cells is present
and therefore its variations over time, rather than its absolute
value, to provide clinically useful information about the
effectiveness of therapy or the onset of relapse. In the second
case, i.e., ctDNA mutational analysis, diagnostic applications are based on the fact that mutations in ctDNA correspond exactly to mutations in the primary tumor, including
both point mutations and copy number variations and
rearrangements.
The most variable stage is preanalytical, particularly sam-
ple collection, handling, and storage. It is preferable to use
EDTA-plasma rather than serum since coagulation may
result in the breakdown of leukocytes with subsequent
release of wild-type DNA and further dilution of
ctDNA.Commercially available methods typically require
1.5–2mL of plasma. The stability of ctDNA is limited by the
presence of circulating DNA activity, so sample processing
should occur within hours of collection. Subsequent quanti-
Technologies forctDNA Isolation
cation is also not without criticality. In addition, other non-
oncologic diseases may affect cfDNA levels.
Theoretically, since the tumor-specic mutations sought in
ctDNA are not present in normal cells, it could represent an
excellent opportunity to identify the presence of a tumor
ctDNA Mutational Analysis
mass noninvasively. However, ctDNA analysis to date is not
helpful for diagnosis, detection of minimal residual disease,
or prognostication because the sensitivity of currently avail-
The mutational analysis of ctDNA can be carried out basi-
cally by two approaches: a targeted one, i.e., aimed at nding
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471
known mutations that fall in mutational hot spots of specic
genes known to be associated with cancer (e.g., KRAS,
EGFR, and BRAF in lung cancer, colon, and melanoma,
respectively), and a non-targeted one, based on NGS techniques. In the latter case, knowledge of specic tumorassociated mutations is not requiredHowever,any molecular
alteration in the ctDNA is identied within the limits of the
analytical sensitivity of the method. Among the disadvantages of massive sequencing methods are the limited analytical sensitivity, the still high costs, and the difculties in
interpreting the results forclinical purposes.
Initially, the analysis of known point mutations of ctDNA
(targeted approach) was performed by end-point Polymerase
Chain Reaction (PCR) and real-time PCR. More recently,
digital-PCR, ARMS-PCR, or BEAMing technology have
been developed to achieve higher analytical sensitivity. Most
of these methods, however, allow the analysis of only a few
loci; mutations in genes that do not present mutational hot
spots, such as oncosuppressors, are not detected.
Application ofLiquid Biopsy toNon-smallCell Lung Cancer
Lung cancer is characterized by several driver mutations, the
best known of which are EGFR point mutations, ALK-EML4
translocations, and RAS mutations. There are now targeted
drugs, Tyrosine Kinase Inhibitors (TKIs), which act only if
the tumor has some of these mutations (target therapy).
These therapies have doubled patient survival. However,
after treatment with these drugs, it is possible to observe a
tumor progression caused by developing resistance mechanisms that make these therapies ineffective. In some cases,
the molecular mechanisms of resistance have been identied
and include specic mutations in the molecular targets of the
drug itself. This is the case of EGFR in Non-small-Cell Lung
Cancer (NSCLC). It has been shown that the molecular alterations of these tumors are not homogeneously distributed
(tumor heterogeneity) and that metastases may present a
mutational prole completely different from that of the primary tumor, making molecular analysis crucial when tumor
progression is observed. Until now, tissue biopsy is considered the gold standard for molecular investigations but is
limited by some factors, including its invasiveness, the
impossibility of accessing some sites, tumor heterogeneity,
and reduced patient compliance. In contrast, searching for
specic mutations of the EGFR gene on plasma (ctDNA) is
a noninvasive investigation that allows longitudinal monitoring by serial sampling over time. For these reasons, liquid
biopsy is a valuable tool for selecting those patients with
NSCLC whocould benet from treatment with TKIs.
The EGFR gene encodes for a transmembrane protein
with an extracellular ligand-binding domain (EGF, TGFα), a
single transmembrane hydrophobic domain, and an intracellular domain with tyrosine kinase activity. The activation of
the receptor is followed by dimerization, autophosphorylation, and subsequent activation of several signal transduction
pathways that regulate the processes of cell survival and/or
apoptosis, angiogenesis (PIK3-AKT, JAK-STAT), cell differentiation and migration (RAS-RAF-MEK). The mechanism of action of TKI drugs involves their competition with
ATP for binding to the receptor and blocking signal transduction. However, in unselected patients, the response to
TKIs is limited to 10% of cases. These therapies’ success
depends on activating mutations, including exon 19 deletions
(Ex19Del) and L858R, which account for 85% of EGFR
mutations. These mutations are considered oncogenic. They
constitutively activate the EGFR receptor without ligand,
supporting the survival and antiapoptotic signal via PI3KAKT and ERK-MAPK.
These mutations, in addition to being “activating” against
EGFR, make the cell sensitive to TKIs because they increase
the afnity of EGFR for the drug by 5–10 times compared to
the wild-type receptor. This allows the inhibition of EGFR
without toxicity at an epidermal and gastrointestinal level
due to the blockade of EGFR in healthy cells. As already
mentioned, usingTKIs over time leads to the establishment
of resistance in 60% of treated patients. From a molecular
point of view, a possible cause is the occurrence of some
mutations in EGFR, among which the best known is
T790M.This mutation alters the ATP binding site to which
TKIs also bind, thus canceling the sensitization induced by
the other activating mutations. In addition to the mutations
mentioned above, dozens of other mutations in the EGFR
gene have been described whose effect on the activation status of the EGFR receptor and the action of TKIs has not yet
been fully elucidated. These are uncommon and often associated with more frequent mutations, such as L858R or exon
19 deletions (Table34.1).
In 2014, the European Medicine Agency (EMA) approved
the use of plasma to assess EGFR mutational status in patient
candidates for TKIstreatment. Patients with locally advanced
or metastatic NSCLC and positive for L858R activating
mutations or exon 19 deletions on plasma are eligible for
treatment with TKIs. Patients with TKI-treated NSCLC who
Table 34.1 Mutations affecting EGFR and indications for the TKI use
Mutation Exon Indications for the TKIs use
L858R 21 Yes
Ex19Del 19 Ye s
T790M 20 No
G719X 18 Yes?
Ex20Ins 20 No?
S768I 20 No?
L861Q 21 No?
TKI Tyrosine Kinase Inhibitors
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M. Ciaccio
have the T790M mutation on plasma are eligible for treatment with new third-generation TKIs, which have been
shown to be effective in progression after treatment with
rst- and second-line TKIs caused by the occurrence of the
EGFR T790M mutation.
Application ofLiquid Biopsy toOther
Neoplasms
The study of CTCs and ctDNA has broadened our knowledge of tumor biology, opening new perspectives on the
potential diagnostic applications that can be derived from
them. Among the most promising examples of the clinicalassistance implications are breast, ovarian, prostate, colorectal, hepatocarcinoma, and melanoma.
In breast cancer, it remains essential to identify those
patients undergoing surgery who may benet from adjuvant
therapy. In these cases, identifying ctDNA or CTC after surgery could be decisive in planning appropriate follow-up
programs. In particular, PIK3CA mutations in plasma before
and after surgery have been associated with minimal residual
disease. Moreover, the presence of CTC after the start of
treatment correlates signicantly with a lower PFS
(Progression Free Survival) and OS (Overall Survival) and,
therefore, with a worse prognosis.
Recommended Readings
Alix-Panabieres C, Pantel K (2013) Circulating tumor cells: liquid
biopsy of cancer. Clin Chem 59:110–118
Ashworth TR (1869) A case of cancer in which cells similar to those
in the tumors were seen in the blood after death. Aust Med J
14:146–149
Attard G, Swennenhuis JF, Olmos D, Reid AH, Vickers E, A’Hern R
etal (2009) Characterization of ERG, AR and PTEN gene status in
circulating tumor cells from patients with castration-resistant pros-
tate cancer. Cancer Res 69:2912–2918
Bettegowda C, Sausen M, Leary RJ, Kinde I, Want Y, Agrawa N etal
(2014) Detection of circulating tumor DNA in early- and late-stage
human malignancies. Sci Transl Med 6:224ra24
Bidard FC, Peeters DJ, Fehm T et al (2014) Clinical validity of
circulating tumour cells in patients with metastatic breast can-
cer: a pooled analysis of individual patient data. Lancet Oncol
15(4):406–414
Cho EH, Wendel M, Luttgen M, Yoshioka C, Marrinucci D, Lazar
D etal (2012) Characterization of circulating tumor cell aggre-
gates identified in patients with epithelial tumors. Phys Biol
9:016001
Cohen SJ, Alpaugh RK, Gross S, O’Hara SM, Smirnov DA, Terstappen
LW etal (2006) Isolation and characterization of circulating tumor
cells in patients with metastatic colorectal cancer. Clin Colorectal
Cancer 6:125–129
Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC
etal (2004) Circulating tumor cells, disease progression, and sur-
vival in metastatic breast cancer. N Engl J Med 351:781–791
Danila DC, Heller G, Gignac GA, Gonzalez-Espinoza R, Anand A,
Tanaka E etal (2007) Circulating tumor cell number and prognosis
in progressive castration-resistant prostate cancer. Clin Cancer Res
13:7053–7058
Dawson SJ, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin SF etal
(2013) Analysis of circulating tumor DNA to monitor metastatic
breast cancer. N Engl J Med 368:1199–1209
Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M etal
(2008) Circulating mutant DNA to assess tumor dynamics. Nat Med
14:985–990
Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B (2003)
Trans- forming single DNA molecules into uorescent magnetic
particles for detection and enumeration of genetic variations. Proc
Natl Acad Sci U S A 100:8817–8822
Engelman JA, Settleman J (2008) Acquired resistance to tyrosine kinase
inhibitors during cancer therapy. Curr Opin Genet Dev 18:73–79
Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR (2010)
Analysis of the size distributions of fetal and maternal cell-free
DNA by paired-end sequencing. Clin Chem 56:1279–1286
Gascoyne PR, Noshari J, Anderson TJ, Becker FF (2009) Isolation of
rare cells from cell mixtures by dielectrophoresis. Electrophoresis
30:1388–1398
Gertler R, Rosenberg R, Fuehrer K, Dahm M, Nekarda H, Siewert JR
(2003) Detection of circulating tumor cells in blood using an optimized density gradient centrifugation. Recent Results Cancer Res
162:149–155
Karabacak NM, Spuhler PS, Fachin F etal (2014) Microuidic, marker-
free isolation of circulating tumor cells from blood samples. Nat
Protoc 9(3):694–710
Leon SA, Shapiro B, Sklaroff DM, Yaros MJ (1977) Free DNA in
the serum of cancer patients and the effect of therapy. Cancer Res
37:646–650
Liotta LA, Saidel MG, Kleinerman J (1976) The signicance of hema-
togenous tumor cell clumps in the metastatic process. Cancer Res
36:889–894
Maheswaran S, Sequist LV, Nagrath S, Ulkus L, Brannigan B, Collura
CV etal (2008) Detection of mutations in EGFR in circulating lungcancer cells. N Engl J Med 359:366–377
Mandel P, Metais P (1948) Les acides nucleiques du plasma sanguin
chez l'homme. C R Seances Soc Biol Fil 142:241–243
Ozkumur E, Shah AM, Ciciliano JC, Emmink BL, Miyamoto DT,
Brachtel E et al (2013) Inertial focusing for tumor antigendependent and -independent sorting of rare circulating tumor cells.
Sci Transl Med 5:179ra47
Perkins G, Yap TA, Pope L, Cassidy AM, Dukes JP, Riisnaes R etal
(2012) Multi-purpose utility of circulating plasma DNA testing in
patients with advanced cancers. PLoS One 7:e47020
Pilotto S, Rossi A, VavalaÃÄ T etal (2018) Outcomes of rst-generation
EGFR-TKIs against non-small-cell lung cancer Harboring uncommon EGFR mutations: a post hoc analysis of the BE-POSITIVE
study. Clin Lung Cancer 19(1):93–104
Qian H, Gao F, Wang H, Ma F (2014) The efcacy and safety of
crizotinib in the treatment of anaplastic lymphoma kinase-positive
non-small cell lung cancer: a meta-analysis of clinical trials. BMC
Cancer 19(14):683
Reck M, van Zandwijk N, Gridelli C etal (2010) Erlotinib in advanced
non-small cell lung cancer: efcacy and safety ndings of the global
phase IV Tarceva lung cancer survival treatment study. J Thorac
Oncol 5(10):1616–1622
Siravegna G, Bardelli A (2014) Genotyping cell-free tumor DNA in the
blood to detect residual disease and drug resistance. Genome Biol
15(8):449
Stroun M, Anker P, Lyautey J, Lederrey C, Maurice PA (1987) Isolation
and characterization of DNA from the plasma of cancer patients.
Eur J Cancer Clin Oncol 23:707–712
34 Liquid Biopsy
https://t.me/medicina_free
473
Stroun M, Lyautey J, Lederrey C, Olson-Sand A, Anker P (2001) About
the possible origin and mechanism of circulating DNA apoptosis
and active DNA release. Clin Chim Acta 313:139–142
Stott SL, Lee RJ, Nagrath S, Yu M, Miyamoto DT, Ulkus L etal (2010)
Isolation and characterization of circulating tumor cells from
localized and metastatic prostate cancer patients. Sci Transl Med
2:25ra23
Tu HY, Ke EE, Yang JJ etal (2017 Dec) A comprehensive review of
uncommon EGFR mutations in patients with non-small cell lung
cancer. Lung Cancer 114:96–102
Vona G, Sabile A, Louha M, Sitruk V, Romana S, Schutze K etal (2000)
Isolation by size of epithelial tumor cells: a new method for the
immunomorphological and molecular characterization of circulat-
ing tumor cells. Am J Pathol 156:57–63
Yamada T, Nakamori S, Ohzato H, Oshima S, Aoki T, Higaki N etal
(1998) Detection of K-ras gene mutations in plasma DNA of
patients with pancreatic adeno- carcinoma: correlation with clinicopathological features. Clin Cancer Res 4:1527–1529
Yu M, Stott S, Toner M, Maheswaran S, Haber DA (2011) Circulating
tumor cells: approaches to isolation and characterization. J Cell Biol
192:373–382
Wang JY, Hsieh JS, Chang MY, Huang TJ, Chen FM, Cheng TL etal
(2004) Molecular detection of APC, K-ras, and p53 mutations in the
serum of colorectal cancer patients as circulating biomarkers. World
J Surg 28:721–726
Weight RM, Dale PS, Viator JA (2009) Detection of circulating mela-
noma cells in human blood using photoacoustic owmetry. Conf
Proc IEEE Eng Med Biol Soc 2009:106–109
Biological Fluids
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GaetatoBernardi, CosimoOttomano, andSabrinaBuoro
35
Introduction
Unlike blood, biological uids are not always repeatable
materials that provide clinical information otherwise unavailable to the caregiver. Despite their importance, their diagnostic power is not always known, nor are the pre-analytical
precautions that should be part of the cultural baggage of
anyone requesting a laboratory test. Biological uids are
divided into serous (peritoneal, pleural, pericardial), synovial, and cephalorachidian. For each of these, the biochemistry and cellular analysis tests of major interest in nonspecialist
medicine are briey described.
This chapter only touches on topics such as analytics,
which are fundamental for specialists in laboratory medicine. However, it is worth mentioning here a fundamental
concept in clinical biochemistry: the matrix. This is different between blood and biological fluids; for this reason, the determination of a measurand in the former
creates different problems in the latter, forcing the specialist to verify the reliability of analytical methods in
each area.
Reference values (RV) are critical to classify the individual health status. Unfortunately, the literature is not completely univocal in this regard, so it is highly advisable to
always refer to the RV of the laboratory.
Pathophysiology
The serous (pericardial, peritoneal, and pleural) and articular
cavities physiologically contain minimal quantities of uid,
similar to plasma in color and chemical-physical characteristics, whose function is essentially lubricating, favoring the
endocavitary sliding of the viscera, covered by a serous
membrane like the cavities that contain them.
The serous liquids are continuously formed by ultraltration of the plasma and, in part, are reabsorbed through the
capillaries of the serosae, so that the quantity and composition remain constant. Liquids result from a dynamic process
of formation and reabsorption in a game of pushes and
counter- pushes of hydrostatic and colloid-osmotic pressure.
In addition to this mechanism, there is also capillary permeability and lymphatic drainage.
Excess production or reduced reabsorption results in disease. These phenomena are pathognomonic of each cavity
(pleural, pericardial, peritoneal, and articular).
In pathological conditions, for inammatory, neoplastic,
or traumatic lesions of the endocavitary organs, sometimes
for an inammatory or neoplastic pathology of the serosae
themselves, the amount of intracavitary uid increases,
resulting in an effusion whose chemical-physical and cytological characteristics are modied according to the pathogenic noxa, with common etiopathogenic features.
It is good practice to obtain the concentrations of the measurands useful for diagnosis in the affected uid and on the
serum/plasma at the same time.
G. Bernardi (*)
IRCSS Fondazione Carlo Besta Neurological Institute, Milan, Italy
C. Ottomano
Research Laboratory, Istituto di Ricerca e Cura a Carattere
Scientico (IRCCS) Synlab SDN Spa, Naples, Italy
S. Buoro
Regional Reference Center for the Quality of Laboratory Medicine
Services, Milan, Italy
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Ciaccio (ed.), Clinical and Laboratory Medicine Textbook, https://doi.org/10.1007/978-3-031-24958-7_35
475
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G. Bernardi et al.
Types ofAnalysis
The main laboratory tests useful for assessing any alterations
in the endocavitary organs are macroscopic analysis and
cytometric and biochemical examination.
Macroscopic Analysis
The color and appearance of uids in pathologic effusions
may vary based on their different etiologies. The appearance
is usually clear and straw yellow in transudates.
Other colorations of pathological effusions include red,
brown, green, white, and black. The presence of blood gives
the uid a red coloration, erythrocyte lysis, and oxidation of
hemoglobin to methemoglobin result in a brownish (brown)
coloration. If the uid has a frankly hematic appearance, the
determination of the hematocrit, or rather the count of the
hematocrits, can help to distinguish between massive bleeding and serous effusion. A greenish coloration indicates the
presence of bile. Fluids with a whitish coloration indicate
pus or chyle; in subjects with melanoma, a blackish coloration may be observed. The ascitic uids of jaundiced
patients assume a particularly pronounced yellow
coloration.
The appearance, in addition to being clear, maybe turbid or opalescent. The turbid appearance of the uids is
caused by increased cellularity or triglyceride concentration, while the chylous effusion may have an opalescent
appearance.
Differential cell counting is performed by light microscopy on cytocentrifugate, after panoptic staining (MayGrünwald Giemsa MGG or Wright). The cytocentrifugation
allows the concentration of the cells, dispersed in the liquid,
in a small circular area of the slide, where they appear in
monolayer.
Each individual cell population is expressed as an absolute value and/or as a percentage, depending on the type of
liquid examined and the decision thresholds.
Cytometric analysis can also be performed by automatic
analyzers, reducing costs and reporting times. The dispersion of the replicates of the measurements (imprecision) also
improves.
The cellular analysis of biological uids represents one of
the main diagnostic criteria in the evaluation of the effusion
etiopathogenesis.
The cellular component of the uids is generally made up
of leukocytes (neutrophil granulocytes, lymphocytes, monocytes, more rarely eosinophilic and basophilic granulocytes),
macrophages/histiocytes, mesothelial cells in cavity effusions, and synoviocytes in joint effusions (Figs.35.1, 35.2,
35.3, and 35.4).
Mesothelial cells are large cells with basophilic cytoplasm on MGG staining (the intensity of basophilia varies
according to the state of the cell), regular, slightly eccentric
nucleus; sometimes it is possible to see the nucleolus.
However, the morphology of mesothelial cells in effusions
is varied and is closely inuenced by the time of onset of
Cytometric Examination
The cytometric examination consists in counting the total
nucleated elements in the serous uids and their differentiation according to morphological characteristics.
For cytometric examination of liquids, with the exception
of CSF, the sample must be collected in tubes with anticoagulant (EDTA), mixed both after collection and before analysis. The liquid should be stored at room temperature, and
the maximum time that can elapse between collection and
analysis is about 2hours.
In synovial uid, the presence of hyaluronic acid, at a
strongly acidic pH, causes the formation of a mucin clot that
invalidates cell counts. The pre-treatment of the sample with
hyaluronidase and the execution of the analysis at slightly
acidic pH, or preferably neutral, improve the analysis.
The gold standard procedure for cell counting in biological uids is light microscopy using a counting chamber
(Burker or Thoma chambers for uids of suspected high cellularity, or Fuchs-Rosenthal and Nageotte for low cellularity
samples).
Fig. 35.1 Pleural effusion of an exudative nature in cytospin preparation with May-Grünwald Giemsa stain (400×), where it is possible to
recognize neutrophilic granulocytes, lymphocytes, and monocytes. In
addition, well-preserved mesothelial cells are present (large cells with
abundant cytoplasm, strongly basophilic, and, in some cases, with small
pericytoplasmic vacuoles; the nucleus is regular, slightly eccentric, and
sometimes characterized by the presence of a nucleolus), malignant
mesothelial cells conserved with numerous vacuoles, andnally macrophages (large cells, cytoplasm with many vacuoles, eccentric kidney
nucleus and strongly thickened chromatin). Sometimes the morphological distinction among mesothelial cells, macrophages, and monocytes can be difcult and is inuenced by the duration of the effusion
and/or the time that has elapsed from the time of sampling to the execution of the cytometric analysis
35 Biological Fluids
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Fig. 35.2 Peritoneal effusion of a subject with cirrhosis and spontaneous bacterial peritonitis. Cytospin preparation with May-Grünwald
Giemsa staining (1000×), where it is possible to recognize neutrophilic
granulocytes, a lymphocyte in the upper right corner, while in the leftmost neutrophil granulocyte, in the central group of the image, the
phagocytosis of some bacteria
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Fig. 35.3 Pericardial uid from a healthy subject undergoing surgery.
Cytospin preparation with May-Grünwald Giemsa staining (400×),
where it is possible to recognize rare neutrophil granulocytes, lymphocytes, and monocytes. The predominant cell population is represented
by mesothelial cells
Fig. 35.5 Pleural effusion of a subject with multiple myeloma evolved
into plasma cell leukemia. There are lymphoplasmacytic and plasma
cells in the pleural effusion, as in the peripheral blood. The cells present
in the pleural effusion are CD138, CD38 positive, and CD56 negative.
Cytospin preparation with May-Grünwald Giemsa staining (1000×)
the effusion. It is possible to nd mesothelial cells in mitosis or binucleated if the hyperplasia is benign. This heterogeneity sometimes makes it difcult to correctly identify
mesothelial cells morphologically with respect to macrophages and neoplastic cells, so much so that cytochemical,
immunocytochemical, or immunouorescence techniques
must be used. Finally, in the case of neoplastic effusions,
malignant cells of various kinds are also present (Figs.35.5
and 35.6).
Fig. 35.4 Joint effusion of a subject with inammation, characterized
by a high number of well-preserved neutrophils and by some rare
monocytes, synoviocytes with morphology similar to mesothelial cells.
Cytospin preparation with May-Grünwald Giemsa staining (400×)
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G. Bernardi et al.
Fig. 35.6 Peritoneal effusion of undetermined diagnosis; the preparation contains neutrophil granulocytes, monocytes, lymphocytes, mesothelial cells, and atypical cells of probable neoplastic nature that make
up the group of central cells. Subsequent cytological examination conrms the presence of undifferentiated ovarian cancer metastatic cells.
Cytospin preparation with May-Grünwald Giemsa stain (400×)
Pleural Fluid
The pleural cavity is an almost virtual space between the
mesothelium of the parietal and visceral pleura, containing a
modest amount of uid sufcient to facilitate movement
between the two membranes.
Its increase, called pleural effusion, is due either to overproduction, mainly in congestive heart failure, or to reduced
reabsorption, mostly from lymphatic obstruction during neoplasms. In summary, the most common conditions causing
pleural effusion are heart failure, tumors, tuberculosis, and
pulmonary embolism.
In the case of suspected pleural effusion, the patient’s
clinical picture can be correctly framed by radiological and/
or ultrasound examinations to conrm its presence. In this
case, thoracentesis is performed for both diagnostic and therapeutic purposes.
The distinction between exudate (increased capillary permeability or decreased lymphatic reabsorption), transudate
(increased hydrostatic pressure or decreased oncotic pressure), and chylous effusion (caused by trauma or neoplasm,
usually lymphoma involving the thoracic duct) is a crucial
diagnostic step.
Transudates are generally bilateral because they have a
systemic etiology.
Exudates are frequently unilateral, associated with disorders that increase capillary permeability or interfere with
ipsilateral lymphatic reabsorption.
Effusions from malignant neoplasms behave ambiguously: unlike what we might expect, they are unlikely to be
exudative due to the concomitant presence of congestive
heart failure.
Table 35.1
related interpretative criteria
Light’s
criteria
Additional
criteria
Biochemical analysis performed on the pleural uid and
Parameter Diagnostic cutoff for exudate
Pleural uid/serum
protein ratio
Pleural uid/serum
LDH ratio
Pleural uid LDH >2/3 upper limit of the
Pleural liquid
cholesterol
Pleural liquid
cholesterol/serum
ratio
Pleural uid/serum
bilirubin ratio
≥0.50
≥0.60
reference range of serum
LDH
>45mg/dL
>0.30
≥0.60
The main causes of a transudate are congestive heart failure, liver cirrhosis, hypoproteinemia (e.g., nephrotic syndrome), infections (e.g., bacterial pneumonia, tuberculosis,
or other granulomatous diseases, such as sarcoidosis and histoplasmosis; viral or mycoplasma pneumonias), neoplasms
(bronchogenic carcinoma, metastatic carcinomas, lymphomas, mesotheliomas), non-infectious inammatory diseases
(rheumatoid arthritis, systemic lupus erythematosus [SLE]),
accumulation of uid of extrapleural origin (e.g., from pancreatitis with increased lipid-lowering lipid), and exudate
(from pancreatitis with increased pancreatic lipase and amylase, from esophageal rupture with increased pancreatic
enzymes and acid pH, and nally, urinothorax).
The rst step in the biochemical evaluation of the effusion
is to determine its origin, either transudative or exudative.
For this purpose, Light’s criteria have been widely used in
clinical practice over the past four decades (Table35.1).
In their initial work, Light etal. reported a sensitivity of
99% and a specicity of 98% in identifying the type of effusion, although in subsequent studies the specicity was lower
(65–86%). Despite this limitation, Light’s criteria are considered superior to clinical judgment alone in characterizing
effusions.
Macroscopic Evaluation andCellular Analysis
In pleural effusions of transudative origin, the uid is generally clear, light yellow, whereas in those of probable exudative origin (neoplastic or infected), the uid is turbid
(Table 35.2). The appearance of the uid may be hemorrhagic, due to a sample collection not performed well or
bleeding. In the latter case, the color of the uid may vary
from red to brown depending on the time elapsed between
the start of the hemorrhage and the collection.
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