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Plasma Cell Neoplasms
Michael A. Linden • Robert W. McKenna
Editors

Plasma Cell Neoplasms

A Morphologic, Cytogenetic
and Immunophenotypic Approach

1  3
Editors
Michael A. Linden Robert W. McKenna
Department of Laboratory Medicine Department of Laboratory Medicine
and Pathology and Pathology
University of Minnesota University of Minnesota
Minneapolis Minneapolis
MN MN
USA USA

ISBN 978-3-319-10917-6    ISBN 978-3-319-10918-3 (eBook)

DOI 10.1007/978-3-319-10918-3

Library of Congress Control Number: 2014955544

Springer Cham Heidelberg New York Dordrecht London

© Springer International Publishing Switzerland 2016
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the
editors give a warranty, express or implied, with respect to the material contained herein or for any errors
or omissions that may have been made.

Printed on acid-free paper

Springer International Publishing AG Switzerland is part of Springer Science+Business Media

(www.springer.com).
Preface

Plasma cell neoplasms, including plasma cell myeloma, did not start to appear in
the medical literature until the 1840s [1]. In 1847, Dr. Henry Bence Jones described
the features of a urine precipitate in a patient that likely had plasma cell myeloma
[1]. Nearly 100 years later the field was revolutionized by the invention of immu-
noelectrophoresis (1953) and immunofixation (1964) [1]. These tools improved the
way in which plasma cell neoplasms are diagnosed and monitored. In the last 20
years, there have been remarkable changes in the treatment approach to myeloma
patients, including bone marrow transplantation and innovative chemotherapy (im-
munomodulatory drugs and proteasome inhibitors), that have increased the median
survival of standard risk patients to greater than 10 years [2].
This book is primarily intending for a pathology audience, including trainees
and practicing pathologists. While these neoplasms may comprise a minority cases
in our practices, improved patient outcomes means that we are continuously see-
ing a greater proportion of bone marrow biopsies from patients with a diagnosis of
plasma cell neoplasm. Moreover, as new ancillary diagnostic testing is continuously
introduced to our practice, it is important for us to be familiar with the right tools
to make an accurate diagnosis and to guide our clinician allies on test utilization.
There are multiple pieces of data necessary to render a diagnosis of plasma cell
neoplasm and to provide important prognostic and predictive information. These
data include clinical findings, laboratory data, morphologic features, immunophe-
notype, and cytogenetics. The first two chapters of this book approach how we
detect and enumerate paraproteins, by electrophoretic and/or immunoturbidimetric/
nephelometric methods. Next, we review how a careful bone marrow examination
is a key component of the diagnosis, often with the aid of immunohistochemical
stains. Building on the first three chapters, we next look at the current 2008 WHO
classification of plasma cell neoplasms, including monoclonal gammopathy of un-
determined significance, solitary plasmacytoma, primary amyloidosis, and plasma
cell myeloma [3]. Chapters 5 and 6 provide an overview of cytogenetic and flow
cytometric features of plasma cell neoplasms, and their role in diagnosis and prog-
nosis. The final two chapters may be the most important—while this book is pri-
marily intended for pathology trainees and practicing pathologists, it’s important to
recognize that patients and clinicians depend on our timely, high quality diagnoses

v
vi Preface

to guide care. Chapter 7 will give a perspective on how a clinician would approach
the treatment of a patient with a plasma cell neoplasm, and Chapter 8 will provide
guidelines for how pathologists can most effectively summarize and communicate
their findings in a diagnostic report.
1. Kyle RA. Multiple myeloma: an odyssey of discovery. Br J Haematol.
2000;111(4):1035–44.
2. Rajkumar SV, Gahrton G, Bergsagel PL. Approach to the treatment of multiple
myeloma: a clash of philosophies. Blood. 2011;118(12):3205–11.
3. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO
classification of tumours of hematopoietic and lymphoid tissues. 4th edn. Lyon:
International Agency for Research on Cancer; 2008.

Michael A. Linden, MD, PhD

Robert W. McKenna, MD
Contents

Clinical Protein and Immunofixation Electrophoresis���������������������������������    1

Jonathan R. Genzen

Serum Free Light Chain Analysis������������������������������������������������������������������ 25

Rajeevan Selvaratnam, Jing Cao and Amy B. Karger

Plasma Cell Neoplasms: Morphology and Immunohistochemistry������������ 43

Garth Aasen and Robert W. McKenna

Classification of Plasma Cell Neoplasms������������������������������������������������������� 65

Sophia L. Yohe

 onventional and Molecular Cytogenetics in Plasma Cell Neoplasms�������

C 79
Michelle Dolan

 ole of Flow Cytometry in Plasma Cell Neoplasms������������������������������������� 101

R
Beenu Thakral, Kristy Wolniak and Michael A. Linden

Plasma Cell Neoplasms, A Therapeutic Approach ��������������������������������������� 123

Brian L. McClune and Sagar S. Patel

 tandardized Synoptic Reports for Plasma Cell Neoplasms:

S
Integration of Laboratory and Clinical Data������������������������������������������������ 143
Elizabeth L. Courville, Zohar Sachs and Michael A. Linden

Index������������������������������������������������������������������������������������������������������������������ 151

vii
Contributors

Garth Aasen Department of Pathology, Borgess Medical Center, Kalamazoo,

MI, USA
Jing Cao Department of Laboratory Medicine and Pathology, University of
Minnesota, Minneapolis, MN, USA
Elizabeth L. Courville Division of Hematopathology, Department of
Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN,
USA
Michelle Dolan Department of Laboratory Medicine and Pathology, University
of Minnesota Medical School, Minneapolis, MN, USA
Jonathan R. Genzen Department of Pathology, University of Utah School of
Medicine/ARUP Laboratories, Salt Lake City, UT, USA
Amy B. Karger Department of Laboratory Medicine and Pathology, University
of Minnesota, Minneapolis, MN, USA
Michael A. Linden Division of Hematopathology, Department of Laboratory
Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA
Brian L. McClune Department of Medicine, Division of Hematology,
Oncology, and Transplantation, University of Minnesota Medical Center,
Minneapolis, MN, USA
Robert W. McKenna Department of Laboratory Medicine and Pathology,
University of Minnesota, Minneapolis, MN, USA
Sagar S. Patel Department of Medicine, University of Minnesota Medical
Center, Medicine Education Office, Minneapolis, MN, USA
Zohar Sachs Division of Hematology, Oncology, and Transplantation,
Department of Medicine, Medical School, University of Minnesota, Minneapolis,
MN, USA
Rajeevan Selvaratnam Department of Laboratory Medicine and Pathology,
University of Minnesota, Minneapolis, MN, USA
ix
x Contributors

Beenu Thakral Department of Laboratory Medicine and Pathology, University

of Minnesota, Minneapolis, MN, USA
Kristy Wolniak Department of Pathology, Northwestern University Feinberg
School of Medicine, Chicago, IL, USA
Sophia L. Yohe Division of Hematopathology, Department of Laboratory
Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA
Clinical Protein and Immunofixation
Electrophoresis

Jonathan R. Genzen

Introduction

Electrophoresis is a laboratory method fundamental to the diagnosis and manage-

ment of plasma cell disorders. Serum protein electrophoresis (SPEP) is commonly
used to detect the presence of circulating monoclonal proteins. Urine protein elec-
trophoresis (UPEP) is used to detect the presence of monoclonal proteins (usually
free immunoglobulin light chains) in the urine. In conjunction with total protein
measurement and scanning densitometry, both methods can be used to quantify
the amount of abnormal protein present. Immunofixation electrophoresis (IFE), a
related technique, is used to confirm that the restricted bands observed by protein
electrophoresis are monoclonal, as well as to characterize the types of monoclonal
antibody present. Information gathered from SPEP, UPEP, and IFE is used in the
assessment of disease categorization, severity, and prognosis.
This chapter reviews the history and principles of electrophoretic techniques, fo-
cusing on clinical applications in the diagnosis and management of plasma cell dis-
orders. Both gel-based and capillary-based electrophoretic methods are reviewed.
Issues surrounding pattern interpretation, protein quantification, and correlation
with other commonly ordered laboratory tests are also addressed. Frequently en-
countered technical, regulatory, and diagnostic problems are highlighted.

Immunoglobulins

Protein electrophoresis can provide valuable information regarding serum and urine
proteins in both health and disease. One particularly important use in clinical prac-
tice, however, is in the detection and monitoring of monoclonal immunoglobulins.

J. R. Genzen ()
Department of Pathology, University of Utah School of Medicine/ARUP Laboratories,
500 Chipeta Way, Mail Code 115, Salt Lake City, UT 84108, USA
e-mail: jonathan.genzen@path.utah.edu
© Springer International Publishing Switzerland 2016 1
M. A. Linden, R. W. McKenna (eds.), Plasma Cell Neoplasms,
DOI 10.1007/978-3-319-10918-3_1
2 J. R. Genzen

In the context of protein electrophoresis, these monoclonal immunoglobulins are

also referred to as monoclonal antibodies, monoclonal proteins, M-proteins, and/
or M-spikes.
Immunoglobulins are synthesized by plasma cells, which are differentiated B-
lymphocytes. There are five distinct classes of immunoglobulins (antibodies) in
humans: IgG, IgM, IgA, IgD, and IgE. Individual antibodies are composed of two
heavy chain and two light chain subunits. The heavy chains are designated by Greek
letters gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε). The Greek letter of
the heavy chain also corresponds to the Latin capital letter used in the antibody
class name—γ in IgG, μ in IgM, α in IgA, δ in IgD, and ε in IgE. Light chains are
designated by the Greek letters kappa (κ) and lambda (λ). While there are multiple
types of heavy chains and light chains, a single antibody will have only one type
of heavy chain and one type of light chain. For example, IgG-kappa contains two γ
heavy chains and two κ light chains. IgM-lambda contains two μ heavy chains and
two λ light chains.
Both heavy chains and light chains have variable regions and constant regions.
Variable regions are critical for antigen binding. Constant regions are largely re-
sponsible for the “effector functions” after antigen binding, such as complement
fixation or binding of the antibody to immunoglobulin receptors. IgG, IgA, and IgE
circulate primarily as monomers, while IgA can be secreted across mucosal surfaces
as a dimer. IgD exists as a monomer, primarily serving as a B-cell antigen receptor
(also a function of IgM), while circulating IgM exists in pentameric form. A simpli-
fied diagram illustrating antibody structure is presented in Fig. 1.

Fig. 1 Prototypic structure of

an immunoglobulin molecule.
a An intact immunoglobulin
consists of two heavy chains
( blue) and two light chains
( red). Variable regions (V;
lighter shading) are involved
in antigen binding. There
is one variable region on
each heavy chain ( VH) and
light chain ( VL). Each heavy
chain contains three constant
regions ( CH), whereas each
light chain contains one con-
stant region ( CL). b Depiction
of antibodies in monomeric
(e.g., IgG, IgD, IgE), dimeric
(e.g., IgA), and pentameric
(e.g., IgM) forms
Clinical Protein and Immunofixation Electrophoresis 3

Antibody diversity, including antigen specificity and affinity, is the result of a re-
markable process of somatic gene recombination, hypermutation, and class switch-
ing [1]. The result of this diversity is vast combination of circulating antibodies
directed against a wide range of targets. There is typically not a single predominant
antibody clone in normal individuals. During antigen exposure, infection, and/or
inflammation, many new antibody clones may arise. This is a natural part of im-
mune defense in primary (initial) and secondary (subsequent) immune response.
While this may result in faint or transient oligoclonal patterns observed on SPEP,
it typically does not lead to predominant monoclonal peaks. In clonal plasma cell
and lymphoproliferative disorders, a proliferation of antibody-producing cells can
lead to the appearance of a predominant monoclonal antibody in serum. If present
in large enough concentration, these antibodies may be detected and quantified as
an M-spike on SPEP or UPEP and clonally characterized by IFE.
It is worth emphasizing that such information is critically important in the
workup of plasma cell and lymphoproliferative neoplasms. For example, the pres-
ence and quantification of monoclonal antibodies are essential in the application of
WHO diagnostic criteria for disorders such as monoclonal gammopathy of undeter-
mined significance (MGUS), multiple myeloma, and Waldenström’s macroglobu-
linemia [2]. They are also valuable in assessing the risk of disease progression from
MGUS and smoldering asymptomatic myeloma [3]. Clonal characterization (such
as IgA type) is even a factor in myeloma risk stratification [4]. While subsequent
chapters in this book incorporate laboratory information in discussing the diagnosis
and management of plasma cell disorders, the focus of the remainder of this chapter
is to provide a practical introduction to the principles of electrophoretic testing in
support of this endeavor.

History of Electrophoresis

Modern clinical electrophoresis is the result of many decades of research on sepa-

rating chemical and biological substances in solution. An early pioneer in this field
was Arne Tiselius, who established the moving boundary electrophoresis method of
protein separation in solution using a U-tube apparatus [5]. Tiselius’ moving bound-
ary method resulted in a clear visualization of albumin and multiple globin fractions
in serum. Protein boundaries were observed in this system by Schlieren imaging,
a technique that detects changes in refractive index observed with concentration
gradients [6]. A more common example of Schlieren imaging that the reader may
have encountered is in classic photographs of air shock waves in the aeronautics and
ballistics industries. In recognition of his work on electrophoresis, adsorption, and
serum protein separation, Tiselius received the 1948 Nobel Prize in Chemistry [7].
Subsequent advances in electrophoretic techniques included the introduction of
support media. This permitted the development of zone electrophoresis, in which
ions (such as proteins) can be discretely and permanently separated into distinct
zones [8]. In moving boundary electrophoresis, however, such fractionation only
4 J. R. Genzen

occurs at the ends of the U-tube and in solution [8]. Early forms of zone electropho-
resis used a variety of support media, including filter paper, starch beds, and starch
gels [8, 9]. Of particular note, Oliver Smithies discovered that starch gel electro-
phoresis allowed the visualization of many additional protein fractions in serum [9].
Agar gels were also found to facilitate the separation of large proteins, although
advantages of using agarose instead of agar in electrophoretic gels was described
by Dr. Stellan Hjertén [10]. Briefly, agarose has a relatively neutral charge when
compared to agaropectin, the other major component of agar. Use of agarose was
therefore found to decrease the electro-endosmotic force (EOF) in the gel when a
voltage is applied (see section “Clinical Electrophoresis”, below). Additional gel
media commonly used in protein separation include polyacrylamide and cellulose
acetate [8, 11]. As most clinical SPEP systems for the detection of monoclonal pro-
teins use agarose gels, these other materials will not be discussed further.
Finally, capillary zone electrophoresis (more commonly referred to as capillary
electrophoresis) is a method in which protein separation takes place in a narrow
diameter glass tube [12]. Capillary electrophoresis gained prominence due to tech-
nological advances in 1970s and 1980s. It is now also a method commonly used
(along with agarose gels) in clinical SPEP.

Clinical Electrophoresis

Principles

Electrophoresis is defined as the movement of charged particles in an electric field.

As proteins contain both acidic and basic amino acid groups, they can have either
a net positive or net negative charge, depending on the combination of constitu-
ent amino acids and the pH of the solution in which they are dissolved. The pH
at which a protein has no net charge is also known as its isoelectric point (pI).
Charged proteins will therefore move (migrate) when subjected to the electromotive
force (EMF) in an applied voltage. Negatively charged proteins move toward the
positively charged electrode, or anode. Positively charged proteins move toward the
negatively charged electrode, or cathode.
Agarose gels contain pores through which proteins migrate when the electric
field is applied. As the pores are large enough in clinical electrophoresis gels to not
impede the migration of most non-denatured proteins, charge (more specifically
charge-to-size ratio) is a much more important factor for protein mobility than is
q
size. An ion’s electrophoretic mobility is defined by the equation , where r is
6πηr
its radius, η is the viscosity of the medium (buffer), and q is its net charge [13, 14].
In practice, at the pH of clinical electrophoresis buffer solutions, most proteins will
have net negative charges and will therefore migrate toward the anode, separated by
their relative electrophoretic mobilities.
Clinical Protein and Immunofixation Electrophoresis 5

EMF, however, is not the only factor that causes proteins to move in an elec-
tric field. Another important force is electro-endosmosis, also known as endosmotic
force (EOF). As the supporting gel matrix typically has a fixed negative charge,
positive ions in the buffer solution will be attracted to, and essentially coat, the gel
surface and interior. When a voltage is applied to the gel, these positive ions in the
buffer (cations) will migrate toward the cathode. This movement of cations, how-
ever, also “pulls” surrounding water molecules in the same direction, thus resulting
in a flow of water toward the cathode—a phenomenon also known as endosmosis.
EOF in clinical gel electrophoresis works to (a) slow the movement of negative
proteins toward the anode, (b) increase the movement of positively charged proteins
toward the cathode, and (c) serve as the driving force for the movement of neutral
proteins toward the cathode. As immunoglobulins are generally neutral or slightly
positively charged at the pH of clinical electrophoresis buffers, they migrate toward
the cathode when a voltage is applied. In clinical SPEP and UPEP, the loading zone
(specimen application point) is therefore not at the “top” or “bottom” of the gel as
one might assume, but rather near the “middle” of the gel, near the interface of the
beta and gamma fractions (Fig. 2a). Proteins ultimately migrate toward the anode or
cathode based on the opposing EMF and EOF.

Gel Electrophoresis

As noted earlier, most clinical SPEP and UPEP are performed using agarose-con-
taining gels. These gels (as kit components) are usually purchased preformed, as
this helps to streamline laboratory workflow and ensures consistent gel quality and
electrophoretic performance. The agarose gel is manufactured using a buffer solu-
tion, frequently Tris-barbital or Tris-barbital/MOPS, along with additional stabiliz-
ers and/or preservatives [15, 16]. The buffer has a typical pH in the range of 8.5–9.5,
depending on the manufacturer and desired separation characteristics.
In research settings (the environment where most scientists and/or physicians
have experience with protein and DNA electrophoresis), specimens are usually
loaded into preformed “wells” that were created during gel formation. In clinical
electrophoresis, however, the presence of such wells could potentially create visual
artifacts that might interfere with specimen interpretation. Clinical protein electro-
phoresis systems therefore usually incorporate a process of loading patient speci-
mens onto applicators made of either paper or mylar substrates. These applicators
are then gently applied to the gel surface at the defined loading zone. This allows
specimens to transfer into agarose without the need for wells and therefore reduces,
but does not altogether eliminate, the possibility of loading zone artifacts.
After the protein migration protocol is completed, gels are dried, stained (us-
ing dyes such as Acid Violet, Amido Black, or Coomassie Brilliant Blue), washed,
and dried again. An image of the gel is then made using either a scanner or an
integrated digital imaging system. An electropherogram is then generated. This is
a visual representation of the specimen migration pattern, in which the area under
6 J. R. Genzen

Fig. 2. Serum protein electrophoresis. a Protein electrophoresis of a normal serum specimen.

Normal albumin, alpha-1, alpha-2, beta, and gamma fractions are observed. b An electrophero-
gram derived from the same specimen as in a (oriented with albumin to the left and gamma fraction
to the right). c An electropherogram derived from the serum of a patient with a distinct monoclonal
protein in the gamma region. Note the gate ( shading) applied over the M-spike, used in quantifica-
tion. Electropherograms from capillary-based platforms demonstrating: d normal pattern; e hypo-
gammaglobulinemia; f polyclonal hypergammaglobulinemia; and g beta–gamma bridging (due to
increased polyclonal IgA in liver disease). Electrophoresis conducted using Sebia HYDRAGEL
PROTEIN(E) 15/30 gels (a–c) and a Sebia MINICAP instrument (d–g)
Clinical Protein and Immunofixation Electrophoresis 7

the curve (AUC) of specific fractions and/or abnormal bands is proportional to the
band intensity on the gel (Fig. 2b, c). Note that the gamma fraction in normal serum
specimens shows a rounded polyclonal response (Fig. 2b), whereas our abnormal
serum specimen shows a distinct abnormal band in the gamma fraction (Fig. 2c).
The shading superimposed on this abnormal peak is called a gate used to quantify
the amount of abnormal protein present. An operator manually sets and refines the
start and end points of this gate, so that the AUC selected is neither under nor over-
quantified. Operator training is particularly important in this step, both to prevent
nonmonoclonal fractions from being falsely “tagged” as monoclonal, as well as to
maintain consistency in how gates are placed. Marked inconsistency in gating be-
tween operators could in theory impact clinicians’ perception of disease progression
and therefore decisions regarding therapeutic management.
To calculate the quantity of fractions in mass concentration (e.g., g/dL), the rela-
tive percent of individual AUCs is multiplied by the serum total protein concentra-
tion performed separately using chemical methods or refractometry. This method is
used to quantify the M-spike at baseline and to monitor a patient’s disease course
over time.
The UPEP is performed similarly to the SPEP procedures outlined above. Urine
concentration protocols, however, are frequently conducted on specimens prior to
electrophoresis. This improves the sensitivity of UPEP for detecting monoclonal
bands in dilute urine specimens. While random urine specimens are frequently used
in screening for monoclonal proteins, in patients with known monoclonal disease,
a 24-h urine collection permits the reporting of monoclonal protein excretion not
just in mass concentrations (e.g., mg/dL), but also in amount per day (e.g., mg/day).
Quantification in 24-h urine specimens allows for a more standardized assessment
of monoclonal protein excretion in the urine, as it is less affected by dilute or con-
centrated random collections.

Capillary Electrophoresis

As outlined above, gel-based protein electrophoresis can be a very manual and

technical procedure. Specialized training and proficiency is required on the part of
laboratory technologists in order to provide consistent, quality results. Many labo-
ratories have therefore moved toward adopting capillary-based systems for clinical
electrophoresis, as these options (a) provide a greater potential for automation in
an otherwise high-complexity process and (b) produce electropherograms that are
quite similar to gel-based methods [17].
In capillary electrophoresis, protein migration is performed in solution (not in a
gel) using a narrow diameter glass tube during high-voltage application. As the in-
ner surface of the glass tube has a strong net-negative charge, EOF is much stronger
than EMF. Positively charged proteins therefore migrate the fastest, followed by
neutral and then negatively charged proteins. A detector window near the distal
end of the capillary tube allows the detection of protein using methods such as UV
8 J. R. Genzen

absorbance. As there is no gel, interpretation of capillary electrophoretic patterns is

performed exclusively by reviewing derived electropherograms (Fig. 2d, e, f and g).
Several manufacturers do, however, maintain the software capability of generating
a “pseudo gel” created from the electropherogram for display purposes.
It should be noted that capillary electrophoretic detection methods may also rec-
ognize some nonprotein substances which appear as unexpected peaks on electro-
pherograms. Such materials include contrast media, certain antibiotics, and plasma
substitutes (see Ref. [18]). Follow-up clonal characterization methods can be used
to exclude the possibility of monoclonal proteins, although such interference (while
rare) may lead to clinical confusion and unnecessary diagnostic workup.

Protein Patterns

Serum

Clinical electrophoresis systems separate serum into multiple protein fractions.

Major fractions include albumin, alpha-1, alpha-2, beta (some gels separate into
beta-1 and beta-2), and gamma. A faint prealbumin fraction can also sometimes be
observed anodal to albumin. While these fractions also follow Greek letter naming
convention, they should not be confused with the nomenclature for immunoglobu-
lin heavy chains and light chains. Common protein constituents of serum electro-
phoretic fractions are presented in Table 1. Comprehensive information on these
and other proteins is provided elsewhere [18–20].
It is important to note there are hundreds of proteins in the circulation, and the
fractions observed by clinical electrophoresis reflect only those at the highest con-
centration and in a largely overlapping manner. More precise separation and vi-
sualization can be achieved through techniques such as isoelectric focusing—ei-
ther alone [21], in the context of 2D gel electrophoresis [22], or by incorporating
immunofixation [23]. Such methods, however, are technically challenging, labor-
intensive, and generally not ideal for the clinical laboratory setting. Furthermore,
the additional resolution provided by high-resolution electrophoretic techniques can
actually confuse and hinder the ability to easily recognize and quantify monoclonal
proteins. The relatively limited resolution of standard SPEP is therefore preferable
for its clinical use in routine screening for monoclonal proteins.

Urine

In unconcentrated urine specimens from healthy individuals, little to no protein may

be observed by UPEP [18]. In laboratory-concentrated specimens from healthy in-
dividuals, a distinct but nonprominent albumin band is often apparent. The baseline
Clinical Protein and Immunofixation Electrophoresis 9

Table 1 Protein constituents of serum electrophoretic fractions [16, 18–20, 56]

Fraction Proteinsa
Prealbumin Prealbumin (transthyretin)
Albumin Albumin
Alpha
Alpha-1 Alpha-1 antitrypsin
Alpha-1 acid glycoprotein (orosomucoid)
G-C globulin (vitamin D binding protein)
Alpha-2 Alpha-2 macroglobulin
Haptoglobin
Ceruloplasmin
Betab
Beta-1 Transferrin
Beta-2 C3 complement
CRP
Gammac,d Immunoglobulins
a
This list is not inclusive
b
“Split-beta” gels separate beta fractions into two distinct peaks. In non split-beta gels, proteins
may be largely overlapping
c
Fibrinogen migrates near the beta-gamma interface in plasma specimens
d
Monoclonal immunoglobulins can migrate in other fractions depending on their charge. IgA
(polyclonal and monoclonal) typically migrates near the beta-gamma interface. Elevated poly-
clonal IgA can therefore lead to “beta gamma bridging” seen on electrophoresis in the context
of liver disease

urine protein concentration, however, would not be elevated. A prominent albumin

band is visible in the context of glomerular proteinuria and may be accompanied
by additional lesser (but distinct) bands in the beta fraction and alpha-1 fraction
due to the passage of proteins such as transferrin and alpha-1 antitrypsin [18, 20,
24]. In non-selective proteinuria, additional peaks (somewhat reflective of serum
specimens) will be present. Tubular proteinuria is frequently noted by the presence
of multiple peaks in the alpha-2 fraction (usually in combination with a distinct
albumin band) [18, 20]. Mixed proteinuria refers to the combination of glomerular
and tubular patterns [24]. Finally, overflow proteinuria is a term used to describe the
excess synthesis of low molecular weight proteins which are released into the urine
and surpass the tubules’ capability to resorb them [20, 25]. Overflow proteinuria is
observed in monoclonal gammopathies, but may also be seen in other conditions
such as inflammation or hemolysis [18, 20, 25]. Monoclonal free light chains in the
urine are also commonly referred to as Bence Jones proteins, named after Dr. Henry
Bence Jones (born, 1813; died, 1873). The fascinating history around the discovery
and characterization of monoclonal light chains in the urine can be found in numer-
ous excellent resources [18, 26–28]. Examples of UPEP patterns are included in
Fig. 3.
10 J. R. Genzen

Fig. 3 Urine protein electrophoresis. A representative serum specimen control is shown at the left
to indicate the position of electrophoretic fractions. The subsequent urine protein migrations dem-
onstrate the following: a normal urine; b glomerular proteinuria; c Kappa Bence Jones proteinuria
(39 mg/day); d Kappa Bence Jones proteinuria (980 mg/day); e Lambda Bence Jones proteinuria
(134 mg/day); f Lambda Bence Jones proteinuria (236 mg/day). Electrophoresis conducted using
Sebia HYDRAGEL 15 HR gels

Clonal Characterization

Immunofixation—Gel Electrophoresis

While protein electrophoresis permits the identification of discrete abnormal bands

in serum and urine, IFE can be used to confirm whether such bands represent mono-
clonal immunoglobulins, and if so to provide clonal characterization [29]. Clinical
laboratory IFE methods share initial steps with protein electrophoresis, except each
patient specimen is initially migrated in multiple consecutive lanes (often six) on
the gel. After protein migration, a template grid of channels is placed on top of the
gel, so that each channel is aligned directly above a migration lane (Fig. 4a). In
the first channel, an acid solution is applied to precipitate all serum proteins and
create what is essentially the patient’s SPEP pattern used for comparison purposes.
In subsequent channels, antibodies directed against heavy chains (γ, α, μ) and/or
light chains (κ, λ) are applied separately. Antisera to δ and ε heavy chains can also
be used when an IgD or IgE monoclonal protein is suspected. At least one initial
Clinical Protein and Immunofixation Electrophoresis 11

Fig. 4. Serum Immunofixation Electrophoresis ( IFE). a Reagent application grid used for serum
IFE on the Sebia Hydrasys platform. The grid is aligned over a HYDRAGEL 9 IF gel (underneath,
partially visible) such that channels are created above corresponding lanes. For this kit, antibody-
containing reagents are color-coded to provide visual of correct pipetting. In this example, reagents
for acid fixation ( yellow) and γ heavy chains ( red) have been applied over nine patient specimens.
b Serum IFE from four patients using the Helena SPIFE 4000 instrument and gels. Note that on
these gels, albumin is oriented toward the bottom whereas the gamma region is oriented toward
the top. Patients 1 and 4 show normal patterns of polyclonal immunoglobulin expression. Patient
2 has a monoclonal IgM-kappa. Patient 3 has a monoclonal IgG-kappa, with suppression of other
polyclonal immuoglobulins

δ- and ε-containing IFE is recommended in patients where free monoclonal κ or λ

light chains are observed in the absence of γ, α, or μ heavy chain restriction. Other
reagents that may also be used in IFEs include mixtures of five antibodies (against
γ, μ, α, κ, and λ), three antibodies (against γ, μ, and α), and/or antibody reagents
against free (or free and bound) κ and λ light chains [16, 18, 30] .
12 J. R. Genzen

When reagent antibodies bind to patient antibody, complexes precipitate in the

gel (i.e., immunofixation occurs). After precipitation, the gel is washed (to remove
any unbound reagent antibody and nonprecipitated serum proteins), dried, stained,
washed, and dried again. Clonal characterization is then conducted based on pat-
terns evident on the gel (Fig. 4b). As the staining procedure labels all precipitated
proteins, including complexes containing both patient and reagent antibodies, IFE
is not useful in quantification of monoclonal proteins. In general, however, staining
intensity is reflective of the amount of immunoglobulin present and marked discor-
dances with other laboratory results warrant further investigation to resolve any dis-
crepancy. As with other antibody-based assays, hook effect (where target antigen is
in excess of reagent antibody, thus interfering with normal complex formation and/
or precipitation) can also lead to discordant appearance of staining intensity on IFE.

Immunosubtraction—Capillary Electrophoresis

Monoclonal proteins can be characterized by capillary electrophoresis in a manner

analogous to IFE [31, 32]. This technique is referred to as immunosubtraction, im-
munotyping, or immunodisplacement. In this method, a baseline electropherogram
is generated from a patient specimen. The same patient’s specimen is also migrated
after preincubation with reagent antibodies against heavy chains (γ, α, or μ) or light
chains (κ or λ). Complexes of patient and reagent antibodies migrate more slowly
than the unbound patient antibody and are therefore “subtracted” from the baseline
electropherogram. Clonal characterization is conducted by reviewing the electro-
pherograms from each reagent antibody condition and identifying peaks that have
disappeared or are markedly reduced (Fig. 5). To date, immunosubtraction reagents
for δ and ε heavy chains are not commercially available.

Commercial Platforms

In the USA, most clinical platforms used for gel and/or capillary-based electropho-
resis, as well as their corresponding reagents, are marketed by one of two diagnostic
companies: Sebia, Inc. (Norcross, GA; www.sebia-usa.com) and Helena Laborato-
ries (Beaumont, TX; www.helena.com). An additional company, Interlab (Rome,
Italy; www.interlab-srl.com) manufactures two electrophoretic platforms, one of
which is now available in the USA and is distributed by Grifols, Inc. (Los Ange-
les, CA; www.grifolsusa.com). Instrumentation offers significant variability in the
character and degree of automation, barcode traceability, reagent handling, software
design, and user experience. Thorough instrument demonstration and evaluation
are recommended before deciding upon which platform best fulfills a laboratory’s
unique clinical, financial, workflow, and staffing needs.
Sebia sells both gel- and capillary-based electrophoresis systems. Their gel-
based electrophoresis and immunofixation platform, HYDRASYS®2, can be
Clinical Protein and Immunofixation Electrophoresis 13

Fig. 5 Clonal characterization by capillary electrophoresis. Illustration of representative serum

capillary electropherograms at baseline ( upper left) and after incubation with reagent antisera
against immunoglobulin heavy chains (γ, α, µ) light chains (κ, λ) in a patient with monoclonal
IgG-kappa. In the presence of reagent antisera against either γ heavy chains or κ light chains, the
prominent electropherogram peak in the gamma region disappears (absence indicated by asterisk).
The reagentrpatient antibody complex migrates anodal ( left) of the albumin bands

combined with a preanalytic ASSIST unit for auto-sampling, dilutions, and antisera
pipetting. A SCAN module can be used for integrated digital imaging and densitom-
etry. Sebia also sells two capillary electrophoresis systems, MINICAP, and CAPIL-
LARYS™2, both of which allow for protein electrophoresis and immunotyping.
The MINICAP is designed primarily for small to moderate size workflows, whereas
CAPILLARYS™2 is designed for higher-volume settings.
14 J. R. Genzen

Helena Laboratories also provides both gel- and capillary-based electrophoresis

systems. The SPIFE® 3000 provides gel-based electrophoresis and immunofixa-
tion. A front-end Auto Sample Handler can be used to automate preanalytic pro-
cessing. The larger SPIFE® 4000 is designed as a walkaway, automated solution
for gel-based electrophoresis and immunofixation. Small-scale gel electrophoresis
can also be conducted using their manual QuickGel™ system. Finally, Helena also
provides capillary electrophoresis and immunosubtraction through their Velocity8
(V8™) platform.
Interlab sells the G26 system for gel-based protein and IFE . This platform pro-
vides a benchtop automated solution that is barcode-traceable. It also incorporates
preanalytic processing and densitometry. A smaller platform (Pretty Interlab) is also
available internationally.
While gel and capillary electrophoresis have been discussed separately, many
laboratories incorporate both techniques. Instrument connectivity (at least within-
vendor) facilitates integration of both technologies into a laboratory’s workflow.
For example, a laboratory may prefer protein electrophoresis to be run by a capil-
lary method, while gel-based IFE may be their method of choice for clonal charac-
terization. Some prefer having the ability to visualize bands on gels, while others
are comfortable reviewing electropherograms and interpreting immunosubtraction
patterns. Furthermore, many laboratories that have moved toward immunosubtrac-
tion have also maintained a backup IFE platform for challenging cases. In the end,
decisions on platform and methodology are based on numerous factors that may
be unique to the laboratory’s workflow, finances, staffing, and operator preference.

Specimen Considerations

Serum

Serum (not plasma) is the appropriate clinical specimen when screening for circu-
lating monoclonal proteins by SPEP and IFE. Plasma is not an acceptable specimen
type, as fibrinogen appears as a discrete (and potentially abnormal) band near the
interface of the beta and gamma fractions. When a fibrinogen band is suspected, the
laboratory has several options for potential follow-up. If the primary specimen tube
is available, reverifying tube type by visual inspection of the label is warranted. A
fibrinogen band would also not yield a monoclonal characterization on IFE. Anoth-
er option includes adding a small amount of thrombin to the specimen before repeat
SPEP. If visible clot formation is observed, and the suspected band disappears on
subsequent electrophoresis, the presence of fibrinogen has been confirmed. Finally,
others have advocated for using a gamma fraction to IgG ratio for distinguishing po-
tential fibrinogen peaks [33]. With increased use of alternative oral anticoagulants
in clinical practice (e.g., direct thrombin inhibitors), it is possible that detection of
faint fibrinogen bands may become more common in electrophoretic screening,
even in specimens properly collected in primary serum tubes.
Clinical Protein and Immunofixation Electrophoresis 15

Hemolyzed specimens can also yield a potentially abnormal band near the beta
fraction. As with fibrinogen, a hemoglobin band would not yield a monoclonal char-
acterization on IFE. Along with visual inspection for marked specimen hemolysis,
follow-up may include suggesting repeat testing on a non-hemolyzed specimen and/
or IFE to rule out any possibility of a monoclonal protein.
Clinical conditions can also lead to specimen-related problems in electropho-
retic assays. Patients with large amounts of monoclonal protein (particularly IgM
in Waldenström’s macroglobulinemia) may have elevated serum viscosity. In some
cases, transfer of specimen from the applicator into the gel may be incomplete, lead-
ing to inaccurate quantification of serum fractions. In such cases, prewarming and
thoroughly mixing the specimen before electrophoresis may improve the quality of
results.
It also should be noted that many (if not most) laboratories batch electrophoretic
assays to be run during the day shift. Specimens are therefore frequently refriger-
ated before testing. This can be particularly problematic when analyzing specimens
from patients with known or undiagnosed cryoglobulinemia. Maintaining warm
temperatures during the delivery and processing of these specimens can help to
prevent inadvertent precipitation before electrophoresis.

Urine

As monoclonal free light chains can migrate to the gamma, beta, or even alpha frac-
tions, it is often impossible to definitively exclude the possibility of monoclonal
proteinuria by UPEP alone in the context of other background proteins. Indeed, a
host of nonmonoclonal proteins can be found in glomerular, tubular, mixed, and/
or overflow proteinuria. Urine IFE should therefore be considered in cases where a
monoclonal protein is either suspected or cannot be definitively excluded. As such,
it remains an exceedingly valuable tool, even in an environment of increased utili-
zation of serum-free light chain assays [34].

Reporting and Interpretation

M-Spike Identification and Quantification

Once a monoclonal protein is detected, clinicians need to evaluate any trend in M-

spike concentration over time in order to assess disease progression, response to
therapy, and/or relapse. Along with providing text-based interpretations, interfacing
the numeric M-spike quantification as a unique data element to the electronic health
record (EHR) allows clinicians to automatically graph any trend in M-spike quanti-
fication. Such an approach is certainly ideal, but is hindered somewhat in how to ad-
dress results in patients with multiple M-spikes (some of which may have different
16 J. R. Genzen

clonal characterizations). While the author is not aware of any written consensus on
how to handle complex multi-clonal quantifications in diagnostic criteria, a “Sum
of All M-Spikes” approach would certainly be beneficial for automated trending
in the EHR. Individual bands could still be quantified and described separately in
interpretative comments. Finally, some laboratories are now providing expanded
reports including M-spike trends, gel images, electropherograms, quantitative im-
munoglobulins, and serum-free light chain results in order to integrate relevant
information for the clinical providers. See the Standardized Synoptic Reports for
Plasma Cell Neoplasms: Integration of Laboratory and Clinical Data chapter for
how such an approach can benefit clinical care.
Another challenge that laboratories face is how to quantify monoclonal proteins
that overlap (either partially or completely) with other fractions on the SPEP. For
example, Fig. 6 shows a broad monoclonal IgA-kappa that completely overlaps

Fig. 6 Monoclonal IgA-kappa in the beta fraction. a Example SPEP from a normal specimen
( left) and a specimen with an abnormally large beta fraction ( right). b Corresponding serum IFE
from the abnormal specimen demonstrates a broad monoclonal IgA-kappa that completely over-
laps with the beta fraction on SPEP. Serum electropherogram (c) and corresponding fractional
AUC (%) and mass concentrations (d), calculated using the patient’s serum total protein concen-
tration of 8.5 g/dL. Serum total protein was measured using a biuret method (UniCel DXC, Beck-
man Coulter, Inc., Brea, CA)
Clinical Protein and Immunofixation Electrophoresis 17

with the beta fraction. In this case, quantification of the entire beta fraction would
yield an M-spike of approximately 2.9 g/dL. Some laboratories would use this
quantification, but indicate with a comment that it is influenced by normal proteins
in the beta fraction. An alternative approach is to subtract an “average normal beta
fraction” from the beta fraction in this patient. Assuming ones laboratory had an av-
erage normal beta fraction of 0.8 g/dL, this approach would yield a revised M-spike
quantification of 2.1 g/dL. Others may decide to place a gate in the beta fraction
that is somewhat narrow, in an attempt to exclude the normal protein present. While
there are no definitive guidelines as to the best approach, what is absolutely clear is
that a laboratory should be consistent in how overlapping M-spikes are quantified.
This ensures that erroneous assumptions about disease progression and/or improve-
ment are not made based on alternative gating strategies between specimens.

Interpretations

Each laboratory tends to provide interpretations and reporting in a slightly different

manner and verbiage. As some patients receive clinical care from multiple provid-
ers who may utilize different laboratories, this can introduce confusion in rectifying
interpretations and discrepant M-spike quantification. Indeed, a call for standard-
ization of SPEP reporting has been published addressing such concerns [35].
As a general rule, the most important information should be presented in the
clinical interpretation first. Clinicians are unlikely to read long interpretations, par-
ticularly if they contain excessive and/or extraneous information. Most laborato-
ries also use templates for routine SPEP, UPEP, and IFE interpretations. Templates
facilitate the process of working through large volumes of tests during sign-out.
More importantly, they provide consistency in how clinicians see results over time
and across patients. Complex interpretations may require deviation from standard
templates to more accurately reflect the specimen’s unique electrophoretic pattern.
When an abnormal band is detected by SPEP, it should be quantified and de-
scribed in the interpretation. Ideally, IFE may be reflexively performed if it is part
of the clinician order and/or the approved laboratory reflexive strategy or panel.
This ensures that clonal characterization is provided to the clinician at the time of
initial report. It also ensures that any nonmonoclonal suspicious peaks on the SPEP
have been excluded. As not all electrophoresis is ordered according to reflexive
strategies and/or panels, results may also be reported with recommendations for
further testing (e.g., “Discrete abnormal band (suspected M-spike) measuring 1.5 g/
dL observed in the gamma fraction. Serum immunofixation electrophoresis is rec-
ommended for further characterization. Work-up for monoclonal gammopathy is
recommended.”).
Verbal communication of newly detected monoclonal proteins to the ordering
clinician also provides a clinically valuable service, but may not be practical or even
possible in all laboratory settings. In the author’s experience, rapid communication
of new findings for hospitalized (and particularly emergency department) patients
significantly improves the time to appropriate consultation and treatment. Rapid
18 J. R. Genzen

communication is also essential when hyperviscosity is suspected. These cases

often reveal themselves to the laboratory as “specimen clot errors” on automated
instruments, even before the possibility of monoclonal gammopathy has been in-
cluded on the differential diagnosis.

Personnel

As SPEP, UPEP, and IFE are categorized as high-complexity tests, in the USA,
they can only be performed by personnel who meet CLIA qualifications for high-
complexity testing [36]. Given the need for quality and consistency, technologists
who perform electrophoresis are frequently specially trained/dedicated to perform-
ing such testing, or they may do so in combination with other laboratory respon-
sibilities. Greater automation in electrophoresis platforms will certainly decrease
the manual complexity of handling and processing specimens. It will also decrease
interoperator variability in assay performance.
Regarding who is qualified to interpret SPEP, UPEP, and IFE results, a CAP
Checklist item (CHM.10800) notes that “all tests that include an interpretation must
be reviewed by the laboratory director or qualified designee before release from the
laboratory” [37]. Most laboratories have a doctoral-level laboratory scientist or phy-
sician review and approve interpretations. Many laboratories include technologists
and or pathology residents in generating preliminary interpretations. While there
does not appear to be a strict prohibition for having the “qualified designee” be a
non-doctoral-level technologist who meets CLIA qualifications for high-complexi-
ty testing, having a qualified laboratory director or pathologist/physician review in-
terpretations makes sense, from both a medical and liability standpoint. Regardless,
extensive experience in differentiating normal from abnormal is required and comes
only with experience and repetition. Finally, only a medical doctor (pathologist or
other; not a PhD-level scientist or laboratory technologist) can bill for a profes-
sional component according to CMS regulations for Medicare and Medicaid [38].

Additional Considerations

Correlation with Other Laboratory Results

M-spike quantification is one of the several methods that clinicians use in monitor-
ing patients with monoclonal gammopathies. Along with protein electrophoresis,
other tests that are frequently ordered include quantitative immunoglobulins (IgG,
IgA, and IgM) and serum-free light chain assays. Both of these techniques utilize
nephelometric and/or turbidimeteric measurements. Serum total protein is most
Clinical Protein and Immunofixation Electrophoresis 19

frequently measured by chemical methods (such as biuret) and/or refractometry.

Urine total protein is frequently measured by chemical/dye binding methods (such
as pyrogallol red) or turbidimetry. It is important to note that protein assays do not
measure all proteins equally, and paradoxically immunoglobulins are often under-
quantified in relation to albumin. Furthermore, urine dipstick methods are remark-
ably insensitive to immunoglobulins and free light chains, making them inadequate
for identifying and quantifying proteinuria due to monoclonal gammopathy [39].
The accuracy of nephelometric and turbidimetric assays for quantifying immu-
noglobulins is also limited. Quantification using these techniques is performed by
comparing patient specimens to standard curves derived from known concentra-
tions of analyte in solution. These standard curves, however, are often created us-
ing normal polyclonal materials. Furthermore, the analytical measurement range of
these assays may be optimized to assess the range of polyclonal responses observed
in clinical practice, as opposed to the unique characteristics of a myeloma patient’s
predominant clonal immunoglobulin. Ultimately, the absolute quantification of an
M-protein may be discordant (sometimes markedly) between methodologies [40].
The results of each individual method, however, should still be trendable.
Finally, it should be noted that monoclonal proteins themselves can interfere
with a variety of other laboratory assays [41, 42]. One well-characterized example
is artificially elevated total bilirubin on Roche/Hitachi platforms [43–45], although
numerous other potential interferences exist and may be unique to an individual’s
monoclonal protein. An appreciation of this phenomenon—including repeat testing
on alternative platforms when a laboratory result seems discordant with the pa-
tient’s clinical presentation—can prevent unnecessary and even invasive diagnostic
workups in patients with monoclonal gammopathy.

Oligoclonal Responses to Therapy

While monoclonal proteins observed in conditions such as multiple myeloma can

be very distinct, fainter atypical and/or oligoclonal responses have been observed
after chemotherapy, immunomodulatory therapy, and stem cell transplantation. This
phenomenon has been characterized by several names, including atypical serum im-
munofixation patterns (ASIPs) [46], abnormal protein bands [47], and secondary
MGUS [48]. In myeloma, the presence of these proteins may correlate with a high
degree of response to therapy [46] and improved overall survival [47, 48].
How to interpret these bands (which are usually faint and sometimes difficult
to fully characterize) can be a challenge for laboratories, which often do not have
clinical information regarding patient therapy. Furthermore, the exercise of trying to
characterize and quantify faint complex patterns (vs. supplying a general comment,
e.g., “trace oligoclonal response observed”) may have unclear benefit for patient
care. Communication with the clinical providers as to the level of information they
require is advisable, as it may save time and energy on the part of laboratory staff.
20 J. R. Genzen

Fig. 7 IFE from a patient on rituxan therapy. A faint cathodal IgG-kappa is observed ( arrows)

Monoclonal Antibody Therapeutics

With the increased use and diversity of monoclonal antibody therapeutics (MATs)
in clinical practice [49, 50], these drugs (often human and/or humanized immu-
noglobulins) can also potentially be detected by IFE and/or SPEP when present
in high-enough concentrations. Detection of siltuximab, rituximab, trastuzumab,
bevacizumab, infliximab, cetuximab, adalimumab [51], as well as ofatumumab [52]
have previously been described. While it is occasionally possible to infer that a faint
monoclonal band could be a MAT (e.g., a new trace cathodal IgG-kappa in a patient
with no prior M-spike, ordered by a provider who specializes in treating chronic
lymphocytic leukemia), in the absence of clinical and pharmacological history it is
impossible to make that determination. As an example, the serum IFE from such a
patient on rituxan therapy is shown in Fig. 7.
Furthermore, other providers who may not be aware of a patient’s MAT therapy
may view electrophoresis and immunofixation results in the EHR during subse-
quent clinical consultation and assume that the patient has MGUS. Thus, a previ-
ously identified monoclonal protein due to MAT therapy may trigger unnecessary
laboratory follow-up. Fortunately, therapeutic concentrations of MATs are far lower
than the M-spike levels that might warrant an extensive and invasive (i.e., bone
marrow biopsy) workup.

Conclusion

From early advances in protein separation techniques through the development

of automated clinical platforms, electrophoretic and immunofixation techniques
have proven to be invaluable in the diagnosis and management of human disease,
Another random document with
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 CHAPTER V.
THE NAVAL BRIGADE LANDS.

W HEN I went on deck the following morning, I found that the

steam-pinnace had towed the Flying-fish to an anchorage in
the roadstead not many cable lengths from the Rattler. I at once
observed the strong resemblance she bore to the Snapping Turtle,
and I no longer felt surprise at the mistake Mr. Osborne had made at
Santiago de Cuba.
Mr. Thompson was standing by the taffrail narrowly observing her
through a telescope.
“I should like to see a race between her and the Snapping Turtle,
sir,” I said to him. “I’m not surprised the Yankee skipper was so keen
on it, for they’re wonderfully alike in build and rig.”
“Indeed they are,” answered the lieutenant; “one might almost call
them twin-vessels. The main difference is that the American’s masts
rake more.”
“It’s a curious thing about the cargo having been all taken out of
the Flying-fish, sir; and by all accounts it’s a valuable one.”
The gunnery lieutenant turned and looked at me keenly.
“You young rascal,” he said, “you’re trying to pump me; but you do
it in such a clumsy way that I can’t help seeing through you.”
I felt rather confused.
“Well, sir,” I said, “I do hope that I may be allowed to go on the
expedition up country if it is true that a force is to be landed.”
“It will be no secret in an hour’s time, Darcy, so I may as well tell
you that to-morrow morning a naval brigade is to be landed in order
to hunt down the mutineers and rebels; and I think there is a very fair
chance of your being able to go. The captain, I believe, has
permission from the captain-general to take any steps he may think
necessary to bring the delinquents to justice.”
I begged the gunnery lieutenant, who had always shown me great
kindness, to try to get me appointed to the expedition, and he
promised to use his influence in that direction. I then ran off to the
sick-bay to see my friend Charlie and tell him the news, which I felt
sure he had not as yet heard. I found him much better; and the
surgeon, who was just leaving the sick-bay as I entered, told me that
I need have no fear as to his recovery.
This was very good news; but I found that I had been forestalled
as news-carrier by Dr. Grant, and that Charlie was as well informed
on the subject of the expedition as I was myself.
“It’s jolly hard lines that I can’t go, old chap,” he said to me; “but
the surgeon says I must be on the broad of my back and nurse this
wretched old head of mine for some time to come. Pleasant
prospect, eh?”
“I’m very sorry indeed,” I answered; “and you must try to console
yourself with the fact that you’ve still a head screwed tight and fast
on your shoulders. Poor Lobb had his taken off by a round-shot.”
“Oh, I’m as grateful as anything, of course, Jack; not only on my
own account, but because as an out-and-out patriot I have the best
interests of my country at heart. What an irreparable loss it would
have been to Great Britain if my brains had bespattered the battle-
field! National mourning for a fortnight, eh, and messages to my
bereaved relatives from the Queen and the other members of the
royal family, to say nothing of minute guns, half-mast flags, and a
tomb near Nelson’s in the crypt of St. Paul’s? By Jove! it makes me
quite excited to think of it.”
“Has Grant ordered you any soothing draught?” I asked, hunting
about with pretended anxiety amongst a whole brigade of medicine-
bottles that stood upon a table at my elbow.
“Yes; Mother Gimcrack’s soothing syrup!” said my chum with a
laugh. “Good for teething babes; and do you know, Jack”—this very
solemnly—“I lost two or three of my front teeth in that nasty
somersault I took yesterday. My beauty is gone for ever and ever!”
I had noticed the disfigurement my friend referred to, but had not
alluded to it for fear of hurting his feelings.
“There is always a silver lining to the cloud,” continued Charlie
more cheerfully. “That rascal of a gunroom steward won’t be able to
palm off on me any longer his wofully tough salt horse and brickbat
biscuit. No; he’ll have to feed me on a special diet of Brand’s beef
jelly, Benger’s food, turtle soup, and jams of all sorts, varied
occasionally by oysters (real natives of course), tipsy cake, and fruit
jellies. Not a bad idea, eh? I’ll give you a tuck-in now and again,
Jack, as you’re a good chum to me!”
“Thanks, awfully!” I said; “but I’m certain the steward would rather
go to the expense of buying you a new set of teeth from a London
dentist, than feed you up on all the delicacies of the season for the
rest of the commission. Now I’m certain you oughtn’t to talk any
more, Charlie, so I’m going to make myself scarce; and you must try
to sleep till dinner-time, when I shall come and see you again.”
Half an hour later the Rattler was a scene of great excitement, for
orders had gone forth that immediate preparations were to be made
for landing a powerful naval brigade. I was very quickly caught up in
the whirl of excitement, for Ned Burton, the coxswain of my boat,
came hurrying to me to say that he had received orders from the first
lieutenant to get the second cutter in readiness to assist in landing
men, stores, and ammunition.
“It’s to be a picked force, sir,” said the seaman in conclusion, “and
I’m glad to say that we’re both detailed for service.”
“I’m delighted to hear it,” I answered, “for I was half afraid that
midshipmen would be excluded. When do we land, Ned?”
“I think in the evening, sir, so as to be ready for a start in the
morning. We can’t take no field-guns, more’s the pity, for they say
the country is a sight too hilly for anything but mountain guns.”
“How about the commissariat, ammunition, tents, and so forth?” I
asked; “we shall require transport animals of some kind.”
 “I believe the Spanish Government is going to let us have a lot of
mules that are accustomed to that sort of work,” said my coxswain.
“Oh, we shall pig it out somehow, I dare say,” I exclaimed with a
laugh, “and it would be rather fun to rough it a bit.”
That evening we occupied the fort in force. The dead had been
buried at an early hour in the morning, and so there was little or no
trace of the struggle that had taken place so recently, except in the
fort itself, where the dismounted and spiked guns told their own tale.
In all we numbered one hundred officers and men, well supplied with
all that was necessary for a short campaign. At the time of my story,
machine-guns had not been invented, and that underhand weapon
of warfare, the torpedo, was unknown. A few of the former would
have been extremely serviceable to our brigade on this occasion; but
still we were extremely well armed in accordance with the ideas of
that day, each man being supplied with a breech-loading rifle, a
cutlass which could be used as a sword-bayonet if necessary, and a
revolver. An ammunition-pouch, a blanket, a water-bottle, and a pair
of leggings for each man completed the equipment, nothing being
showy, but everything extremely serviceable.
As before, Mr. Thompson was appointed to command the brigade,
as he had had a great deal of experience in shore-going expeditions
in a previous commission on the west coast of Africa. Two
lieutenants, the captain of marines, Dr. Grant, four sub-lieutenants,
the gunner, Fitzgerald, and myself made up the list of officers; and
about seventy picked blue-jackets and twenty marines composed the
rank-and-file. No commanding officer could have wished for finer
men. Not only was their physique splendid, but they were tried,
trustworthy fellows who had all seen service on previous occasions,
and could be relied on to do their duty in the direst emergency.
Tenacious bull-dogs! that’s what they were. It would be impossible
to describe them better in a couple of words.
 CHAPTER VI.
“COLD PIG” AND “SLING THE MONKEY.”

I WAS effectually roused from my slumbers on the following

morning by the shrill bugle-calls which the drummer seemed to
take a delight in blowing as near the gunroom tent as possible. On
murderous thoughts intent, and clad in very scanty apparel,
Fitzgerald and I made a desperate sortie, one carrying a huge bath-
sponge saturated with water, and the other a well-knotted towel.
“What a lark!” exclaimed Fitzgerald, capering about with delight;
“cold pig for the drummer, and a lambasting afterwards to warm him
up and prevent any possibility of his catching cold whilst so far away
from his mammy’s protecting care!”
Dawn had scarcely broken, and it was almost dark outside the tent
and rather unpleasantly chilly. The bugle-calls had ceased, but we
thought we distinguished the drummer some yards away just upon
the point of raising his instrument of torture to his lips again.
“I’ll put a stopper on his little game,” said Fitzgerald hastily to me.
“Ready! present! fire!” and he hurled the heavy sponge with
admirable aim straight at the dusky little figure; whilst I darted
forward with a sort of Red Indian war-whoop, waving the knotted
towel over my head.
The sponge landed with a splosh full upon the head of the
individual it was intended for, and the latter staggered and gave a
shout of dismay and disgust as the highly-unpleasant projectile came
into contact with him.
“Good shot!” I cried exultingly. The next moment I recoiled in
horror, and Fitzgerald turned deadly pale, for we recognized in our
unlucky victim the short but sturdy Mr. Triggs, the gunner, who, being
a very early riser, had taken it into his head to emerge from his tent
and endeavour to make out the Rattler through a pair of night-
glasses. How would he take our explanation that we had mistaken
him for the drummer-boy tooting on a bugle?
Before we had time to think or apologize for our mistake, the
sponge was sent hurtling back through the air by the muscular arm
of Mr. Triggs. I was relieved to see that it was aimed at the real
delinquent, Fitzgerald, and not at me.
“O you mischievous middies!” shouted the gunner, running
towards us; “you’re always up to some tomfoolery or other!”
Fitzgerald saw the sponge flying towards him, and tried to dodge
it, but as ill luck would have it trod with his bare foot upon a sharp
stone. The pain was so great that it brought him to the ground; but in
trying to save himself he threw out his arms and they unfortunately
encountered me, and I felt myself seized in a grip which there was
no shaking off. In a moment we were both sprawling upon the
ground, arms and legs inextricably mixed up in a sort of “limb hotch-
potch.”
The gunner, chuckling with delight at our misadventure, now came
running up, his hair and face dripping from the effects of his lately-
inflicted “cold pig.”
“If I don’t pay you youngsters out, my name ain’t Timothy Triggs!”
he exclaimed; “and ’tis a grand opportunity I’ve got,” and so saying
he snatched the knotted towel out of my hand, and began
belabouring us both with it with remarkable muscular energy.
“Stop, stop, stop!” I yelled; “we mistook you for the drummer, and
are awfully sorry, Mr. Triggs!”
Whack, whack, whack! The blows fell with wonderful regularity and
with marvellous impartiality, first on Fitzgerald and then on me.
All this time the gunner was chuckling with suppressed laughter,
for he was thoroughly enjoying the joke, being at heart a most good-
natured man.
“You can just imagine you’re playing ‘sling the monkey,’” he
exclaimed; “’tis a right good game and no mistake!”
 Fitzgerald and I, however, had by this time managed to
disentangle our arms and legs, and we were on our feet again in a
moment. We did not at all appreciate this novel kind of “sling the
monkey.”
“Is that the enemy coming over the hill?” I exclaimed in an alarmed
voice, and pointing away to the rising ground which, beyond the
confines of the fort, rose steep and dark against the primrose-tinted
sky.
Mr. Triggs promptly turned his head to look, and in an instant I had
snatched the towel from his hand.
“Cut and run, Fitz!” I cried; “I thought I’d gammon him,” and so
saying I fled precipitately in the direction of the gunroom tent, my
brother-middy hobbling after me as fast as his wounded foot would
allow.
Mr. Triggs, however, did not attempt to give chase, feeling, I
suppose, that his skylarking days—now that he was on the shady
side of fifty—were over. So the worthy warrant-officer contented
himself with keeping up a hot and strong running fire of anathemas
upon us as long as we remained in sight.
“The bath-sponge, Fitz, the bath-sponge!” I gasped out, as I ran
panting into the tent and flung myself upon the ground, which formed
the only flooring.
“By Jove! I forgot all about it,” said my hobbling messmate; “I hope
old Triggs won’t appropriate it.”
At that moment the real drummer-boy passed our tent whistling a
merry air.
I promptly stopped him.
“Do you mind seeing, like a good fellow, if there’s a bath-sponge
lying just over there by that tent?” I said.
“All right, sir, I’ll have a look,” answered the drummer-boy, good-
naturedly, and off he went.
In a minute or two he returned with it.
 “Here you are, sir. Been playing Aunt Sally with it, I suppose?”
“No, Uncle Triggs,” I said laughingly. “You’ve had an awfully
narrow escape, bugler, only you don’t know it. I should strongly
advise you not to come near the gunroom tent in the early morning,
for Mr. Fitzgerald there always gets a violent attack of homicidal
mania about that time.”
An hour later the tents were struck and we had started on our
march up country to the tune of “Rule Britannia,” played with
tremendous energy by our fife-and-drum band.
Little did I anticipate what was before me—such adventures as
even in my wildest dreams had not occurred to my mind.
 CHAPTER VII.
NED AND THE MULE-DRIVER.

W E had two sets of native auxiliaries. One consisted of a fine lot

of Spanish baggage-mules, strong hardy beasts, thoroughly
acclimatized, and remarkably sure-footed; and the other a little bevy
of guides, interpreters, and spies, without whose aid we could have
accomplished little or nothing, for we were entirely ignorant of the
country we were about to traverse, and our knowledge of Spanish
was confined to about a dozen words or so.
The spies, some of whom were negroes and the others half-
castes, assured us that they had tracked the mutineers for some
distance, and were well acquainted with the route they had taken,
which was a beaten track leading straight into the interior. These
swarthy fellows also asserted that a body of insurgents had
accompanied the lawless crew of the Flying-fish in their retreat. We
questioned them as to any knowledge they might have acquired with
regard to the whereabouts of the valuable cargo which it was the
object of our expedition to recover. About that they declared that they
knew nothing whatever, although they confessed to having heard
rumours that large bodies of men were passing and repassing
between the shores of the creek and the spurs of the inland hills
during the whole of the day before the Rattler’s arrival upon the
scene.
“’Tis a good thing we’ve no field-guns and limber-waggons with
us,” said Ned Burton to me as we marched along; “they’d have
delayed us terribly, and prevented our making forced marches.”
“You think we’ll soon come up with them then?” said I. “For my
own part I hope the fun won’t be over too soon. If we returned
victorious in a couple of days, the fellows left on board would be sure
to jeer at us, and say we had only gone for a sort of picnic into the
mountains.”
 “Ah, ’twill take more than a couple of days even under the most
favourable circumstances,” answered Ned. “I take it these merchant-
service fellows haven’t got marching-legs, so to speak, and are
perhaps encumbered with wounded men, but still they’ve got a pretty
fair start, you see, and that ain’t a thing to be sneezed at.”
“The difficulty will be to find where they have hidden away the
booty,” I said; “no doubt the insurgents have put them up to a wrinkle
or two, knowing every inch of the country as they do.”
“Doesn’t the Rattler look jolly?” exclaimed an enthusiastic voice at
my elbow.
I turned and beheld Fitzgerald, who still had a slight limp as a
legacy from the morning’s fracas.
“Poor old ‘hop-and-go-one,’ what’s he trying to say?” I asked in a
jocose tone, and clapping him on the shoulder rather harder than
was altogether necessary.
“‘Do you bite your thumb at me, sir?’” demanded Fitzgerald,
tapping his sword-hilt with his left hand, and trying hard but very
unsuccessfully not to laugh.
“‘I do bite my thumb, sir,’” I answered promptly, and trying to put on
a swashbuckler air; “but I need not say that I should infinitely prefer
to bite yours or even Mr. Triggs’s.”
“Then old ‘hop-and-go-one’ and old ‘hop-o’-my thumb’ would be
sworn chums for ever and ever,” laughed Fitzgerald; “but at this
moment I don’t want to fall out with you, honour bright! I want you to
look back at that magnificent view, and the dear old Rattler in the
middle of it. I never saw a more lovely picture!”
Fitz was an artist of no mean capacity, and I strongly suspected
that he had at that moment a paint-box and brushes in his pocket.
Hand-cameras would have enchanted him, but they had not then
been invented.
It certainly was a lovely view, and I felt grateful to my brother-
middy for calling my attention to it.
 We had been winding gradually along the summit of a low range of
hills, on the outermost spur of which was situated the fort we had just
evacuated. The gradient was upwards, though in no place steep,
and we had now reached a somewhat extensive plateau covered
with short springy sward. From this point of vantage we had a full
and extensive view of the winding tortuous creek; the hills, clad with
palm groves, which enclosed it; and the broad blue sea beyond,
glittering in the sunshine, and here and there barred with purple
cloud-shadows. For the primrose streaks of colour in the sky had
melted away as if by magic, and the glorious sun had recalled a
sleeping world to life. In the roadstead our beautiful frigate lay calmly
and serenely at anchor, her guns frowning from the portholes, and
her shapely hull and taut spars and rigging reflected with
extraordinary fidelity in the waters which appeared to sleep in the
warm rays of the sun. Astern lay the Flying-fish, which, though a
well-built vessel, lacked the trim appearance and impressiveness of
the British man-of-war. Above, the blue vault of heaven stretched
away into limitless infinity, its tint of deepest azure only broken here
and there by a few sluggishly-moving clouds and the white wings of
innumerable sea-gulls.
As we gazed admiringly at our floating home we saw the proud
white ensign slowly ascend to her gaff, drooping listlessly in the
stagnant air; and the distant strains of “God save the Queen” came
faintly to our ears through the still, clear atmosphere of a Cuban
early morning.
“Eight bells!” I cried. “If we were on board the old hooker, Fitz, we
should be just sitting down to eat salt-junk and swill gunroom catlap.”
“Instead of which we’re out upon the war-path,” said Fitzgerald,
“and, like Fenimore Cooper’s Indian braves, are dying to scalp the
enemy.”
A halt was called just at this moment on account of a stampede
amongst some of the baggage-mules.
The gunnery lieutenant, who was very anxious to push on and find
traces of the enemy, was exceedingly angry at this unlooked-for
delay.
 “Mr. Darcy,” he sang out to me, “ascertain at once the cause of
that stampede among the mules; and if it was due in any way to the
cruelty of the Spanish drivers, have the delinquents brought before
me, and I’ll give them a lesson they won’t forget in a hurry.”
I touched my cap and ran off to the rear to make inquiries,
expecting endless difficulties in having to conduct an investigation
with native mule-drivers who were most probably as ignorant of the
English language as I was of Spanish.
Meanwhile about a dozen of the mules were careering about wildly
in the neighbouring ravines, pursued by their shouting and
screaming owners. Some of the frightened animals had already rid
themselves of their burdens, and the ground was strewn with bags of
biscuit, preserved provisions, and cases of ammunition.
The worthy Mr. Triggs proved to be a friend in need to me, for on
reaching the spot where the main body of the baggage-animals was
collected, I found him firmly holding a swarthy Cuban by the scruff of
the neck and administering to another portion of his body some
hearty kicks.
“This is the rascal that caused all the mischief with the mules, Mr.
Darcy,” he exclaimed in rather breathless tones as I ran up. “The
cruel brute broke several sticks over the back of a poor mule that
had gone dead lame, and the wretched animal was in such pain and
so frightened that it broke away, and seems to have infected a lot of
the others with its terror.”
I promptly seized the culprit by one arm.
“You come along with me,” I said; “our chief is going to have you
tried by a drumhead court-martial, and perhaps shot, according to
the regulations of war.”
I do not know if the wretch understood what I was saying, but he
commenced to struggle and shout defiantly in his native tongue.
Mr. Triggs, however, seized him by the other arm in an iron grip,
and, in spite of his writhings and kickings, we hurried him forward to
the spot where the gunnery lieutenant was standing awaiting events.
 The gunner related to his superior in a few words how he had
caught the culprit in the very act of brutally ill-treating a helpless
lame mule.
“Is there an interpreter there?” demanded Mr. Thompson.
A respectable-looking elderly Spaniard stepped forward and took
off his sombrero with a sweeping bow.
“Be good enough to tell this fellow that he is a heartless cowardly
brute,” said the lieutenant sternly, and pointing to the still defiant-
looking mule-driver; “ask him what he means by such conduct.”
The Spaniard interpreted the officer’s words, but the culprit
obstinately and sullenly refused to answer a word.
“Where is the stick with which he belaboured the poor mule?”
demanded the gunnery lieutenant.
“Here it is, sir,” said Ned Burton, coming up at that moment with a
long, business-like cane in his hand.
“We’ll now give him a taste of what the poor mule felt,” said the
lieutenant. “A couple of you smart blue-jackets tie the fellow up to
that stump of a tree.”
The culprit resisted with all his strength, and attempted to bite,
scratch, and kick; but the two brawny seamen made short work of
his struggles, and soon had him securely lashed to the tree.
“One dozen,” said Mr. Thompson, nodding to Ned Burton
significantly.
My coxswain touched his cap, grinned, and rolled up his sleeve in
a workmanlike manner.
“Trust me to polish him off!” I heard him mutter to himself; “I can’t
abide them furriners that wreaks their bad temper on dumb animals
that can’t ’it you back agin—smother me if I can!”
As soon as the fellow’s flogging was over, he was turned out of the
camp and told that his services were no longer required. Then, the
scattered mules having been secured again, we once more set out
on our march towards the interior.
 The sun had now attained to a considerable altitude in the
heavens, and as there was an absence of wind, even upon the
heights, the heat and glare became intense. Not a single grumble
was heard, however, the men being much too gay and light-hearted
to care whether they were baked like salamanders or not. Our spirits
were kept up by the novelty and excitement of active service on
shore and the assurances of the guides that ere long we should
reach the outskirts of a forest, which it would be necessary to
traverse, and where plenty of shade would shield us from the sun’s
overpowering rays.
“Give me old Father Sol and an open country,” observed Ned
Burton to me, “in preference to jungle and the shade of trees. I’d
sooner chance a sunstroke than the ambush of a skulking enemy!”
“You think they may lie in wait for us,” I said. “If they do we shall
give them a drubbing.”
“I wouldn’t put it past them,” said my coxswain. “These Cubans, I
believe, are as wily as sarpents; and as to drubbing them and their
mutinous pals, it’s just a question of whether they’ve the sperrit to
meet us in the open or not. If they have, well, we shall just eat ’em
up. Trust the Rattler boys for an out-and-out shindy, Mr. Darcy.”
I was on the point of replying to my coxswain, when my attention
was entirely absorbed by the sudden apparition of a large and
compact cloud of horsemen emerging from behind some steep
scarped rocks immediately in front, and some four or five hundred
yards distant from the head of our column. They appeared to be
about to charge us.
“Cavalry, as I’m a living sinner!” exclaimed Ned, slipping a
cartridge into his rifle. “I’m jiggered if that don’t beat everything!”
It was certainly strange to find that the enemy had already secured
some mounted allies. It looked as if we should find this expedition no
child’s play—in fact, a great deal more like catching a Tartar.
“Prepare for cavalry!” thundered the gunnery lieutenant. “Keep
steady, men, and we’ll soon send them to the right about.”
 The horsemen were evidently provided with carbines, for as they
wheeled up into position they fired a wild volley at us, and then
dashed forward at full gallop straight in our direction.
 CHAPTER VIII.
“PREPARE FOR CAVALRY!”

T HE Rattler’s officers had reason to be proud of their little brigade

of seamen and marines. In this sudden emergency they were
calm, cool, and self-reliant. Their discipline and the celerity of their
movements were beyond praise. It was a severe test, and they came
out of it with flying colours.
As the enemy’s irregular cavalry came thundering down towards
us over the broken ground, we formed square, and, with loaded rifles
and fixed bayonets, stood ready to receive them. There was no time
to get the baggage-animals and native drivers into the centre of the
square, and so they were forced to remain huddled together in the
rear—a squad of marines being told off to guard them to the best of
their ability.
The horsemen seemed nothing daunted by our steadiness and
military formation, but swept on at a gallop. Two of their steeds,
however, stumbled badly on the rough ground and threw their riders,
after which they rushed away in the direction from which they had
come like mad creatures.
I was all excitement at the idea of this unexpected brush with the
enemy, and drew my loaded revolver from my belt. Ned Burton was
standing up just in front of me in the square, looking the essence of
determination and tenacious valour. The outer ranks were kneeling.
The rays of the tropical sun flashed on the serried lines of bayonets
and glinted on the less polished rifle barrels.
On came the cavalry with desperate bravery. Even on that rocky
ground they raised a cloud of dust. The horsemen had slung their
carbines and drawn their sabres, the blades of which flashed
ominously over their heads like the gleams of sheet lightning.
 “Give the swabs a volley,” muttered Ned Burton, “and we’ll empty
some of their saddles for ’em.”
At that very moment the order to fire was given.
Little tongues of flame and puffs of grey smoke darted from the
muzzles of the rifles defending one side of the square, and the crash
of a volley of musketry rang out into the air with almost deafening
effect. Amid it all I seemed to hear distinctly the thunder of the
chargers’ hoofs.
“Give them another volley!” shouted the gunnery lieutenant; and
darting about, hither and thither, amid the blinding, choking smoke,
we juniors repeated his order.
It was too late.
They were dashing horsemen these irregular cavaliers of the
enemy, for our steady, well-directed fire did not check them or smash
up their formation as we had expected. Though many of their
saddles had been emptied, a cheer of defiance arose from their
ranks as they dashed on into the curtain of smoke which enveloped
us, and which the sluggish air seemed powerless to disperse. When
they were almost upon us, their wings wheeled to right and left and
hurled themselves upon the two side faces of the square, while the
centre squadron dashed in upon the ranks where Ned Burton and I
were standing awaiting the onset.
“No more cartridges; it’s steel to steel!” said my coxswain grimly,
as he gripped his rifle firmly and prepared for the shock.
In another moment it came. Through the slowly-dispersing
vaporous smoke I saw the towering forms of the snorting chargers
and the fierce-looking, swarthy faces of their riders. Shouts of
defiance and rage arose on all sides, together with the angry clash of
steel as sabre and bayonet contended for the mastery, though these
sounds were almost drowned in the sharp stinging reports of the
revolvers brought into use at close quarters by the officers.
One naturally hesitates to speak of one’s own acts on an occasion
of this sort for fear of being thought egotistical; but being bound to
describe what actually occurred, I cannot avoid stating that I saved
my coxswain’s life on the occasion of this cavalry charge, and I am
very thankful that I had it in my power to do him that service.
It was a simple matter. Just at the point where Ned was standing
and forming part of the hedge of steel, the full brunt of the cavalry
charge seemed to fall, and for a few seconds I really almost feared
that the face of the square would be driven in. Certainly there was a
little disorder for a moment, and a swaying motion in the ranks which
told its own tale. However, it was only for a moment; for it is just at
these critical times that the tenacity of the British bull-dog comes into
play.
Amidst the hurly-burly of the mêlée I caught sight of Ned Burton
hard pressed by two horsemen. He seemed to have been brought to
his knees almost under the hoofs of one of the horses, while the two
horsemen were bending forward in their saddles and aiming terrific
blows at his head with their sabres. Ned was endeavouring to the
best of his ability to protect himself, but there was no doubt that he
was in great jeopardy.
My revolver was fortunately still loaded with three cartridges, and I
immediately took steady aim at the horseman nearest to Ned. No
bullet ever went truer, for it pierced the man’s heart, and he fell from
the saddle without even a groan and lay dead at my coxswain’s feet.
His steed, recognizing that there was no longer a restraining hand on
the bridle, took to his heels, and, with distended nostrils and wildly
tossed mane, galloped away from the battle-field.
Almost at the moment that I had accomplished this feat, the
charger of the other horseman was shot dead by one of our seamen,
and his rider was thrown to the ground with some violence. I instantly
rushed forward, seized the man, and demanded his surrender. Not
liking the look of my revolver, the barrel of which was within a couple
of inches of his temples, the fellow sullenly acquiesced, and I had
him disarmed and sent into the square under an escort. Ned had
nearly been crushed by the falling horse, but had fortunately
escaped with a few bruises.
The square remained unbroken. On three of its faces the
squadrons of horsemen had dashed like little whirlwinds, but in no
case had an entrance been forced. A fierce hand-to-hand struggle
had taken place for a few moments, but on every side our men were
triumphant. The cavalry charge was fairly repulsed, and as the
horsemen beat a hasty retreat they were terribly harassed by the
withering fire of our riflemen. We had not gone unscathed, however,
for one of our sub-lieutenants and a marine had been killed, and we
had one man seriously and three slightly wounded. In the volley from
their carbines the enemy had wasted their ammunition sadly, for not
a single shot took effect, all the casualties having occurred during
the hand-to-hand struggle. The enemy, we found, had suffered
severely, having lost eleven men killed outright and seven horses,
whilst we found upon the field eighteen wounded men, of whom five
or six were mortally injured. We had also secured half a dozen
prisoners, all of whom were Cuban insurgents. Needless to say, we
questioned these fellows closely; but they obstinately kept their lips
sealed and would divulge no secrets, though we tried to impress
upon them how foolish they were to league themselves with such
disreputable scoundrels as the mutineers of the Flying-fish.
The spot where this skirmish took place was not more than four
miles distant from the creek where we had landed, although any
view of the latter was shut out by an intervening ridge. We could see
the distant blue ocean stretching away to the horizon line, and dotted
here and there with the sails of passing vessels, but the Rattler and
the Flying-fish were invisible.
We at once told off a party to convey the wounded back to the
shores of the creek, that they might be taken on board the frigate as
quickly as possible. Mr. Triggs was placed in charge of this
detachment, which included the prisoners, and had orders to rejoin
the main body as expeditiously as he could, so that there might be
no delay.
A fatigue party was also told off to bury the dead—a mournful duty
which brings forcibly to one’s mind the horrors of warring with one’s
fellow-creatures.
Fitzgerald and I felt this most acutely, for we had lost a very dear
messmate, and it was part of our sad task to assist to lay him in his
narrow grave in this foreign land far from his home and kindred.
“It will break his mother’s heart,” said a mournful voice near us, as
we began to fill up the poor fellow’s last resting-place with the sand
which we had dug out.
I turned and saw that it was Dr. Grant who had spoken.
“You know her?” I said interrogatively.
The surgeon nodded assent. Then he quoted,—

“We laid him in the sleep that comes to all,

And left him to his rest and his renown.”