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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 immunoelectrophoresis (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 (immunomodulatory 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 seeing 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, immunophenotype, 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 undetermined 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 prognosis. The final two chapters may be the most important—while this book is primarily intended for pathology trainees and practicing pathologists, it’s important to recognize that patients and clinicians depend on our timely, high quality diagnoses
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
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
Conventional and Molecular Cytogenetics in Plasma Cell Neoplasms 79 Michelle Dolan
Role of Flow Cytometry in Plasma Cell Neoplasms 101
Beenu Thakral, Kristy Wolniak and Michael A. Linden
Plasma Cell Neoplasms, A Therapeutic Approach 123
Brian L. McClune and Sagar S. Patel
Standardized Synoptic Reports for Plasma Cell Neoplasms: Integration of Laboratory and Clinical Data 143
Elizabeth L. Courville, Zohar Sachs and Michael A. Linden
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
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 management of plasma cell disorders. Serum protein electrophoresis (SPEP) is commonly used to detect the presence of circulating monoclonal proteins. Urine protein electrophoresis (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, focusing on clinical applications in the diagnosis and management of plasma cell disorders. 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 encountered 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 practice, 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
M. A. Linden, R. W. McKenna (eds.), Plasma Cell Neoplasms, DOI 10.1007/978-3-319-10918-3_1
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 Blymphocytes. 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 responsible 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 simplified 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 constant region ( CL). b Depiction of antibodies in monomeric (e.g., IgG, IgD, IgE), dimeric (e.g., IgA), and pentameric (e.g., IgM) forms
Antibody diversity, including antigen specificity and affinity, is the result of a remarkable process of somatic gene recombination, hypermutation, and class switching [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 immune 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 presence and quantification of monoclonal antibodies are essential in the application of WHO diagnostic criteria for disorders such as monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma, and Waldenström’s macroglobulinemia [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 separating 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 boundary 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
occurs at the ends of the U-tube and in solution [8]. Early forms of zone electrophoresis 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 electrophoresis 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 proteins 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 technological 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 constituent 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 size. An ion’s electrophoretic mobility is defined by the equation , 6 q rπη where r is 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.
EMF, however, is not the only factor that causes proteins to move in an electric 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, however, 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-containing 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 solution, frequently Tris-barbital or Tris-barbital/MOPS, along with additional stabilizers 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 electrophoresis systems therefore usually incorporate a process of loading patient specimens 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 (using 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
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 electropherogram 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 quantification. Electropherograms from capillary-based platforms demonstrating: d normal pattern; e hypogammaglobulinemia; 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)
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 overquantified. 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 between 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 relative percent of individual AUCs is multiplied by the serum total protein concentration 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 concentrated 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 laboratories 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 inner 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
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 recognize some nonprotein substances which appear as unexpected peaks on electropherograms. 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 immunoglobulin heavy chains and light chains. Common protein constituents of serum electrophoretic 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 concentration and in a largely overlapping manner. More precise separation and visualization can be achieved through techniques such as isoelectric focusing—either alone [21], in the context of 2D gel electrophoresis [22], or by incorporating immunofixation [23]. Such methods, however, are technically challenging, laborintensive, 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 individuals, a distinct but nonprominent albumin band is often apparent. The baseline
Table 1 Protein constituents of serum electrophoretic fractions [16, 18–20, 56]
Fraction
Prealbumin
Albumin
Alpha
Alpha-1
Alpha-2
Betab
Beta-1
Beta-2
Gammac,d
Proteinsa
Prealbumin (transthyretin)
Albumin
Alpha-1 antitrypsin
Alpha-1 acid glycoprotein (orosomucoid)
G-C globulin (vitamin D binding protein)
Alpha-2 macroglobulin
Haptoglobin
Ceruloplasmin
Transferrin
C3 complement
CRP
Immunoglobulins
aThis 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
cFibrinogen migrates near the beta-gamma interface in plasma specimens
dMonoclonal immunoglobulins can migrate in other fractions depending on their charge. IgA (polyclonal and monoclonal) typically migrates near the beta-gamma interface. Elevated polyclonal 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 numerous excellent resources [18, 26–28]. Examples of UPEP patterns are included in Fig. 3.
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 demonstrate 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 monoclonal 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
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, antibodycontaining 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] .
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 patterns 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 discordances with other laboratory results warrant further investigation to resolve any discrepancy. 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, immunotyping, 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 electropherograms 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 electrophoresis, as well as their corresponding reagents, are marketed by one of two diagnostic companies: Sebia, Inc. (Norcross, GA; www.sebia-usa.com) and Helena Laboratories (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 Angeles, 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 gelbased electrophoresis and immunofixation platform, HYDRASYS®2, can be
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 densitometry. Sebia also sells two capillary electrophoresis systems, MINICAP, and CAPILLARYS™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.
Helena Laboratories also provides both gel- and capillary-based electrophoresis systems. The SPIFE® 3000 provides gel-based electrophoresis and immunofixation. A front-end Auto Sample Handler can be used to automate preanalytic processing. 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 provides 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 withinvendor) facilitates integration of both technologies into a laboratory’s workflow. For example, a laboratory may prefer protein electrophoresis to be run by a capillary method, while gel-based IFE may be their method of choice for clonal characterization. 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 immunosubtraction 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 circulating 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. Another 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 potential 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.
Hemolyzed specimens can also yield a potentially abnormal band near the beta fraction. As with fibrinogen, a hemoglobin band would not yield a monoclonal characterization 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 electrophoretic 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, leading 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 refrigerated 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 fractions, 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 utilization 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 Mspike 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 quantification. Such an approach is certainly ideal, but is hindered somewhat in how to address results in patients with multiple M-spikes (some of which may have different
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 immunoglobulins, 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 overlaps 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 concentration of 8.5 g/dL. Serum total protein was measured using a biuret method (UniCel DXC, Beckman Coulter, Inc., Brea, CA)
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 average 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 improvement 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 providers who may utilize different laboratories, this can introduce confusion in rectifying interpretations and discrepant M-spike quantification. Indeed, a call for standardization 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, particularly if they contain excessive and/or extraneous information. Most laboratories 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 described 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 recommended 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
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 included 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 highcomplexity testing [36]. Given the need for quality and consistency, technologists who perform electrophoresis are frequently specially trained/dedicated to performing such testing, or they may do so in combination with other laboratory responsibilities. 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 physician 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-complexity testing, having a qualified laboratory director or pathologist/physician review interpretations 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 professional 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 monitoring 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
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 underquantified in relation to albumin. Furthermore, urine dipstick methods are remarkably 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 immunoglobulins is also limited. Quantification using these techniques is performed by comparing patient specimens to standard curves derived from known concentrations of analyte in solution. These standard curves, however, are often created using 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 patient’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 immunofixation 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.
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 immunoglobulins) 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 subsequent clinical consultation and assume that the patient has MGUS. Thus, a previously 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,
Fig. 7 IFE from a patient on rituxan therapy. A faint cathodal IgG-kappa is observed ( arrows)
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your old acquaintance, Captain Lenox—he will be Major Lenox immediately, I hear,—is coming to us. He forsook us quite shamefully when we went to Yorkshire, but he has written to apologise, and my son has invited him here for a week's furlough from next Wednesday. It is rather surprising that he should have leave again so soon: but it seems that he has been unwell, and we hear that he is a great favourite with the Colonel of his regiment, which may facilitate matters.
"I shall be glad to see him, for he really is a most agreeable young man. It is rather a pity that you will be away, if it would be any pleasure to you to renew the acquaintance. Of course Yorkshire is tempting still to all you young people. Captain Lenox writes that he is tired of England, and that he hopes to exchange soon into a regiment going to India or on foreign service. So I fancy this is the last time we may see him for a good while. I wonder he does not marry. He seems very much alone in the world, poor man, with so few ties in the way of relationship."
And that is all. She writes easily, not as if she in the least suspected the true state of affairs. I do not suppose that she does suspect. Sometimes I have felt that I could almost tell her all,—only never quite. For she has never sought or invited my confidence in this matter: and I cannot give confidence unasked.
Next Wednesday, for a week's furlough! That means—if we go to Glynde on Tuesday the 20th, he will be at The Park
one night after our arrival.
But our journey may be delayed; or his going away may be hastened. And even if we do reach Glynde on the day named, and he is there, is it likely that we shall meet?
Hardly,—unless he wishes it.
I dread the next fortnight of suspense. But I must hold myself strongly in. No one must see what I feel. To Lady Denham I can only intimate in most general terms a polite hope or willingness to see him again. Some in my place might perhaps say more, confidentially, but I cannot.
How do I know that Arthur Lenox would go to Glynde at all just now, but for the fact that he expects my absence?
CHAPTER
XXIX.
AND YET—!
THE SAME.
October 13. Tuesday Night.
THEY have come! I may as well write, for I am in no mood for sleep.
I suppose reunions after long partings seldom pass off exactly as one expects beforehand. Imagination sees only the poetry and delight of meeting. But the reality includes a good deal that is by no means poetical, or perhaps delightful.
When the travellers arrived, at the close of long waiting on our part, there was, of course, a general rushing of everybody into everybody's arms,—exclusive of Miss Millington and myself. Maggie was foremost in the rush: her face beaming.
Everybody said how well everybody else was looking; and then Mrs. Romilly grew a little hysterical, and a glass of water had to be fetched; and talk came spasmodically, as if no one knew exactly what subject to venture on next. Mr. Romilly, as usual, appeared upon the scene with a continuous murmur of small complaining tones: "Such a long journey—er; and the luggage not arrived yet—er; and the dear girls all so blooming; he only wished he could say as much for himself—er: and the dear boy absent—er; such
a trial—er; but after all so much to be thankful for—er!—" in the dolefully unthankful tone which good men do sometimes adopt when talking of their "mercies."
Mrs. Romilly has lost her young looks. She might be ten years older than when I saw her last, and she is worn, thin, faded, though still graceful, for nothing can do away with the charm of her bearing. Nellie is not precisely what I expected, not at all pretty or graceful, but perfectly ladylike, with a kind good sensible face. I like her much. There is such a charm in her absolute naturalness, her complete forgetfulness of self.
It was very curious to watch Maggie. At first I almost thought her extreme delight must be a little put on. But no: as the evening passed, I became convinced that it was entirely genuine, that in fact, this is the true Maggie. She has evidently reverted at once, and almost instantaneously, from her later to her earlier love. "Millie" has for a time filled the gap in her life: but no gap now exists to be filled. Millie has dropped from the position of necessary prop; Mrs. Romilly and Nellie being at once installed side by side in their old position.
I do not suppose Maggie means to be fickle or unkind. But it is very plain that "Millie" has ceased to be of any importance to Maggie.
For there were no wandering looks after Miss Millington, as Maggie sat on a stool by her mother's side, clasping one of Mrs. Romilly's hands, and gazing up in her face with eyes of sweetest content. It was a look which I have not seen in Maggie's face all these months. Can it be that in her own fashion, she really has suffered far more than I have believed, and has flown to perpetual engagements, tennis and "Millie," as a distraction from loneliness?
I could not but be sorry for Miss Millington, forsaken by her especial ally, and left outside the charmed circle, a forlorn nobody. The children had no eyes for any one but Nellie, and Maggie seemed glued to her Mother. There was in Mrs. Romilly's manner, when she spoke to Miss Millington, a certain slight air of distance and dissatisfaction, which I could not but notice: and Miss Millington plainly felt unhappy under it.
That gave me no pleasure. I am glad to be able to say so honestly. I have not sunk so low as to rejoice in another's pain, even though this "other" has been in a sense my enemy.
Towards me, Mrs. Romilly was all sweetness and affection. She brought me forward, held me lovingly, thanked me again and again for all I had done, bade her husband and Nellie unite their expressions of gratitude, told the girls how dear I was to her. And I—well, I could not but feel her kindness, even while oppressed by it. I had such a stupid longing to slip away from the flow of words, and to be let alone.
Nobody would imagine this evening that Maggie does not like me. She has dropped in the most easy and marvellous manner into her Mother's tone. Instead of glowering or averted glances, I meet softly smiling grey eyes. Instead of rushing off to Millie, she slides her arm quietly through mine, as she stands by her Mother.
Is it genuine?—Or is it assumed? Has she been all these months under an unnatural strain, and in bondage to "Millie," and is this the real Maggie, set free from trammels? Or is she so utterly weak and pliable a character, as to be heart and soul under the dominion of any one present for whom she most cares?
I cannot solve the riddle? I only know that it is shallowness, not depth, which usually results in such a riddle. Lake waters are transparent, while a little pond will be muddy; and nothing is more difficult to see through than mud. But in this mood, Maggie is so lovable, that I can hardly wonder at her Mother's devotion.
At all events, Gertrude Romilly is satisfied. "My Maggie is sweeter than ever," she said, when bidding me goodnight; and for her sake, I was pleased that she saw no farther below the surface. I cannot see to the bottom of the pond myself; but, alas! I know there is mud.
"And the other dear girls, so improved," she went on. "Thyrza and Elfie especially. Your doing, dearest Constance!"
I ought to be gratified, but I can hardly say that I am. Everything is a burden just now. I only feel thankful that the long evening has come to an end.
Friday. October 16.—Plans are unchanged. We go south next Tuesday, starting early, and arriving before night. Nellie had a letter from Gladys to-day, in which she writes with delight of her friend's return, and mentions in passing that Arthur Lenox will be at The Park,—"till Wednesday evening." Nothing more.
Nellie read aloud a few sentences from the letter; and when Captain Lenox' name was mentioned, Miss Millington's eyes came straight to my face. I did not look up, but I was keenly conscious of her fixed stare. I felt myself turning slowly cold and pale; and I knew that she must see it. No remarks were made at the time; only somewhat later, Mrs.
Romilly said affectionately, "Constance, my dear, I don't like to see you so worn-out. I shall have to send you soon to your sister for a holiday." But I made nothing of it.
Then all at once she spoke to me about Miss Millington, expressing a grave sense of doubt, and begging to hear confidentially my honest opinion. Was Miss Millington, or was she not, a desirable companion for the girls, and a fit person to train the children?
I do not know how much or how little my friend has heard. Nellie, being everybody's confidante, is generally well acquainted with everything, and doubtless she has used her own discretion in passing on facts to her Mother.
I declined to give any advice in the matter. This was the only line open to me; and I said so. I had not succeeded in winning the affection of Miss Millington: and I did not think I ought to count myself a fair judge.
Mrs. Romilly looked at me in questioning silence. Then she said, "That is enough. You would never fail to say what you conscientiously could in favour of anybody."
This may be true: I hope it is. But I would rather have no hand or voice—even tacitly—in the dismissal of Miss Millington: and I cannot but expect that to be the consummation.
Saturday. October 17.—About an hour after lunch today, I was in the back-garden, half-reading, half-dreaming. The girls had all started together for a long ramble, from Nellie down to Pet.
A footstep made me look up: and I saw Miss Millington hurrying along the path, her face aflame, her eyes glazed with tears. Finding me there alone, she stopped short in front of me, and burst out
"It is your doing!"
"What is?" I asked, though indeed I could guess. She tossed her head, and bit her lips, glaring at me.
"Oh, you needn't pretend! You know very well! It's what you've been scheming for ever since you came! I know well enough. But I'll be even with you yet. I'll have my revenge."
"It would be idle to pretend that I cannot guess what you may mean," I said seriously. "But you are mistaken, Miss Millington. I have not moved in the matter."
"Oh, I dare say! When your very words show it! You knew she was going to get rid of me! And you persuaded her."
"I did not know it," I answered. "I knew only that Mrs. Romilly did not seem satisfied: and I could guess that she might have spoken to you. That is all."
"Oh, of course! It's all very fine When you can twist Mrs. Romilly any way you please! And everybody knows it!" she said, jerking out short sentences With gasps of passion between. "I understand! You've gone and told her! That stupid cooked-up tale about your accident! Such a fuss about nothing! And it's all untrue! A downright lie! And she has told me I'm to go! Doesn't like my influence! I know what that means! But I'll be even with you yet!"
"You are wronging me," I said, and I found it difficult to control myself. "I have told Mrs. Romilly nothing. She questioned me, and I declined to answer."
"Oh, I dare say! With a virtuous air, just showing what you meant! And I'll have my revenge!"
"Is that Christian, Miss Millington?" I asked. It was grievous to see her look. "I cannot pretend to think that you have acted rightly towards me, or with the girls. But I have done my best to keep from influencing Mrs. Romilly. If you go, it is not my doing."
"But I say it is," she retorted violently. "And I'll never forget,—never! I've got you in my power too, though you mayn't think it, and I'll make you feel my power. I tell you I will."
"How am I in your power?" I asked.
A kind of chill ran through me at the words. I thought of her stealthy peep at my journal. But I could not tax her with that; I had promised not to compromise Elfie.
She burst into angry sobs. "It doesn't matter how. You've nothing to do with what I mean. I know, and that's enough. I'll never forget,—never!—what I owe to you. I'm to leave, and I'm not to be recommended, and it is all your doing. If my mother and sister come to want, that's all your doing too! And I'll be revenged! Sneaking and telling lies like that! But I declare I'll have my revenge! I'll be even with you!"
"Miss Millington!" an astonished voice said close behind her, and Mrs. Romilly appeared, passing round the clump of bushes which shut off the house. "Is this Miss Millington speaking? I can hardly believe my own ears."
The girl looked down sullenly, crimson and sobbing.
"Miss Millington is under a mistake," I said. "She believes that I have influenced you, Mrs. Romilly, in deciding to part with her."
"And if you had!—What then? You are my friend. When I asked your advice, you declined to give it: but I had a right to ask."
A short silence followed. I did not say another word. Miss Millington stirred as if to escape.
"Stay! One moment, if you please," Mrs. Romilly said coldly, and she reined up her head in her graceful way. I do not think I should admire the gesture in anybody else; it is so seldom graceful: but it suits Mrs. Romilly.
"Stay!" she repeated. "This settles the matter, Miss Millington. One who can speak in such a manner to my friend is no fit companion for my children. You will go to your own home on Monday, instead of accompanying us to Glynde. Of course you will receive three months' salary in full, from to-day: and I will also undertake your travelling expenses to London. I hope you will take warning, and learn a different spirit for the future. And remember,—I am able to recommend you as companion to a lady, but not as a governess."
One scowl of positive hate was cast sidelong at me, and Miss Millington fled. I do not think Mrs. Romilly saw that parting glance. She sat down by my side, and I found her to be trembling.
"Anything agitating tries me," she said. "But it must be so. My husband will agree with me, fully." And when I would have pleaded for some slight relaxation of the sentence, she
refused to listen. "No, no, not another word, Constance! I cannot sacrifice my darlings' good to her feelings. She has done harm enough already. Have you not seen?"
"I have feared," I said.
"My Maggie used to be so scrupulously true," she said in a low voice of positive anguish.
I could not deny the change, but I spoke comfortingly, foretelling that under her influence and Nellie's, there would soon be a difference. "Maggie loves you devotedly," I added.
"Yes: but is that all?" she asked, her lips quivering. "I thought my Maggie was the one of them all who had most truly given herself to the service of Christ. And now—Yes, I see it in Thyrza and in Elfie,—the fight going on. But Maggie —my Maggie—could I have been mistaken in her before?"
She broke down, and cried bitterly. I did my best to comfort her. No doubt she is seeing daily more and more those faults and weaknesses which have most markedly developed in Maggie during the last few months. I would not suggest that her whole past estimate of Maggie's character has been a mistaken estimate. If her eyes must be opened, I would rather leave them to open naturally.
Tuesday Evening. October 20.—Only time for a few words. It is very late. The journey has been long, and we did not reach Glynde House till past seven. Since arrival, all of us have been hard at work, unpacking for the night.
Miss Millington left us in London. Her good-byes were hurried and cold. She looked no one in the face, and she would not shake hands with me.
Shall we ever meet again? Our intercourse has been far from happy. Yet I cannot but feel a kindly interest in one with whom I have lived for so many months,—the more so, as I fear she will bring sorrow on herself, wherever she may be.
Maggie has not seemed troubled at losing Miss Millington, being entirely absorbed in her mother—or, if her mother is not present, in Nellie. It is very curious to watch Maggie now, and to contrast the state of things only ten days ago. Then she and "Millie" were inseparable; if "Millie" was pleased, Maggie was pleased; if "Millie" was out of temper, Maggie was out of temper. Now all is changed.
I suppose Maggie cannot stand alone. She must be supported by—must be under the dominion of—somebody else. When Millie was her prop, Maggie thought, felt, acted, in unison with Millie. Mrs. Romilly being now her prop, Maggie thinks, feels, acts, in unison with Mrs. Romilly. This is the real love; that was only a spurious attachment. But the character which can undergo such phases is scarcely to be admired.
Maggie's affectionate manner to myself is amazing. It seems to be quite natural, not assumed; and no doubt she does at the moment honestly feel what she expresses. At all events, it gives Mrs. Romilly pleasure. So I accept the warmth, and I make no remarks; only the affection of Thyrza and Elfie is worth more to me.
I hardly know why I write all this. My mind is full of other matters.
A note from Lady Denham to Mrs. Romilly mentions Sir Keith's intention of calling to-morrow morning, "with their guest, Captain Lenox."
Somehow I feel very calm; not shaken or tremulous. Things will be well, however they turn out. I love the thought that all my life is in a Father's keeping.
"Thy way, not mine, O Lord, However dark it be; Lead me by Thine own Hand, Choose out the path for me.
"Smooth let it be, or rough, It will be still the best; Winding or straight, it leads Right onward to Thy rest.
"I dare not choose my lot; I would not, if I might; Choose Thou for me, my God, So shall I walk aright."
I think I can say these words from my heart to-night.
Wednesday Evening. October 21.—I can still say the same. I would not choose for myself. But the disappointment has fallen heavily.
Sir Keith called alone, not long after twelve. He said that Captain Lenox had suddenly found it needful to go off by an early train, instead of waiting till this evening. A letter by post had caused the change of plan.
"A singular fellow—Lenox!" Sir Keith said musingly. "One never knows what he will do next. Curiously reserved too."
That was all, or nearly all, said about the matter. Nobody seemed to count Captain Lenox' defection a thing of any moment. I of course made no remark: and Miss Millington's inquisitive eyes were absent: while Sir Keith, usually very observant, was absorbed in Thyrza.
I will not allow myself to think who may have written that letter. What use? I cannot know, and I must not run the risk of suspecting unjustly. Better to take the pain straight from my God. Nothing comes, not permitted by Him.
But will life ever again seem worth the trouble of living?
CHAPTER XXX. A REAL FIVE-SHILLING BOOK!
GLADYS HEPBURN'S DIARY.
October 6. Tuesday.
THE first copy of my book arrived by this evening's post. Eleven more copies are to follow by rail. We have been looking out for it very anxiously.
October 7. Wednesday. I think my book looks very nice; so prettily bound: and Mother is so pleased. It seems strange to have written at last a real five-shilling tale,—my childish dream come true. What a long while I have been looking forward to this! I don't know exactly when I first began to expect to publish, though I can remember writing little stories at nine years old,—others say, at seven. But at fourteen, I had quite made up my mind to bring out a fiveshilling book some day,—if I could, I mean. And now I am nineteen!
After all, such things don't really affect one's happiness. I am very glad and thankful, but I feel quiet about it,—not as I should have expected.
October 10. Saturday.—Such delightful news! The Romillys are coming home!
Eustace goes to a clergyman in the country, for study; and Mr. and Mrs. Romilly and Nellie travel to Beckdale for just one week, and then the whole party comes south.
Delicious!
Mrs. Romilly seems really stronger, they say,—almost as if the accident and illness had done her good in the end, instead of harm. I suppose troubles often do that. The doctors think she may safely spend the winter at home.
A few words from Nellie tell me this. I am so happy at the thought of seeing Nellie again.
People seem pleased with my book, on the whole. A great many kind things are being said about it,—some of course only out of politeness, but others I fancy are real. Aunt Anne complains that my two stories are exactly alike, because, she says, there are three boys in each, and she is afraid I am "in danger of the usual fault of repetition, common to all young authors." But I don't know what she means, or how she counts, for there really are five boys in one, and six in the other. Ramsay declares that aunt Anne only thinks a little dose of criticism wholesome for me. I don't call that real criticism though; if it were, I should like it, because I do want to be shown real faults in my writing.
October 15. Thursday.—Not a week now before I expect to see Nellie again! I am counting the hours. And that dear Miss Con too! I hope she has quite got over her accident. I am working at a little story for children, which I think of calling "Winnie." When it is done I mean to offer it to the same Society that accepted my first book: and afterwards I shall most likely write another tale for Mr. Willis.
Captain Lenox is actually at The Park again. I do wonder if it means anything. He is to stay till next Wednesday, so he and Miss Con might meet. He called to-day with Sir Keith, but not a word was said about Miss Con. I took very
good care not to bring her name forward this time, I was so afraid of making mischief. Mother says I was right. It almost seems to me, from something he said, that he doesn't know yet about the Romillys and Miss Con coming before he leaves.
October 21. Wednesday.—Mother would not let me go to Glynde House this morning. She was sure the Romillys would be too busy: and of course she is right; only I did not know how to keep to my thinking of Nellie being so near. But still I stuck to work; for it does not do to be mastered.
After lunch, in came Lady Denham, for a long talk. She always seems so pleased to see Mother. A good deal was said first about the Romillys: and then she told us that Captain Lenox had left by an early train, directly after breakfast. I felt very disappointed, thinking of Miss Con. Lady Denham laughed a little, and said, "He is a curious man, particularly agreeable, but erratic."
Mother asked, "Would he have objected to meet the Romillys?"
"Why, no, I think not," Lady Denham said. "My son proposed last night that they should call together on Mrs. Romilly to-day, and he seemed to fall in with the proposal; but a letter by this morning's post altered his plans."
Mother said, to my surprise, only I know she and Lady Denham don't mind what each says to the other—"I have sometimes fancied there might be something between him and Miss Conway."
"So I imagined at one time," Lady Denham answered. "But I think it is a mistake. Miss Conway speaks of him with complete indifference; and he never mentions her."
Lady Denham hesitated, and looked at me, before going on—"Gladys is safe, is she not? Between ourselves, the letter this morning was in a lady's hand. He left the envelope on the table, close to my plate, so I could not help seeing it. I know he has no near relatives. One has no business to build on so slight a foundation, but he looked very strange over it,—so strange that I asked if he had had any bad news. He gave an odd short laugh, and said —'Nothing of consequence: only he found it needful to leave by an early train.'"
Lady Denham must feel very friendly towards Mother, to say so much. It is not her way to speak out generally. But I don't think Mother is so sure as she is that we are mistaken about Miss Con and Captain Lenox.
As soon as she was gone, I hurried off to Glynde House. The first person I saw was Mr. Romilly. He was in the hall; and he gave me such a kind welcome, that really I felt ashamed of not liking him more. He smiled at me, and took my hand in his, and he seemed such a pretty little bit of dainty old china! Only I do wish he could be kept under a glass shade, for he is fit for nothing else.
And then I saw Mrs. Romilly. She looks years and years older than before her illness: but she reared up her head and squirmed herself about, just as she always does; and she made me turn so fearfully shy, I had not a single word to say, till Nellie came. And then I forgot Mrs. Romilly's existence, and I was all right.
One very good piece of news I heard the first thing: and that is that Miss Millington has gone home, and is not to return. I don't understand exactly how or why. We shall hear more later. What a relief that she has vanished!
Nellie looks so well: and she is just the same as always, the darling! She has enjoyed being abroad, and now she enjoys coming home. Somehow Nellie always enjoys everything. Mother says it is because she has that rare gift —"a mind at leisure from itself." And I do think Mother is right.
Maggie is exactly what she was before she went away: always flying round after Mrs. Romilly; or if Mrs. Romilly is not within reach, always hanging on to Nellie. Mother says she has grown prettier: but I do not see it. I never cared very much for Maggie.
Thyrza is changed. I never supposed she could grow so handsome! The first moment I saw her, I felt quite startled. Her eyes used to look at every one in a kind of grim way, as if she wanted to fight: and now they are beautifully soft. And when she kissed me, instead of stooping stiffly, like a young fir-tree trying to bend, and giving a poke like a bird's peck, she was gentle and almost affectionate. I always used to think she could not endure me, but perhaps it was shyness and not being happy. I am sure Miss Con has made a great difference in Thyrza's life.
I did not see Miss Con, as I had hoped. Thyrza said she was tired, and had gone to her room to rest. That looks as if she were not strong yet.
October 22. Thursday.—The first notice of my book came this morning: a short one, but good; all praise and no blame.
I have just finished "Winnie." I hope to get it off tomorrow; and to start a new tale next day.
Miss Con came in before lunch, and oh, she is so altered! It has made me really unhappy. She is much thinner, but that is not the worst. It is the look in her eyes that I mind most,—such a sad sweet look, as if she had been through a great stretch of trouble and pain, and were not out of it all yet. And her cheeriness of manner is gone. Mother told her she looked tired, and put her into an easychair; and though Miss Con laughed, she did not resist, but sat listening, hardly speaking at first, only giving a little smile, if her eyes met ours.
Presently Mother asked her how she was; and Miss Con smiled again, and said, "Lazy, rather! Beckdale has used up my reserve-powers."
"You will have to get away for a holiday," Mother said.
"At Christmas, perhaps," she answered. "Lessons must go on regularly for a while first."
Then she asked about my writing, and was so kind; not merely polite, but full of real interest. It was the only time she brightened up.
When she was gone, Mother said, "If they do not take care, she will break down altogether. Too much has been put upon her."
I believe Miss Millington has been Miss Con's greatest bother.
