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Protein Electrophoresis, Immunofixation, and Immunodisplacement in Clinical Diagnosis. 2nd Edition, May 2011
Protein Electrophoresis, Immunofixation, and Immunodisplacement in Clinical Diagnosis. 2nd Edition, May 2011
by
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2
Contents
Preface 1
Acknowledgements 3
Introduction 5
(i)
3
Section C: Double and Biclonal Gammopathy 56
4
(ii)
Case#22 Smoldering Myeloma vs Symptomatic Myeloma 128
5
(iii)
Preface
Four years ago, Frank N. Bever, MD, Medical Director of Laboratories, St.
John Detroit Riverview Hospital, Detroit, Michigan, asked me (Zia Uddin) to assay
the serum of few clinically significant patients for serum protein electrophoresis
(SPE) and immunofixation (IF) employing the agarose gel on the semi-automated
HYDRASYS system (Sebia, France); and this was compared with the SPE and
immunotyping results obtained from the capillary zone electrophoresis (CZE) using
the semi-automated CAPILLARYS system (Sebia, France).
Permission for the research project (Study # SJ 1207-06) was obtained from
the Institutional Review Board (IRB) of St. John Health System, Detroit, Michigan,
and a formal authorization was issued by Peter Nickles, MD, Chairperson, IRB.
http://www.scribd.com/doc/14114106
In 2009, David G.Grenache, Ph.D. (an expert in this field) and I discussed
the possibility of putting together a similar compendium comparing the two most
commonly used agarose gel systems for SPE and IF of Sebia, France
(HYDRASYS) and Helena Laboratories, USA (SPIFE 3000). We also decided to
compare the SPE and immunotyping (IT) results of CZE (CAPILLARYS System,
Sebia, France) with the SPE and immunodisplacement (ID) results of a recently
introduced CZE system (V8) of Helena Laboratories, USA. In view of his joining
ARUP Laboratories, Salt Lake City, Utah, where he no longer directed this service
(SPE, IF, immunodisplacement, etc.), David G. Grenache, Ph.D. recommended the
name of his associate Christopher R. McCudden, Ph.D., Department of Pathology
and Laboratory Medicine, University of North Carolina School of Medicine, Chapel
Hill, NC, to collaborate with me on this project. A formal approval for this project was
obtained by Christopher R. McCudden, Ph.D. from the University of North Carolina
(Chapel Hill) Human Research and Ethics Board (IRB No. 09-2209).
During the course of our work on this new compendium we consulted more
than one hundred internists, oncologists, pathologists, hematologists, surgeons, etc.
to provide their expertise in understanding of the laboratory results and the eventual
clinical diagnosis. We are highly indebted to all these professionals for their
contribution in this compendium. As per the instructions of the IRB, we are not
allowed to disclose the names of these physicians (HIPPA), as this might
inadvertently disclose the identity of the patient, in view of the clinical diagnosis
associated with a particular patient and his or her physician.
September 2010
Several readers contacted us since the release of the first edition of this
compendium, and made comments and suggestions. In the second edition, we
have tried to accommodate them.
May 2011
Acknowledgements
The Medical Records and Medical Informatics staff at St. John Providence
Health System, Detroit, Michigan played a significant role in providing us the data of
more than 2000 patients reviewed for this compendium. We sincerely appreciate
their contribution in this project.
The technical staff (Ruthann Stewart and Leela Tansamrit) at the Research &
Development Division of Helena Laboratories, Beaumont, Texas, performed the
experiments on the Helena SPIFE 3000 and Helena CZE System (V8). Mr. John
O’Keefe, Manager, Immunodiagnostics and Electrophoresis, Helena Laboratories,
Beaumont, Texas, played a pivotal role in coordinating the laboratory aspects of the
data collection related to SPIFE 3000 and CZE System (V8), and we appreciate
their efforts.
We are highly appreciative of and thankful to Mr. Tipton Golias, President &
CEO, Helena Laboratories, Beaumont, Texas, for his encouragement and approval
of this project, and providing us the instruments, reagents and the facilities for the
collection of data presented in this compendium. Without the indispensible help of
Mr. Tipton Golias, this project was not feasible.
Finally we would like to thank the following persons at our institutions for facilitating
our work:
In 2009, Helena Laboratories, USA introduced their CZE system for SPE and
immunosubtraction.
Sebia, France
Parc Technologique Leonard de Vinci
CP 8010 LIsses-91008 EVRY Cedex-France
e-mail: sebia@sebia.com
Tel: 33(0) 1 69 80 80
Sebia, USA
1705 Corporate Drive
Norcross, GA 30093-2991
Tel: 770-446-3707
Fax: 700-446-5511
http://www.sebia-usa.com
Serum Protein Electrophoresis (Sebia): Sebia Hydragel Beta1 – Beta2 was used
to separate serum proteins on alkaline buffered agarose gels (pH 8.5) into six
fractions, i.e. albumin, alpha-1 globulin, alpha-2 globulin, beta-1 globulin, beta-2
globulin, and gamma globulin. The kits were used in conjunction with the semi-
automated HYDRASYS System (Sebia, France). Amidoblack was used to stain the
SPE gels, and the percent fractions were quantified using the scanner.
Serum Immunofixation (Helena): Helena SPIFE 3000 was used for the qualitative
identification of monoclonal gammopathies, and this procedure consists of first SPE
(see above), then followed by IF. Here again there were six tracks as employed by
the Sebia IF procedure (see above).
University of North Carolina Samples: Sera were salvaged from patient samples
after completion of physician-ordered electrophoresis testing. By necessity, these
samples were stored refrigerated for 1-2 days while testing was done before being
frozen for transport and testing by the Helena methods. Samples were transported
on dry ice. The reader should note that because of the storage times, it is probable
that there was degradation of some proteins (e.g. C3). It is also probable that there
was differential overall storage times/thawing times when analyzing the samples
using different methods and different institutions.
St. John Health System Samples: Sera for SPE were collected at eight hospitals
and > 125 drawing stations affiliated with the system. Here again by necessity,
these samples were stored refrigerated for 1-2 days while testing was done before
being frozen for transport and testing at the University of North Carolina McLendon
Clinical Laboratories, and Helena Laboratories. We anticipate similar possibilities of
degradation of samples as mentioned above for the specimens of the University of
North Carolina.
We therefore reiterate that this was not designed as a method comparison
study, but an educational endeavor and readers should not focus on minute
differences between traces, but the main point of each case. Specific method
comparison studies are likely to appear in the literature in the future.
Presentation of Clinical Cases and the Electrophoretic Data: For each case
presented in this compendium, we have presented a brief medical history, prior
diagnoses (if available), diagnosis at the time of the admission to the hospital (if
applicable), and any other information that might be helpful in the diagnosis of the
disease and the understanding of the electrophoretic patterns. The electrophoretic
data and other laboratory results for each case are presented as follows (except in
few cases where this protocol is deviated, e.g., Case #7, Case #13, Case #20,
Case #22, Case #23, and Case #26).
Immediately after the case history for each patient, we have provided the
concentration (g/dL) of the total protein, albumin, alpha-1 globulin, alpha-2 globulin,
beta-globulin and gamma globulin obtained from the manual agarose gel SPE
procedure (Sebia, HYDRASYS), and the quantitative values (mg/dL) of IgG, IgA,
and IgM (Beckman Coulter Image). In situations, where urine protein
electrophoresis and immunofixation were performed by the manual agarose gel
method (Sebia, HYDRASYS), the final interpretation was provided.
We have presented on the top half of the first page for each case the
agarose gel SPE scan (Sebia, HYDRASYS), and on top of the scan we have
embossed the agarose gel IF picture (Sebia, HYDRASYS). Besides the IF picture
we have also presented (left side corner of the first page for each case) the picture
of the separation (SPE) by the agarose gel procedure (Sebia, HYDRASYS).
On the bottom half of the first page, we have presented the agarose gel SPE
scan (Helena, SPIFE 3000), and on top this scan we have embossed the agarose
gel IF picture (Helena, SPIFE 3000). Besides the IF picture, we have also
presented the picture of the separation (SPE) by the agarose gel procedure
(Helena, SPIFE 3000) on the left side of the bottom half of first page for each case.
On the top half of the second page for each case, we have presented the
enlarged SPE scan obtained from CZE (Sebia, CAPILLARYS).
On the bottom half of the second page for each case, we have presented the
enlarged SPE scan obtained from CZE (Helena, V8).
On the third page for each case we have presented the following six scans
obtained from the immunotyping (Sebia, CAPILLARYS) of the serum proteins:
Note: The SPE scan (CZE) of Reference Track (green color) is superimposed on
the remaining five CZE scans of pretreatment sera (red color).
On the fourth page, we have presented the following six scans obtained from
the immunodisplacement (Helena, V8) of the serum proteins:
Note: The SPE scan (CZE) of Reference Track (green color) is superimposed on
the remaining five CZE scans of pretreatment sera (red color).
The majority of the readers of this compendium are familiar with the
interpretation of the serum protein electrophoresis and immunofixation patterns
(agarose gel). The interpretation of the serum protein electrophoresis by CZE is
virtually identical to scanning densitometry used in the agarose gel serum protein
electrophoresis, and thus needs no further comments.
The difference between the two curves (green minus red) is plotted in violet
color. This violet colored curve is plotted only to exhibit symmetrical
polyclonal immunoreduction process in a normal person, and is not actually
shown on the immunotyping scans of any of the five tracks (anti-IgG, anti-IgA,
anti-IgM, anti-kappa, and anti-lambda). A smooth and symmetrical shaped curve
after the immunoreduction step (red curve) indicates a polyclonal reduction of the
immunoglobulins from the serum. This kind of smooth and symmetrical shaped
curve (red color) is most commonly seen in a normal person after the liquid phase
reaction with anti-IgG. Uniform immunoreduction in the green curve, Fig No. A,
cannot be construed as a monoclonal band, as polyclonal immunosubtraction
results in the reduction of the gamma globulin fraction in a symmetrical manner.
In a typically healthy person the serum IgA is lower than the serum IgG,
therefore a very small difference is observed between the serum CZE scan and the
serum CZE scan after the anti-IgA reaction. This immunoreduction after reaction
with anti-IgA is primarily witnessed in the beta-globulin region. Similarly in a normal
person the serum IgM is even lower than IgA, therefore again a very little reduction
without change of the symmetry of the fraction is observed (virtually mirror image of
each other).
The situation in the case of kappa and lambda chains is slightly different.
Kappa chains are normally present in an approximate 2:1 ratio to lambda chains.
Therefore in a normal person after liquid phase reaction with anti-kappa, one should
expect 2/3 reduction in the gamma region. Conversely, in a normal person after
liquid phase reaction with anti-lambda, one should expect 1/3 reduction in the
gamma region. The magnitude of the reduction in the gamma globulin region for
both the kappa and lambda chains is significantly lower as compared to the IgG,
and this is obviously due to the high concentration of serum IgG as compared to
kappa and lambda chains in serum.
iii) Bone marrow clonal plasma cells <10% and low level of plasma cell
infiltration in a trephine biopsy (if done)
MGUS is also called pre-malignant plasma cell disease in view of the fact
that there is a risk of progression in some individuals to multiple myeloma, but in
vast majority of cases of MGUS a lymphoproliferative malignancy will never
develop,d and the patient dies of other causes. The reason for this discrepancy in
the progression of MGUS to MM is based on the “Risk Stratification Model” of
Rajkumar et al.d This model is based on three characteristics or risk factors, a)
serum M protein concentration, b) IgG isotype, and c) Free Light Chains ratio in
serum, and the four levels of the risk of progression of MGUS to MM are
summarized in Table 1.
In summary, a MGUS patient with all the three abnormal risk factors
[abnormal serum light chain ratio, non-IgG MGUS and a high serum M-protein level
(> 1.5 g/dL)] shall have a 58% absolute risk of developing MM after 20 years of
follow-up; as compared to a MGUS patient in whom all the risk factors are normal,
there is only 5% risk of progression to MM in the same 20 years.
Table 1
Absolute risk of
Risk stratification model
Absolute risk of progression progression to MM at 20
incorporating all three
to MM at 20 years, % years accounting for death
predictive factors:
as competing risk, %
Low Risk [serum M protein
<1.5g/dL, IgG subtype, normal 5 2
FLCs ratio (0.26-1.65)]
Low-intermediate risk (any
21 10
one factor abnormal)
A 65 year-old male was admitted to hospital with complaints of chest pain radiating
to the left and shortness of breath with a diagnosis of unstable angina. The patient
had cardiac catheterization, successful PTCA of the circumflex artery with stent
deployment.
A 63 year-old female fell at home in bathroom and the X-ray obtained in the
emergency department of the hospital showed right femoral hip fracture. The
patient underwent a right hip hemiarthroplasty and then sent for physical therapy
and rehabilitation to an extended care facility.
A 69 year-old female presented at the physician office with pleuritic left-sided chest
pain and dyspnea on minimal exertion. The patient was admitted after the chest CT
indicated the presence of bilateral pulmonary emboli, and the ultrasound of venous
duplex lower extremities indicated findings positive for left lower extremity deep
venous thrombosis.
Interpreters should be aware of potential fibrinogen artifacts and ensure that they
use immunofixation to confirm apparent SPE abnormalities. Interpreters should also
be cautioned that fibrinogen has been reported to cross-react with IgA (Snyder et al.
Clinical Chemical Acta 2006; 368: 192-194).
c. Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features
of fibrinogen and fibrin. Ann NY Acad Sci 2001; 936: 11-30.
Case #5: Acute Phase Reaction without Serum Iron Deficiency
Past Medical History: The patient had a history of pulmonary embolism, rheumatoid
arthritis, hypertension, gout, deep venous thrombosis, arteriosclerotic coronary
artery disease, and myelodysplastic syndrome with recent diagnosis of acute
myelogenous leukemia status post-induction chemotherapy.
Past Surgical History: Spinal fusion L4-L5 in 2009, left knee arthroscopy, right
rotator cuff repair, and bilateral carpal tunnel release.
Comment: Please compare the serum electrophoretic patterns of this case with
Case #6, in which the transferrin band is increased due to serum iron deficiency.
Case #6: Acute Phase Reaction (Bacterial Meningitis) with
Concomitant Serum Iron Deficiency
A 69 year-old female was brought to the emergency department appearing quite ill
with shortness of breath, ear pain and mental status changes (positive Kernig
sign). The patient was intubated upon admission for respiratory failure as well as
mental status changes and airway protection, and was treated with multiple
antibiotics. A lumbar puncture was performed, revealing pustular CSF. The CSF
culture was positive for Haemophilus influenzae. A blood culture was positive for
Haemophilus influenzae and Streptococcus pneumoniae and a urine culture was
positive for Klebsiella pneumoniae. A CSF culture was negative for acid-fast bacilli
(AFB) growth after six weeks.
MRI showed abnormal signal in T6 and T7 with destruction of the intervertebral disc
and residual paraspinal soft tissue abnormalities suggestive of abscess with
paraspinal extension.
Comment: SPE four weeks after the initiation of antibiotic therapy, showed no
monoclonal band, therefore confirming the disappearance of elevated fibrinogen
restriction (see page 52-54).
Another way to confirm the fibrinogen band is to perform IF using anti-fibrinogen.
Section C: Double and Biclonal Gammopathy
a Weinstein S, Jain A, Bjagavan NV, Scorrolini AG. Biclonal IgA and IgM
gammopathy in lymphocytic lymphoma. Clin Chem 1984; 30: 17110-1712.
b Uddin Z. Comparison of Agarose Gel Serum Protein Electrophoresis and
Immunofixation with Capillary Electrophoresis and Immunotyping.
http://www.scribd.com/doc/14114106, pp29
c Kyle RA, Robinson RA, Katzman JA. The clinical aspects of biclonal
gammopathies. Review of 57 cases. Am J Med 1981; 71: 999-1008.
rd
d Rajkumar SV, Kyle RA, Therneau TM, Melton LJ 3 , Bradwell AR,
Clark RJ, Larson DR, Plevak MF, Dispenzieri A, Katzman JA. Serum free
light chain ratio is an independent risk factor for progression in
monoclonal gammopathy of undetermined significance. Blood 2005; 106:
812-817.
e Ramasamy I. Letter to the Editor: Serum Free Light Chain Analysis in
B-cell Dyscrasia. Am Clin Lab Sci 2007; 37: 291-94
Case #8 Double Gammopathy: a) IgA-Lambda (major band)
b) IgG-Lambda (minor band)
Comment: The patient was treated for the urinary tract infection and discharged
back to the nursing home. No further treatment details were available after
discharge to the nursing home.
Case #10 IgM-Kappa and IgM-Lambda Biclonal Gammopathy
A 79 year-old male was admitted to the hospital, where his EKG showed a right
bundle branch block with atrial flutter. He was placed on telemetry and was found to
be quite bradycardic, where his rate dropped into the 20s while he was sleeping. A
pacemaker was inserted stabilizing his clinical condition. His comprehensive
workup indicated serum calcium of 14.5 mg/dL (Reference Value: 8.4-10.5 mg/dL)
and also elevated urinary total protein of 399 mg/24 hr (Reference Value: 0-150
mg/24Hr). The patient had a parathyroid hormone concentration of 387 pg/mL
(Reference Value: 15-65 pg/mL) prompting a CT scan of neck to screen for a tumor
as the source for the hyperparathyroidism; the CT did not show evidence of soft
tissue tumor. Chest X-ray showed no pneumothorax or pneumoperitoneum. Ultra-
sound showed kidneys within normal limits. X-ray of the spine cervical indicated no
evidence of traumatic spondylolisthesis.
Past Medical History: Hypertension, chronic kidney failure, coronary artery disease,
myocardial infarction, hyperlipedemia, benign prostatic hypertrophy, hernia,
cataract and carotid stents.
Comments: No further treatment data was available and the patient expired in the
nursing home.
Note: The reader will note the inferiority of immunosubtraction to immunofixation in
this case. Although, both bands are difficult to identify by immunosubtraction, there
would be no clinical significance to missing the second band.
Section D: Polyclonal Gammopathy
Case #11 Polyclonal Gammopathy: Increased IgG, IGA, and IGM with
Beta-Gamma Globulin Bridging
Discussion: The patient recovered from urinary tract infection and was returned to
the nursing home. The most probable cause for the beta gamma bridge is the
polyclonal increase in IgA that migrates in this region, and there is also increase in
IgG and IgM (though borderline) levels that completes the ‘bridge’ and is indicative
of broad increase in gamma globulin. In general, a polyclonal increase in gamma
globulin relates to immune response to enteric antigens (UTI in this case).
Case #12 Polyclonalgammopathy: Pancytopenia with Bone Marrow
Containing <5% Plasma Cells
A 54 year-old female was admitted to the hospital with complaints of upper quadrant
pain, fatigue, lower abdomen pain, and was found to be pancytopenic. CT scan of
the abdomen and pelvis were essentially normal. X-Ray of the hip showed no
acutely displaced bone fractures and without hip joint space narrowing.
Comment: Patient was followed by the family physician and we had no access to
her laboratory data.
*There should be a reduction in the polyclonal IgG to baseline. This is shown to
exemplify what happens if the sample is not appropriately diluted to account for
high immunoglobulin concentrations.
Section E: Hypogammaglobulinemia/Light Chain Multiple Myeloma
A 58 year-old female was admitted to the hospital with an acute deep venous
thrombosis involving her left lower extremity. CT scan of her abdomen confirmed
the presence of significant splenomegaly with generalized intra-abdominal
lymphadenopathy. Surgical pathology (left axillary lymph node) studies along with
flow cytometry, and bone marrow examination led to a diagnosis of follicular center
cell lymphoma. The patient was successfully treated with chemotherapy.
Five years later the patient was again admitted to the hospital, this time with sepsis.
A hepatobiliary imino-diacetic acid (HIDA) scan did not show definite gallbladder
activity. Severe hypogammaglobulinemia was detected and the patient had an
anaphylactic reaction in response to intravenous immunoglobulin infusion
(Gammaguard).
Serum Immunofixation: IgG, IgA, IgM, IgD, IgE, Kappa and Lambda chains (free &
total) were not detected.
Urine Immunofixation: No monoclonal band was detected.
No clinical or theoretical reason has been proven scientifically for the observation of
profound hypogammaglobulinemia 5-7 years after cessation of the chemotherapy
for lymphoma, except for the possible relationship to the genotype FCGR3-158V/F
polymorphism.b
Chest X-Ray: Abnormality in the right upper lobe. Chest CT: Two-centimeter mass
against the left chest wall and a 5-centimeter mass in the right inferior hilum.
Surgical Pathology: Left chest wall mass: Plasmacytoma, consistent with
involvement with multiple myeloma. Lung Biopsy: Moderately differentiated
adenocarcinoma. Bone Marrow Biopsy: 41% plasma cells, consistent with bone
marrow involvement. Flow Cytometry: CD56 positive monoclonal kappa positive
plasma cell population (approximately 15% of the leukocytes) indicative of plasma
cell dyscrasia.
Treatment: Alternating treatment every week for multiple myeloma and lung cancer
restored normal renal function; chest X-rays did not show any worsening of his right
lung adenocarcinoma.
Case #15 Hypogammaglobulinemia and Light Chain (Lambda)
Multiple Myeloma
A 58 year-old male presented to the emergency department after onset of left arm
pain, right knee pain, double vision, and severe headache. The patient was found to
have a fractured left humerus.
Chest X-Ray: Soft tissue lesion associated with the left ninth rib. Thoracic CT:
Multiple lytic lesions including the left transverse process of T6, ribs, left scapula,
left humerus, right AC joint, right clavicle and right glenohumeral joint.
Bone Marrow Biopsy and Aspirate: 31% plasma cells; trilineage differentiation and
adequate iron stores. Flow Cytometry-Bone Marrow Aspirate:
Increased CD56 positive monoclonal plasma cells (approximately 32% of the
leukocytes) indicative of plasma cell dyscrasia.
Bone Biopsy Right Femur: Portions of the bone with sheets of plasma cells within
the bone marrow. Consistent with multiple myeloma (immunoperoxidase stain
positive for CD 138).
Discussion: Treatment of light chain multiple myeloma was rendered for six months,
and according to the oncologist there was improvement in his clinical condition but
the patient expired due to other medical reasons. Additional information was not
available.
No monoclonal band (Free Lambda Chains) was detected in serum from CZE (V8
and CAPPILLARYS), but both the serum IF procedures (HYDRASYS and SPIFE
3000) were positive for free Lambda chains.
Note: As with the earlier case (#14), it is more difficult to identify low concentration
monoclonal light chains by immunosubtraction.
Case #16 Light Chain (Lambda) Multiple Myeloma
with normal Gammaglobulins
Comment: (1) Please compare this with another case of “Light Chain Multiple
Myeloma” (Case #15) where the gamma globulin was decreased and no
monoclonal band was detected from SPE (V8 and CAPILLARYS). (2) The free
lambda chains in the serum are quantified as 968.0 mg/dl. It should be noted that
this concentration is however highly overestimated because the entire gamma-
globulin region has a quantity of only 800.0 mg/dl. This means that the
nephelometric FLC concentration overestimates the ‘true’ FLC concentration with a
factor of 6 to 10. In a recent publication it is demonstrated that serum FLC are in
fact consistently overestimated using immunophelometry (ref de Kat Angelino et al.
2010)
Section F: Multiple Myeloma and other B-Cell Dyscrasia
CT scan of the brain was normal. CT scan of the chest was consistent with the
bilateral lower lobe pneumonia. The pelvic CT images were indicative of pathologic
fracture involving the left hip, and a small amount of non-specific pelvic fluid. A bone
scan showed abnormal uptake in the left hip. The patient underwent a closed
reduction and intermedullary nailing of the peritrochanteric fracture of the left femur.
She had a complicated postoperative course including respiratory failure,
subsequent intubation. She had vent-dependent respiratory failure with subsequent
tracheostomy placed in the ICU.
Bone marrow biopsy revealed a plasma cell neoplasm consistent with multiple
myeloma. Marrow had 80-90% plasma cells with CD138 staining. Flow cytometry
showed around 40% plasma cells, with expression of CD45.
The legal guardian of the patient was advised about her extremely poor medical
condition, and that treatment of her underlying hematological neoplasm would not
be of any benefit to her in terms of her quality of life. She was transferred to a
hospice, where she died.
2. Feyler S, O’Connor SJ, Rawstron AC, Subash C, Ross FM, Pratt G, Drayson
MT, Ashcroft J, Cook G, Owen RG. IgM myeloma: a rare entity characterized
by a CD20-CD56-CD117- immunotype and the t(11;14). Br J Haematol. 2008
Mar; 140(5): 547-51.
Case #18 IgG-Lambda Multiple Myeloma
A 78 year-old male was admitted for pneumonia, COPD exacerbation, rapid arterial
fibrillation. No evidence of hypercalcemia, and renal failure.
Peripheral Blood Smear: Red blood cell, white blood cell, and platelet morphology
were within normal limits.
Immunofixation:
IgD-Lambda monoclonal gammopathy
was confirmed by immunofixation studies.
Discussion: IgD multiple myeloma is a relatively rare type of plasma cell dyscrasia.
IgD accounts for ~1% of all cases of multiple myeloma and is associated with a
poor prognosis (median survival is 12-17 months). Occurring more commonly in
men between 55-60 years of age, the clinical features of IgD MM are bone pain,
fatigue, and weight loss. Typically, patients with IgD MM have relatively low
concentration monoclonal proteins. These features are reflected in this case, where
the patient presented with bone pain and the SPE revealed
hypogammaglobulinemia with a monoclonal protein concentration <0.3 g/dL. The
presence of associated kappa light chains is also common in this type of MM. This
patient was treated with chemotherapy and eventually underwent successful
autologous stem cell transplant.
Case #20 IgE-Lambda Multiple Myeloma
A 71 year-old female presented with low back pain, and arthralgia of the knees and
shoulders. No abnormalities concerning heart, lung, and abdomen were apparent
upon physical examination. There was no evidence of hepatosplenomegaly. No
pathological lymph nodes were palpable. Skeletal X-ray showed diffuse osteopenia
(patient was diagnosed with osteoporosis in 2008), and multiple lytic lesions were
detected in the skull, spine, and both femurs. Bone marrow aspirate indicated 25%
plasma cells; and flow cytometry and genetic studies were not performed. No
abnormalities of the skin were observed, and also there was no indication of allergy.
Serum Immunofixation:
IgE-Lambda monoclonalgammopathy (approximately 1.0 g/dL from
densitometry of SPE)
Urine Protein Electrophoresis & Immunofixation:
Free lambda chains were detected.
Acknowledgement:
We are highly indebted to Dr. Joannes F.M. Jacobs, Ph.D., MD (Laboratory Medical
Immunology, Radbound University Nijmegen Medical Center, Geert Grooteplein 10,
6525 GA Nijmegen, The Netherlands) for providing us the case history and the
laboratory data (Clin Chem 2010; 56:1368).
Case #21 Light Chain (Lambda) Multiple Myeloma
A 49 year-old female was brought to the emergency department with severe back
pain and shortness of breath. Chest CT showed a large right posterolateral chest
wall mass involving the right back musculature and destroying the tenth rib.
Additionally, there were multiple lytic lesions of the ribs and the thoracic and lumber
spine. CT guided biopsy of the chest wall mass revealed a poorly differentiated,
pankeratin-negative, non-epithelial infiltrating neoplasm confirmed with CD 138
immunostain to be a plasmacytoma.
Bone Marrow Biopsy and Aspirate: Three days after CT guided biopsy of the chest
wall mass (see above):
Trabeculae: Adequate
Comments: The patient underwent radiation and chemotherapy treatment, and the
SPE and quantitative immunoglobulins were as follows two months after the start of
the treatment.
Discussion:
Small abnormal protein bands (APBs) are frequently observed after high dose
chemotherapy and stem cell transplantation for multiple myeloma. It is proposed
that the bands appear as a result of transient dysregulation of B-cells as the
immune system recovers from therapy. The emergence of these bands can make
treatment monitoring somewhat challenging, as it can be difficult to differentiate the
original disease clone from newly emerging monoclonal bands. It is reported that
the presence of APBs after hematopoietic cell transplantation is a positive
prognostic indicator of overall survival. a, b
a Hall, SL, Tate J, Gill D, Mollee P., Significance of Abnormal Protein Bands in
Patients with Multiple Myeloma following Autologous Stem Cell
Transplantation. Clin Biochem Rev. 2009 Aug;30(3):113-8.
b Zent CS, Wilson CS, Tricot G, Jagannath S, Siegel D, Desikan, KR, Munshi
N, Bracy D, Barlogie B, Butch AW., Oligoclonal bands and lg isotype
switching in multiple myeloma treated with high-dose therapy and
hematopoietic cell transplantation. Blood. 1998 May 1;91(9):3518-23.
Case #24 Waldenstrom’s Macroglobulinemia
A 60 year-old male complained of severe back pain in 1999, and since then
underwent extensive examination including visits to a cardiologist, rheumatologist,
hematopathologist, neurologist, oncologist, and neurosurgeon.
Bone Marrow Biopsy, Aspirate & Peripheral Blood (2007): Hypercellular marrow for
age with trilineage hematopoietic maturation and focal lymphoplasmacytic infiltrate
along with small population of lambda positive monoclonal B-cells that lacked CD5
and CD10 expression, consistent with low level marrow involvement by
lymphoplasmacytic lymphoma.
Surgical Pathology (right ileum biopsy, 2007): Scant portions of fibrous tissue with
small lymphoid cells.
Discussion: The patient consulted two other physicians, who also advised him that
his chronic back pain was not related to Waldenstrom’s macroglobulinemia. It was
the opinion of his hematologist/oncologist that he required no treatment for his
Waldenstrom’s macroglonulinemia, as he has remained asymptomatic since
diagnosis.
Bone Marrow Biopsy & Aspirate: Cytogenetic studies performed on the bone
marrow aspirate showed a normal karyotype. Flow cytometry did not demonstrate
any evidence of monoclonality. The fragmented marrow showed a focal area of
fibrosis without any distinct Reed-Sternberg cells or variants were identified,
however the CD30 stain showed focal staining. The T-cells appeared phenotypically
normal and have a CD4:CD8 ratio of 2.4. The B-cells were polytypic and had a
kappa:lambda ratio of 1.5. These morphologic features, along with the
immunohistochemical stains were suggestive of involvement by Hodgkin’s disease.
Note:
1. Serum immunofixation revealed a monoclonal component typed as IgG-
Kappa.
Discussion: Plasma cell leukemia (PCL) is a clinical variant of plasma cell myeloma.
Defined by the presence of >2x109 clonal plasma cells in peripheral blood (and/or
>20% of the leukocyte differential), PCL develops in 2-5% of myeloma cases; PCL
also occurs as a primary disorder. As evident in this case, plasma cell leukemia is
commonly associated with light chains (or IgD). In contrast to this case, PCL
typically does not express CD56. The prognosis of PCL is poor, with a median
survival time of 7-11 months.
Case
#27 Solitary Plasmacytoma of Bone:
A 71 year-old male presented to the emergency department after a femur fracture, and
underwent a nailing of the left femur.
Surgical Pathology Report: Reemed Bones Left Hip: CD138 immunohistochemical
stain for plasma cells performed on three separate blocks showed a mild plasmacytosis
involving bone marrow with a cell count of about 7%.
Bone Marrow Biopsy: Normal to slightly mild hypercellular marrow with normal myeloid
erythroid activity, elements of plasma cells were seen, but there was no evidence of
tumor, lymphoma or granuloma throughout the biopsy material.
Bone Marrow Aspirate: 6% plasma cells, some of the plasma cells had nucleoli,
suggesting of myeloma cells.
Flow Cytometry: Small population of CD56 positive monoclonal plasma cells
(approximately 2% of the leukocytes) indicative of a plasma cell dyscrasia. No flow
cytometric evidence of acute leukemia, or lymphoproliferative disorder.
Cytogenetic Studies: No clonal abnormality was apparent. Normal male karyotype:
46,XY[14].
MRI and X-ray: No evidence of bone lesion.
This female patient was first examined in 1971 at the age of 25 years by a
rheumatologist, and a diagnosis of rheumatoid arthritis was made. She was initially
put on gold therapy, and subsequently showed microscopic hematuria without
proteinuria. She was examined by a urologist, and urinalysis in his office showed
some micropyuria but was negative for red blood cells. Endoscopic studies showed
a severe chronic trigonitis that very well could be responsible for her cellular
findings.
From 1977-97, she was examined by several physicians. She also had several
surgical procedures performed on her (joint replacement surgery in 1988, multiple
foot surgeries related to her arthritis, bilateral breast biopsies for benign disease, a
total abdominal hysterectomy and bilateral salpingo-oophorectomy for
endometriosis, and a tendon repair to her right hand). Her diagnoses in 1997 were
rheumatoid arthritis, osteoporosis, Felty’s syndrome, chronic microhematuria and
previous possible myasthenia gravis after using Penicillin.
64 years old male with symptoms of dizziness, loss of balance, fatigue, leg edema,
and previous history of Hepatitis C (genotype 2b) was examined at the out-patient
clinic, and was found to have severe hyponatremia. Patient was admitted to the
hospital and the following abnormal laboratory results were noticed:
Hemoglobin: 10.2 (12-18 g/dL), Hematocrit: 30.4 (36.0-54.0%), RBC: 2.77 (4.0-5.5
M/uL), MCV: 109.7 (80-94 fL), MCH: 36.7 (27-31 pg), Platelet: 57 (130-400 Th/UL),
Sodium: 103(135-145 mmol.L), Potassium: 3.0 (3.5-5.4 mmol/L, Chloride: 75 (98-
109 mmol/L), CO2 Content: 19 (23-34 mmol/L), Protime: 22.2 (11.3-14.5 sec),
APTT: 55.1 (22.5-35.0 sec), Fibrinogen: 62 (245-498 mg/dL), Aldosterone-serum:
2.5 (supine: 16.0 ng/dL or less) Renin Activity: 0.1 (supine: 0.2 -1.6 ng/ml/Hr).
Bone-Marrow Aspirate:
Microscopic Examination: 3-4% plasma cells. Non-Diagnostic. No evidence of
tumor, lymphoma or granuloma.
Flow Cytometry: Small population of monoclonal plasma cells (approximately 1% of
the leucocytes) consistent with a plasma cell dyscrasia. There is no evidence of
acute leukemia or lymphoproliferatiive disorder.
Test Result Reference Value
In view of the above stated criteria the diagnosis of IgM WM is ruled out in this
patient. It is also obvious that this patient does not have IgM multiple myeloma
(which is very rare, please see case # 17).
The Paneld proposed a term IgM-related disorders, in view of the fact that these
patients may have symptoms (such as cryoglobulinemia, amyloidosis, or
autoimmune phenomena such as peripheral neuroparthy, cold agglutinin disease.
etc.) attributable to the IgM monoclonal protein, but no overt evidence of lymphoma.
This patient does not meet the criteria for the proposed IgM-related disorder.
a
Novara F, Arcaini L, Merli M, et al. High-resolution genome-wide array
comparative hybridization in splenic marginal zone B-cell lymphoma. Hum
Pathol 2009; 40: 1628-37.
b
De Renzo A, Perna F, Persico M, et al. Excellent prognosis and prevalence
of HCV infection of primary hepatic and splenic non-Hodgkin’s lymphoma.
Eur J Haematol 2008; 81(1): 51-7.
c
Matutes E, Oscier D., Montalban C, et al. Splenic marginal zone lymphoma
proposals for a revision of diagnostic, staging and therapeutic criteria.
Leukemia 2008; 22(3): 487-95
d
Owen RG, Treon SP, Al-Katib A, Fonseca R, Greipp PR, McMaster ML,
Morra E, Pangalis GA, San Miguel JF, Branagan AR, Dimopoulos MA.
Clinicopathological definition of Waldenstrom’s Macroglobulinemia:
Consensus panel recommendations from the second international workshop
on Waldenstrom’s Macrglobulinemia. Sem in Onc 2003; 30:110-115.