The following case is an interesting overlap of
Hematopathology and Molecular Diagnostics, and shows the utility of sequencing
to detect a cancer before biopsy could.
A 63 year old gentleman presented to a heme/onc physician
with six months of intractable anasarca, fatigue, and a recent mild
thrombocytopenia (Table 1). They were otherwise in healthy condition. The
physician initiated a lymphoma work-up that included a bone marrow biopsy. The
tests were negative for M-protein.
The bone marrow biopsy was somewhat limited, but the core contained multiple marrow elements. After a thorough review by a Hematopathologist, no evidence of dysplasia or other irregularities could be detected (Image 1). Flow cytometry detected no aberrant blast population. Cytogenetics detected 20del [16/20] and 5del [3/20]. These findings did not clearly indicate a specific diagnosis.
As the clinical suspicion for a malignancy was high, the
bone marrow specimen was sent for sequencing on a 1385-gene panel test. The
test included tumor-normal matched DNA sequencing (“tumor” sample: bone marrow,
normal: saliva), RNA whole transcriptome sequencing on the bone marrow, and
Copy Number Variant (CNV) analysis. Tumor-normal matched sequencing helps rule
out variants that are normal and present in the patient.
Somatic mutations were determined as those that were present
in the “tumor” sample and not in the matched normal sample. The somatic
variants found are listed below with their variant allele frequency (VAF) in
parenthesis. Recall that a VAF of 40% means that a mutation is present in the
heterozygous state in 80% of cells.
IDH2 (p.R140Q, 46%)
SRSF2 (p.P95T, 51%)
CBL (p.R499*, 47%)
KRAS (p.K117N, 12%)
The mutations in these genes are commonly found in myeloid
cancers including myselodysplastic syndrome. Activating mutation in IDH2 (isocitrate dehydrogenase 2)
increase the production of the oncometabolite 2-HG, which alters methylation in
cells taking them to an undiffereitiated state. SRSF2 (Serine And Arginine Rich Splicing Factor 2) is a part of the
spliceosome complex, which regulates how sister chromatids separate from each
other. Failures in the proper function of the complex creates genomic
instability. CBL (Casitas B-lineage
Lymphoma) is a negative regulator of multiple signaling pathways, and loss of
function mutations (as seen here) lead to increased growth signals through
several tyrosine kinase receptors. KRAS
(Kirsten RAt Sarcoma virus) is an upstream mediator of the RAS pathway, which
acquires mutations that lead to constitutive activation and sends growth
signals to cells causing them to proliferate.
Furthermore the CNV analysis
also found the heterozygous loss of chromosome 20 as reported in cytogenetics.
CNV analysis did not detect chromosome 5 deletion, as it was below the limit of
detection (20% for CNV analysis).
These mutations are all individually common in MDS, but the co-occurance of each gives very strong evidence that MDS is the diagnosis (Figure 3). There have also been studies that provide prognostic implications for several of the genetic mutations present. Some mutations like SRSF2 or CBL at high VAF (>10%) indicate a poor prognosis, but mutations in IDH2 or TP53 at any frequency have not only a high chance of progression, but also a faster time to onset of disease. Another non-genetic risk factor for developing MDS is an elevated RDW, which we saw in our patient.
All of these high-risk factors together led us to push for a diagnosis of MDS based off of molecular findings, and the patient was started on treatment with Azacitadine. Our assessment was confirmed 3 months later when, the patient’s follow up bone marrow biopsy showed significant progression with megakaryocytic and erythroid dysplasia and hyperplasia and reticulin fibrosis MF2 (Image 2). Aberrant blasts were detected (1-2%), but not elevated. This demonstrates how molecular findings predicted and predated the patient’s rapid progression to morphologic disease.
In summary, multiple molecular mutations indicative of MDS
were found in a symptomatic patient’s unremarkable bone marrow biopsy months
before a rapid progression to MDS.
Steensma DP, Bejar R, Jaiswal S et al. Blood 2015;126(1):9-16.
Sellar RS, Jaiswal S, and Ebert BL. Predicting progression to AML. Nature Medicine 2018; 24:904-6.
Abelson S, Collord G et al. Prediction of acute myeloid leukemia risk in healthy individuals. Nature 2018; 559:400-404.
Desai P, Mencia-Trinchant N, Savenkov O et al. Nature Medicine 2018; 24:1015-23.
Becker PM. Clonal Hematopoiesis: The Seeds of Leukemia or Innocuous Bystander? Blood.2016 13(1)
-Jeff SoRelle, MD is a Chief Resident of Pathology at the
University of Texas Southwestern Medical Center in Dallas, TX. His
clinical research interests include understanding how the lab intersects
with transgender healthcare and improving genetic variant
A 66 year old man with past medical history of recently diagnosed Clostridioides difficile colitis presented to emergency department with diarrhea, weight loss of 52 pounds in 4 months, and occasional night sweats. CT imaging revealed dilation of small bowel with thickened mucosal folds. The duodenum was subsequently biopsied to reveal diffuse intestinal lymphangiectasia containing PAS positive and Congo red negative eosinophilic material and lamina propria foamy macrophages. Laboratory investigations revealed normocytic anemia, proteinuria, and peripheral IgM kappa monoclonal gammopathy.
marrow aspirate shows increased plasma cells and mast cells. H&E stained
sections demonstrate a normocellular bone marrow with trilineage hematopoiesis
and involvement by 35% plasma cells. By immunohistochemistry, CD138 highlights clusters
of plasma cells that predominantly express kappa light chain restriction.
and Mutation Analysis
demonstrated loss of chromosome 11 and gain of chromosome 15, which was
consistent with plasma cell dyscrasia. MYD88 mutation analysis did not
detect the mutation.
findings of the patient’s normocytic anemia, IgM monoclonal gammopathy, and
intestinal lymphangectasia with an associated plasma cell dyscrasia involving
the bone marrow favor a lymphoplasmacytic lymphoma/Waldenström
macroglobulinemia (WM) is a malignant B-cell lymphoproliferative disorder characterized by lymphoplasmacytic infiltration of the bone
marrow and peripheral IgM monoclonal gammopathy.1 It is rare with an
overall incidence of 3 per million persons per year, accounting for 1-2% of
hematologic cancers.1 It
occurs predominantly in Caucasian males, with a median age of 63-68 years old
may be asymptomatic for years and require observation or experience a broad
spectrum of signs and symptoms. These symptoms may be attributable to the tumor
infiltration of the bone marrow and lymphoid tissues, IgM circulating in the
blood, and IgM depositing into tissues. The most common clinical presentation
of WM is fatigue and nonspecific constitutional symptoms, such as fever, night
sweats, and weight loss, due to normochromic, normocytic anemia. 20-30% of
patients may exhibit lymphadenopathy and hepatosplenomegaly due to infiltration
of peripheral tissues. High concentration of IgM in the circulation may lead to
hyperviscosity, resulting in oronasal bleeding, gingival bleeding, blurred
vision due to retinal hemorrhages, and neurological symptoms, including
headache, ataxia, light-headedness, dizziness, and rarely, stroke.2-3 The gastrointestinal manifestations are rare; however, IgM monoclonal
protein may deposit into the lamina propria of the GI tract, causing diarrhea,
steatorrhea, and GI bleeding.4 Other
IgM-related manifestations include cold agglutinin hemolytic anemia,
cryoglobulin, and amyloid deposition in tissues.3
of WM includes evidence of IgM monoclonal gammopathy
and at least 10% of bone marrow infiltration by lymphoplasmacytic cells.5 Monoclonal
gammopathy can be detected by the monoclonal spike, or M-spike, on serum
protein electrophoresis.3 Serum immunofixation may be performed to identify
the type of monoclonal protein and the type of light chain involved.3
In terms of immunophenotype, neoplastic cells express surface IgM, cytoplasmic Igs, CD38, CD79a, and
pan B-cell markers (CD19, CD20, and CD22). CD10 and CD23 are absent. Expression
of CD5 occurs in approximately 5-20% of cases.6 Recent
studies have reported two most common somatic mutations in WM, which are MYD88
L265P mutations (90-95% of cases) and CXCR4 (30–40% of cases).7 Absence
of these mutations, however, do not completely exclude the diagnosis of WM.
The International Staging System for WM
identifies five factors associated with adverse prognosis, including age older
than 65, hemoglobin < 11.5g/dL, platelet count < 100K/μL,
beta-2-microglobulin > 3mg/L, and monoclonal IgM concentration > 7g/L.3 Patients
younger than the age of 65 years with 0 or 1 of these factors are in the
low-risk category with a median survival of 12 years.3 In contrast, patients
with 2 or more risk factors are in the intermediate- and high-risk categories and
have a median survival of almost 4 years. 3
Management of WM depends on the patient’s
clinical manifestations.Furthermore, patients with minimal symptoms
should be managed with rituximab, whereas patients with severe symptoms related
to WM should receive more aggressive treatment, including dexamethasone,
rituximab and cyclophosphamide. Hyperviscosity syndrome is an oncologic
emergency that requires removal of excess IgM from the circulation via
Neparidze N, Dhodapkar MV. Waldenstrom’s Macroglobulinemia: Recent advances in biology and therapy. Clin Adv Hematol Onco. 2009 Oct;7(10): 677-690.
Leleu X, Roccaro AM, Moreau AS, Dupire S, Robu D, et al. Waldenstrom Macroglobulinemia. Cancer Lett. 2008 Oct;270(1):095-107.
Tran T. Waldenstrom’s macroglobulinemia: a review of laboratory findings and clinical aspects. Laboratory Medicine. 2013 May;44(2):e19-e21.
Kantamaneni V, Gurram K, Khehra R, Koneru G, Kulkarni A. Distal illeal ulcers as gastrointestinal manifestation of Waldenstrom Macroglbulinemia. 2019 Apr; 6(4):pe00058.
Grunenberg A, Buske C. Monoclonal IgM gammopathy and Waldenstrom’s macroglobulinemia. Dtsch Arztebl Int. 2017 Nov;114(44):745-751.
Bhawna S, Butola KS, Kumar Y. A diagnostic dilemma: Waldenstrom’s macroglobulinemia/plasma cell leukemia. Case Rep Pathol. 2012;2012:271407.
Varettoni M, Zibellini S, Defrancesco I, Ferretti VV, Rizzo E, et all. Pattern of somatic mutations in patients with Waldenstrom macroglobulinemia or IgM monoclonal gammopathy of undetermined significance.
Oza A, Rajkumar SV. Waldenstrom macroglobulinemia: prognosis and management. Blood Cancer Journal. 2015;5:e394.
-Jasmine Saleh, MD MPH is a pathology resident at Loyola University Medical Center with an interest in dermatopathology and hematopathology. Follow Dr. Saleh on Twitter @JasmineSaleh.
–Kamran M. Mirza, MD, PhD, MLS(ASCP)CM is an
Assistant Professor of Pathology and Laboratory Medicine, Medical
Education and Applied Health Sciences at Loyola University Chicago
Stritch School of Medicine and Parkinson School for Health Sciences and
Public Health. A past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza
was named to The Pathologist’s Power List of 2018 and placed #5 in the
#PathPower List 2019. Follow him on twitter @kmirza.
I belong to a Hematology Interest Group and always enjoy
seeing the case studies and questions that other techs post. This group is multinational
so I see posts from techs all over the world. It’s interesting to see the
similarities and differences in standard operating practices and the roles
techs play in different areas and different countries. It’s also interesting to
see that we all come across the same types of problems and difficult specimens!
In the last few months in this
Hematology Interest Group, I have seen many questions and comments about
resolving clumped platelets, and am therefore using this opportunity to shed
some light on these tricky specimens. The case I am presenting, and the photos,
are courtesy of Abu Jad Caesar, who is a Lab manager at Medicare Laboratories – Tulkarm branch, in
The patient had a CBC performed
on a Nihon Kohden 6410. WBC was 12.7 x 103μL, impedance platelet count was 20,000/μL on initial run, other parameters appeared within normal
limits. The sample was warmed and a Na Citrate tube was requested to rule out pseudothrombocytopenia.
After warming, the EDTA was rerun with a platelet count of 0/μL. The Na Citrate tube was run, and platelet count from the
instrument was 189,000/μL. (Figure 1)
Because of the blood:anticoagulant ratio in the Na Citrate tube, a multiplier
of 1.1 was applied, thus making the Na Citrate platelet count 207,900/μL. Slides were made, stained and examined. Image 1 shows
the clumping in the EDTA tube. Image 2 shows the smear from the Na Citrate
tube, with no visual clumping.
The CBC was reported with the following comments:
Platelet clumping observed, 2 samples drawn to rule out thrombocytopenia. EDTA
whole blood smear had many platelet clumps noted (EDTA induced
thrombocytopenia). Conclusion: Platelets are adequate and estimated to be about
Platelet counts in the normal range don’t usually give us too
much trouble in reporting, even if some clumping is present, mainly because
they are normal. Adequate platelet counts fall within a typical reference range
of about 150- 450 x 103/μL.
If there are instrument flags for a platelet abnormal scattergram or platelet
clumps, it is recommended to repeat testing by another method. If the initial
count is performed by impedance counting, many analyzers can also report
optical or fluorescent platelet counts. With impedance counting, very small
RBCs or fragments may be counted as platelets, thus giving a falsely increased
platelet count. With optical counting, large platelets can be counted as RBCs,
thus giving a falsely decreased count. Some Sysmex hematology analyzers use
impedance and optical counts and also feature fluorescent platelet counts which
use a platelet specific dye and give accurate platelet counts without the
interferences of other methods. A normal platelet count, even with clumping
seen on a smear, is still usually estimated to be normal (or may occasionally be
Thrombocytopenia, on the other hand, can be a challenge in
the hematology laboratory. With thrombocytopenia, physicians need an accurate
count to diagnose, treat or monitor patients. Even a small increase or decrease
can be significant when there is a severe thrombocytopenia. With fewer
platelets, every platelet counts!
One of the first questions we must ask with an apparent
thrombocytopenia is if this is a true thrombocytopenia or if it is pseudothrombocytopenia
(PTCP). A true thrombocytopenia represents a patient with a low platelet count
who may need monitoring or medical intervention. It can be dangerous to miss
true thrombocytopenia but is also dangerous to report a low platelet count in a
patient with a spurious thrombocytopenia who is not actually thrombocytopenic. Pseudothrombocytopenia,
or spurious thrombocytopenia, is defined as an artificially or erroneously low
platelet count. In PTCP, the low platelet count is due to clumps that are
counted as 1 platelet. (These large clumps can also be counted as WBCs, thus
giving a falsely increased WBC count.)
We can divide PTCP into 2 categories Platelet
clumping is most commonly caused by pre-analytic errors such as over-filled or
under-filled EDTA tubes, clotted specimens, or a time delay between sample
collection and testing. Techs should check the tube for clots and sample volume
and do a delta check to help differentiate thrombocytopenia and PTCP. But, with
an apparent ‘good’ sample, the next step would be a smear review. If there are
clumps seen on the smear, then we need to decide what caused the clumps. Is it the
first category, one of these common pre-analytical issues, or is it the 2nd
category of PTCP, an in vitro agglutination of platelets? Conditions that can
cause this in vitro agglutination of platelets include cold agglutinins,
multiple myeloma, infections, anticardiolipin antibodies, high immunoglobulin
levels, abciximab therapy and EDTA induced pseudothrombocytopenia. (EDTA-PTCP) Of
these, EDTA induced pseudothrombocytopenia is the most common cause. (Nakashima,
When techs talk about platelet clump issues, it is usually
because we are looking for ways to resolve or to accurately estimate the
platelet count in these samples, and there doesn’t seem to be one easy answer.
The clumping makes precise counting impossible and even estimates can be very
tricky. How can we estimate these counts? Do we simply report the presence of
clumping with “appear normal”, “decreased” or “increased”? Or, should we break
our estimates into more ranges to give physicians more valuable information?
And, what if the provider wants an actual count in order to give the patient
the best care possible and we can’t resolve the clumping? What can we do to
provide a count? Some of the first steps recommended include vortexing the
sample for 2 minutes to break up platelet clumps, then re-analyzing. Warming
samples may also help to resolve platelet clumps, particularly in samples with
cold agglutinins or that have had a delay in testing and have been transported
or stored at room temperature or below. If clumps persist and recollecting the
sample still yields platelet clumping, then pre-analytical error can be ruled
out an EDTA induced pseudothrombocytopenia may be suspected. Many labs will
have an alternate tube drawn or use another method to help resolve the
So, what is EDTA induced thrombocytopenia
(EDTA-PTCP)? This is not representative of a particular
clinical picture, and is not diagnostic for any disorder or drug therapy, but
is a laboratory phenomenon due to presence of EDTA dependent IgM/IgG autoantibodies.
These antibodies bind to platelet membrane glycoproteins in presence of EDTA. EDTA
induces and enhances this binding by exposing these glycoproteins to the antibodies.
(Geok Chin Tan, 2016) Though it is an
in vitro phenomenon, patients with certain conditions, such as malignant
neoplasms, chronic liver disease, infection, pregnancy, and autoimmune
diseases, do have increased risk of EDTA-PTCP. However, EDTA-PTCP has also been
observed in patients who are disease free. (Zhang, 2018)
What are some alternate methods to help resolve EDTA induced
platelet clumping challenges? Probably the most common is to redraw the sample
in a Na Citrate tube. Both EDTA and Na Citrate tubes should be drawn. In a true
EDTA-PTCP, as seen in our case study, you should see clumps on the smear made
from the EDTA tube and no clumps on the smear made from the Na Citrate tube. Because
of the volume of the anticoagulant in the Na Citrate tube you must also apply
the dilution factor of 1.1 to the count from the Na Citrate tube to get an
accurate platelet count. Note, however, that hematology analyzers are FDA
approved and validated for use with EDTA tubes. If you wish to use a different
anticoagulant, the method must be validated in your laboratory. Note also that
alternate methods will generally only resolve EDTA -PTCP, and not clumping due
to other cold agglutinins, medication or disorders. In addition, anticoagulant
induced thrombocytopenia is not limited to EDTA. It can also occur with citrate
and heparin. In a study, it was found that up to 17% of patients with an EDTA -PTCP
also exhibited this phenomenon with citrate. In fact, researchers have found,
and we have found in our own validations, that some samples that do not clump
in EDTA actually DO clump in Na Citrate. Thus, alternate tubes may not resolve
all platelet clumping. (Geok Chin Tan, 2016)
Some labs have validated ACD (Citric acid, trisodium citrate,
dextrose) anticoagulant tubes for EDTA-PTCP. Using this method, the EDTA tube
and ACD must be run in parallel and a conversion factor applied, reflecting the
difference in sample dilution in the 2 tubes. A parameter such as the RBC must
be chosen to make this comparison. Using a formula that divides the RBC in EDTA
by the RBC in ACD gives a ratio that reflects the dilutional differences
between anticoagulants. This ratio can then be multiplied by the ACD platelet
count to obtain the ACD corrected platelet count. (CAP Today, 2014). Some
sources have recommended ACD tubes because the incidence of clumping with Na
Citrate can be frustratingly high. It is theorized that the more acidic ACD
tube may prevent platelet clumping better than Na Citrate. (Manthorpe, 1981)
Less commonly used tubes are CTAD (trisodium citrate,
theophylline, adenosine, dipyridamole) and heparin. CTAD acts directly on
platelets and inhibits platelet factor 4 thus minimizing platelet activation. Downsides
to CTAD tubes are that they are light sensitive and must be stored in the dark,
and can be costly. They also alter the blood/additive dilution ratio so
calculations must be used, as seen with Na Citrate and ACD. Heparin tubes are
less commonly found to be beneficial in resolving platelet clumping issues
because heparin can active platelets. Heparin tubes are also more expensive, so
have not generally been a first choice for EDTA-PTCP.
I have heard from techs that their labs have very good
results using amikacin added to EDTA tubes to prevent spuriously low platelet
counts in patients with EDTA-PTCP. Amikacin should be added to the EDTA tube
within 1 hour after draw and testing is stable for up to 4 hours at room
temperature. Results of a study done in 2011 showed that the addition of
amikacin to the EDTA tube produced rapid dissociation of the platelet clumps
with little or no effect on morphology or indicies. This method has proved very
promising for reporting accurate platelet counts in patients with
multianticoagulant induced PTCP. (Zhou, 2011)
The last anticoagulant tube that I have seen mentioned by
many techs in the hematology interest group are Sarstedt ThromboExact tubes. I
have seen many posts from techs who use these and they seem to have a very good
success rate. ThromboExact tubes contain magnesium salts and are specifically designed
to determine platelet counts in cases of PTCP. They are currently validated
only for platelet counts and samples are stable for 12 hours after collection.
Interestingly, before automated hematology analyzers, magnesium was the
anticoagulant of choice for manual platelet counts. EDTA-PTCP has been
recognized since EDTA automated platelet counts were introduce in the 1970s. A
2013 study in Germany used ThromboExact tubes with excellent results for
resolving multianticoagulant induced PTCP. These tubes became commercially
available during the study, in 2013. (Schuff-Werner, 2013) Unfortunately for us
in the United States, these tubes are not available in the US. I was recently
at a conference and went up to the Sarstedt representatives and asked about
these tubes. I was told that they are available in parts of Europe and Asia but
are not FDA approved in the US. I asked very hopefully if they were looking at
getting FDA approval and was unfortunately told that “they didn’t think they
had the market for them to pursue approval.”
Whichever alternative method your lab chooses to use, it is
recommended to draw an EDTA and the alternate tube together. This way the 2
counts and the presence or absence of clumping in the tubes can be compared. We
have many patients who had one incidence of clumping, yet when the provider orders
a Na Citrate platelet count, we get a new draw of both EDTA and Na Citrate tubes
together, and there is no flagging or clumping seen with EDTA. In these cases
it is appropriate to result the EDTA results as there is no evidence of
When a patient has a low PLT count without any
hematologic disease, family history, and/or bleeding-tendency identified, and
pre-analytical errors have been ruled out, PTCP should be considered. This
does not mean that a patient with PTCP will have a normal platelet count after
the clumping is resolved. As stated above, many patients with EDTA-PTCP have
hematological or other disorders and may be truly thrombocytopenic. Resolving
the clumping in these patients allows us to give the provider an accurate
platelet count, which is very important in thrombocytopenic patients.
The flow chart below (Figure 4) shows some things to
consider when dealing with platelet clumping. It is our goal to resolve
clumping so that we can report an accurate platelet count in a timely fashion. In
the laboratory where I work, I have validated Na citrate tubes, but these seem
to resolve clumping in less than 50% of patients. As a last resort, to get an
accurate platelet count, some articles have suggested collecting a fingerstick
and performing manual counts. I did include this in the chart as an option for
multianticoagulant PTCP, however, due to the difficulty in collecting a good
specimen and the subjectivity of counts, along with problems associated with
necessary calculations, our pathologists have decided that we will not do
manual platelet counts. For this reason, I am currently involved in platelet
clumping monitoring and will be conducting a small internal study to compare
ACD, CTAD and Na Citrate tubes in parallel. Depending on those results we may
also then test amikacin. If we come to any enlightened conclusions I’ll write
another short blog with our results!
Thanks again to Abu Jad Caesar, lab manager at Medicare Laboratories – Tulkarm branch, in Palestine, who provided me with this textbook perfect
case of PCTP, which was easily resolved by collecting in Na Citrate. We wish
they all read the textbooks and were as cooperative!
R, Kofod B, et al. Pseudothrombocytopenia, In vitro studies on the underlying
mechanisms. Scand J Haematol 1981; 26:385-92
MO, Kottke-Marchant K. Platelet Testing: In: Kottke-Marhchant K, ed. An
Algorithmic Approach to Hemostasis Testing, 2nd ed. CAP Press;
Schuff-Werner,Peter, et al.
Effective estimation of correct platelet counts in pseudothrombocytopenia using
an alternative anticoagulant based on magnesium salt. Brit J of Haematol Vol
162, Issue 5. June 29, 2013
Geok Chin et al. Pseudothrombocytopenia due to platelet clumping: A Case Report
and Brief Review of the Literature. Case Reports in Hematology. Volume 2016
Lixia Zhang, MMed,* Jian Xu, MD,* Li
Gao, MMed, Shiyang Pan, MD, PhD. Spurious Thrombocytopenia in Automated
Platelet Count. Laboratory Medicine 49:2:130-133. 2018
et al. Amikacin can be added to blood to reduce the fall in platelet count. Am
Journal of Clinical pathology, Vol 136, Issue 4, Oct 2011.
-Becky Socha, MS, MLS(ASCP)CM BB CM graduated
from Merrimack College in N. Andover, Massachusetts with a BS in
Medical Technology and completed her MS in Clinical Laboratory Sciences
at the University of Massachusetts, Lowell. She has worked as a Medical
Technologist for over 30 years. She’s worked in all areas of the
clinical laboratory, but has a special interest in Hematology and Blood
Banking. When she’s not busy being a mad scientist, she can be found
outside riding her bicycle.
This last month, I rotated through our Children’s hospital,
which included reviewing hemoglobin electrophoresis tests. I’d learned about
them before in residency, but they can be quite more interesting (complicated)
than I expected.
Hemoglobin electrophoresis is a blood test to look at
different types of hemoglobin to determine if there are any abnormalities. In a
children’s hospital it is frequently ordered as a reflex for an abnormal
newborn screen or when a child is incidentally found to be anemic. The test is
performed in 2 stages. 1st lysed blood samples are run on gel
electrophoresis and different types of hemoglobin are separated as they move at
different speeds. Several types of hemoglobin will run within the same region,
so a secondary method of separation is always employed.
Below, you can see how some bands in the same area of an acidic gel (agarose) are actually very
different on the alkaline gel (cellulose
acetate) and vice versa.
At our hospital, we use HPLC
and measure retention times of the hemolysate to quantify and identify
different hemoglobin types present. As a basic primer you should recall that hemoglobin is a tetramer with a pair of alpha globin + a pair of either beta, delta or gamma globin
(each separate genes).
Alternative hemoglobins are enriched in populations where
malaria is endemic as these variants may provide improved fitness by promoting
resistance to the malarial parasite that reproduces inside red blood cells. Thus,
many people of African or south east Asian descent may carry these variants.
Our case is that of a 2 year old girl with anemia who had
testing sent by her primary care doctor for the following CBC:
This is indicative of microcytic anemia, but unlike some Thalessemias
the RBC isn’t very high. More on this later.
Looking at the gel result, there is a large band in the area
coinciding with Hgb C. We also see the normal Hgb A2 and a small amount of Hgb
F. We know Hgb F can be increased in Hgb SS and thus could also be present if
she had Hgb C trait or disease.
Looking at the next HPLC result, we see there is a similar very high level of Hgb C (68%) with corresponding levels of Hgb F and Hgb A2 (note: acetylated Hgb F and Hgb F are added together). Thus, this fits with a homozygous C with some compensatory A1 and F, right?
Remember Hgb C is a β -globin variant and
you only have 2 β -globin genes, so if you are homozygous
for the C variant on the β-globin gene (HBB), then Hgb A1, which is
made of normal β-globin would be impossible to produce.
Also you might be bothered by all of these small peaks. However, there are
often small peaks that can’t be definitively identified and are likely
post-translationally modified hemoglobin. But in the context of an abnormal Hgb
A1 that shouldn’t be there, we dug deeper.
One of the most common hemoglobinopathies is Beta Thalassemia (β-Thal), which clinically manifests when less of
the beta hemoglobin protein is produced. Heterozygous mutations lead to Beta Thalassemia
minor with minimal symptoms, while homozygous mutations lead to β-thal major with symptoms of anemia. Mutations in the β -globin gene, HBB,
can lead to complete loss of β-globin (β0 variant)
or partial of β-globin (β+ variant).
As this patient has less than 50% of Hgb A present (expected
amount), they could also have a β+ variant
as well. This would make them compound heterozygous for C and β+.
One of the hallmarks of Thalassemia is an increase in Hgb A2 (normal 2.5-3.5%).
Hemoglobin A2 is a normal variant of A that is composed of two alpha and two
delta chains (δ2α2). We see in our
case that the Hgb A2 is normal at 2.5%. So it seems the patient doesn’t display
a typical Thalassemia picture.
One condition that could create this scenario is if there is
a variant in the delta chain of A2
that causes it to elute differently. Indeed, there is a delta variant that
creates hemoglobin A2 prime (A2’)
that moves near the S region of the
HPLC. And when we look back at our unknown hemoglobins, Hgb X is marked at 1.03 of the S region and has an abundance of
3.9%. This supports it being the Hgb A2’ and if we add this together with the
Hgb A2 we get an elevated 6.6% A2 total,
which would be consistent with Beta Thalassemia.
Lastly, one would wonder if we could find this
third hemoglobin variant A2’ on the alkaline gel. Previous studies have shown
the A2’ variant is more negatively charged, so on a basic gel, it should move
further from the negative anode than the other hemoglobins. We don’t see
anything to the left of the HgbC, but if we flip the gel over and look under the patient label, you can see a
faint band that is likely the A2’!
In summary this case arose from 3 separate mutations in a
single patient. She was compound
heterozygous for a Hgb C and β+ variants in the β-globingene and she was heterozygous
for an A2’ variant on the delta-globin
gene. This was certainly a case
where paying close attention mattered.
Abdel-Gadir D, Phelan L, and Bain BJ.
Haemoglobin A2′ and its significance in beta thalassaemia diagnosis. Int J Lab
Hematol. 2009 Jun;31(3):315-9. doi: 10.1111/j.1751-553X.2008.01038.x. Epub 2008
-Dr. Charles Timmons MD PhD is a pediatric pathologist at Children’s Medical Center in Dallas, TX. His responsibilities include signing out hemoglobin electrophoresis, HPLC and globin sequencing, and has been residency director for 17 years.
-Jeff SoRelle, MD is a Chief Resident of Pathology at the
University of Texas Southwestern Medical Center in Dallas, TX. His
clinical research interests include understanding how the lab intersects
with transgender healthcare and improving genetic variant
An 80 year old man presented with rapid onset of cervical adenopathy over a period of few months. The largest lymph node measuring 6 cm was biopsied and sent for histopathological evaluation.
from the lymph node showed effacement of the lymph node architecture by a
fairly monotonous population of medium to large sized lymphoid cells arranged
in vague nodular pattern. Focally, a starry sky pattern was observed. The cells
were 1.5-2 times the size of an RBC, with high N:C ratio, irregular angulated
nuclei and small nucleoli. A high mitotic rate of 2-3 mitoses/hpf was seen.
stains showed that the lymphoma cells were positive for CD20, CD5, SOX-11, and
negative for Cyclin D1, CD10, CD23, CD30, BCL-1, and BCL-6. Ki67 index was
of Mantle cell lymphoma, pleomorphic variant was made.
lymphoma is a peripheral B cell lymphoma, occurring in middle aged or older
adults, with a male: female ratio of 7:1. Although Cyclin D1 expression is
considered a hallmark of mantle cell lymphoma, yet about 7% cases are known to
be Cyclin D1 negative. In these cases, morphological features and SOX-11
positivity helps in establishing a definitive diagnosis.
assessment of morphological features of lymphoma, the cell size is an important
starting point. In this case, the lymphoma cells ranged from medium to large
sized. The following differential diagnoses were considered:
This case showed a “starry sky” pattern focally. A medium sized
population of cells, high mitotic rate and a high Ki67 index (70%) favoured a
Burkitt lymphoma. However, although commonly seen in Burkitt lymphoma, a
“starry sky” pattern is not specific for this type of lymphoma. Also, the lack
of typical “squaring off” of nuclei, basophilic cytoplasmic rim were against
the diagnosis of Burkitt lymphoma. The nuclei in this case showed 0-1 small
nucleoli, unlike the typical basophilic 2-3 prominent nucleoli of Burkitt
lymphoma. Moreover, Ki67 index, even though high was not enough for Burkitt
lymphoma where it approaches 100%. The cells were negative for CD10 and Bcl-6, which
are almost always found in a Burkitt lymphoma. Hence, a diagnosis of Burkitt
lymphoma was ruled out.
Diffuse Large B cell Lymphoma
The presence of interspersed large cells with nucleoli, irregular
nuclei, high mitotic rate, and a high Ki67 index with a history of very rapid
enlargement of lymph node suggested a diagnosis of Diffuse Large B cell
lymphoma. However, the scant cytoplasm, lack of bizarre cells, and absence of
CD10, BCl-2, BCl-6 were against a diagnosis of DLBCL.
A diagnosis of lymphoblastic lymphoma was favoured by the irregularly
angulated nuclei, and presence of nucleoli. However, the cells of lymphoblastic
lymphoma have a more delicate nuclear chromatin, higher mitotic rate as against
the relatively condensed chromatin and the low to high variable mitotic rate of
Mantle cell lymphoma. Also, lymphoblastic lymphomas are more commonly of the T
cell subtype and occur commonly in younger individuals. In this case, B cell
markers were positive (CD 20), and the patient was 80 year old, disfavouring a
lymphoblastic lymphoma. The blastoid variant of mantle cell lymphoma is
practically indistinguishable from lymphoblastic lymphoma, except that it is
D1 negativity in Mantle cell lymphoma
cases of Cyclin D1 negative mantle cell lymphomas, morphology plays a critical
role in coming to a diagnosis of mantle cell lymphomas. In this case, points
that favoured the diagnosis of mantle cell lymphoma were clinical features such
as older age (80 years), and male gender, and morphological features such as a
vaguely nodular pattern of growth, irregular nuclei, and 0-1 small nucleoli.
Due to the presence of variably sized cells with distinct nucleoli, a
pleomorphic variant was considered. Even though Cyclin D1 was found to be
negative, the cells were positive for SOX-11.
SOX-11 is a
transcription factor that is not normally expressed in B cells, but is
sensitive and fairly specific for mantle cell lymphomas. It is important to
note that SOX-11 is also positive in 25% Burkitt lymphoma, 100% lymphoblastic
lymphoma, and 66% T-prolymphocytic leukemia. Herein lies the importance of
recognising morphological features, as all of these lymphomas that may express
SOX-11 were ruled on the basis of morphology. A more specific antibody, MRQ-58
may be used for greater specificity. The presence of SOX-11 is considered a
specific biomarker for Cyclin-D1 negative mantle cell lymphomas. In these
cases, there is upregulation of Cyclin D2 or D3 that may substitute for Cyclin
D1 upregulation. But, immunohistochemical detection of Cyclin D2 or D3 is not
helpful for establishing a diagnosis, as other lymphomas are commonly positive
for these markers. Hence, it is important to perform SOX-11
immunohistochemistry to diagnose the Cyclin D1 negative variant of mantle cell
be used not just for the diagnosis, but also for determining prognosis of
mantle cell lymphoma. Indolent MCL usually lack SOX-11 expression. The pattern
of SOX-11 staining has also been used a marker of prognosis. Cytoplasmic
expression of MCl, seen in only a few cases was associated with a shorter
survival as compared to the more common nuclear staining of SOX-11.
age, lymphoma diagnosis relies heavily on the use of immunohistochemical
markers. However, this case highlights the importance of morphological features
in diagnosing lymphomas with unusual immunohistochemical marker profile.
Although, this case was negative for Cyclin D1, considered a hallmark of Mantle
cell lymphoma, yet, the combination of morphological features with SOX-11
staining helped in clinching the diagnosis. To avoid a misdiagnosis, it would
be prudent to perform SOX-11 staining in all lymphoma cases morphologically
resembling MCL, but lacking Cyclin-D1.
-Swati Bhardwaj, MD has a special interest in surgical pathology and hematopathology. Follow her on Twitter at @Bhardwaj_swat.
–Kamran M. Mirza, MD, PhD, MLS(ASCP)CM is an Assistant Professor of Pathology and Laboratory Medicine, Medical Education and Applied Health Sciences at Loyola University Chicago Stritch School of Medicine and Parkinson School for Health Sciences and Public Health. A past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza was named to The Pathologist’s Power List of 2018 and placed #5 in the #PathPower List 2019. Follow him on twitter @kmirza.
4 year old child was brought to the pediatrician by her mother with a complaint
of new onset of severe bruising on her legs. The mother could not recall any
falls or bumps that would have caused the bruising. On exam, the physician also
noted mucosal bleeding in the oral cavity. Questioning revealed that the
patient had experienced flu like symptoms several weeks earlier. The physical
exam was normal except for the bleeding. There was no family history of
bleeding disorders. A CBC was ordered.
RBC, Hgb, Hct, RBC indicies normal
count 26 x 103/μL
22% (reference range IPF% 1.0-7.0%)
The physician evaluated the results, noting the
normal CBC but decreased platelet count. The above results also show the
immature platelet fraction (IPF), an additional Advanced Clinical Parameter reported
from the Sysmex XN hematology analyzer. A low platelet count, as seen in this
patient, will reflex a fluorescent platelet count (PLT-F). The impedance count (PLT-I)
can be falsely increased if small RBCs or fragments are counted as platelets. On the other hand, in an optical
platelet count, when measuring platelets by size (PLT-O), large platelets can
be missed, giving a falsely low count. In this case there was a low
platelet count and an instrument flag for an abnormal platelet scattergram. The PLT-F, on the other hand, uses a
platelet specific dye which eliminates interference seen with other methods.
The fluorescent dye labels the RNA, and forward scatter is used to determine
size while side fluorescence is used to measure RNA content. With gating set
based on cell volume and RNA content, the PLT-F can be measured. Therefore,
the reflexed and more reliable
PLT-F was the reported count.
Additionally, when there is an abnormal
scattergram or a low platelet count, the IPF% and IPF# are also reported. The immature
platelet fraction is a measure of the youngest platelets, or reticulated
platelets. These are the first circulating platelets, right out of the bone
marrow. An increased IPF indicates an increase in platelet production, yet this
child’s platelet count was very low. This suggests that the thrombocytopenia
may be due to excessive destruction of platelets; the bone marrow was actively
making platelets, but they were being destroyed, causing the low platelet
Immune Thrombocytopenia- ITP.
Primary immune thrombocytopenia (ITP), formerly
known as idiopathic thrombocytopenic purpura or immune
thrombocytopenic purpura, is one of the most common bleeding
disorders of children. In most cases, it presents with sudden onset of bruising
and petechiae in an otherwise healthy child, with normal WBC and hemoglobin. ITP is an
autoimmune bleeding disorder in which the immune system makes anti-platelet
antibodies which bind to platelets and cause destruction. Even though the exact
cause of ITP remains unknown, it is recognized that it can follow a viral
infection or live vaccinations. While there are some similarities between
pediatric ITP and ITP in adults, in children this tends to be an acute disease
which is self-limiting and resolves itself in several weeks, with no treatment.
However, in a small number of children, the disorder may progress to a chronic
ITP. In contrast to ITP in children, a chronic form is more commonly seen in
adults. It is usually a diagnosis of exclusion, does not follow a viral illness
and requires treatment.
patient recovered in a few weeks. One month after the initial episode, her PLT
was 174 x 103/μL and her IPF% was 6.0%
An IPF reported with a CBC, in combination with a low platelet count, is fast, inexpensive, and can be extremely beneficial in aiding in a timely diagnosis. As the child’s platelet count recovered, the IPF% returned to normal range. ITP can therefore be monitored with a CBC. Thus, the IPF can be used not only to help diagnose but also as an indicator of remission.
Arshi Naz et al. Importance of Immature platelet
Fraction as a predictor of immune thrombocytopenic purpura. Pak J Med Sci 2016
Vol 32 No 3:575-579
Briggs,C. Assessment of an immature plateletfraction
(IPF) in peripheral thrombocytopenia. Br J Haematol 2004Jul;126(1):93-9
Sysmex White Paper. The role of the ImmaturePlatelet
Fraction(IPF) in the differential diagnosis of thrombocytopenia. www.sysmex.com/us
D-Orazio, JA, Neely, J, Farhoudi,N. ITP in children: pathophysiology and current
treatment approaches.J Pediatr Hematol Oncol.2013 Jan;35(1): 1-13
-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.
The patient is a 69 year old male who presented to the
hospital with a 3-month history of drenching night sweats, weight loss, fatigue,
and generalized lymphadenopathy. He also endorsed a very itchy rash all over
his body. He denied smoking. There was no other relevant social or family
Physical examination confirmed diffuse lymphadenopathy,
hepatosplenomegaly and a mild diffuse skin rash. Notably, there was a 2.5 cm
level-1 lymph node palpated in the left neck. This was subsequently biopsied.
Biopsy of the level-1 neck lymph node revealed a 2.3 x 1.5 x
1.2 cm mass pink-tan and firm mass. Sectioning revealed a glossy white-tan cut surface.
H&E staining revealed a polymorphic lymphocytic infiltrate of in the interfollicular
zones. The infiltrating lymphocytes ranged from small to large cells with abundant
cytoplasm, eosinophils, and plasma cells. There was also a notable increase in
the number of high endothelial vessels lined by lymphocytes with irregular
nuclear borders and clear cytoplasmic zones.
Further characterization by immunohistochemical staining showed
the majority of the interfollicular cells to be CD3 and CD5 expressing T cells.
These were a mix of CD4 and CD8 positive cells but with marked CD4
predominance. CD7 appeared positive in a smaller population of T-cells compared
to CD3 (consistent with loss of this pan-T-cell marker). Varying numbers of the
interfollicular cells were positive for CD10, BCL-6, CXCL-13, and PD-1 with a
strong positivity for ICOS, phenotypically consistent with an expansion of Tfh
(T-follicular helper cell) cells.
Interspersed between the T cells were numerous CD20 positive
cells with prominent nucleoli that also revealed CD30 positivity. CD21 staining
revealed expanded follicular dendritic cell meshworks. EBER ISH was positive in
a rare subset of cells. Kappa and lambda ISH showed an increased number of
polytypic plasma cells.
Flow Cytometry showed the presence of a small population of
T-cells that were CD4 positive but CD3 negative. There was no evidence of
B-cell clonality. TCR-G PCR was positive.
A final diagnosis of Angioimmunoblastic T-cell lymphoma
(AITL) was rendered.
AITL is a relatively rare neoplasm of mature T follicular
helper cells, representing about 1-2% of all non-Hodgkin lymphomas. It is;
however, one of the more common subtypes of peripheral T-cell lymphomas,
accounting for 15-30% of this subgroup. The condition was first reported in
1974 in Lancet as a non-neoplastic abnormal immune reaction1. It was
first recognized as a distinct clinical entity in in 1994 in the Revised
European American Lymphoma Classification2. The disease shows a
geological preference to Europe (28.7%) over Asia (17.9%) and North America
(16%). AITL occurs primarily in middle aged and elderly individuals and shows a
slight predominance of males over females.
The disease has a strong association with EBV infection, but
the neoplastic T-cells are almost always EBV negative, creating an interesting
question of EBV’s function in the etiology of AITL. AITL most often presents
late in the disease course with diffuse systemic involvement, including
hepatosplenomegaly, lymphadenopathy and other symptoms such as rash with
pruritis and arthritis. Lab findings include cold agglutinins, rheumatoid
factor and anti-smooth muscle antibodies. There also tends to be
immunodeficiency secondary to the neoplastic process. The clinical course of
AITL is variable, but the prognosis is poor, with the average survival time
after diagnosis being < 3 years. The histological features and genetic
findings have not been found to impact clinical course.
Microscopically, AITL presents with either partial or total
effacement of the normal lymph node architecture with perinodal infiltration.
The cells of AITL are small to medium-sized lymphocytes with clear to pale
cytoplasm, distinct cell membranes and very minimal cytological atypia. These
cells often congregate around the high endothelial venules. The T-lymphocytes
are present in a largely polymorphous inflammatory background of other
lymphocytes, histiocytes, plasma cells and eosinophils. There are 3 overlapping
sub-patterns of AITL. The first of these is similar to a reactive follicular
hyperplasia, and can only be distinguished from normal hyperplasia by use of
immunohistochemical stains to differentiate the neoplastic cells from normal
reactive cells. The second pattern has retained follicles, but they show
regressive changes. The third pattern has completely or sub totally effaced.
These three patterns seem to be on a spectrum with one another, given that
progression from the first to the third pattern has been seen on consecutive
biopsies in the same patient.
Cytologically, AITL cells express pan-T-cell markers
including CD2, CD3 and CD5 and the vast majority are CD4 positive. CD3 may be
quantitatively decreased or absent by flow cytometry. There are a variable
number of CD8 positive T-cells. The tumor cells also show the immunophenotyping
of normal T follicular helper cells including CD10, CXCL13, ICOS, BCL6 and PD1
in 60-100% of cases. CXCL13 and CD10 are the most specific, whereas PD1 and
ICOS are the most sensitive.
Horne, C., Fraser, R., & Petrie, J. (1974).
Angio-Immunoblastic Lymphadenopathy With Dysproteinemia. The
Lancet, 304(7875), 291. doi:10.1016/s0140-6736(74)91455-x
Harris, N.l. “A Revised European-American
Classification of Lymphoid Neoplasms: a Proposal from the International
Lymphoma Study Group.” Current Diagnostic Pathology, vol. 2, no. 1, 1994,
pp. 58–59., doi:10.1016/s0968-6053(00)80051-4.
Swerdlow, Steven H. WHO Classification of
Tumours of Haematopoietic and Lymphoid Tissues. International Agency for
Research on Cancer, 2017.
-Zachary Fattal is a 4th year medical student at the Central Michigan University College of Medicine. He is pursuing a career in pathology and has a special interest in hematopathology, cytopathology and blood bank/transfusion medicine. You can follow him on Twitter @Paraparacelsus.
–Kamran M. Mirza, MD, PhD, MLS(ASCP)CM is an Assistant Professor
of Pathology and Medical Education at Loyola University Health System. A
past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza was named to
The Pathologist’s Power List of 2018. Follow him on twitter @kmirza.