1.

Case Report:Severe Refractory Immune Thrombocytopenia Successfully Treated with High-Dose Pulse
Cyclophosphamide and Eltrombopag

Case Presentation:

A 43-year-old female was diagnosed with ITP 6 years ago during pregnancy (male fetus) and had been
treated with steroids. Over the years, she underwent a number of treatments for “refractory” ITP
including, in addition to corticosteroids, splenectomy, IVIG, Rho(D) immune globulin, rituximab,
cyclosporine, azathioprine, vincristine, mycophenolate mofetil, romiplostim, and eltrombopag (Figure
1). She responded to romiplostim; however, she developed peripheral neuropathy, which is one of the
common adverse effects of romiplostim, and treatment was halted. She subsequently was treated with
eltrombopag combined with danazol; however, the regimen was stopped because of intolerance even
though her platelets improved on this regimen. She was being treated according to the regimen
proposed by Arnold et al. [5] but did not achieve a good response. She experienced a left occipital
hemorrhage when her platelet count was less than 5 × 109/L. She underwent 12 days of plasmapheresis
with no improvement and was transferred to our center.

Figure 1: Platelet response to sequential treatments for ITP.

Upon transfer, a marrow examination showed normal cellularity with megakaryocyte hyperplasia,
multilineage hematopoiesis, and no dysplasia. She received high-dose cyclophosphamide (50
mg/kg/day) for 4 days for the first cycle, followed by four more cycles of 500 mg cyclophosphamide IV
4–6 weeks apart based on count recovery. Eltrombopag (25 mg daily) was added with cycle 2 of
cyclophosphamide and dose was increased up to 50 mg and 75 mg after cycle 3 and cycle 5 of
cyclophosphamide, respectively. She is currently on eltrombopag 75 mg daily and maintaining normal
platelet counts (Figure 1). Except neutrophils (ANC) decline to 0.96 × 109/L after 1st cycle of high-dose
cyclophosphamide, she has had no significant alterations of ANC and platelets nor has she had any
clinical evidence of infections.

Discussion:

Various therapeutic modalities have been shown to be effective in ITP [6, 7]. Splenectomy (complete
response (CR) rate 70%) works by reducing the clearance of autoantibody-coated platelets [8],
intravenous (IV) anti-D and IV immunoglobulin (IVIG) cause extravascular hemolysis of anti-D or
autoantibody-sensitized red blood cells (RBCs) by splenic macrophages, which results in decreased
splenic sequestration of autoantibody-sensitized platelets [9] and an increase in platelet counts, and
rituximab [10] depletes B cells; intravenous immunoglobulin also contains anti-idiotypic antibodies and
decreases autoantibody production but the exact mechanism is still not clear [11]. Corticosteroids
suppress lymphocytes and macrophages (via apoptosis) and reduce the destruction of platelets [12].
Thrombopoietin and thrombopoietic agents stimulate megakaryocyte progenitors and increase
production of platelets [13].
Immunosuppression with agents such as azathioprine [14] (via blockade of the pathway for purine
synthesis), cyclosporine [15] (by decreased production and release of IL-2 and IL-2-induced activation of
resting T lymphocytes), and mycophenolate mofetil (by inhibiting inosine monophosphate
dehydrogenase which inhibits de novo guanosine nucleotide synthesis pathways that are required for T
and B lymphocytes) results in suppression of cell and antibody mediated immunity. Platelet transfusions
provide transient replacement for urgent and selective medical situations. Other selective antibodies
(anti-CD44, anti-CD154) have shown activity for ITP therapy [16, 17] as well. Most of ITP patients
undergo treatments with corticosteroids, and if there is no lasting response, splenectomy is performed.
60–70% of patients with ITP achieve stable responses with this strategy, defined as platelet counts >30 ×
109/L or not requiring additional therapy for symptomatic thrombocytopenia.

A rare patient will undergo hematopoietic stem cell transplantation for refractory ITP [18–20]. High-dose
(HD) cyclophosphamide given over multiple days as used for conditioning before allogeneic stem cell
transplantation [21, 22] and for graft versus host disease prophylaxis [23] is profoundly
immunosuppressive. It has also shown activity in the treatment of aplastic anemia [24], severe
autoimmune hemolytic anemia [25], and other autoimmune diseases [26]. HD cyclophosphamide has
also been described for the treatment of refractory ITP [27]. Eltrombopag is an FDA approved
thrombopoietin receptor agonist for the treatment of patients with chronic ITP [28, 29]. Standard
treatment with single agent glucocorticoid therapy and immunosuppressive agents is effective to induce
remissions in patients with chronic refractory ITP [30]. Combining various agents has shown good
activity in patients with refractory chronic ITP.

In the present case pulsed HD cyclophosphamide in combination with a thrombopoiesis stimulating

agent led to a long-lasting remission of ITP without causing neutropenia or infections. Our patient
continues to have a stable range of platelets with more than 12 months of follow-up. Thus, while the
contributions of cyclophosphamide and eltrombopag, respectively, cannot be differentiated in the
present case, eltrombopag was well tolerated in combination with HD cyclophosphamide in contrast to
the intolerance observed in an earlier treatment attempt with danazol. Cyclophosphamide may modify
the immunological milieu, resulting in an enhanced response to the thrombopoietin receptor agonist.
4.) The Case of the Pyrogenic Platelet Product

Jackie is administering six units of pooled random donor platelets to Stuart, a patient with
myelodysplastic syndrome. The transfusion was started at 120 ml per hour, per hospital policy. When
Jackie rechecked Stuart’s vital signs at 15 minutes, she noted that his temperature had increased from
37.1°C to 38.1°C. In addition, Stuart was chilling.

What Would You Do?

Fever associated with the administration of blood products is generally referred to as a febrile non-
hemolytic transfusion reaction. It is defined as an increase in temperature of 1°C or more, with or
without chills and is estimated to occur in up to 30% of all platelet transfusions but is likely under-
reported.

Fever can be caused by bacterial contamination of platelet products; however, a more common etiology
is the presence of white blood cells or leukocytes in the product. These “stowaway” cells produce
increasing levels of cytokines IL1-beta, IL6, IL8, and TNF-alpha as platelets are stored, which result in a
pyrogenic response. Prostaglandin E2 is also stimulated by way of circulating soluble CD40 ligand.
Leukocytes can be filtered, although not all white cells are removed. There is currently no method to
remove cytokines once they have contaminated the product.

Apheresed products can be leukoreduced during collection, reducing the production of cytokines. In
some cases, removing most of the plasma from the product is necessary, because the plasma (not the
platelets) is the source of the reaction.

Jackie stopped the transfusion and notified the patient’s medical provider. Acetaminophen was given to
reduce the fever. The product was discarded, and a new product was ordered.
2.) Recurrent Immune Thrombocytopenic Purpura

Case Report: A 9 year old boy presented with complaints of petechaeal rash over body for five days and
bleeding from nose for two days. No history of fever or pallor was elicited. Nose bleed comprised only 5
to 6 drops of fresh blood and subsided spontaneously or with local pressure. The child was active and
playful. On examination, petechiae and purpura were present over the trunk, extremities, face and oral
mucosa (Figure 1).

There was no organomegaly and lympadenopathy. His vitals were stable and blood pressure was within
normal range for the age. On laboratory investigations hemoglobin was 11 gm%, total leukocyte count
was 9.25 x 109 /L, with Polymorphs 66%, Lymphocytes 32% and Eosinophils 02%, erythrocyte
sedimentation rate was 14 mm in 1st hour and platelet count was 11.2 x 109 /L. Renal function test,
liver function test and coagulation profile were within normal limits. Anti- nuclear antibody and serology
for HIV and hepatitis B surface antigen were negative. On account of compatible history and supportive
examination findings a presumptive diagnosis of acute Immune thrombocytopenic purpura was made.
Bone marrow biopsy was done before starting treatment. Bone marrow examination showed increased
number of megakarocytes without any other abnormality. Since the platelet count was less than 20 x
109 /L with mucosal bleed, the decision to treat the child was taken. Patient was started on Tablet
Prednisolone, in a dose of 2 mg/kg/day for 4 weeks, with weekly follow up. Patient responded well to
the treatment and his platelet count increased to 26.4 x 109 /L after first week. After 4 weeks, his
platelet count reached 221 x 109 /L and glucocorticoid therapy was tapered over next 4 weeks and
finally stopped.

After 6 months of remission, at the age of 10 years and 8 months patient again presented with
petecheal rash over body and a diagnosis of ITP was made on similar ground. This time his platelet count
was 9.0 x 109 /L, which increased to 153 x 109 /L after 4 weeks of treatment with prednisolone. Patient
presented with 4 more recurrences following this episode, after 4, 8, 9 and 6 months of respective
previous episodes at the ages of 11 years and 2 months, 12 years, 12 years 11 months and 13 years and
7 months respectively. On each occasion patient was subjected to the same investigations, (except ANA
and HIV/HBs serology) and his platelet count were 14.2, 21.6, 11.8 and 16.9 x 109 /L respectively. Only
on one of these occasions, patient had mucosal bleed with a platelet count of 11.8 x 109 /L (at the age
of 12 years and 11 months) and for this episode it was decided to treat the patient with prednisolone.
For remaining 3 episodes patient was only followed up weekly without any treatment. After eight weeks
patient had normal platelet count for all of these 4 episodes.

Now the child is 16 year of age and is being followed up for the last two and a half years. No further
recurrence has been documented yet. Discussion: ITP is an autoimmune disease in which anti-platelet
immunoglobulins bind to platelet membranes leading to premature death. The antibody coated
platelets are rapidly removed by spleen and liver macrophages. An estimated 5% of paediatric patients
have recurrent episodes of thrombocytopenia followed by variable periods of remission. This is
presumed to reflect a chronic compensated state of ITP [14]. During periods of remission, increased
platelet production balances the increased rate of platelet destruction, but during exacerbations,
platelet production by the marrow is suppressed by viral infections or other factors and is unable to
offset the rate of destruction

Treatments options available for recurrent ITP patients, not responding to glucocorticoids range from
rituximab, azathioprine, cyclophosphamide, danazol, vinca alkaloids, ascorbic acid, colchicine,
interferon-α, combination chemotherapy, protein A immunoadsorption, cyclosporine, ε-aminocaproic
acid, plasma exchange, to recently approved thrombopoietin receptor agonists [12]. Splenectomy is
considered in patients who do not respond to these drugs. Corticosteroids were the mainstay of
treatment in our patient. The main mechanism of action has been hypothesized as decrease
autoantibody production; [15] improved integrity of leaking capillaries [16] and impaired clearance of
antibody-coated platelets by mononuclear macrophages [17]. Although recurrent episodes of ITP have
been previously reported in literature but these patients often don’t respond to first line
immunosuppressive therapy. To our best of knowledge this is the first case of ITP having five
recurrences, thoroughly followed up and all episodes treated successfully with mere prednisolone.

Conclusion :

Idiopathic Thrombocytopenic Purpura (ITP) can present as multiple episodes of recurrences in children
and can be treated satisfactorily with prednisolone and a trial of prednisolone should be given before
resorting to other costly treatments.

3.) Pseudothrombocytopenia due to Platelet Clumping: A Case Report and Brief Review of the Literature

Clinical Presentation:

A 14-year-old female presented to our hospital with complaint of abdominal pain. Physical examination
was unremarkable. She had no bleeding symptoms. The patient’s past medical history was significant for
Klippel-Feil syndrome and hearing loss. Prior platelet counts had been within normal ranges. The clinical
concerns of her current low platelet count included idiopathic thrombocytopenic purpura and bone
marrow suppression by a viral illness.

Laboratory Tests and Findings of Peripheral Blood Smear The platelet count obtained on sample
collected in ethylenediaminetetraacetic acid (EDTA) anticoagulant was 80,000 mm3. Hemoglobin (Hgb)
and WBC were normal. Upon examination of peripheral blood (PB) smear, clumping of platelets was
observed. Repeat testing on a sample collected in sodium citrate showed similarly low platelet count
and PB smear showed platelet clumps as well. A photomicrograph of her stained PB smear is provided in
Figure 1.

EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) was suspected and the clinician was advised to
send a sample in a heparin tube. Repeat testing of the new sample presumed to be collected in heparin
showed normal platelet count. The CBC data/platelet counts obtained over prior months of past follow-
up care were reviewed; see Table 1.
Discussion EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is a common laboratory
phenomenon with estimated prevalence of 0.1%–2% in hospitalized patients [1, 2]. It is due to in vitro
agglutination of platelets in the blood collection tube caused by IgM/IgG autoantibodies directed against
epitopes on platelet surface glycoprotein (GP) IIb/IIIa. EDTA induces a conformational change in
GPIIb/IIIa, exposing these epitopes and resulting in platelet agglutination [3]. The use of an alternate
anticoagulant, such as citrate or heparin, may be helpful. However, up to 17% of patients with EDTA-
PTCP also show this phenomenon with citrate [2, 3].

Bizzaro conducted a large study of EDTA-PTCP cases and found that 83% had antiplatelet antibodies.
The phenomenon was not age-related or gender-related, nor was it associated with any particular
pathology or use of specific drugs. It showed that EDTA-dependent PTCP is a phenomenon related to the
presence of natural autoantibodies with antiplatelet activity and is not associated with any pathological
significance [4].

It is important to differentiate EDTA-associated thrombocytopenia from that seen in type 2B von

Willebrand disease (vWD type 2B). Kumar and colleagues reported a case of vWD type 2B in a child that
was misconstrued as EDTA-PTCP [3]. The patient presented with extensive bruising. CBC showed
thrombocytopenia, baseline coagulation profile was normal, and PB smear showed platelet clumping.
Due to the severity of bruising, child abuse was suspected as thrombocytopenia was initially
misconstrued as being caused by EDTA-related platelet clumping. Further coagulation work-up revealed
low von Willebrand factor antigen and ristocetin cofactor activity, and molecular testing confirmed vWD
type 2B [3]. The latter is an in vivo consumption of platelet, which results in true thrombocytopenia.
Additionally, due to the consumptive nature and compensatory regenerative activity in megakaryocytic
cell line, causing a platelet “left shift,” the mean platelet volume (MPV) is increased in vWD type 2B. This
morphologic observation may help further separate the two conditions presumptively upon
examination of PB smear; refer to Figure 2 for morphologic comparison and Table 2 for comparative
features.

Other possible preanalytical factors to consider upon investigating platelet clumps include the collection
method, that is, capillary venous or line draws. Capillary collections are prone to clotting and formation
of platelet clumps. Viral infection, drugs, and medications, especially chemotherapeutic agents, are all
possible inducers of platelet clumping [5, 6]. Clumping can also be due to a combination of more than
one of the above factors, and it is possible that a transient viral infection was a confounding cause in our
patient (note the atypical lymphocyte suggestive of a viral infection seen in Figure 1 and the CBC results
listed in Table 1 showing the fluctuation in WBC, RBC, and Hgb levels coinciding with episodes of
clumping and returning to normal levels along with the platelet count).

Recommendations:

From a practical laboratory point of view, investigation of platelet clumping may include the following
steps until a nonclumping smear is obtained, noting that Steps 3 and 4 are reserved for the rare
instances, where Steps 1 and 2 do not resolve the platelet clumping.
Step 1. Verify method of blood draw (e.g., finger stick versus venipuncture versus line draw) and exclude
collection method related clotting.

Step 2. Test a blood sample collected in sodium citrate. If clumping persists, continue to Step 3.

Step 3. Test a sample collected in heparin. If Step 3 is not possible, proceed to Step 4.

Step 4. Obtain a sample in ammonium oxalate, and count platelets utilizing a hemocytometer grid, if
available, as per described methods [4]. Modern hematology analyzers “flag” platelet clumps, and this
should prompt manual verification by examination of a stained PB smear. Reporting platelet counts on
samples that show platelet clumps can be a challenge. A recommended approach is not to give a
platelet result value for a sample with clumps if the instrument’s count is below the lower limits of
normal, report the clumping, and recommend one of the steps described above. If the instruments’
platelet count of a sample is within or above normal range, a count may be given with an added
comment noting the presence of platelet clumps and suggesting that the true count is likely higher than
reported.

Pseudothrombocytopenia due to Platelet Clumping: A Case Report and Brief Review of the Literature :
Figure 1

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Pseudothrombocytopenia due to Platelet Clumping: A Case Report and Brief Review of the Literature :
Table 1

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Pseudothrombocytopenia due to Platelet Clumping: A Case Report and Brief Review of the Literature :
Table 2

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Pseudothrombocytopenia due to Platelet Clumping: A Case Report and Brief Review of the Literature :
Figure 2

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