VOLUME - XII

ISSUE - LXX
JUL/AUG 2015

Polycythemia or Polycythaemia vera (PV, PCV) (also known as erythremia, primary

polycythemia and polycythemia rubra vera) is a neoplasm in which the bone marrow
makes too many red blood cells. It may also result in the overproduction of white blood
1 Editorial cells and platelets.
Disease
2 Most of the health concerns associated with polycythemia vera are caused by the blood
Diagnosis being thicker as a result of the increased red blood cells. It is more common in the elderly
8 Interpretation and may be symptomatic or asymptomatic. Common signs and symptoms include itching
(pruritus), and severe burning pain in the hands or feet that is usually accompanied by a
reddish or bluish coloration of the skin. Patients with polycythemia vera are more likely to
10 Bouquet
have gouty arthritis. Treatment consists primarily of phlebotomy.

11 Trouble
Shooting Polycythemia veras occurs in all age groups, although the incidence increases with age.
One study found the median age at diagnosis to be 60 years. Polycythemia vera (PCV),
12 Tulip News being a primary polycythemia, is caused by neoplastic proliferation and maturation of
erythroid, megakaryocytic and granulocytic elements to produce what is referred to as
panmyelosis. In contrast to secondary polycythemias, PCV is associated with a low serum
level of the hormone erythropoietin (EPO). Instead, PCV cells have a mutation in the
tyrosine kinase (JAK2), which acts in signaling pathways of the EPO-receptor, rendering
those cells hypersensitive to EPO.

For complete clinico-diagnostic approach, please turn over. The DISEASE DIAGNOSIS
section reveals all. INTERPRETATION clearly outlines the morphologies of all blood cells
with colour pictures, sizes and features etc. Identification hereafter would be much easier.
The TROUBLESHOOTING component highlights the erroneous results with their
reasons that we often get while using automated cell counters.

Amidst all this BOUQUET proudly occupies its place. Happy READING!!

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different forms of MPDs need to be elucidated. Thrombosis and bleeding

DISEASE DIAGNOSIS are frequent in persons with polycythemia vera, as a result of the
disruption of hemostatic mechanisms because of (1) an increased level
of red blood cells and (2) an elevation of the platelet count. There are
POLYCYTHEMIA VERA findings that indicate the additional roles of tissue factor and
Background polymorphonuclear leukocytes (PMLs) in clotting, the platelet surface as
Polycythemia vera (PV) is a stem cell disorder characterized as a a contributor to phospholipid-dependent coagulation reactions, and the
panhyperplastic, malignant, and neoplastic marrow disorder. Its most entity of platelet microparticles. Tissue factor is also synthesized by
prominent feature is an elevated absolute red blood cell mass because blood leukocytes, the level of which is increased in persons with MPD,
of uncontrolled red blood cell production. This is accompanied by which can contribute to thrombosis. Rusak et al evaluated the
increased white blood cell (myeloid) and platelet (megakaryocytic) hemostatic balance in patients using thromboelastography and also
production, which is due to an abnormal clone of the hematopoietic stem studied the effect of isovolemic erythrocytapheresis on patients with
cells with increased sensitivity to the different growth factors for polycythemia vera. They concluded that thromboelastography may help
maturation. to assess the thrombotic risk in patients with polycythemia vera.
Pathophysiology Hyperhomocystinemia is a risk factor for thrombosis and is also widely
Normal stem cells are present in the bone marrow of patients with prevalent in patients with MPD (35% in controls, 56% in persons with
polycythemia vera (PV), but also present are abnormal clonal stem cells polycythemia vera). Acquired von Willebrand syndrome is an
that interfere with or suppress normal stem cell growth and maturation. established cause of bleeding in persons with MPD, accounting for
Evidence indicates that the etiology of panmyelosis is unregulated approximately 12-15% of all patients with this syndrome. von Willebrand
neoplastic proliferation. The origin of the stem cell transformation syndrome is largely related to the absorption of von Willebrand factor
remains unknown. See the image below. onto the platelets; reducing the platelet count should alleviate the
bleeding from the syndrome.
Epidemiology
Frequency: Polycythemia vera (PV) is relatively rare, occurring in 0.6-
1.6 persons per million population.
Race: Many studies have shown that this condition occurs in all ethnic
groups.
Sex: Polycythemia vera has no sex predilection, although the
Polycythemia Vera Study Group (PVSG) found that slightly more males
than females are affected.
Age: The peak incidence of polycythemia vera is age 50-70 years.
However, this condition occurs in persons of all age groups, including
Bone marrow film at 100X magnification demonstrating early adulthood and childhood, albeit rarely.
hypercellularity and increased number of megakaryocytes. History: Symptoms of polycythemia vera (PV) are often insidious in
Progenitors of the blood cells in these patients display abnormal onset, and they are often related to blood hyperviscosity secondary to a
responses to growth factors, suggesting the presence of a defect in a marked increase in the cellular elements of blood. Subsequent sludging
signaling pathway common to different growth factors. The observation of blood flow and thrombosis lead to poor oxygen delivery, with
that in vitro erythroid colonies grow when no endogenous erythropoietin symptoms that include the following: Headache, Dizziness, Vertigo,
(Epo) is added to the culture and the presence of a truncated Epo Tinnitus, Visual disturbances, Angina pectoris, Intermittent claudication.
receptor in familial erythrocytosis indicate that the defect is in the Bleeding complications, seen in approximately 1% of patients with PV,
transmission of the signal. The sensitivity of polycythemia vera include epistaxis, gum bleeding, ecchymoses, and gastrointestinal (GI)
progenitors to multiple cytokines suggests that the defect may lie in a bleeding. Thrombotic complications (1%) include venous thrombosis or
common pathway downstream from multiple receptors. Increased thromboembolism and an increased prevalence of stroke and other
expression of BCLX suggests an additional decrease in cellular arterial thromboses. Abdominal pain due to peptic ulcer disease may be
apoptosis. Several reasons suggest that a mutation on the Janus present because PV is associated with increased histamine levels and
kinase-2 gene (JAK2) is the most likely candidate gene involved in gastric acidity or possible Budd-Chiari syndrome (hepatic portal vein
polycythemia vera pathogenesis, as JAK2 is directly involved in the thrombosis) or mesenteric vein thrombosis. Early satiety can occur in
intracellular signaling following exposure to cytokines to which patients with splenomegaly, because of gastric filling being impaired by
polycythemia vera progenitor cells display hypersensitivity. A recurrent the enlarged spleen or, rarely, as a symptom of splenic infarction. Weight
unique acquired clonal mutation in JAK2 has been found in most loss may result from early satiety or from the increased
patients with polycythemia vera and other myeloproliferative diseases myeloproliferative activity of the abnormal clone. Pruritus results from
(MPDs), including essential thrombocythemia and idiopathic increased histamine levels released from increased basophils and mast
myelofibrosis. A unique valine-to-phenylalanine substitution at position cells and can be exacerbated by a warm bath or shower. This occurs in
617 (V617F) in the pseudokinase JAK2 domain has been identified. The up to 40% of patients with PV.
substitution, called JAK2V617F, leads to a permanently turned-on Physical
signaling at the affected cytokine receptors. How these mutations Physical findings in patients with polycythemia vera (PV) are due to the
interact with the wild-type kinase genes and how they manifest into myeloproliferative process and excess concentrations of the cellular

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elements of blood with extramedullary hematopoiesis. Splenomegaly is that overlap with polycythemia vera (PV): Essential thrombocytosis
present in 75% of patients at the time of diagnosis. Hepatomegaly is (ET), Chronic myelogenous leukemia (CML), Agnogenic myeloid
present in approximately 30% of patients. Plethora or a ruddy metaplasia (AMM). Abdulkarim et al studied the long-term rate (15
complexion is characteristic of PV and results from the marked increase years) of leukemic transformation (acute myelogenous leukemia [AML])
in total red blood cell mass. This manifests in the face, palms, nailbeds, in 795 unselected patients with Ph– MPD in the regions of Gothenburg,
mucosa, and conjunctiva. Hypertension is common in patients with PV. Sweden, and the Côte d'Or, Burgundy, France. Fifty-six patients (7%)
Measurement of the red blood cell mass should differentiate this with Ph– MPD demonstrated transformation to AML, of whom six had
condition from Gaisbock syndrome, which is hypertension and never received cytoreductive agents and two had been exposed to
pseudopolycythemia (i.e. high hemoglobin levels due to low plasma interferon. The annual rate of AML transformation was 0.38% in patients
volume). with PV, 0.37% in those with ET, and 1.09% in individuals with idiopathic
Causes myelofibrosis (IMF). The average time from diagnosis to AML
The causes of polycythemia vera (PV) are unknown, but a number of transformation was 88 +/- 56 months in the PV patients compared with
approaches are now being studied to define the molecular lesion or 76 +/- 57 months in the ET patients and 42 +/- 33 months in those with
lesions. The JAK2 V617F mutation can give rise to a turned-on cytokine IMF (significantly shorter than the PV and ET patients), and the
receptor, leading to pancytosis similar to the PV phenotype. This is investigators noted that it appeared to be a continuous event in all 3
similar to the biologic properties of the BCR/ABL abnormality in that they MPDs. Another finding was that 17 of 18 patients with PV whose
both mimic cytokine signaling. Clonality studies using a rare condition transformed to AML were females despite the fact that the
polymorphism in the G6PD gene demonstrate predominant expression male-to-female ratio of the entire PV group was 146:171. The other
of a single allele in all blood cell lines. X-chromosome inactivation conditions (ET and IMF) showed a slight male preponderance (ET, 1.33;
studies have played a pivotal role in establishing current concepts of IMF: 1.13). The average survival for the 56 patients with MPD who
many hematologic malignancies. Approximately 90% of patients with PV developed AML did not differ among the 3 diseases (4.6 +/- 5.5 months).
show a skewed pattern of X inactivation in all their blood cell lines, Differential Diagnoses
indicating support for the concept of a transformed multipotential stem Agnogenic Myeloid Metaplasia With Myelofibrosis, Chronic
cell. Cytogenetic studies show the presence of an abnormal karyotype in Myelogenous Leukemia, Essential Thrombocytosis, Polycythemia,
the hematopoietic progenitor cells in approximately 34% of patients with Secondary.
PV, depending on which stage of the disease the study was performed Approach Considerations
at. Approximately 20% of patients have cytogenetic abnormalities at The Polycythemia Vera Study Group (PVSG) was the first to set rigorous
diagnosis, increasing to more than 80% for those with more than 10 criteria for the diagnosis of polycythemia vera (PV) in the 1970s. With the
years of follow-up care. The following genetic abnormalities, which are establishment of polymerase chain reaction (PCR)–based methods for
similar to the abnormal karyotypes observed in patients detecting the JAK2V617F mutation, this may become the first molecular
with myelodysplastic syndromes and other MPDs, have been observed diagnostic marker for PV, similar to BCR/ABL for chronic myelogenous
in patients with PV: Deletion of 20q (8.4%), Deletion of 13q (3%), Trisomy leukemia (CML). However, because of a paucity of centers doing red
8 (7%), Trisomy 9 (7%), Trisomy of 1q (4%), Deletion of 5q or monosomy blood cell mass measurements, demonstrating an elevated red blood
5 (3%), Deletion of 7q or monosomy 7 (1%). Spivak and colleagues cell mass continues to become more difficult. The diagnostic criteria set
analyzed gene expression in CD34+ peripheral-blood cells from 19 by the PVSG are organized into two categories, A and B. The diagnosis
patients with PV and found twice as many up-regulated or down- of PV is established if all three category A criteria are present, or if criteria
regulated genes in men as in women. In addition, these researchers A1 plus A2 plus any two criteria from category B are present.
found 102 genes with differential regulation that was concordant in men Category A criteria are as follows:
and women and that could be used to divide patients into two 1. Total red blood cell mass ≥ 36 mL/kg in males or ≥ 32 mL/kg in
phenotypical groups. The groups differed significantly with respect to females
disease duration, clinical manifestations and prognosis. 2. Arterial oxygen saturation ≥92%
Diagnostic Considerations 3. Splenomegaly
Diagnostic laboratory tests have been developed to increase the ability Category B criteria are as follows:
to diagnose primary myeloproliferative diseases (MPDs) and to Thrombocytosis, with platelet count > 400,000/ìL
differentiate them from reactive conditions associated with increased Leukocytosis, with a white blood cell count > 12,000/ìL
blood cell levels, which can mimic MPDs. Polycythemia is characterized Increased leukocyte alkaline phosphatase (ALP) > 100 U/L
by increased cell counts in all cell lines in the myeloid series (i.e. red Serum vitamin B-12 concentration > 900 pg/mL or binding capacity >
blood cells, white blood cells [preferentially granulocytes] and platelets). 2200 pg/mL
Thus, if red blood cell levels are increased, several conditions must be Total red blood cell mass is measured by labeling the cells with
excluded, including the following: Conditions that increase red blood chromium 51 (51Cr). Documentation of an elevated total red blood cell
cells secondary to systemic hypoxia or an artificial condition stimulating mass with51 Cr-labeled red blood cells and, ideally, an iodine-131 (131 I)
erythropoietin secretion in the kidneys. Granulocytosis from infections or plasma volume dual technique differentiates true erythrocytosis from
mobilization by secondary causes, as in leukemoid reactions. pseudoerythrocytosis (decreased plasma volume). However, the red
Thrombocytosis from bleeding and iron deficiency. Once an MPD blood cell mass is becoming difficult to obtain because the51 Cr isotope
(Philadelphia chromosome negative [Ph–]) is documented, it must be needed to perform the test is no longer readily available, and institutions
differentiated from the following conditions, which have manifestations willing to perform the test are few as a result of small demand and lack of

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profit in performing the test. Diagnostic criteria for PV as per the 2008 leukocyte alkaline phosphatase (LAP) score is elevated (>100 U/L) in
revised World Health Organization (WHO) guidelines include both major 70% of patients. This technique is only semiquantitative and is
and minor criteria. Diagnosis requires the presence of both major criteria susceptible to interobserver and laboratory errors unless it can be
and one minor criterion or the presence of the first major criterion performed by flow cytometry, which is not routinely available. The
together with two minor criteria. platelet count is elevated to 400,000-800,000/µL in approximately 50%
Major WHO criteria are as follows: of patients. The release of potassium into the serum caused by the
1. Hemoglobin > 18.5 g/dL in men and > 16.5 g/dL in women, or other increased number of platelets during in vitro coagulation may cause a
evidence of increased red blood cell volume pseudohyperkalemia in the serum, whereas the true plasma potassium
2. Presence of JAK2617V F or other functionally similar mutation, such level in vivo is actually within the reference range, as shown by
asJAK2 exon 12 mutation measuring plasma levels and by the lack of electrocardiography (ECG)
Minor WHO criteria are as follows: changes. Morphologic abnormalities in platelets include
Bone marrow biopsy showing hypercellularity for age with trilineage macrothrombocytes and granule-deficient platelets. Abnormal platelet
growth (panmyelosis) with prominent erythroid, granulocytic, and function (as measured by platelet aggregation tests with epinephrine,
megakaryocytic proliferation. Serum erythropoietin level below the adenosine diphosphate [ADP], or collagen) may be demonstrated, but
reference range for normal. Endogenous erythroid colony formation in bleeding time may be normal. Some patients' platelet-rich plasma
vitro. The major diagnostic issue related to PV is distinguishing it from spontaneously aggregates without the addition of any of the above
other forms of erythrocytosis, which are more common than substances. This indicates a propensity for thromboses. Routine
PV. JAK2 V617F mutation and erythropoietin (Epo) level are key in the coagulation test results are normal, with a high turnover rate for
diagnosis of erythrocytosis. If the JAK2V617F mutation is positive and fibrinogen. The prothrombin time (PT) and activated partial
Epo level is low, then it confirms the diagnosis of PV (JAK2 V617F thromboplastin (aPTT) time may be artifactually prolonged, however,
mutation is positive in 97% of PV patients). If the JAK2 V617F mutation is because the erythrocytosis results in the collection of a low amount of
absent but the Epo level is low, then testing forJAK2 exon 12 and 13 plasma in relation to the anticoagulant in the test tube. Thus, the volume
mutations would be helpful is making a diagnosis of PV in the 2-3% of PV of the ratio of anticoagulant to blood must be modified when drawing
patients who are negative for JAK2 V617F mutation. Patients who are blood for coagulation tests in patients who are polycythemic. Vitamin B-
negative for JAK2 mutations and have a normal or high Epo level have 12 levels are elevated to more than 900 pg/mL in approximately 30% of
secondary erythrocytosis. patients, and 75% of patients show an elevation in the unbound vitamin
Laboratory Studies B-12 binding capacity greater than 2200 pg/mL. This is because of
Automated red blood cell counts and hematocrit values (including increased transcobalamin-III, a binding protein found in white blood
hemoglobin levels) may be deceptive with regard to the total red blood cells, and it reflects the total white blood cell counts in the peripheral
cell mass in patients with polycythemia vera (PV). Direct measurement blood and bone marrow. Hyperuricemia occurs in 40% of patients and
of the red blood cell mass should show an increase with a normal or reflects the high turnover rate of bone marrow cells releasing DNA
slightly decreased plasma volume. This is a nuclear medicine test that metabolites. The most important diagnostic tests are JAK2 mutation
uses radiochromium-labeled red blood cells to measure actual red blood analysis and the serum erythropoietin (Epo) level. A
cell and plasma volume. However, patients with hemoglobin positive JAK2 V617F mutation and a low Epo level confirms the
concentrations of at least 20 g/dL or hematocrit values of at least 60% in diagnosis of PV. A low serum Epo level, which is decreased in nearly all
males and 56% in females always have an elevated red blood cell mass. patients with PV who have experienced no recent hemorrhage,
The red blood cells in patients with PV are usually normochromic and distinguishes polycythemia from secondary causes of polycythemia in
normocytic, unless the patient has been bleeding from underlying peptic which the serum Epo level is generally within the reference range or is
ulcer disease or phlebotomy treatment (wherein the cells may be elevated. Each laboratory has its own reference range for serum Epo
hypochromic and microcytic, reflecting low iron stores). See the image levels.
below. Imaging Studies
An enlarged spleen is often palpable and in such cases, imaging studies
are not required. In some patients with posteriorly enlarged spleens or in
those who are obese, ultrasonography or computed tomography scans
may be able to detect splenic enlargement that was not evident on
physical examination.
Other Tests
Measuring arterial oxygen saturation (SaO2) and carboxyhemoglobin
(COHb) levels is important to rule out hypoxia as a secondary cause for
erythrocytosis. Pulse oximetry is the most convenient method for
This blood film at 10,000X magnification shows a giant platelet and an measuring SaO2 however, in people who smoke cigarettes, the COHb
eosinophil. Erythrocytes show signs of hypochromia as a result of must be determined directly and subtracted to give an accurate
repeated phlebotomies. SaO2 value. A value below 92% indicates a causal relationship with
An elevated white blood cell count (>12,000/µL) occurs in approximately erythrocytosis. If the fall is due to increased COHb, this is less likely to
60% of patients. It is mainly composed of neutrophils with a left shift and cause erythrocytosis. Nocturnal oxygen desaturation due to sleep
a few immature cells. Mild basophilia occurs in 60% of patients. The apnea is observed in 20% of patients. Bone marrow studies are not

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necessary to establish the diagnosis of polycythemia vera. If such elements, mainly red blood cells, to improve the circulation of blood by
studies are performed, however, the finding of hypercellularity and lowering the blood viscosity. Because phlebotomy is the most efficient
hyperplasia of the erythroid, granulocytic, and megakaryocytic cell lines method of lowering the hemoglobin and hematocrit levels to the
or myelofibrosis supports the diagnosis of a myeloproliferative process. reference range, all newly diagnosed patients are initially phlebotomized
See the image below. to decrease the risk of complications. Patients can be phlebotomized
once or twice a week to reduce the hematocrit to the range of less than
45%. A recent randomized trial demonstrated a significant difference in
the rate of thrombotic events and cardiovascular deaths (2.7 % vs 9.8%)
when the hematocrit goal was 45% versus 50%. Patients with severe
plethora who have altered mentation or associated vascular
compromise can be bled more vigorously, with daily removal of 500 mL
of whole blood. Elderly patients with some cardiovascular compromise
or cerebral vascular complications should have the volume replaced
with saline solution after each procedure to avoid postural hypotension.
The presence of elevated platelet counts, which may be exacerbated by
phlebotomy, is an indication to use myelosuppressive agents to avoid
Bone marrow film at 400X magnification demonstrating dominance of thrombotic or hemorrhagic complications.
erythropoiesis. Maintenance therapy
Iron stores are decreased or absent because of the increased red blood Once the patient's hemoglobin and hematocrit values are reduced to
cell mass, and macrophages may be masked in the myeloid hyperplasia within the reference range, implement a maintenance program either by
that is present. Fibrosis is increased and detected early by silver stains inducing iron deficiency by continuous phlebotomies (the frequency of
for reticulin. Cytogenetics of the bone marrow cells show a clonal the procedure depends on the rate of reaccumulation of the red blood
abnormality in 30% of patients who are not treated and in 50% of patients cells) or by using a myelosuppressive agent. The choice depends on the
who are treated with alkylating or myelosuppressive agents. These risks of secondary leukemias and the rate of thrombosis or bleeding.
chromosomal abnormalities include deletions of the long arm of Patients must be cautioned to not take iron supplements. The risks for
chromosome 5 or 20 (5q-, 20q-) and trisomy 8 (+8) or 9 (+9). Leukemic secondary leukemia depend on the type of therapy (eg, phlebotomy,
transformation is usually associated with multiple or complex radioactive phosphorus-32 [32 P], chlorambucil) or the type of
abnormalities. Measuring spontaneous growth of erythroid progenitors myelosuppressive agents (eg, hydroxyurea [HU], anagrelide, interferon
in cultures (burst-forming unit, erythroid [BFU-E]) in the absence of Epo alfa) and duration of therapy. The Polycythemia Vera Study Group
is a very sensitive test for polycythemia vera (PV) or familial (PVSG) demonstrated a decreased survival rate and increased mortality
erythrocytosis. However, it is not routinely available for clinical use. The rate from acute leukemia in the first 5 years, and a total of 17% of patients
hemoglobin-oxygen dissociation curve may be useful in rare cases to had leukemia after 15 years with chlorambucil and with32P. An increased
detect a congenital hemoglobinopathy with increased oxygen affinity. incidence of thrombotic complications occurred in the phlebotomy arm.
This condition can occur in families. This indicates that phlebotomy is not ideal for patients with elevated
Medical Care platelet counts and previous thrombosis, as are observed in patients
The long-term risks of polycythemia vera (PV) include leukemic and who are older. In this situation, using HU has decreased these
fibrotic transformation, which occurs in fewer than 5% and 10% of complications. Hydroxyurea has been the mainstay therapy for PV since
patients, respectively, at 10 years. Current treatment modalities do not the PVSG results indicated it is an effective agent for myelosuppression;
change these outcomes. Instead, treatment for PV is intended to however, concerns have been raised regarding long-term risks for
decrease the risk of arterial and venous thrombotic events, which could leukemic transformation.[16] In the PVSG trial, HU therapy reduced the
be approximately 20%. Patients can be risk-stratified for their risk of risk of thrombosis compared with phlebotomy alone; the PVSG
thrombosis according to their age and history of thrombosis. Patients recommended that HU should be the drug of choice for patients older
older than 60 years or with a previous history of thrombosis are than 40 years. The role of HU in leukemic transformation is not clear.
considered to be high risk. Patients younger than 60 years and with no Several nonrandomized studies have supported or refuted a significant
prior history of thrombosis are considered low risk. All patients with PV rise in leukemic conversion with the long-term use of HU in patients with
should undergo phlebotomy to keep their hematocrit below 45% and essential thrombocythemia (from 0% to 5.5%) and in patients with PV
should take aspirin, 81 mg daily. In addition, if a patient is at high risk for (from 2.1% to 10%). The PVSG closed the chlorambucil arm because of
thrombosis, cytoreductive therapy is added to the management plan. increased rates of acute leukemia after 7 years. However, in the 15-year
Hydroxyurea at a starting dose of 500 mg twice daily is the most follow-up of the HU arm compared with the phlebotomy-alone arm, the
commonly used cytoreductive agent. It can be titrated on the basis of trend for leukemic transformation was greater in the HU arm but the
blood counts. In patients who are refractory to or intolerant of differences did not meet statistical significance. Followup for a median of
hydroxyurea, interferon-alpha can be used as an alternative. Busulfan is 8.6 years and a maximum of 795 weeks showed that 5.4% of patients
also an option for patients older than 65 years. developed leukemia in the HU arm compared with 1.5% of patients
Phlebotomy treated with phlebotomy alone. Other case series have reported
Phlebotomy (bloodletting) has long been the mainstay of therapy for secondary leukemia in 3-4% of patients, which is relatively low
polycythemia vera (PV). The object is to remove excess cellular compared with the benefits of preventing thrombotic complications. In an

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open-label study by Huang and colleagues that included 136 patients successful in 38-100% of patients, with follow-up ranging from 9-98
with JAK2V617F mutation–positive PV, treatment with interferon alfa 2b months.
(IFN á-2b) did not produce a superior overall hematologic response, Consultations
compared with HU. However, IFN á-2b provided better 5-year Consultation with a hematologist is recommended in cases of
progression-free survival (66.3% versus 46.7%, P< 0.01) and clinical polycythemia vera (PV). Long-term follow-up care of these patients and
improvement (in vasomotor symptoms, distal paresthesias, and managing complications of the disease and its treatment can be difficult.
erythromelalgia). No severe hematological adverse events were Medication Summary
observed in patients receiving IFN á-2b. Do not administer alkylating One objective of therapy for polycythemia vera (PV) is to control the
agents to younger patients (< 40 y) who need long-term treatment. myeloproliferative activity of this disease. Evidence of an increase in
Alternative nonleukemogenic agents are needed for these patients. levels of white blood cells and/or platelets and organomegaly indicate
Low-dose aspirin suppresses thromboxane biosynthesis by platelets, uncontrolled myeloproliferative activity that requires a
which is increased in PV and essential thrombocythemia. The European myelosuppressive agent. Studies by the Polycythemia Vera Study
Collaboration on Low-dose Aspirin in Polycythemia Vera (ECLAP) found Group (PVSG) have led to the abandonment of long-term therapy with
that low doses of aspirin (40 mg/d) were effective for preventing Phosphorus-32 (32 P) and most alkylating agents (eg, busulfan,
thrombosis and controlling microvascular painful symptoms chlorambucil), and to the use of hydroxyurea (HU) instead. However,
(erythromelalgia), which result from spontaneous platelet aggregation, long-term data seem to indicate a possible slight late increase in cases of
in patients with PV and essential thrombocythemia, without creating a acute leukemia in patients with PV who are treated with HU for more than
bleeding diathesis. Ruxolitinib: Ruxolitinib (Jakafi), a JAK1/JAK2 15 years. Ruxolitinib is now approved in the United States for
inhibitor, was approved by the FDA in December 2014 for the treatment intermediate- or high-risk myelofibrosis, including primary myelofibrosis,
of patients with polycythemia vera who have had an inadequate post-PV myelofibrosis, and post–essential thrombocythemia
response to or are intolerant of hydroxyurea. Approval was based on myelofibrosis.
data from the Phase III RESPONSE trial. In this trial, patients treated Antimetabolites: Class Summary: HU is a nonalkylating agent that
with ruxolitinib demonstrated superior hematocrit control and reductions inhibits DNA synthesis and cell replication by blocking the enzyme
in spleen volume compared to best available therapy. A greater ribonucleoside diphosphate reductase. Hydroxyurea (Droxia,
proportion of patients on the ruxolitinib treatment arm achieved complete Hydrea): Inhibitor of deoxynucleotide synthesis and DOC for inducing
hematologic remission (ie, hematocrit control, lowering platelet and hematologic remission in CML. Less leukemogenic than alkylating
WBCs). Hematologic adverse reactions are prevalent with ruxolitinib agents such as busulfan, melphalan, or chlorambucil. Myelosuppressive
(incidence >20%) and include thrombocytopenia and anemia. effects last a few days to a week and are easier to control than those of
Ruxolitinib was initially approved in the United States in 2011 for patients alkylating agents; busulfan has prolonged marrow suppression and can
with intermediate- or high-risk myelofibrosis including primary cause pulmonary fibrosis. Can be administered at higher doses in
myelofibrosis, post-polycythemia vera myelofibrosis, and patients with extremely high WBC counts (>300,000/µL) and adjusted
post–essential thrombocythemia myelofibrosis. accordingly as counts fall and platelet counts drop. Dose can be
Surgical Care administered as a single daily dose or divided into 2-3 doses at higher
Consider splenectomy in patients with painful splenomegaly or repeated dose ranges. Droxia, available in smaller tabs of 200, 300, and 400 mg,
episodes of thrombosis causing splenic infarction. Budd-Chiari is for patients with sickle cell disease. Ruxolitinib (Jakafi): JAK1/JAK2
syndrome occurs in patients with myeloproliferative disease (MPD) and kinase inhibitor indicated for polycythemia vera in patients who have had
most frequently in young women. Surgical approaches to the an inadequate response to or are intolerant of hydroxyurea. Janus-
management of Budd-Chiari syndrome are, therefore, relevant to associated kinases (JAKs) JAK1 and JAK2 mediate the signaling of a
patients with polycythemia vera. Budd-Chiari syndrome is a liver-related number of cytokines and growth factors that are important for
condition associated with large-vessel thromboses and outflow hematopoiesis and immune function. Imidazole Quinazolines: Class
obstruction with inferior vena cava or portal vein thrombosis. This is Summary: Imidazole quinazolines have been demonstrated to have
associated with the development of ascites, hepatosplenomegaly, powerful anti-aggregating effects on platelets and to cause
abdominal pain, and gastrointestinal bleeding, but 20% of patients are thrombocytopenia. Anagrelide hydrochloride (Agrylin): Primary
asymptomatic. The diagnosis is made by using ultrasonography to activity is to lower platelet levels but shows slight decrease in mean
identify portal vein patency. In addition to the standard computed hemoglobin and hematocrit while WBC counts maintained. Effective in
tomography (CT) scan and magnetic resonance imaging (MRI), patients polycythemia vera with elevated platelet counts. Adjust dosage to lowest
with Budd-Chiari syndrome may need invasive angiographic imaging to effective dose to reduce and maintain platelet counts, WBC count, and
determine the hemodynamics of the liver and the intrahepatic and vena hemoglobin levels within reference range. Interferons: Class
caval gradients to determine the best surgical procedure. The histology Summary: Recombinant interferon alfa is a biologic response modifier
of the liver helps determine the acuteness of the problem, the presence with myelosuppressive activity. Recombinant alfa-2a (Roferon) or
of chronic changes, and the degree of cirrhosis. This determines alfa-2b (Intron) interferon: Protein product manufactured by
whether a patient requires a shunt or a liver transplant. The following recombinant DNA technology. Can lower counts and shrink enlarged
procedures have been used in patients with Budd-Chiari syndrome: spleens.
Transjugular intrahepatic portosystemic shunt (TIPS). Side-to-side Further Inpatient Care
portocaval shunt or mesocaval shunt, portocaval/cavoatrial shunt, or The optimum management of polycythemia vera (PV) remains elusive
mesoatrial shunt. These procedures have been reported to be despite the findings of the Polycythemia Vera Study Group (PVSG).

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However, certain diseases, such as familial erythrocythemia, secondary anticoagulation with warfarin (see below).
polycythemia, and relative polycythemia (a benign condition), should be
differentiated, and the exact diagnosis of PV should be established.
General principles in the management of PV include the following: Tailor
therapy to suit the clinical needs of the patient; consider the status of the
formed elements of the blood, bone marrow, and organomegaly.
Normalize red blood cell mass with phlebotomy as rapidly as clinically
possible (250-500 mL every other day); patients who are elderly or have
cardiovascular compromise should be phlebotomized cautiously, and
smaller amounts should be removed. Suppress myeloproliferative
activity with chemotherapy (hydroxyurea) in all patients older than 50
32
years. In general, phosphorus-32 ( P) should be reserved for patients
older than 80 years or patients with comorbid conditions in whom life This blood film at 1000X magnification shows a giant platelet and an
expectancy is less than 5-10 years and the convenience of 32 P dosing eosinophil. Erythrocytes show signs of hypochromia as a result of
outweighs the substantial risks of developing acute leukemia 5-15 years repeated phlebotomies.
after 32 P administration. Patients with thrombotic tendencies or those Complications
who develop thrombocytosis following phlebotomy should be treated Bleeding complications (1%) in patients with polycythemia vera (PV)
with marrow suppression; consider anagrelide in younger patients (aged include epistaxis, gum bleeding, ecchymoses, and GI bleeding.
50-70 y). Maintain blood values at reference range levels by regular Thrombotic complications (1%) include venous thrombosis or
examination and treatment. Avoid overtreatment and toxicity by careful thromboembolism and an increased prevalence of stroke and other
and judicious use of chemotherapy and radiation; supplemental arterial thromboses. In young patients and during pregnancy, interferon
phlebotomy is preferred over excess marrow suppression. Postpone alfa may be useful when HU is unsuitable. Anagrelide may be useful
elective surgery until long-term control of the disease is established. when interferon alfa is not tolerated. In the very elderly patients for whom
Women of childbearing age should be treated with phlebotomy only. In regular clinic attendance is impractical,32 P or intermittent busulfan may
young males, myelosuppressive therapy can lead to aspermia; thus, still be used.
evaluate treatment carefully before using any chemotherapy or Prognosis
radiotherapy. The PVSG no longer recommends the use of alkylating Polycythemia vera (PV) is a chronic disease and its natural history of 1.5-
agents because of the associated increased incidence of leukemia and 3 years of median survival in the absence of therapy has been extended
certain types of cancer. Treat hyperuricemia with allopurinol (100-300 to at least 10-20 years because of new therapeutic tools. The major
mg/d) until remission has been attained; for acute gouty attacks, causes of morbidity and mortality are as follows: Thrombosis has been
colchicine or other anti-inflammatory agents are indicated. reported in 15-60% of patients, depending on the control of their disease.
Further Outpatient Care It is the major cause of death in 10-40% of patients. Venous and arterial
Thrombosis in polycythemia vera (PV) is substantially more frequent in thromboses have resulted in pulmonary emboli, renal failure from renal
patients treated with phlebotomy alone without myelosuppression. This vein or artery thrombosis, intestinal ischemia from mesenteric vein
risk is believed to be related to thrombocytosis, which was not observed thromboses, or peripheral arterial emboli. Hemorrhagic complications
in the study. Platelet numbers alone are not likely to be the primary factor occur in 15-35% of patients and lead to death in 6-30% of these patients.
responsible for the increased risk of thrombosis; the presence of Bleeding is usually the consequence of vascular compromise resulting
abnormal platelets is more likely. The initial PVSG study using from ischemic changes from thrombosis or hyperviscosity. Peptic ulcer
antiplatelet drugs also used aspirin at 300 mg 3 times a day plus disease is reported to be associated with polycythemia vera (PV) at a 3-
dipyridamole at 75 mg 3 times a day. This showed an increase in the to 5-fold higher rate than that of the general population. This has been
incidence of hemorrhage. Lower doses of aspirin have been suggested attributed to increased histamine serum levels. Myelofibrosis and
to be more effective without increasing bleeding complications, although pancytopenia occur in 3-10% of patients, usually late in the disease,
this has not yet been demonstrated in a prospective randomized trial. A which is considered the spent phase of polycythemia vera (PV). In these
syndrome specific to polycythemia vera (PV) and other MPDs is termed patients, infections and bleeding complications may be the most serious
erythromelalgia, and it is associated with an increased risk of health threats, and red blood cell transfusions may be required to
thrombosis. The symptoms are burning pain in the feet, hands, and maintain adequate red blood cell counts and to improve fatigue and
digits, sometimes associated with pallor, erythema, or cyanosis of the other anemia-related symptoms. Acute leukemia or a myelodysplastic
distal portions of the extremities. Occasionally, it may progress to frank syndrome develops in 1.5% of patients treated with phlebotomy alone.
gangrene. In some instances, this is treated with aspirin (50-300 mg/d) The transformation risks increase to 13.5% within 5 years with treatment
and dipyridamole (75 mg orally 3 times a day). Myelosuppressive using chlorambucil and to 10.2% within 6-10 years in patients treated
therapy plus phlebotomies, with the intent of normalizing the erythrocyte with 32 P. At 15 years, the transformation risk for HU is 5.9%, which,
and platelet counts, also decreases or eliminates these symptoms. although not statistically significant, is a worrisome trend.
Proven thrombotic complications warrant the use of long-term

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Fig. 1.2: Erythropoiesis

INTERPRETATION

DEVELOPMENT OF BLOOD CELLS SITES OF BLOOD

FORMATION
Normal Sites
Foetus: Less than 2 months—yolk sac.
From 2-7 months: Liver, with minimal haemopoiesisin spleen.
After 3 months: Haemopoiesis starts in bone marrow.
Full-term infant: Bone marrow is the only site for production of
granulocytes and monocytes. Occurs mainly in the spleen, lymph nodes
and other lymphoid tissues, though liver and bone marrow produce
these in much less numbers.
After birth: Same as above except that the monocytes are provided by
the bone marrow, spleen and lymphoid tissues contribute minimally.
Abnormal Sites
Extramedullary haemopoiesis (myeloid metaplasia): In certain disorders
the foetal organs revert to their old function supported by the reticulum
cells which retain their potential haemopoietic activity. This occurs when
bone marrow cannot any further fulfil the requirements or demand
imposed upon it, e.g. in Growing children with haemolysis,
Myelosclerosis, Secondary carcinoma of the bone.
Development of Blood Cells (Chart 1.1)
Blood formation has to undergo three stages: (1) Multiplication of
precursor cells (1% of all marrow cells are in dividing phase). (2) Gradual
maturation (both structural and functional).
Chart 1.1: Development of blood cells

(3) Release into the peripheral circulation. The exact release mechanism Late normoblast: 8-10 ìm, cytoplasm is acidophilic,nucleus becomes
is ill understood, granulocytes achieve this by their motility and RBCs by much smaller, later it becomes pyknotic and is eccentrically placed,
diapedesis. ultimately it is lost by extrusion.
Erythropoiesis (Fig. 1.2) Reticulocyte: Flat, non-nucleated, disc shaped, slightly larger than
Erythroblast is a nucleated red cell. Normoblast implies normal mature RBC. It shows diffuse pale basophilia which appears in the form
(reaction) erythropoiesis. Normoblastic maturation involves: Reduction of a reticulum with supravital stains (brilliant cresyl blue or new
in cell size. Ripening of cytoplasm, i.e. haemoglobinisation. Maturation methylene blue). In 1-2 days, it loses its basophilia and becomes a
time from pronormoblast to RBC is 7 days. Mitotic division occurs till the mature erythrocyte.
intermediate normoblast stage. Control of erythropoiesis: Erythropoietin (formed in kidneys) is released
Pronormoblast: 12-20 ìm, large nucleus surrounded by a rim of deep in response to lowered tissue oxygen tension. Erythropoietin is a
basophilic cytoplasm and has a perinuclear halo. Nucleus is round and glycoprotein and stimulates primitive cell differentiation to
has several nucleoli. pronormoblasts. It affects the rate of multiplication and maturation. It
Early normoblast: 10-16 ìm, nucleus still large, chromatin coarser and acts up to early normoblast stage and also affects the rate of
deeply staining nucleoli disappear. haemoglobinisation.
Intermediate normoblast: 8-14 ìm, nucleus smaller, haemoglobinisation Erythropoietin levels are reduced in:
commences, cytoplasm takes an acidophilic tint, chromatin becomes • Acute starvation. • Hypophysectomy.
coarser and very deeply staining. • Transfusion-induced polycythaemia.

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Erythropoietin levels are increased in: granules which are peroxidase positive. Nuclear chromatin becomes
• All anaemias except those of renal origin. condenser and nucleoli are less well defined.
• Aplastic anaemia. Myelocyte: Specific neutrophilic granules appear, nucleus shows no
• Polycythaemia. nucleoli. N:C ratio reduces, cytoplasm is pale pink, chromatin thicker and
Leucopoiesis (Fig. 1.3) deeply stained.
The Myeloid Series: Specific granules are developed at the myelocyte Metamyelocyte: Nucleus is smaller and indented, cytoplasm is pink with
stage which determine the nature of the mature cell. neutrophilic granules (purplish).
Band or stab form: Cell becomes still smaller, nucleus has a deep
indentation, chromatin is coarsely clumped. Cytoplasm is pink with
purplish granules.
Segmented neutrophil: 12-14 ìm in size, nucleus shows 2-5 lobes,
chromatin in dark purple clumps, cytoplasm has numerous, fine, evenly
distributed purplish granules. In female at least 6 neutrophils/500 should
show drumsticks.
The mature eosinophil: 16 mm in size, granules are acidophilic and
larger. Nucleus is bilobed and is not masked by granules.
The mature basophil: It usually has a bilobed nucleus, but the nucleus is
masked by about 10 large basophilic granules.
Lymphocytic Series (Fig. 1.4)
Lymphoblast: 15-20 ìm in size, resembles myeloblast, cytoplasm is
agranular and moderately basophilic. Nuclear chromatin gives fine
reticular appearance with up to 2 nucleoli. It is peroxidase negative.

Fig. 1.3: Leucopoicsis

Development of a Mature Neutrophil: Maturation involves: (1)
Development of specific granules. (2) Loss of basophilia of the
cytoplasm. (3) Nuclear ripening till the segmented stage. (4) Ability to be
motile and to phagocytose. (Mitotic division occurs till the myelocyte
stage only).
Myeloblast: 15-20 ìm has a large round or oval nucleus, evenly stained
chromatin in strands or granules with reticular appearance, 1-6 nucleoli.
The cell is peroxidase negative.
Promyelocyte: It is like myeloblast except that it contains azurophilic Fig. 1.4: Lymphocytic series

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Large lymphocyte: 12-16 ìm in size, has abundant pale sky blue Promegakaryocyte: 30 ìm in diameter, cytoplasm is intensely basophilic
cytoplasm with a few purplish red granules seen in about 33% of the with fine azurophilic granules. Nucleus may show mild lobulation and
cells. chromatin appears denser.
Small lymphocyte: 9-12 ìm in size, has scanty cytoplasm. Nucleus is Megakaryocyte: 30-90 ìm in diameter, it contains a single multilobulated
usually round and shows heavily clumped chromatin. or indented nucleus. Nuclear lobes may vary from 4-16 in number.
Monocytic Series (Fig. 1.3) Cytoplasm is bulky, light blue with fine azurophilic granulation. The
Monoblast: It resembles myeloblast. margin is irregular and may show fragmentation or budding, precursor of
Promonocyte: Up to 20 ìm in size, has a large convoluted nucleus, circulating platelets.
chromatin is seen in skein like strands. Cytoplasm is dull grey-blue and Mature platelet: 1-4 ìm. It is formed by fragmentation of megakaryocytic
may contain a few azurophilic granules. pseudopods. In circulation they acquire a discoid shape. Cytoplasm
Monocyte: 15-20 ìm in size, has abundant dull grey-blue cytoplasm with stains light blue and contains purple reddish granules which may be
a ground glass appearance and may show vacuolation and fine clumped centrally.
azurophilic granules. It has a kidney-shaped nucleus. Control of platelet production: Perhaps by a humoral factor called
Thrombopoiesis (Fig. 1.3) thrombopoietin, acts by a feedback mechanism.
Megakaryoblast: 20-30 ìm in size, has a large, oval or kidney-shaped
nucleus with several nucleoli. It possesses relatively small amount of
agranular cytoplasm.

BOUQUET Wisdom Whispers

Thoughts to Introspect
In Lighter Vein
A Lot Of Trouble Would Disappear If Only People Would Learn To Talk To One Another
There's this guy who had been lost and walking in Instead Of Talking About One Another .....
the desert for about 2 weeks.
One hot day, he sees the home of a missionary. When People Walk Away From You, Let Them Go. Your Destiny Is Never Tied To
Tired and weak, he crawls up to the house and Anyone Who Leaves You. It Doesn't Mean They Are Bad People. It Just Means That
collapses on the doorstep. Their Part In Your Story Is Over.. !
The missionary finds him and nurses him back to
People nowadays are like Bluetooth, If you stay close they stay connected, If you go
health. away they find new devices...
Feeling better, the man asks the missionary for
directions to the nearest town. Human Life Would Be Perfect If... Anger Had A STOP Button
On his way out the backdoor, he sees this horse. He Mistakes Had A REWIND Button Hard Times Had A FORWARD Button And Good Times
goes back into the house and asks the missionary, A PAUSE Button !!
"Could I borrow your horse and give it back when I
reach the town?” Always Welcome Your Problems, Because Problems Gives You Dual Advice, Firstly,
The missionary says, "Sure but there is a special You Can learn How To Solve Them, Secondly, You Learn How To Avoid Them In Future,
thing about this horse. You have to say 'Thank God' Have Faith In GOD And Yourself…!
to make it go and 'Amen' to make it stop.”
Reflection Cannot Be Seen In Boiling Water, In The Same Way,
Not paying much attention, the man says, "Sure,
Truth Cannot Be Seen In A State Of Anger! Analyze Before You Finalize.
ok.”
So, he gets on the horse and says, "Thank God" and Success Is Like A Beautiful Lover It Will Leave Us At Anytime,
the horse starts walking. Then he says, "Thank God, But Failure Is Like A Mother It Will Teach Us Some Important Lessons Of Life!
Thank God, " and the horse starts trotting.
Feeling really brave, the man say, "Thank God,
Thank God, Thank God, Thank God, Thank God" Brain Teasers Identify the species of the malarial parasites by looking
and the horse just literally takes off. at the images of their micro-gametocytes
Pretty soon he sees this cliff coming up and he's
doing everything he can to make the horse stop.
"Whoa, stop, hold on!!!!”
Finally he remembers, "AMEN!!”
The horse stops 4 inches from the cliff.
The man leans back in the saddle and says, "Thank
God" 1 2 3 4
1. P. vivax 2. P. malariae 3. P. falciparum 4 P. ovale
ANSWERS.　

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TROUBLESHOOTING well,　 such　 as　 heparin,　 citrate　 or　 oxalate.　 Because　 the　 generated　 platelet　
aggregates　 are　 large,　 the　 automated　 counters　 do　 not　 recognize　 them　 as　
platelets,　 leading　 to　lower　 platelet　 counts.　 In　 some　 cases　 the　 aggregates　
are　 large　 enough　 to　 be　 counted　 as　 leukocytes,　 causing　 a　 concomitant　
ISSUES　
FACED　
WITH　
AUTOMATED　
CELL　
COUNTERS pseudoleukocytosis.　 The　 aggregation　 in　 pseudothrombocytopenia　 is　
Measurement　 of　 complete　 blood　 cell　 count　 is　 one　 of　 the　 essential　 time-dependent　 and　 usually　 temperature-sensitive,　 with　 maximal　 activity　
laboratory　 tests.　 Electronic　 blood　 cell　counters　 simplify　 and　 speed　 up　 the　 at　 room　 temperature.　 The　 EDTA-induced　 pseudothrombocytopenia　 is　
performance　 of　blood　 counts　 and　 the　 calculation　 of　 red　 cell　 indexes.　 mediated　 by　 autoantibodies　 of　 IgG,　 IgM　 and　 IgA　 subclasses　 directed　 at　
Because　 of　 their　high　 precision,　 physicians　 tend　 to　 accept　 their　 results　 an　 epitope　 on　 glycoprotein　 IIb.　 This　 epitope　 is　 normally　 hidden　 in　 the　
as　accurate.　 However,　 these　 counters　 can　 be　 “fooled”　 by　 changes　 in　cell　 membrane　GP　IIb/IIIa.　Ionized　calcium　has　an　important　role　in　
size　with　 platelet　 clumping,　 agglutination　 of　erythrocytes,　 or　 precipitation　 maintaining　 the　 heterodimeric　 structure　 of　the　 GP　 IIIb/IIa　 complex.　 The　
of　abnormal　 proteins.　 Failure　 of　 the　 physician　 to　 recognize　 these　 errors　 EDTA,　 through　 its　 chelating　 effect,　 dissociates　 the　 GP　 IIIb/IIa　 complex　
may　 lead　 to　 patients　 being　 subjected　 to　unnecessary　 procedures　 and　 with　 epitope　 exposure.　 In　 Glanzmann’s　 thrombasthenia,　 a　 disorder　
therapy.　 In　 this　 issue　 of　 Crux,　 we　 present　 four　 patients　 in　 whom　 the　 characterized　by　the　quantitative　and/or　qualitative　abnormality　of　
results　 of　 red　 blood　 cell　 indexes　 measured　 by　 an　 automated　 counter　 glycoprotein　IIb/IIIa,　pseudothrombocytopenia　does　not　occur.　
were　incompatible　and　unreasonable.　These　patients　had　cold　 Interestingly,　 in　 recent　 years,　 Abciximab-a　 GP　 IIb/IIIa　 antagonist　 has　
agglutinins:　two　of　them　had　a　respiratory　infection,　one　had　 been　associated　with　pseudothrombocytopenia.　If　anticoagulant-
cytomegalovirus　mononucleosis,　and　the　fourth　had　a　B　cell　 induced　pseudothrombocytopenia　is　suspected　a　peripheral　blood　
lymphoproliferative　 disease.　 One　 of　 the　 patients　 also　 had　 a　 clinically　 smear　 should　 be　 examined　 for　 platelet　 clumping.　 Platelet　 satellitism　 is　
overt　 autoimmune　 hemolytic　 anemia.　 Cold　 agglutinins　 are　 polyclonal　 or　 similar　 to　 anticoagulant　 pseudothrombocytopenia.　 In　 the　 presence　 of　
monoclonal　autoantibodies,　usually　of　immunoglobulin　M　subtype,　 EDTA,　 platelets　 bind　 to　 leukocytes　 and　 form　 rosettes.　 The　 binding　 is　
directed　 against　 I　or　 i　antigens　 and　 preferentially　 binding　 erythrocytes　 at　 usually　 to　 neutrophils　 but　 binding　 to　 other　 white　 blood　 cells　 has　 been　
cold　temperatures.　These　autoantibodies　may　be　associated　with　 reported.　 The　 automated　 analyzers　 do　 not　 correctly　 recognize　 platelet-
malignant　 or　 benign　 disorders　 (e.g.,　 B　 cell　 neoplasm,　 post-infection,　 neutrophil　clumping,　resulting　in　pseudothrombocytopenia.　Platelet　
collagen　vascular　disease)　and　can　be　manifested　by　transient　 satellitism　is　mediated　by　autoantibodies　of　IgG　type.　These　
laboratory　 abnormalities　 up　 to　 severe　 autoimmune　 hemolytic　 anemia.　 autoantibodies　 are　 directed　 at　 GP　 IIIb/IIa　 on　 the　 platelet　 membrane　 and　
With　 automated　 analyzers,　 cold　 agglutinin　 laboratory　 abnormalities　 to　an　Fc　gamma　receptor　III　on　the　neutrophil　membrane.　
typically　 present　 as　 a　 discrepancy　 between　 the　 red　 blood　 cell　indexes.　 Pseudothrombocytopenia　 occurs　 with　 giant　 platelets.　 Because　 of　 their　
The　 agglutinated　 erythrocytes　 may　 be　 recognized　 as　 single　 cells　 or　 may　 size,　 the　 giant　 platelets　 are　 excluded　 from　 electronic　 platelet　 counting.　
be　too　large　to　be　counted　as　RBC;　therefore,　measured　mean　 Platelet　cold　agglutinin-induced　pseudothrombocytopenia　is　a　rare　
corpuscular　 volume　 is　 falsely　 elevated　 and　 the　 red　 blood　 cell　 count　 is　 condition.　The　platelet　agglutination　is　anticoagulant-independent,　
disproportionately　 low.　 While　 the　 measured　 hemoglobin　 is　 correct,　 the　 occurs　 at　 maximal　 activity　 at　 48　 degrees　 C,　 and　 is　 mediated　 by　 IgM　
calculated　 indexes　 are　 incorrect:　 the　 hematocrit　 (red　 cell　 count　 x　MCV)　 is　 autoantibody　 directed　 against　 GP　 IIb/IIIa.　 Because　 this　 autoantibody　
low,　while　 the　 mean　 corpuscular　 hemoglobin　 (hemoglobin/red　 cell　count)　 has　 little　 activity　 at　 temperatures　 above　 30　 degrees　 C,　 no　 clinical　
and　the　mean　corpuscular　hemoglobin　concentration　(hemoglobin/　 complication　occurs.　Other　technical　problems　and　less　known　
hematocrit)　 are　 elevated.　 By　 rewarming　 the　 blood　 sample　 to　 37　 degrees　 situations　 may　 cause　 abnormal　 cell　 counts　 and　 indexes　 with　 automated　
C,　the　 erythrocyte　 agglutination　 is　 abolished　 and　 correct　 values　 will　 be　 analyzers.　These　include　clots　or　overfilling　of　tubes,　
read.　 In　 the　 blood　 sample,　 hemagglutination　 may　 be　 visible　 to　 the　 hypertriglyceridemia,　 hyperbilirubinemia,　 and　 extreme　 high　 white　 blood　
unaided　 eye　 and　 examination　 of　 the　 peripheral　 blood　 smear　 may　 reveal　 count　 　+　 any　 of　 which　 may　 interfere　 with　 cell　 counting　 and　 cell　indexes.　
erythrocyte　 clumping.　 Another　 problem　 might　 appear　 with　 the　 presence　 Severe　 microcytosis,　 microorganisms,　 and　 cytoplasmic　 fragments　 of　
of　cryoglobulins.　 Cryoglobulins　 are　 immunoglobulins　 that　 precipitate　 at　 leukocytes　 may　 cause　 spurious　 elevation　 of　 the　 platelet　 counts.　 These　
temperatures　 below　 37　 degrees　 C,　 producing　 high　 molecular　 weight　 conditions　 are　 characterized　 by　 small　 particles　 that　 are　 wrongly　 counted　
aggregates.　 The　 first　 clue　 to　 a　 diagnosis　 of　 cryoglobulinemia　 could　 be　 as　 platelets.　 Larger　 particles　 may　 be　 recognized　 as　 leukocytes,　 e.g.,　
laboratory　 artifacts　 detected　 in　 the　 automated　 blood　 cell　 counts.　 The　 circulating　 normoblasts,　 giant　 platelets,　 and　 erythrocytes　 with　 more　
precipitated　cryoglobulin　particles　of　various　sizes　may　falsely　be　 resistance　 to　 lysis.　 The　 latter　 occurs　 in　 automated　 analyzers　 when　
recognized　 as　 leukocytes　 or　 platelets　 causing　 pseudoleukocytosis　 and　 leukocyte　 counting　 is　 based　 on　 prior　 erythrocyte　 lysis.　 Erythrocyte　
pseudothrombocytosis.　At　the　same　time,　the　RBC　indexes　are　 resistance　 to　 lysis,　 causing　 interference　 with　 leukocyte　 counting,　 was　
generally　 unaffected.　 Reliable　 automated　 counts　 can　 be　 obtained　 by　 reported　 in　 hemoglobinopathies　 (e.g.,　 hemoglobin　 C　 trait,　 CC,　 SC,　 and　
warming　 the　 blood　 to　 37 degrees C　 or　 by　 keeping　 the　 blood　 at　 37　 SS)　 and　 fetal　 (cord)　 red　 cells.　 EDTA-dependent　 leukoagglutination　
degrees　 C　 from　 the　 time　 of　 venipuncture　 to　 analysis.　 May-Grunwald- (similar　to　platelet　satellitism)　and　cold-induced　leukoagglutination　
Giemsa-stained　 blood　 films　 are　 usually　 normal,　 extracellular　 material　 is　 uncommonly　cause　pseudoleukopenia.　Rarely　does　severe　
occasionally　 seen,　 and　 leukocyte　 cytoplasmic　 inclusion　 is　 rarely　 found.　 hyperglycemia　cause　spurious　macrocytosis.　The　hyperosmolar　
Another　 important　 laboratory　 artifact　 seen　 with　 the　 automated　 analyzers　 glucose-“loaded”　 erythrocytes　 become　 swollen　 when　 they　 are　 diluted　
is　pseudothrombocytopenia.　This　condition　is　caused　by　diverse　 into　 a　 relatively　 hypotonic　 counting　 medium,　 but　 after　 hyperglycemia　 is　
m e c h a n i s m s , 　i n c l u d i n g : 　a n t i c o a g u l a n t - i n d u c e d 　 corrected　 the　 MCV　 returns　 to　 normal.　 To　 conclude,　 in　 our　 modern　 era,　
pseudothrombocytopenia,　 platelet　 satellitism,　 giant　 platelets,　 and　 cold　 automated　 analyzers　 are　 able　 to　 increasingly　 recognize　 pathologic　
agglutinin-induced　platelet　agglutination.　The　anticoagulant-induced　 conditions　 and　 artifacts.　 First,　 the　 results　 are　 presented　 in　 numbers,　
pseudothrombocytopenia　 is　 an　 in　 vitro　 platelet　 agglutination　 generally　 histograms,　 and　 scatter　 plots　 with　 or　 without　 flags　 for　 internal　 laboratory　
seen　 in　 specimens　 collected　 into　 EDTA.　 It　 has　 been　 reported　 both　 in　 review.　 The　 results　 are　 then　 transferred　 to　 the　 clinician,　 usually　 as　
healthy　 subjects　 and　 in　 patients　 with　 various　 diseases　 (e.g.,　 collagen　 numbers　 only.　 Still,　 undetected　 artifacts　 occur　 and　 go　 unnoticed.　 The　
vascular　 disease,　 neoplasm,　 and　 in　 severely　 ill　patients)　 and　 has　 an　 clinician　 should　 be　 alert　 to　 those　 artifacts,　 thus　 avoiding　 unnecessary　
overall　 incidence　 of　 approximately　 0.1%　 Although　 the　 agglutination　 is　 investigations　 and　 therapies.　
most　 pronounced　 with　 EDTA,　 it　
may　 occur　 with　 other　 anticoagulants　 as　

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INTRODUCING

S P R AY
SIMPLIFIES PHLEBOTOMY IN STYLE
SPRAY CLEAN DRAW

*
- Benefits :
• Dual acting antiseptic effective against wide
spectrum of microbes.

• Facilitates adequate wetting of phlebotomy

site for effective antiseptic activity.

• Convenient to handle in laboratory settings,

home sample collection and medical camps. The images reflect skin preparation technique only &

• Convenient for distribution within

not the complete Phlebotomy protocol.

departments in hospitals and collection

centres.

…Inspires Safe Phlebotomy !

*
Printed and published by D.G. Tripathi, Edited by Dr. Ramnik Sood, M.D. (Path.) for and on behalf of Tulip Diagnostics Private Ltd., Gitanjali, Tulip Block, Dr. Antonio Do Rego Bagh, Alto Santacruz, Bambolim Complex Post Office, Goa - 403 202, INDIA.
Fax: (0832) 2458544. E-mail: [email protected] Website: www.tulipgroup.com

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