Thrombocytopenia is a common finding in complete blood counts (CBCs) performed on inpatients and may also occur, less frequently, in outpatient samples. In most instances, the clinical context provides the physician with a clear explanation for the thrombocytopenia. In some cases, however, the cause is unclear and, less commonly, the thrombocytopenia is the dominant laboratory finding. Especially careful evaluation of the cause is necessary and further laboratory testing is often required for clarification.

What is Thrombocytopenia?
Thrombocytopenia is usually defined as the observation of a platelet concentration (count) of less than some arbitrary concentration limit, commonly 140-150 x 109/L. This data is generated from automated particle counters in which platelets are regarded as particles in whole blood with a size (volume) of between 2 and 35 fL depending on the analyzer. Lowering this lower threshold to 1 fL can increase the platelet count and, to a lesser extent, increase the upper threshold. Hence, the concept of a normal range (actually reference interval) is highly dependent on these analyzer thresholds.

An additional consideration is the effect of gender and race on the lower limit of the platelet count. Women have slightly higher platelet counts than men and African-Americans have lower platelet counts than European-American men. A platelet count of 115 x 10⁹/L may be an appropriate lower platelet limit for African-American men.

Evaluation of Platelet Size
Platelets also vary in size: In general, the mean size (geometric mean) is about 9 fL - about 1/10 the volume of a red cell. The mean size (MPV) shows an inverse relation to the platelet count in healthy subjects: Higher platelet counts (> 300 x 10⁹/L) have lower MPVs than those with lower platelet counts (< 180 x 10⁹/L). In thrombocytopenia, low MPV indicates a problem in marrow production and a high MPV is indicative of peripheral destruction or early regeneration post chemotherapy.

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In addition to assessing mean size, the platelet size histogram typically shows a slight right skew. Inspection of the histogram can be important in the evaluation of thrombocytopenia. Fig. 1 shows three histograms: the middle histogram is normal while the top histogram shows smaller than normal platelets and the bottom one shows larger platelets. The right skew is more pronounced at the bottom.

In addition to the size and histogram, a Romanowsky stained blood smear should be evaluated. Typically in a high power field (x1000) 15-40 platelets are visible when the platelet count is within the reference interval (140-440 x 10⁹/L). Smear examination can also show platelet size. Fig. 2 shows large platelets in a case of congenital thrombocytopenia; large platelets are often not enumerated since they exceed the 28-35 fL thresholds. Hence, the analyzer reports thrombocytopenia, but inspection of the MPV, histogram and blood smear may indicate a normal count. Smear examination is helpful in cases of pseudo thrombocytopenia where platelet clumps can be seen or single platelets attached to neutrophils (satellites).

Triple Digit Thrombocytopenia
Thrombocytopenia can be separated into triple-digit thrombocytopenia (platelet count 100-140 x 109/L); double digit thrombocytopenia (platelet count 10-99x 10⁹/L) or single digit thrombocytopenia (platelet count 1-9 x 10⁹/L). Platelet counts in the range of 100-140 x 109/L need careful consideration since gender and race (vide supra) can cause platelet counts to be in this range. In healthy subjects, the MPV will be high (typically > 10 fL); such individuals do not require any further investigation. Platelet counts closer to 100 x 10⁹/L may be seen in myelodysplastic states or other hematologic conditions, rarely with marrow infiltration. Where doubt remains, bone marrow aspiration and biopsy will often be the most useful investigation in these patients. These patients are not known to be an increased risk of bleeding, either spontaneously or in association with invasive diagnostic or therapeutic procedures.

Double Digit Thrombocytopenia
Double digit thrombocytopenia (platelet count 10-99 x 10⁹/L) is commonly seen in hospitalized patients. The cause is usually evident from the clinical context. Common causes are hematologic malignancies, liver disease, splenomegally, dilutional from blood transfusion, drug associated thrombocytopenia, post cardiac surgery, immune thrombocytopenia (ITP), neonates, congenital thrombocytopenias and the (gestational) thrombocytopenia of pregnancy. These patients are not at risk for spontaneous bleeding, but may be at risk for provoked bleeding in association with surgery or invasive procedures and, as such, may be transfused prophylactically with allogeneic platelets.

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The threshold for platelet transfusion has never been established, but is likely in the 30-50 x 10⁹/L range. The cause of thrombocytopenia is sometimes unclear in these patients.

Additionally important, but less frequent, causes of thrombocytopenia in this range include:

• Idiopathic thrombocytopenic purpura (ITP), a bleeding disorder due to platelet autoantibodies that is largely a diagnosis of exclusion;
• Thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder due to a severe deficiency of a von Willebrand ­factor cleaving protease, ADAMTS-13;¹ atypical hemolytic uremic (aHUS), a hereditary disorder of complement regulation resulting in renal failure;²
• Heparin-induced thrombocytopenia (HIT), a thrombotic disorder due to the presence of an antibody to a complex of heparin-platelet factor 4.³

The laboratory is frequently called upon to separate the above entities, since these conditions can be life- or limb-threatening and have different treatments. ITP is treated with steroids, IVGG, rituximab (anti-CD20) or thrombopoietic agents; TTP is treated with plasma exchange; aHUS is treated with eculizumab (Soliris) and HIT is managed by discontinuing heparin and short-term use of a direct thrombin inhibitor (DTI) such as argatraban

Tests
There is no specific laboratory testing for ITP. Patients with aHUS may be found deficient in plasma proteins or may have genetic mutations in cell-bound complement regulatory proteins. Hence, the laboratory may be requested to have these factors assayed in plasma or DNA analyzed by techniques such a PCR-SSCP. Both the plasma assays and DNA techniques are more suitable for specialized reference laboratories, resulting in a turnaround time (TAT) from weeks to months.

HIT, however, is more common among these disorders. HIT is a clinical diagnosis primarily and laboratory testing is supplemental. Nevertheless, clinicians rely heavily on laboratory testing, mostly to exclude the diagnosis. A rapid TAT is often requested and expected; the most common test performed is an ELISA assay. Though it's very sensitive, it has a poor specificity and most positive results are false positives. In addition, the ELISA assay is run in batches and typically on a daily basis or less frequently.

TTP is a life-threatening disorder in which thrombosis occurs in small arterioles leading to acute end-organ ischemia. The cause of TTP has been clarified in recent years as relating to a severe deficiency (< 5%) in ADAMTS-13, either congenital or more commonly acquired due to an autoantibody. Prompt institution of plasma exchange (TPE) is the treatment of choice.

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Several conditions can mimic TTP that do not respond to TPE and clinicians are increasingly looking to the measurement of ADAMTS-13 to separate TTP from other related entities. Several types of assay are available, but functional assays are preferable. In addition, a short TAT is desirable, particularly to exclude TTP and avoid the wasteful TPE regimen.

Measurement of the activity of this enzyme requires considerable technical skill and sophisticated equipment, such as a fluorimeter. The problem is separating very low levels of this enzyme (< 5%, indicative of TTP) from the modest reductions in activity (15-50%) seen in a variety of conditions, some of which mimic the features of TTP.

Single Digit Thrombocytopenia
Single digit thrombocytopenia (platelet count < 10 x 10⁹/L) is commonly only seen in hospitalized patients with hematologic conditions or bone marrow transplant patients. These patients are at risk for spontaneous bleeding and platelet transfusions commonly administered; further laboratory investigation is not usually indicated. Outside of the context of hematologic malignancies that undergo chemotherapy, the major causes are the rarer entities of ITP and TTP, discussed above. HIT rarely, if ever, shows a platelet count in this range.

Another very rare bleeding condition, post transfusion purpura (PTP), occurs in a small number of transfusion recipients 5-15 days post transfusion who present with very low platelet counts. This condition is an immune thrombocytopenia in which an alloantibody to platelet antigens can be demonstrated in the plasma. Treatment is similar to ITP but IVIG is the treatment of choice.

Christine A. Guertin is senior coagulation specialist, Rhode Island Hospital. Dr. Sweeney is professor of pathology and laboratory medicine, Brown University.

 References

1. Scully M, Hunt BJ, Benjamin S, et al. Guidelines on the diagnosis and Management of TTP and other thrombotic microangiopathies. Brit J Haem. 2012.
2. Caprioli J, Noris M,Brioschi S, et al: Genetics of HUS: The impact of MCP, CFH and IF mutations on clinical presentation, response to treatment and outcome. Blood. 2006;108:1267-1279.
3. Cuker A. Heparin Induced thrombocytopenia (HIT):in 2011: An epidemic of overdiagnosis. Thromb Haemostas 2011;106:993-994.