Recurrent severe thrombocytopenia in critical illness complicated by hemolysis

Division of Hematology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada Trillium Health Partners, Etobicoke, Ontario, Canada Transfusion Medicine, Hamilton Regional Laboratory Medicine Program, Hamilton, Ontario, Canada Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada Department of Pathology & Molecular Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada Service of Benign Hematology, Hamilton Health Sciences, Hamilton General Hospital, Hamilton, Ontario, Canada


| CASE PRESENTATION
subsequently, his platelet counts gradually declined, and during the second and third weeks of hospitalization, they were within the normal range. However, 24 days following admission, his platelet count fell from 315 (Day 22) to 139 Â 10 9 /L (Day 24). At this time, vancomycin was restarted, and initial doses of cefazolin were given, for concern regarding septicemia.
Thrombocytopenia among critically ill patients has a prevalence of 35% to 45%. 1 Although the etiology is usually multifactorial, common mechanisms include platelet consumption with or without overt disseminated intravascular coagulation (DIC) and hemodilution (administration of fluid including blood products); less common explanations include platelet activation from heparin-induced thrombocytopenia (HIT), platelet clearance by drug-dependent antibodies, or decreased platelet production by the bone marrow secondary to a severe infection or toxic effect of drugs. 1,2 The platelet counts gradually increased to 299 Â 10 9 /L (day 34).
However, over the next 4 days, and in association with further administration of vancomycin and cefazolin, the platelet count precipitously declined to 6 Â 10 9 /L ( Figure 1). The patient did not exhibit any signs or symptoms of bleeding. Laboratory investigations revealed a normal coagulation profile, the absence of fragments in peripheral blood, and a negative HIT serology.
Multiple clues can be helpful in identifying the cause of thrombocytopenia in the critical care setting, including timing of onset (in relation to potential triggers), severity (degree of thrombocytopenia), and rapidity of decline. For example, drug-induced immune thrombocytopenia (D-ITP) is an important explanation for abrupt-onset and severe thrombocytopenia that typically begins approximately a week after starting the implicated drug. 3,4 A "clinical pearl": whereas D-ITP typically causes the platelet count to fall to less than 20 Â 10 9 /L, such a severe magnitude of platelet count decline is uncommonly seen in sepsis or DIC. 2 The consultant hematologist suspected D-ITP secondary to either vancomycin or cefazolin; drug cessation usually results in platelet count recovery within 4 to 14 days (median, 7 days). 4 Platelet transfusions were given, and both antibiotics were held; the platelet count recovered to >100 Â 10 9 /L over the next week.
However, cefazolin was readministered on Day 48, and the platelet count abruptly fell to 1 Â 10 9 /L. The patient was treated with high-dose intravenous immunoglobulin (IVIg), 1 g/kg (80g; one dose), as well as prednisone, 1 mg/kg administered for two days.
The second episode of apparent D-ITP was characterized by an even more rapid platelet count decline to an even lower nadir value.
Thus, additional treatments (high-dose IVIg and corticosteroids) were given, although the efficacy of these agents in D-ITP remains uncertain. 4 Once again, the platelet count recovered to >100 Â 10 9 /L over 4 days.
Unfortunately, the patient was inadvertently re-exposed to cefazolin (along with a first dose of ampicillin) 12 days later, with a recurrence of severe thrombocytopenia (1 Â 10 9 /L), which was successfully managed with platelet transfusions alone. Later during the hospitalization, the patient received vancomycin without precipitating thrombocytopenia.
A review of the serial platelet counts in relation to vancomycin, cefazolin, and ampicillin, as summarized in Figure 1  rare side effects of cefazolin. [7][8][9][10] The mechanism by which this happens is unclear; however, some studies suggest that cephalosporins can act as haptens, which are small molecules that can result in a hapten-specific immune response when attached to a larger carrier protein. 11 D-ITP usually occurs 5-7 days after starting the offending drug; however, in cases of previous exposure and sensitization, repeat exposure can trigger thrombocytopenia within hours. 12 Fortunately, despite generally severe thrombocytopenia often accompanied by overt bleeding (e.g., petechiae, oral mucosal blood blisters), fatal outcomes 13,14 or long-term sequelae are uncommon. 7 Indeed, full recovery was seen in this patient following all three episodes of D-ITP. These uneventful outcomes are in marked contrast with HIT, The patient formed anti-E antibodies, suggesting that his Rh genotype was R 0 R 0 (i.e., CDe/CDe), which is the most common Rh+ phenotype that can be complicated by the formation of anti-E (and often concomitant anti-C antibodies). Although three of the eight initially transfused RCC units were E+ and thus susceptible to alloimmune hemolysis, the stable hemoglobin levels, absence of free hemoglobin, and normal haptoglobin levels indicate that despite the positive direct Coombs test, clinically significant hemolysis appeared unlikely over this time period.
Following transfusion of 3 sets of group O plateletsadministered because of the first episode of D-ITP (platelet count, 6 Â 10 9 /L)-the patient developed abrupt and progressive anemia, to a value of 5.7 g/dL. At this time (Day 16), testing for hemolysis was positive, that is, free hemoglobin was detected, haptoglobin was absent, and in addition, anti-K antibodies were detected. Further, anti-A antibodies were eluted from his red cells, and numerous spherocytes were seen on examination of the peripheral blood film.
Abrupt increases in lactate dehydrogenase (LDH) and absolute reticulocyte counts were documented on days 19 and 21 (Figure 3, inset).  formerly, BloodCenter of Wisconsin). 7 (All but two of the drugs listedhaloperidol and simvastatin-appeared in both drug lists. 6,7 ) Note that the drug implicated in our patient case-cefazolin-is not included in the above list, but was cited in the Versiti laboratory paper 7 as being definitively implicated (per frequency category, "1-3 cases") as a cause for D-ITP identified in their laboratory.
Following a 10-day period with relatively stable and rising hemoglobin values, the patient's hemoglobin began to fall once again, from 10.1 to 6.6 g/dL (Day 37). This began a short time before his third episode of D-ITP, which was managed with transfusion of group-specific (i.e., group A platelets) as well as high-dose IVIg (80g on two consecutive days). At this time, the direct Coombs test was negative, haptoglobin levels were normal, and free hemoglobin was not detected (Day 37). He received four units of group O+, EÀ/KÀ RCCs, with a rise in hemoglobin to 9.9 g/dL.
The laboratory studies showed that this abrupt hemoglobin fall could not plausibly be explained by hemolysis. This raised the issue of whether occult, intermittent gastrointestinal bleeding could be responsible for the otherwise unexplained episode of anemia.
Upper gastrointestinal endoscopy was performed, which showed friable tissues in the gastric fundus with localized areas of bleeding requiring hemoclips and local injections of epinephrine.
Dual antiplatelet therapy with aspirin and clopidogrel was also held.
With these measures, resolution of anemia occurred.
Review of serial laboratory studies, summarized in The patient was eventually discharged home with the following blood counts at discharge: hemoglobin, 11.8 g/dL; WBC, 7.5 Â 10 9 /L; platelet count, 209 Â 10 9 /L. However, 4½ years post-discharge, the hematology service was re-consulted for thrombocytosis (maximum   examples. 8,9 Our observations include an initial false-negative test for cefazolin-dependent antibodies when the blood sample was taken during the first episode (perhaps because nascently formed antibodies were primarily bound to platelets rather than free in plasma); Figure 1 also supports a more abrupt platelet count decline with the second and

| SUMMARY
Thrombocytopenia is a common condition in hospitalized patients, particularly those with critical illness. D-ITP is a dramatic adverse effect of medications such as antibiotics that can cause profoundly reduced platelet counts and bleeding. The typical clinical scenario is a severe decline in platelet count with a temporal relationship (approximately 1-week delay) following initiation of a drug known to cause D-ITP (in our case, cefazolin). Besides having two subsequent abrupt D-ITP recurrences (due to cefazolin readministration), this case is noteworthy for the laboratory demonstration of cefazolindependent D-ITP antibodies (with initial false-negative testing) and the occurrence of passive alloimmune hemolysis due to transfusion of group O platelets (containing anti-A) to a severely thrombocytopenic blood group A recipient.

ACKNOWLEDGMENT
We thank Ms. Jo-Ann I. Sheppard for preparing the three figures, and Ms. Hina Bhakta for performing the flow cytometry studies for D-ITP.

FUNDING INFORMATION
No funding was used for this study.

CONFLICT OF INTEREST
Theodore E. Warkentin has received lecture honoraria from Alexion and Instrumentation Laboratory (Werfen); has provided consulting services to Aspen Canada, Aspen Global, CSL Behring, Ergomed, Instrumentation Laboratory (Werfen), Octapharma, Paradigm Pharmaceuticals, and Veralox; has received research funding from Instrumentation Laboratory (Werfen), and has provided expert witness testimony relating to HIT and non-HIT thrombocytopenia. S. Piran and N. A. declare no relevant conflicts of interest.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this published article.

PATIENT CONSENT STATEMENT
The patient provided written permission for this report (institutional approval is not required for case reports in our medical community).