Pseudoreticulocytosis by the ADVIA 2120 Hematology Analyzer and Other Hematologic Changes in a Cynomolgus Macaque (Macaca fascicularis) With Malaria

Abstract

Important hematologic changes can be observed in nonhuman primates with malaria, including inaccurate reticulocyte counts by the ADVIA 2120 hematology analyzer. A 5-year-old male purpose-bred cynomolgus macaque ( Macaca fascicularis) imported from a commercial source in Cambodia was enrolled in a nonclinical toxicity study investigating the effects of an immunomodulatory pharmaceutical agent. On study day 22, an increase in large unstained cells (LUCs), due to increased monocytes (2.20 × 103/µl, reference interval: 0.17-0.76 × 103/µl), was reported by the analyzer during a scheduled hematologic evaluation, which prompted blood smear review and revealed that the macaque had a high burden of Plasmodium spp.. The macaque did not have clinical signs for the infection at this time point. Progressively higher parasite burdens and persistently increased monocytes (markedly increased by study day 56, 10.38 × 103/µl) were observed at subsequent hematologic evaluations. New Methylene Blue stain manual reticulocyte counts were performed on study day 43 and at later time points, and showed that the analyzer reported erroneous higher reticulocyte counts (study day 43: +6.7%, +266.2 × 109/L; study day 50: +18.9%, +409.8 × 109/L) compared with the manual reticulocyte counts (pseudoreticulocytosis). The magnitude of regenerative response was considered inadequate for the severity of anemia at these time points. Atypical reticulocyte scatter plot distributions from the analyzer were also observed at time points with high parasite burdens, and combined with increased LUCs, may suggest high burden parasitemia. Verification of automated reticulocyte counts is important in cases with high malarial parasite burdens and the recognition of pseudoreticulocytosis is prudent in assessing appropriateness of the regenerative response. Increases in monocytes correlated with higher parasite burdens and marked increases may be an indicator of advanced disease.

Impact of Staining Methods and Human Factors on Accuracy of Manual Reticulocyte Enumeration

“…In addition, updated evidence has shown that automated reticulocyte enumeration does not render manual counting obsolete. Not only did a recent investigation demonstrate comparable accuracy in reticulocyte enumeration between the automated approach using the Sysmex XT-2000i analyzer and manual counting on freshly prepared (i.e., less than six hours) blood smears [ 29 ], but a previous animal study also reported the need for manual verification because of malaria-related pseudoreticulocytosis from the automated approach [ 30 ]. Therefore, manual reticulocyte counting using supravital dyes remains the gold standard for reticulocyte enumeration.…”

Special Issue on the Pathobiology of Laboratory Nonhuman Primates: A Review of Species, Substrain, Geographical Origin, Age, and Modality-Related Factors

“…Skeletal abnormalities in laboratory macaques are discussed in a report based on a series of cases investigating coxofemoral joint abnormalities that have a similar presentation to idiopathic femoral head chondrolysis, 18 while a case of pseudoreticulocytosis in a cynomolgus macaque with malaria draws attention to the clinical pathology presentation of this plasmodial disease, demonstrating that erroneously high reticulocyte counts may occur in cases with high parasitic burden when evaluated by hematology analyzers utilizing fluorescence measurement. 19 Two articles then discuss organ system responses of NHPs to emerging biotherapeutic modalities. In the first from the Special Interest Group in Neuropathology (SIGN) of the Society of Toxicologic Pathology (STP), an in-depth discussion of neuropathologic findings associated with the administration of candidate biotherapeutics (nucleic acids, proteins, viral gene therapy vectors, etc.)…”

“…Skeletal abnormalities in laboratory macaques are discussed in a report based on a series of cases investigating coxofemoral joint abnormalities that have a similar presentation to idiopathic femoral head chondrolysis, 18 while a case of pseudoreticulocytosis in a cynomolgus macaque with malaria draws attention to the clinical pathology presentation of this plasmodial disease, demonstrating that erroneously high reticulocyte counts may occur in cases with high parasitic burden when evaluated by hematology analyzers utilizing fluorescence measurement. 19…”