Automated Hematology Parameters

Vol. 24 • Issue 9 • Page 28


The utility of hematologic indices has long since been established, such that to practice routine medicine without them is all but unthinkable. The earliest complete blood count (CBC) was limited to a manual count of red blood cells (RBCs), white blood cells (WBCs) and platelets (Plts). This was a time- and labor-intensive approach that was also limited by human error. Alternate techniques and advances in automation have brought us far, providing more information with much less time and effort while helping to limit manual review of peripheral blood smears to cases in which it is specifically indicated.

A number of indices beyond those basic cell counts have become routinely available and even considered standard-of-care. Today’s typical CBC also includes indices such as hemoglobin (Hb) concentration, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW) and at least a five-part WBC differential – including neutrophils, lymphocytes, monocytes, eosinophils and basophils. Here, we focus on new and upcoming parameters and their potential clinical utility.

Red Blood Cells

Nucleated red blood cells (NRBCs) can be present in a number of clinical settings, but their importance in automated hematology stems from their historical misidentification as leukocytes, spuriously increasing WBC counts. Most hematology analyzers (HAs) on the market today have addressed this issue using various combinations of optical, impedence and staining properties with ðvarying degrees of success, but limitations include underestimation of NRBCs by automated methods.1,3,5,8,9

The immature reticulocyte fraction (IRF), an assessment of reticulocyte maturation, has been proposed as a marker of bone marrow recovery that may be superior to the absolute neutrophil count (ANC). One study showed detection up to three days earlier in most patients, and contrary to the ANC, the IRF is unaffected by infection.11 Defined as reticulocytes with the highest RNA content, this is generally determined by use of fluorescent RNA dyes and/or light scatter properties.6

The reticulocyte hemoglobin concentration can be used to identify different populations of reticulocytes, which can provide information about recent erythropoiesis and response to iron therapy. This can be measured using flow type systems, which determine cell volume and hemoglobin content based on light and/or forward scatter properties. The percent hypochromic cells and low hemoglobin density are of similar clinical utility but have been less well studied.6,9,14

Manual microscopic counts of schistocytes are notorious for their wide interobserver variability. In an attempt to address this, some HAs have added algorithms to further analyze the small RBC fraction and generate a flag if a threshold number of suspected fragments is present. While the flag is nonspecific and requires microscopic confirmation, the absence of such a flag has excellent negative predictive value (i.e., that schistocytes are not present in significant numbers).9

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The microcytic-to-hypochromic ratio (Siemens %HYPO, Sysmex %HYPO-He) can help to distinguish thalassemia from iron deficiency anemia (IDA), as the former tends to show more prominent microcytosis, while severe hypochromasia is more characteristic of the latter. An elevated ratio has a relatively good positive predictive value, but a normal or decreased value must be interpreted with caution. Iron deficiency superimposed on certain thalassemia variants can result in marked hypochromia, yielding a ratio similar to that of a non-thalassemic patient with IDA.9,14

Spherocytes are most prominent in hereditary spherocytosis and, to a lesser extent, autoimmune hemolytic anemia. Some HAs make use of light scatter properties to detect hyperchromic RBCs, which have been shown to correlate well with the presence and number of spherocytes. Furthermore, this method has proven more sensitive than traditional osmotic fragility tests. This is best assessed in fresh specimens, as the number of spherocytes has been shown to decrease significantly in blood stored at room temperature longer than six hours. The eosin-5′-maleimide test, an adaptation of a proven benchtop method, is also available on some HAs with flow cytometry capabilities.12

White Blood Cells

The presence of blasts in peripheral blood is nearly always cause for concern, and most HAs have developed mechanisms for their detection.3 Some HAs will only generate a blast flag once a threshold number has been detected; these threshold values can be adjusted to alter the sensitivity and specificity.5,8,9,10

Immature granulocytes (IGs; promyelocytes, myelocytes and metamyelocytes but not blasts) can also be detected by some HAs, using light scatter and fluorescence to identify the granulocytic cells with larger nuclei. This method is not highly sensitive, but a significant increase in IGs is relatively specific for bacterial infection over noninfectious inflammatory conditions.3,5,8,9,10

Another parameter with the potential to aid in diagnosing sepsis is neutrophil volume (NV). Although this can be increased in either immature or reactive neutrophils, significant increases in NV correlate reasonably well with the likelihood of sepsis, even in neonates. In fact, the superior sensitivity over C-reactive protein levels (79 percent vs. 72 percent) makes it a useful screening tool.2,9

The neutrophil granularity index can aid in detection of both the toxic granulation of infectious conditions and the hypogranularity of myelodysplastic syndromes. One study of the Sysmex XE-5000 indicates sufficient correlation with microscopy to justify abandoning manual slide-based assessment of toxic granulation, particularly given its subjective nature.3, 15, 16


The immature platelet fraction (IPF), or reticulated Plts, contain RNA and can be detected using nucleic acid dyes. The IPF are the larger, strong-staining subset and are typically expressed as a percent of total Plts. This parameter is increased in the setting of platelet destruction or consumption and decreased with bone marrow failure.4, 6, 13

Future Direction

Image analysis technologies have the potential to supplant much of the manual slide review that occurs in hematology laboratories. The main challenges include obtaining a monolayer of cells and the appropriate classification of abnormal cells.

Drs. Harrison and George are with the University of New Mexico Health Sciences Center and TriCore Reference Laboratories, Albuquerque, New Mexico.


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2. Celik IH, Demirel G, Aksoy HT, Erdeve O, Tuncer E, Biyikli Z, Dilmen U. Automated determination of neutrophil VCS parameters in diagnosis and treatment efficacy of neonatal sepsis. Pediatr Res. 2012 Jan;71(1):121-5.

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6. George T. Automated hematology instrumentation. In: UpToDate, Post TW (ed), UpToDate, Waltham, MA. (Accessed on July 21, 2015.)

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8. Kim H, Hur M, Choi SG, Moon HW, Yun YM, Hwang HS, Kwon HS, Sohn IS. Performance evaluation of Sysmex XN hematology analyzer in umbilical cord blood: a comparison study with Sysmex XE-2100. Clin Chem Lab Med. 2014 Dec;52(12):1771-9.

9. Lecompte TP, Bernimoulin MP. Novel Parameters in Blood Cell Counters. Clin Lab Med. 2015; 35:209-24.

10. Meintker L, Ringwald J, Rauh M, et al. Comparison of automated differential blood cell counts from Abbott Sapphire, Siemens Advia 120, Beckman Coulter DxH 800, and Sysmex XE-2100 in normal and pathologic samples. Am J Clin Pathol 2013;139:641-50.

11. Raja-Sabudin RZ et al. Immature reticulocyte fraction is an early predictor of bone marrow recovery post chemotherapy in patients with acute leukemia. Saudi Med J. 2014 Apr;35(4):346-9.

12. Rooney S, Hoffmann JJ, Cormack OM, McMahon C. Screening and confirmation of hereditary spherocytosis in children using a CELL-DYN Sapphire haematology analyser. Int J Lab Hematol. 2015 Feb;37(1):98-104.

13. Sachdev R, Tiwari AK, Goel S, Raina V, Sethi M. Establishing biological reference intervals for novel platelet parameters (immature platelet fraction, high immature platelet fraction, platelet distribution width, platelet large cell ratio, platelet-X, plateletcrit, and platelet distribution width) and their correlations among each other. Indian J Pathol Microbiol. 2014 Apr-Jun;57(2):231-5.

14. Winichagoon P, Kumbunlue R, Sirankapracha P, Boonmongkol P, Fucharoen S. Discrimination of various thalassemia syndromes and iron deficiency and utilization of reticulocyte measurements in monitoring response to iron therapy. Blood Cells Mol Dis. 2015 Apr;54(4):336-41.

15. Zimmermann M, Cremer M, Hoffmann C, Weimann K, Weimann A. Granularity Index of the SYSMEX XE-5000 hematology analyzer as a replacement for manual microscopy of toxic granulation neutrophils in patients with inflammatory diseases. Clin Chem Lab Med. 2011 Jul;49(7):1193-8.

16. Zimmermann M, Steenhuis P, Linssen J, Weimann A. Detection and quantification of hypo- and hypergranulated neutrophils on the new Sysmex XN hematology analyzer: establishment of an adapted reference interval for the neutrophil-granularity-intensity compared to XE-technology in adult patients. Clin Lab. 2015;61(3-4):235-41.

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