Available online at www.annclinlabsci.org 422 Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017 Analysis of Separation of White Cells in Peripheral Blood as an Indicator of MPO Deficiency

Kinga Rośniak-Bąk1, Agnieszka Jeleń2, and Marek Bąk3

1Department of Laboratory Diagnostics and Clinical Biochemistry, Medical University of Lodz, 2Laboratory of Molecular Diagnostics and Pharmacogenomics, Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Medical University of Lodz, and 3Department of Occupational Diseases and Toxicology, Nofer Institute of Occupational Medicine in Lodz, Poland

Abstract. The purpose of this study was to analyze the white (WBC) percentage pattern of patients with myeloperoxidase disorder. During the 18 months of routine work, 36 blood samples were found with disorders of myeloperoxidase activity: 12 cases of total myeloperoxidase (MPOt) deficiency and 24 cases of partial myeloperoxidase (MPOp) deficiency. In the group with MPOp, according to the results, monocytes (MONO) were the dominant population 33.2%±21.3; however, the microscopic evaluation of leucocytes showed the dominance of neutrophil (NEUT). The average NEUT value was 66.63%±12.31; LYMPH 23.33%±10.08; MONO 6.00%±3.20; EOS 2.04%±2.20; BASO 0.29%±0.62; ATYP 0.83%±1.09. In the group with MPOt, the results of automated leukocyte analysis showed that the dominant group consisted of large unstained cells (LUC) 72.6%±8.64. LUC category reflects large immature cells such as blusts. In the microscopic evaluation: NEUT 67% ±11.40; LYMPH 23%±8.94; MONO 6.17%±3.47; EOS 1.25%±1.06; BASO 0.08%±0.29; ATYP 0.92%±1.38. During microscopic verification, no LUC cells were found. Results of the evaluation of automatic WBC separation according to morphology and functionality of cells led to the conclusion that monocyte dominance in the differential WBC count is associated witha high likelihood of MPOp, and the domination of large unstained cells with MPOt.

Key words: myeloperoxidase, deficiency, MPXi index, large unstained cells, chronic disease.

Introduction preparation of peripheral blood or , which was described in 1965 by Kaplow [9,10]. Myeloperoxidase (MPO) belongs to the family of The presence of MPO was confirmed cytochemi- heme peroxidase enzymes that is also comprised of cally in 1918 by Graham, who indicated brown- eosinophil peroxidase (EPO) and lactoperoxidase colored grains in neutrophils, eosinophils, and in (LPO) [1,2]. It is the product of a single gene lo- their early stages of development by staining with cated on the long arm of chromosome 17 (q21.3 an alcoholic solution of formaldehyde, H2O2, and q23.2) 11 kb (11 intron and 12 exon). Built in the benzidine. In hematology, MPO is commonly used form of tetramer two chains α (60 kDa) and 2 to classify or diagnose acute leukemia [10-12]. chains β (14 kDa) of molecular weight 140 kD Automated determination of myeloperoxidase ac- [3,4]. The protein is located in azurophilic granules tivity is now made feasible using specific mean per- of the circulating leukocytes (neutrophils, mono- oxidase index (MPXi) on a hematology analyzer. cytes, and eosinophils) and tissue macrophages, Several authors have reported the use of the specific Kupffer cells in the liver, microglia, granules of neu- MPXi to assess the degree of MPO deficiency, eg. rons, pyramidal system and the hippocampus [5- Kutter et al. have demonstrated that MPXi values 8]. It is a strongly cationic protein with an isoelec- from +10 to -10 correspond to the normal range tric point higher than 10 [6]. Formerly, it could MPO; -11 to -30 represent mild MPO deficiency; only be confirmed by specific staining of the -31 to -55 denote partial MPO deficiency; while values lower than -55 indicate total MPO deficien- Address correspondence to Agnieszka Jeleń, Laboratory of Molecular Diagnostics and Pharmacogenomics, Department of Pharmaceutical cy. Please note that the ranges are valid for the Biochemistry and Molecular Diagnostics, Medical University of Lodz, Technicon H*3 measurement system [8,13-15]. Muszynskiego 1, 90-151, Lodz, Poland; phone/fax: +48 42 677 9130; e mail: [email protected] Index MPXi is the mean MPO activity, which is

0091-7370/17/0400-422. © 2017 by the Association of Clinical Scientists, Inc. Hematological evaluation of myeloperoxidase deficiency 423

lyses of erythrocytes, thrombocytes, and leukocyte cell Table 1. Detailed characteristics of an automated chemi- membranes except for basophils. Thus, it was possible to cal analyzer. determine total number of white blood cells (WBC-B), the number and percentage of total basophils, mononu- Performance Specifications Within Run Precision clear and polymorphonuclear cells, blasts (# and % BASO, MN, PMN, BLAST) and alarms in the form of Analytical Ranges (Linearity) Mean SD CV morphological flags of +, ++ and +++ intensity due to the presence of BLAST and the rejuvenation of LS. WBC 0.02-400x10^3/µL 7.5 0.2 2.7 According to the principles of laboratory practice, the RBC 0.0-7.0x10^6/µL 5.0 0.06 1.2 final classification of alarmed cells was carried out using PLT 5.0-3500.0 x 10^3/µL 300 8.8 2.93 the reference method (microscopic assessment). Hgb 0.0-22.5 g/dL 15.0 0.14 0.93 Retic 0.2-24.5% 2.0 0.25 12.5 In the PEROX channel pathway, using three reagents supplied in accordance with the procedure, erythrocytic Throughput 120 samples/hr membrane lyses, we have achieved fixation of white blood cells, production of hypertonicity and reaction of detected on the basis of the cytochemical method 4-chloro-1-naphthol with hydrogen peroxide (H2O2) described by Kaplow [16]. Unfortunately, it has and myeloperoxidase found in the granules of neutro- also been shown that this index does not reflect the phils, monocytes, and eosinophils. In the presence of endogenous MPO activity, the 4-chloro-1-naphthol degree of myeloperoxidase deficiency. substrate was oxidized by active oxygen (O*) from hy- Determination of myeloperoxidase activity is not a drogen peroxide. routine method. The laboratory standard is based on a morphological assessment of WBC: conducto- H2O2 + cellular MPO → O*+H2O metric measurements for counting of cells as well as O*+4-chloro-1-naphthol → dark intracellular scattered light measurements to determine the cell’s precipitates nucleus and cytoplasm complexity. A technology combining cytochemistry (determination of MPO The analyzer measuring system evaluates the average en- activity) with baseline methods which are further dogenous MPO activity by measuring the absorption described has allowed us to determine MPO activ- and expresses it as MPXi. The PEROX channel gives to- tal number of white blood cells (WBC-P), and the per- ity as well as the percentage pattern centage of the total number of neutrophils, monocytes, of patients with MPO deficiency. lymphocytes, eosinophils, large immature cells (# and % of NEUT, MONO, LYMPH, EOS, LUC) and a flag Materials and Methods describing virocytes/atypical lymphocytes (ATYP), reju- venation in the pathway (IG), MPO defi- Whole blood cells collected in EDTA-K2 were assessed ciency (MPO+), the presence of erythroblasts (NRBC) using Advia 2120i Hematology System (Siemens, and clumps (PLT-CLM) [16]. Cells classified as Germany) in conjunction with routine determinations ATYP, IG, LUC, NRBC and others were differentiated of peripheral blood morphology characteristics per- by microscope. formed at the Department of Laboratory Diagnostics and Clinical Biochemistry, University Clinical Hospital The result of white blood cell separation (according to Military Memorial Medical Academy - Central Veterans' the principles of laboratory practice) for each individual Hospital in Lodz. This study was ovappr ed by the Local diagnosed as being MPO-deficient was verified by mi- Bioethical Committee (no. RNN/136/15/KB dated 17 croscopic assay - peripheral blood specimen stained by February 2015). More detailed characteristics of the in- May-Grunwald-Giemsa method. Each of the results has strument are included in Table 1. In this measurement also been provided with a comment informing about system, the interpretation of results is by simultaneous myeloperoxidase deficiency. two-track analysis of the white blood cells performed in the PEROX and the BASO channels which are shown in Statistical analyses were performed using STATISTICA the form of histograms (Figure 1). v. 10 from StatSoft, Inc. (2011). The significance of dif- ferences between compared groups was assessed by The assessment of the number and types of leukocytes in ANOVA and post hoc Scheffe test after checking for the BASO channel is based on the analysis of the volume normality by the Shapiro-Wilk test. The level of statisti- and the density of the nucleus after prior cal significance was determined at p<0.05. 424 Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017

Figure 1. Analysis of the whiteblood cells (a) performed in the PEROX channel: analysis of the nucleus volume and peroxi- dase staining of the leukocytes and (b) performed in the BASO channels: analysis of the volume nuclear and the density/ lobularity of mononuclear (MN) and polymorphonuclear (PMN) white cells. LUC: large immature cells, NEUT: neutro- phils, MONO: monocytes, EOS: eosinophils, LYMFO: lymphocytes, PLT-CLM: clusters of , BASO: basophils, BLAST: blasts, MN: mononuclear white cells, PMN: polymorphonuclear white cells.

Figure 2. Analysis of white blood cells (a) from patient with partial deficiency of MPO (MPOp) and (b) from patient with total deficiency of MPO (MPOt) in the PEROX channel.

Study group. We analyzed 36 cases of 18 women aged The determinations of peripheral blood cell counts 58.55±19.32 years, and 18 men, aged 54.11±21.28 years, in both groups was performed for a variety of rea- collected over 18-month. Simultaneous interpretation of sons: follow-up/periodic tests commissioned by G P, the PEROX and the BASO channel histograms resulted in Local Military Medical Commission, gynecolo- diagnosing 12 cases of the total (MPOt) and 24 cases of gists, paid tests, invasive diagnostics, and tests prior partial (MPOp) deficiency of myeloperoxidase. to hospitalization due to acute or scheduled admis- sions. The predominant causes included: heart fail- The MPOt group comprised 5 women and 7 men aged22 ure, hypertension, alcohol dependence syndrome to 84 years (57.33±17.46), while the MPOp consisted of and its consequences, and cancer. More compre- 13 women and 11 men aged from 19 to 87 years hensive characteristics of the test groups are shown (55.83±21.72). in Tables 2 & 3. Hematological evaluation of myeloperoxidase deficiency 425

Table 2. Characteristics of the group with partial myeloperoxidase deficiency (MPOp).

No. Sex age Tests ordered due to: Anamnesis [years]

1F30 SE: pregnancy Negative 2M58 EA: injury –skull injury Negative 3M66 SA: ablation arrhytmiasince 13 yrs; 15 yrs ago splenectomy after road accident, right hydrocele removed twice 4F51 EA: cerebral ischemic stroke arterial hypertension for several years-pharmacotherapy was stopped 3 weeks ago; diabetes 5F31 SA: GP follow-up examination no anamnesis; results show evident microcytic hypochromic anemia 6M79 EA: obstructive jaundice 2012 gastrectomy due to cancer with recurrence and local metastases to bile ducts, liver and lungs; chronic renal disease; left kidney calculosis and cyst; right nephrectomy due to cancer; 7F53 SE: GP follow-up examination nodules on distal phalangeal joints 8F53 SE: paid examination spontaneous primary hypertension; chronic cardiac ischemia; varices of lower extremities without ulceration or inflammation; 9F76 SE: biopsy of left kidney true polycythemia without signs of transformation; arterial hypertension; COPD 10 F 79 SE: glaucoma glaucoma, no information on other diseases 11 M 28 EA: spasmodic seizure alcohol dependence syndrome 12 M 39 SE: arthroscopy knee injury 3 years ago; chronic diseases denied 13 F 87 EA: stroke condition after 4 strokes; recurrent fainting episodes 14 F 54 scheduled pre-surgery testing uterine myomas: acne erythomatosa - managed by dermatologist 15 M 37 EA: hyperglycemic coma type 1 diabetes for 10 yrs; esophagitis; chronic gastritis; megaloblastic anemia; alcoholic liver damage, nicotinism 16 M 64 transfer from another hospital three-vessel IHD; arterial hypertension; paroxysmal atrial fibrillation; nicotinism 17 F 19 SE: pre-employment examination Negative 18 M 33 EA: paralytic ischialgia – no anamnesis paresis of right tarsal joint extensors 19 M 84 SE: GP follow-up examination chronic disease 20 M 20 SE: pre-employment examination Negative 21 F 84 EA: sudden spell of severe vertigo insufficiency of vertebro-basiliar system; accompanied by nausea arterial hypertension;3incidents of myocardial infarct 22 M 66 SE: GP follow-up examination hypothyroidism; abnormal lipid metabolism; undetermined hyperglycemia; idiopathic hypertension 23 F 68 SE: GP follow-up examination chronic inflammation of buccal, nasal and laryngeal mucosa resolving after anti-allergic treatment 24 F 56 EA: epileptic seizure epilepsy; arterial hypertension

Abbreviations. EA - emergency admission; SE - scheduled examination.

A reference group (MPOn) was formed, consisting of 13 the majority of cases ordered by a physician, or due to the women and 12 men, aged from 2 to 87 years (60±22.55) analyzer-suggested presence of atypical lymphocytes, eryth- (Table 4) to confirm that the analyzer counts were in roblasts or blasts, rejuvenation in the WBC system, throm- good agreement with the results of microscopic evalua- bocytopenia or leucopenia. Comparing white blood cell tion of MGG-stained specimen. Microscopic verifica- analysis conducted in the BASO and the PEROX channel tion of the peripheral blood smears of the group was in did not show abnormalities in MPO activity. 426 Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017

Table 3. Characteristics of the group with total myeloperoxidase deficiency (MPOt).

No. Sex age Tests ordered due to: Anamnesis [years]

1F46 EA: exacerbation of primary irritable colon syndrome disease 2M84 SE: coronarography ischemic heart disease; condition after PCI (LAD) 3F48 EA: exercise intolerance alcoholic hepatic insufficiency; megaloblastic anemia; nephrolithiasis; circulatory insufficiency 4M60 EA: coronarography tricuspid and aortic valve insufficiency, paroxysmal supraventricular tachycardia; arterial hypertension; combined hyperlipidemia; obesity; mild anemia; gastric mucositis; hepatopathy; gout; prostate adenocarcinoma; hypothyroidism; schizophrenia 5M45 EA: abdominal complaints post alcohol depression 6M22 SE: recurrent detachment negative of right eye retina 7M46 EA: thoracic pains, auto-psychical alcohol dependence syndrome; psychiatric treatment orientation abnormalities 8F71 SE: electric cardioversion persistent atrial fibrillation; valvular insufficiency; arterial hypertension; thyroid nodules; left hip replacement8 years ago 9F63 SE: left hand stump arterial hypertension for 20 years reamputation and reconstructive operation 10 M 83 EA: suspected alimentary duct chronic bronchitis; right bundle branch block; chronicrenal bleeding insufficiency 11 M 60 SE: inguinal hernia inguinal hernia 12 F 60 EA: post-traumatic epilepsy epilepsy

Abbreviations. EA - emergency admission; SE - scheduled examination.

Results the mature cells, a younger granulocytic cell line was also found: nonsegmental neutrophil – bands In the group of patients with MPOp, the average and metamyelocytes: BAND 0.71%±1.33; META MPXi was -26.01±7.90 (the lowest -34.9, the high- 0.042%±0.20. est up to +1.6). The average values of leukocyte separation analysis using hematology analyzer are as In the group of patients with MPOt, the average follows: WBC-P 9.80x103/µl±4.85; WBC-B MPXi was -1.725±9.99 (lowest -18.1, highest to 9.29x103/µl±3.92; NEUT 22.4%±23.5; LYMPH +15.3). Results of automatic average leukocyte sep- 24.6%±10.4; MONO 33.2%±21.3; EOS aration analysis were as follows: WBC-P 8.01x103/ 1.96%±1.94; BASO 0.4%±0.28; LUC µl±2.30; WBC-B 8.23x103/µl±2.37; NEUT 17.5%±11.4. According to the analysis of average 0.45%±0.47; LYMPH 24.4%±7.41; MONO individual cell groups, monocytes (MONO) were 0.62%±0.33; EOS 1.54%±1.61; BASO the dominant population (Figure 2a). 0.46%±0.28; LUC 72.6%±8.64. So, large un- stained cells (LUC) were the dominant group, Microscopic (morphological) assessment of leuko- while neutrophils were virtually absent (Figure cytes showed a dominance of neutrophils instead of 2b). However, microscopic evaluation showeda monocytes. The average value for NEUT was clear domination of NEUT, which accounted for 66.63%±12.31; LYMPH 23.33%±10.08; MONO 67%±11.40; LYMPH 23%±8.94; MONO only 3.20±6.00%; EOS 2.04%±2.20; BASO 6.17%±3.47; EOS 1.25%±1.06; BASO 0.29%±0.62; ATYP 0.83%±1.09. In addition to 0.08%±0.29; ATYP 0.92%±1.38. BANDS were Hematological evaluation of myeloperoxidase deficiency 427

Table 4. Characteristics of the group without myeloperoxidase deficiency (MPOn).

No. Sex age Tests ordered due to: Anamnesis [years]

1F57 commissioned GP suspected bacterial infection 2F77 BLAST+ negative, verification alarm 3M62 LS +++ bacterial infection, verification alarm 4M52 NRBC anemia, bleeding from the gastrointestinal tract, verification alarm 5F60 commissioned GP Negative 6F2commissioned GP Negative 7F64 NRBC thoracic pains, verification alarm 8M87 BLAST+ verification alarm 9F77 ATYP suspected viral infection, verification alarm 10 M 69 ATYP negative, verification alarm 11 F 21 Individual fee for Anemia laboratory test 12 M 60 commissioned by the suspected viral infection, verification alarm doctor; LS, BLAST+ 13 F 84 leukopenia, virus infection thrombocytopenia, commissioned by the doctor 14 F 29 commissioned GP pregnancy 15 M 48 NRBC verification alarm 16 M 12 commissioned GP Negative 17 F 83 commissioned GP Negative 18 M 66 ATY verification alarm 19 M 66 ATY verification alarm 20 M 62 NRBC anemia, verification alarm 21 F 58 commissioned GP leukopenia family 22 M 64 ATYP verification alarms 23 F 82 ATYP verification alarm 24 M 76 commissioned by the doctor verification leukopenia 25 F 82 commissioned by the doctor suspected bacterial infection

Abbreviations. ATYP – virocytes/atypical lymphocytes; NRBC – presence of erythroblast, LS – left shift, BLAST– presence of blasts; (+, ++, +++ means intensity).

also observed 1.58%±1.56. Low-differentiated LUC Microscopic evaluation showed that NEUT av- cells were not found in the microscopic evaluation, eraged 54.48%±12.78; LYMPH 30.72%±13.05; despite that hematology analyzer clearly showed that MONO 10.08%±3.74; EOS 2.76%±2.22; they were present in large quantities. BASO 0.44%±0.829; ATYP 0.8%±0.96. with band nuclei were observed In the MPOn group, the average MPXi was -3.30±6.70 1.00%±2.40. In two cases we found, one cell (lowest -17.7; highest +9.3). The average values of the blast and one person had hypersegmentation of automatic analysis of leukocyte separation were as fol- granulocyte nuclei. Hematological values for lows: WBC-P 6.24x103/µl±2.11; WBC-B 6.43x103/ normal, partial and total MPO deficiency groups µl±2.25; NEUT 53.7%±13.4; LYMPH 30.1%±12.1; achieved by two different methods: an automat- MONO 8.6%±3.07; EOS 2.83%±1.88; BASO ed chemical analyzer and microscopic evaluation 0.63%±0.27; LUC 4.18%±2.31. are summarized in Table 5. 42 8

Table 5. Hematological values for normal, partial and total MPO deficiency groups achieved by two different methods: an automated chemical analyzer and microscopic Ann evaluation. al s An automated chemical analyzer Microscope evaluation of Cl in

MPX-i WBC-P WBC-B%%%%%%%%%%%%%% ic

NEUT EOS BASO LYMPH MONO LUC MetM BANDS NEUT EOS BASO LYMPH MONO ALY al & La

MPOp bora MEAN -26.01 9.80 9.29 22.39 1.96 0.40 24.58 33.16 17.50 0.04 0.71 66.63 2.04 0.29 23.33 6.00 0.83 to

SD 7.90 4.85 3.92 23.48 1.94 0.28 10.35 21.28 11.42 0.20 1.33 12.31 2.20 0.62 10.08 3.20 1.09 ry

MIN -34.90 4.17 4.40 0.80 0.00 0.10 2.70 3.80 4.30 0.00 0.00 44.00 0.00 0.00 3.00 2.00 0.00 Sc

MAX 1.60 24.39 21.77 64.40 6.50 1.10 43.30 65.80 62.10 1.00 6.00 93.00 9.00 2.00 40.00 14.00 3.00 ie nc

MPOt e, vo

MEAN -1.73 8.01 8.23 0.45 1.54 0.46 24.35 0.62 72.58 0.00 1.58 67.00 1.25 0.08 23.00 6.17 0.92 l.

SD 9.99 2.30 2.37 0.47 1.61 0.28 7.41 0.33 8.64 0.00 1.56 11.40 1.06 0.29 8.94 3.46 1.38 47

MIN -18.10 5.16 5.11 0.10 0.10 0.20 11.80 0.20 57.30 0.00 0.00 44.00 0.00 0.00 7.00 2.00 0.00 , MAX 15.30 13.29 13.20 1.80 6.00 1.10 35.70 1.20 86.50 0.00 4.00 82.00 3.00 1.00 36.00 15.00 3.00 no . 4,

MPOn 20

MEAN -3.30 6.24 6.43 53.67 2.83 0.62 30.10 8.60 4.18 0.00 1.00 54.48 2.76 0.44 30.72 10.08 0.80 17 SD 6.70 2.11 2.25 13.41 1.88 0.27 12.05 3.07 2.31 0.00 2.40 12.78 2.22 0.82 13.05 3.74 0.96 MIN -17.70 2.67 2.51 21.50 0.10 0.20 6.50 2.90 1.40 0.00 0.00 24.00 0.00 0.00 6.00 4.00 0.00 MAX 9.30 10.23 11.06 83.10 7.90 1.20 67.40 15.40 11.70 0.00 10.00 75.00 7.00 3.00 65.00 18.00 4.00

Table 6. Comparison of MPX-i, %NEUT, %MONO, %LUC medians in patients with partial myeloperoxidase deficiency (MPOp), total myeloperoxidase deficiency (MPOt) and without deficiency (MPOn). MPOp (n=25) MPOt (n=12) MPOn (n=24) p MPOp/MPOn MPOt/MPOn MPOp/MPOt

MPX-i Median (Quartile range) -28.40 (8.55) -0.05 (13.65) -1.60 (8.00) 0.000000189424 1.000000000000 0.000005815421 %NEUT Median (Quartile range) 8.50 (43.70) 0.30 (0.45) 53.80 (13.80) 0.002042514764 0.000000001930 0.002079832720 %MONO Median (Quartile range) 42.00(39.75) 0.55(0.50) 8.00(4.30) 0.003252226384 0.000993442268 0.000000001621 %LUC Median (Quartile range) 13.95 (9.10) 72.50 (12.20) 4.60 (3.10) 0.000019672790 0.000000000085 0.009010891410 Hematological evaluation of myeloperoxidase deficiency 429

As we can see in Table 5, results of peripheral blood MPO deficiency may be genetic (primary deficien- smear examination using a microscope are similar cy), or it may be a consequence of a variety of dis- in all investigated groups. Subsequently, differences eases (secondary deficiency) [14,17-20]. According in WBC percentage patterns obtained using hema- to reports, secondary myeloperoxidase deficiency tology analyzer measuring cell morphology and may be observed in patients with renal failure, car- MPO activity, between patients with normal, par- diovascular disease, diabetes, myelodysplastic syn- tially reduced and total lack of activity of this en- dromes, degeneration of the joints, depression, can- zyme was compared. When medians of %NEUT, cer, leukemia [21-28]. MPO deficiency can activate %MONO, %LUC between MPOn, MPOp, the development of diseases but is also likely to be a MPOt groups were compared, statistically signifi- result of the primary disease. Pathomechanisms ex- cant differences were observed among each group. plaining exact biochemical changes associated with However, median MPX-i was similar in both MPOt this fact have been already reported [4]. However it and MPOn group. This parameter value was sig- is believed, that total MPO deficiency does noto- pr nificantly welo r for MPOp only (Table 6). duce adverse health effects, except for predisposi- tion to fungal infections [21,29-32]. Discussion Our observations are also consistent in this regard. Observations of peripheral blood morphological This referred usually to blood count tests ordered findings obtained in the haematological analyzer by gynecologists for frequent intimate infections, show large variations in the percentage of white typically mycotic. The other reports associated with blood cell counts. impaired antibacterial response are much less com- mon [22]. This is a surprising conclusion since itis No reaction or weak reaction of myeloperoxidase known that MPO plays a major role in the oxygen- has resulted in the classification of the laboratory dependent mechanisms of intracellular killing by results into total or partial deficiency groups, which hydrogen peroxide. The result of its activity is the are identical to the MPOp and MPOt study groups formation of hypochlorous acid (HOCL) [33-38]. presented in the Results section. If the most bactericidal system H2O2-MPO- HOCL can not be activated, hydrogen peroxide it- It is quite obvious that in the MPOp group, prob- self is not sufficient to effectively oydestr bacteria, ably because of the quantitative reduction of MPO, but it can be a source of free hydroxyl radical OH*. the analyzer included neutrophils in the monocyte In vitro experiments were carried out which showed category, which would be consistent with the he- that the ability of MPO-impaired granulocytes is matologic information in this field, which clearly merely delayed instead of being canceled [14,20]. characterizes the monocytes as cells displaying This is due to the production of *OH and theacti- physiologically lowest peroxidase activity [5]. vation of oxygen-independent defense mechanisms. Granulocytes of MPOt group subjects did not show Cytoplasm, grains of neutrophils and phagocytes any myeloperoxidase activity. The analyzer, there- include proteins and enzymes which also show bac- fore, could not include any of these cells into the tericidal activity. These include lysozyme, granulo- group defined as the reference pattern. It assigned cytic elastase, cathepsin G, β-glucuronidase, colla- to those cells to the LUC population which, by genase, lactoferrin, BPI (bactericidal permeation definition, represent young, immature cells, which, enhancing agent), defensins - cationic arginine-rich depending on the line, show a residual peroxidase peptides, which are toxic to bacteria and some activity or none at all. Probably, due to no MPO mammalian cells [39]. However, reports indicate activity, the analyzer did not consider those cells to that imperfection associated with myeloperoxidase be derivatives of the myeloid line. As the analyzer defect may be the cause of development of chronic could not define these cells, it shifted the responsi- diseases, including autoimmune diseases [41-43]. bility to make a decision to the human operator. Activation of alkaline proteins of neutrophil gran- Hence the need for microscopic evaluation of ules, or lysosomal enzymes, arachidonic acid deriv- leukocytes. atives (prostaglandins and leukotrienes) can 430 Annals of Clinical & Laboratory Science, vol. 47, no. 4, 2017 severely damage the endothelium and the tissues, the combination of white blood cell results by a leading to the expansion of the inflammatory pro- haematological analyzer measuring cell morpholo- cess and its transfer to other tissues. A common gy and secondary MPO-containing activity may be mechanism of regurgitation, or the reflux of lyso- useful for verifying individual medical causes such somal enzymes from the vacuole during closing, as increased frequency and prolonged infection. results in local destruction of healthy tissues. Some authors have found that it is possible to dis- tinguish between leukemia types using the PANDA When assessing the treatment groups in terms of formula or to predict a neutropenic phase using causes of the ordered peripheral blood count test MPXi in chemotherapy patients [44-47]. Therefore, and medical history, it is difficult to conclude what the skillful use of cameras gives you the chance to may be the cause or the disease characteristic of a gain a more comprehensive insight into many partial or complete myeloperoxidase deficiency. health problems. Therefore, results of white blood The MPOp group, relapse and tumor metastasis cell separation by hematology analyzer that mea- were more frequent. On the other hand, the obser- sures cell morphology and activity of secondary vation can be misleading. Groups are rather small grains containing MPO, may be useful for verifying and, therefore, it is not possible to draw firm con- the individual medical causes such as increased fre- clusions. However, it is evident that in the reference quency and prolonged infections. (MPOn) group, except for one individual with ac- tive bleeding from the gastrointestinal tract, cer- From the results of this study, it was not possible to tainly long-term (because MCV=65 fl, MCH=17.4 determine whether MPO defects and their severity pg, MCHC=26.7 g/dl), nobody was diagnosed were due to a complete absence of physical or only with any chronic disease, and comparative analysis deficiency of the oxidative enzyme in the original of the automatic separation of leukocytes coincided granules, or enzyme (as protein) was present but with the microscopic evaluation. Diagnosis in the not active. reference group in most cases was performed be- Acknowledgments cause of acute conditions (viral or bacterial infec- tions, injuries, follow-up of infections, or simply This study is supported by the Statutory funds of the paid tests extended to include microscopic assess- Department of Laboratory Diagnostics and Clinical Biochemistry, Interdepartmental Chair of Laboratory ment ordered by the person concerned). and Molecular Diagnostics, Medical University of Lodz no. 503/5-020-01/503-51-001. Conclusion. Simultaneous automatic analysis of morphology and functionality of WBC in combi- References nation with results of the peripheral blood smear 1. O’Brien PJ. Peroxidases. Chem Biol Interact and cytochemical detection of MPO activity among 2000;129:113–139. 2. Furtmuller PG, Zederbauer M, Jantschko W, Helm J, Bogner patients with myeloperoxidase disorders lead to M, Jakopitsch C, Obinger C. Active site structure and catalytic some significant conclusions. 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