http://www.revistadechimie.ro REV.CHIM.(Bucharest)♦ 67♦ No. 7 ♦ 20161382

The Importance of Hematoxylin Eosin Staining Techniquein Accurate Diagnosis of Tumors

OANA VIOLA BADULESCU¹, ROXANA HULTOANA²*, MADALINA MOCANU¹*, CRISTINA ELENA IANCU3*,STEFAN OCTAVIAN GEORGESCU4

¹ Grigore T. Popa University of Medicine and Pharmacy, Faculty of Medicine, Department of Pathophysiology, 16 Universitatii Str.,700115, Iasi, Romania² The second Clinic of General Surgery, University Hospital St. Spiridon, 1 Independentei Blvd., 70011, Iasi, Romania.3 Grigore T. Popa University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Science, 16 UniversitatiiStr., 700115, Iaºi, Romania.4 Grigore T. Popa University of Medicine and Pharmacy, Faculty of Medicine, Department of General Surgery, 16 Universitatii Str.,700115, Iasi, Romania

Retroperitoneal tumors, benign or malignant derive from a variety of tissues, so their classification is primarilybased on this histological parameter (tissue origin). Retroperitoneal tumors, although very rare, raise interestsby the specific characteristics from the space where they develop, causing serious problems for diagnosisand treatment. Autoimmune Hemolytic Anemia is a common complication of malignancy, but may besecondary to the retroperitoneal tumors. We present the case of a woman, 42 years old, who is admitted inthe Hematology Clinic for abdominal pain and marked asthenia. The abdominal CT objectified presence ofexpansive tumor on the caudate lobe liver, well defined, with mass effect on portal trunk. She is transferredto a Surgery Clinic where, although the investigations oriented towards a liver tumor, during surgery it wasdiscovered the presence of a tumor that did not belong to the liver parenchyma but retroperitoneal tissue.The pathological anatomy confirmed the diagnosis of benign lesion tumor with conjunctiva behavior. Thereare discussed the relationship between autoimmune hemolytic anemia and retroperitoneal tumors, diagnosticproblems but also the therapeutic methods addressed in this case, represented by the surgical act, performedas a diagnostic and therapeutic purpose. Retroperitoneal tumors are rare injuries, but make delicate problemsof diagnosis and therapeutic attitude.

Keywords: autoimmune hemolytic anemia, retroperitoneal tumors, liver tumors, hematoxylin eosin

Retroperitoneal tumors are developed on account of fattissue, connective fascia, sheaths, vascular tissue, lymphvessels and lymph nodes or embryonic remnants,particularly derived from urinary and other structures. Theyare a heterogeneous group of histologically, rarelyencountered in surgical practice, but the peculiarities ofthe space in which they develop raise serious problems ofdiagnosis and treatment. [1-3]

Autoimmune hemolytic anemia is known as aparaneoplastic syndrome secondary to lymphoproliferativediseases, but have also been reported cases ofautoimmune hemolytic anemia secondary to thedevelopment of solid tumors. [11, 12]

Autoimmune hemolytic anemia secondary to themechanism of tumor growth remains unknown; it appearsthat B lymphocytes produced by the monoclonalimmunoglobulins on their surface are responsible forhemolysis. Hemolysis, mainly with headquarters spleencauses a peripheral chronic anemia with splenomegalyand reticulocytes grown; direct Coombs test positive. [10,13-15]

Experimental partMaterial and method

We present the case of a patient, aged 42 years, female,without pathological personal history, coming to theDepartment of Hematology of University Hospital St.Spiridon for marked asthenia and fatigue. Hematologicalexamination revealed: severe macrocytic anemia (Hb=5.7g/dL, VEM=105.7 µ³) hiper regenerative (reticulocytes

=17.25%), leukopenia with moderate neutrophilic inleukocyte formula (GA=1190/mm³, neutrophils=120/mm³). Peripheral blood smear revealed leukocytes are veryrare, severe neutropenia; erythrocyte series: anisocytosis,macrocytosis, poikilocytosis. Biochemistry revealedcholestasis syndrome and hepatic cytolysis (TGO=37 U/L, GGT =92 U/L), hyperbilirubinemia prominent faction onaccount indirect (total bilirubin=3.6 mg/dL, directbilirubin=0.81 mg/dL) elevated LDH and serum ferritin(LDH=353 U/L ferritin=685 ng/mL). Clinical and laboratorydata indicates diagnosis of hemolytic anemia. To determineif the cause is immune hemolytic anemia we have madethe following determinations: Coombs test–positive. Bonemarrow puncture was performed to exclude a hematologicmalignancies: bone is normal; erythroblasts series is veryrich (53% of total cells), macrocytic and left shift maturingcurve with increased proportion of basophils erythroblastsand pro erythroblast; granulocytic series is slightlyhypoplasia (41%); Neutrophils are macrocytic and have aleft shift of the maturing curve – predominates myelocitesand metha myelocytes 29%.

At the same time, there have been made somemeasurements (markers for rheumatic diseases andvasculitis, viral liver tests, the dosage of thyroid hormones,HIV antibody, toxoplasmosis antibodies, antibodies againstCytomegalovirus, antibodies to infectious mononucleosis,and antibodies for systemic sclerosis, immuneelectrophoresis) with negative or normal results, to specifythe context or etiology.

* email: alicia_roxana@yahoo.com; mocanu_madalina@ymail.com; cristinaelena.iancu@yahoo.com

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To be noted that during hospitalization in Hematology,we could not detect blood group and Rh patient and thelaborator y doctor indicated achievement red celltransfusion therapy group 0 I, Rh negative. The abdominalcomputer tomography performed raised the suspicion of aliver caudate lobe tumor.

Results and discussionsCorroborating clinical data with laboratory results we

establish the diagnosis of autoimmune hemolytic anemia.Excluding other possible causes of this, we considered theassociation between liver tumor and autoimmunehemolytic anemia. This association raised issues inchoosing therapeutic conduct. Thus, they were given 3units of packed red blood cells (group 0 I Rh negative) withclinic – biological evolution favorable (Hb = 10.8 g%). Wedecided to transfer the patient to the Surgery Clinic toachieve surgery and continued corticosteroid therapyinitiated.

The onset of abdominal pain has been described to beabout 2 months ago, initially predominant epigastric andright upper quadrant subsequently prone to expansionthroughout the abdomen; in the past 3 days, the pain iswidening.

Clinic she presents: general state influence; pale skinand mucous membranes, jaundice, hot, dr y;Cardiovascular: blood pressure = 110/65 mmHg, urinarytract = dark urine (urine culture performed in theHematology Clinic was negative). Local clinicalexamination objectified: abdomen increased volume atthe expense of body fat, hollow with the respiratorymovements, painful on shallow and deep palpation inepigastric and right upper quadrant; liver, spleenimpalpable.

Abdominal ultrasound revealed the presence of acaudate lobe tumor hypoechogenyc, homogenous, welldefined, about 41/52/78 mm, compresses the portal vein(normal size); 122/52 mm spleen, splenic vein to the hilum7 mm, free liquid in Douglas space about 26 mm.

The abdominal – pelvic computer tomography made,describes an expansive tumor on caudate liver lobe of 55/

50 mm, native hypodense, heterogeneous, with a centralhypodense area. The tumor is presented well defined, withmass effect on trunk portal (moved forwards, permeable),the inferior cave vein and the left renal vein spills, bothpermeable and the pancreatic head and process uncinated(moved above). The tumor comes into contact with part IIand III of the duodenum, with boundary.

Fig. 1. Ultrasound images of the liver tumor

Fig. 2. CT images into sections

Fig. 3. Aspects of tumor dissection during surgery1. Phase Identification of tumor; 2. Phase of tumor dissection; 3. Final phase of excision/extraction

Biological samples collected in Surgery Clinic:macrocytic anemia (Hb = 11.3 g/dL, VEM=100.6 ì³);leukocytosis (13.740/mm³), lymphocyte (7.4%), hepaticcytolysis syndrome (TGP = 185 mg/dL), hyper-bilirubinemia (total bilirubin = 1.89 mg/dL, direct bilirubin= 0.61 mg/dL). Following explorations that we have made,following diagnoses were established: observation livertumor, severe secondary autoimmune hemolytic anemia,chronic gastritis.

During surgery we have discovered a tumor that did notbelong to the liver parenchyma but retroperitoneal tissue,developed into liver pedicle and inferior cave vein diameter40/26 cm, elastic consistency renitent with induratedzones, well encapsulated. We some difficulty wecompleted the excision of the tumor with hemostasis inalmost near.

Histopathological examination revealed the presenceof a tumor lesions nature conjunctiva with benign behavior,a myofibroblast inflammatory tumor or a solitary fibroustumor.

The technique used in this case was eosin – hematoxylincoloration – method commonly applied for histo-pathological analysis of tissues. Eosin, called tetrabromo

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fluorescein, is a red fluorescence dye, formed by theaddition of bromine on fluorescein. It is a dye used tohighlight the cytoplasm and collagen fibers. Eosin is usedas a contrast medium in staining with hematoxylin, eosinstaining the pink – orange cytoplasm and the nucleus inblue or purple. Eosin stains also in deep red erythrocytes.

Fig. 4. Macroscopic appearance of tumor 1 – morphology and measuring the dimensional in superior

overall view; 2 – lateral view; 3 – dimensional measurement of theside view

ConclusionsAlthough CT scanning is the most important exploration

of the arsenal used to diagnose this tumor, that andabdominal ultrasound, could not specify affiliation organtumor and no vascular or visceral relationships thereof.

The main therapeutic attitude used for retroperitonealtumors is the surgical act, performed in diagnostic andtherapeutic purposes; these are associated for malignanttumors with chemotherapy and radiant treatment [7-9].

The main problems of surgical treatment inretroperitoneal tumors are related to the chosen route ofapproach, the surgical exploration as complete, the needof visceral sacrifices and vascular associated withdifficulties of hemostasis in the remaining space afterremoval of the tumor and difficulties in the limits of excision,safety tumor excision complete (our case) or the purposeof reducing the tumor mass [4, 5].

The bleeding risk in retroperitoneal tumors is importantnot only because of vascular relations but also becausethese tumors determines in the retroperitoneal space thedevelopment of dilated venous vasculature or because thetumors themselves are hipervascularized [6].

The histopathological result in this case was a tumorwith conjunctiva benign behavior, myofibroblastinflammatory tumor or solitary fibrous tumor.

Retroperitoneal tumors are rare injuries, but makedelicate problems of diagnosis and therapeutic attitude.

Anemia is a common complication of malignancyoccurs in almost 50% of patients with solid tumors andmore frequently in those with blood diseases – cancer:myeloma, leukemia and lymphoma. In our case,autoimmune hemolytic anemia was secondar y toretroperitoneal tumor growth, as proof, after the extractionof tumor, at the subsequent checks hemoglobin level wasnormalized in our patient.

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Manuscript received: 22.12.2015

Fig. 5. Appearance before and after sectioning the tumor

Fig. 7. The chemicalstructure of hematoxylin

Fig. 6. The chemicalstructure of eosin

Hematoxylin is a natural dye extracted from the treeHaematoxylum campechianum (colored wood). Byoxidation is converted into haematin, a compound of theblue – violet staining intensity. This is used together with amordant (usually salts of Fe (III) or Al (III))) for staining thenucleus of cells.

Immediate postoperative course of the patient wasfavorable, on postoperative day 9 being discharged.