Kathleen Finnegan MS MT(ASCP)SHCM

Kathleen Finnegan MS MT(ASCP)SHCM

Discuss the history of hematology automation and digital differentials. Discuss the HemoFAXS Hematology Analysis System by Tissue Gnostics. Review automated microscopy analysis with the use of case studies.

1953 Wallace Coulter and the Coulter Principle 1960 Hematologic Evaluations were performed manually 1961 Coulter Electronics Inc, Hialeah, Florida 1970 s Vigorous competition began 1980 s Reporting out 7-parameter blood count and a three part differential 1980 s Introduction of flow based analyzers

Next 20 years grew in complexity 1988 a slide maker was introduced 1990 s the five part differential and multi parameters Early 2000 s the fully automated Reticulocyte was introduced 2011 Bloodhound Integrated Hematology System: Blood printing

For over 100 hundred years have manually viewed blood smears with the use of a microscope 1962: First television based image analyzer utilizing microscopic pictures which were developed by Metals Research. 1976: A cytology study conducted using a microscope, TV camera, an automatic cell finder, and a servo driven computer controlled stage is conducted. 1982: Automated Intelligence Microscopy (AIM) is coined with the advent of the Yellow IRIS urinalysis workstation. 1986: Hirox Co Ltd develops early digital microscope lens in Tokyo, Japan. 2000: Launch of Cellavision s Diffmaster Octavia in Europe. 2011: Bloodhound and a digital differential.

Automated cell differential to detect cell nucleus and cytoplasm Measurement parameters are: Morphology Color Differences Granularity Cell Nuclei Cytoplasm

3 Mega Pixel Color Camera 10 x and 100X Oil objectives Zeiss AxioImager Z2 Microscope 8 Motorized Slide Insert

10X 10X Slide ID is scanned, digitized and presented with a control image Monolayer is localized Leukocytes are localized within the identified monolayer

Image acquisition and preclassification HemoFAXS searches automatically for the optimal working area (monolayer) HemoFAXS detects the predefined number of Leukocytes (10x) HemoFAXS acquires morphology pictures for morphological evaluation (10x) in the same process Insert Slides Acquisition & Classification Validation Reporting

Morphology 10 x morphology images Morphology comments for Thrombocytes, Erythrocytes and Leukocytes 100 x morphology images Easy one-click scoring Insert Slides Acquisition & Classification Validation Reporting

Image acquisition and preclassification The image of each leukocyte is localised and acquired with a 100x oil objective The image analysis algorithms segment the cellular compartments to acquire the dataset Insert Slides Acquisition & Classification Validation Reporting

Classification into leukocyte classes Insert Slides Acquisition & Classification Validation Reporting

Evaluation Slide ID Patient data fields Differential count and percentages 100 x cell images displayed in columns Insert Slides Acquisition & Classification Validation Reporting

Optimal Visualisation Up to 10 columns can be visualized Insert Slides Acquisition & Classification Validation Reporting

All cell images displayed Number and Percent Insert Slides Acquisition & Classification Validation Reporting

Patient History: A 84 year old female was admitted to University Hospital with complaints of exhaustion, heart palpitations and dehydration.

WBC 131.2 RBC 2.44 HGB 8.2 HCT 26.6 MCV 109 MCH 33.6 MCHC 30.8 RDW 20.6 PLT 76 MPV 11.2

Symptoms of long duration May be asymptomatic for a long time Mostly mature cell forms found Usually organ involvement Platelets are normal to increased Anemia develops later on

Majority involve the B Lymphocyte 75% Usually seen in the older population Male Commonly picked up by chance Accumulation of small mature lymphocytes Smudge cells: very fragile lymphocytes Platelet count normal N/N Anemia Progressive anemia and thrombocytopenia

Patient History A 63 year old female was admitted to the hospital for fatigue and anemia. She had been feeling poorly over the course of several weeks. Additional complaints were abdominal fullness.

WBC 22.7 RBC 2.39 HGB 7.7 HCT 24.9 MCV 104.2 MCH 32.2 MCHC 30.9 RDW 18.7 PLT 113 MPV 11.0

Clonal stem cell disorder Marked leukocytosis with all stages of granulocytic maturation Primarily disease of adults rare in children Hepatosplenomegaly Thrombocytosis is common

WBC count ranges from 100,000-200,000 Differential: complete spectrum Increase Eos and Baso N/N Anemia Increased platelets, giant and fragments Bone Marrow: hypercellular 25:1 BCR/ABL gene or fusion gene 95% cases (necessary for pathogenesis) Philadelphia Chromosome arm of 22 is translocated to 9, positive in 90% of the cases

BCR: Breakpoint Cluster Region DNA fragment localized on chromosome 22 Defines the chromosomal break ABL: protein possessing increased tyrosine kinase activity

Patient History A 43 year old male admitted to the Emergency Room for a progressive infection and stagnant healing of a molar tooth extraction.

WBC 27.7 RBC 2.96 HGB 8.4 HCT 24.6 MCV 83.1 MCH 28.4 MCHC 34.1 RDW 14.5 PLT 32 MPV 11.9

Short Duration Onset sudden Many immature cells in the bone marrow and peripheral blood > 30% Blasts in the bone marrow (FAB) >20% Blasts in the bone marrow (WHO) Platelet count usually decreased N/N Anemia

L1- Lymphocytic, childhood Small uniform lymphoblasts L2- Lymphocytic Adult Large pleomorphic lymphoblasts L3- Burkitts, poor prognosis Deeply basophilic and vacuolated

Precursor B lymphoblastic Leukemia/Lymphoma Precursor B cell Pre B - ALL B - ALL Precursor T-Lymphoblastic Leukemia/Lymphoma

Frequent found in adults (64%) Blasts are large, twice the size of L1 Chromatin is fine to course condensed, nuclear clefting Nucleoli present, usually single and large Blasts are confused with M1

Patient History A 42 year old female was being seen by her physician for treatment of a bacterial infection and a progressive anemia.

WBC 3.63 RBC 2.72 HGB 7.8 HCT 24.3 MCV 89.3 MCH 28.7 MCHC 32.1 RDW 15.6 PLT 90 MPV 11.6

Benign inherited autosomal dominant Neutrophil nucleus does not segment beyond the bi - lobed stage Peanut or dumbbell shape Heavy chromatin clumping distinguishes from bands Mutant defect in the lamin B receptor gene Controls the shape of the nucleus 70-90% neutrophils affected The cell functions normal

Heterozygous State Nuclear chromatin densely clumped, dark and coarse 55-93% show bilobed nucleus Homozygous Nucleus is often single, round or slightly indented Can be eccentrically placed in the granulocyte with no nuclear segmentation Dense chromatin pattern Acquired Leukemoid reactions during severe bacterial infections, HIV, TB, MDS, MPN s

Patient History A 19 year old male is seen by his physician for flu like symptoms. Major complaints was progressive fatigue, low grade fever, swollen lymph nodes and a sore throat. He had been feeling poorly for several weeks.

WBC 9.2 RBC 4.27 HGB 12.4 HCT 36.9 MCV 86.4 MCH 29.0 MCHC 33.6 RDW 14.6 PLT 320 MPV 9.7

Young Adult : 14-24 yrs Epstein - Barr virus Transmission: oral through saliva (most common) Relative and Absolute Lymphocytosis Reactive Lymph > 50% lymphs Splenomegaly: 50-75% of the cases Hepatomegaly: 25% of the cases Positive Mono Spot/ Heterophile Antibody

Incubation period 11 days Low grade fever Pharyngitis (sore throat) Enlarged lymph nodes Hematological Complications: auto hemolytic anemia Rare complication: hepatitis Convalescence: few weeks to months

HemoFAXS is in the process of running the clinical trials for FDA clearance