CENTRAL CLINICAL LABORATORY FLAGS AND TROUBLESHOOTS – 3 PART DIFFERENTIAL CELL COUNTERS DR. PRASAD P. SHENOY CENTRAL CLINICAL LABORATORY PANAJI – GOA drpshenoy@gmail.com

COMMON INSTRUMENT IN A HEMATOLOGY LABORATORY 3 PART DIFFERENTIAL CELL COUNTER COMMON INSTRUMENT IN A HEMATOLOGY LABORATORY CBC – ‘BREAD AND BUTTER’ OF HEMATOLOGY LAB RAPID, ACCURATE AND PRECISE BLOOD COUNTS (CONSISTENT)

3 PART DIFFERENTIAL CELL COUNTER HYDRODYNAMIC FOCUSSING ELECTRICAL IMPEDENCE VOLUMETRIC METERING COLORIMETRY (Hb)

PARTICLE 2 – 20 fl: PLATELET 3 PART DIFFERENTIAL CELL COUNTER RBC AND PLATELET COUNTED IN ONE CHAMBER PARTICLE > 35 fl: RBC PARTICLE 2 – 20 fl: PLATELET HEMOGLOBIN AND WBC COUNT IN SECOND CHAMBER

3 PART DIFFERENTIAL CELL COUNTER DIRECTLY MEASURED PARAMETERS AND HISTOGRAMS HEMOGLOBIN CONCENTRATION WBC COUNT RBC COUNT PLATELET COUNT WBC HISTOGRAM RBC HISTOGRAM PLATELET HISTOGRAM

PARAMETERS DERIVED FROM HISTOGRAMS 3 PART DIFFERENTIAL CELL COUNTER PARAMETERS DERIVED FROM HISTOGRAMS LYMPHOCYTE PERCENTAGE MID-SIZE CELL PERCENTAGE GRANULOCYTE PERCENTAGE MCV RDW MPV PDW

CALCULATED PARAMETERS 3 PART DIFFERENTIAL CELL COUNTER CALCULATED PARAMETERS LYMPHOCYTE # MID-SIZED CELL # GRANULOCYTE # HEMATOCRIT MCH MCHC PCT

3 PART DIFFERENTIAL CELL COUNTER MANUAL DIFFERENTIAL COUNT REMAINS A DEFINITIVE TOOL FOR COMPLETE HEMATOLOGIC ANALYSIS PERIPHERAL BLOOD SMEAR REVIEW IS MANDATORY

FLAG SIGNAL TO THE OPERATOR INDICATES A SIGNIFICANT ABNORMALITY IN THE ANALYSED SAMPLE / ANALYSER ITSELF

QUESTIONABLE RESULT SAMPLE RELATED ANALYSER RELATED ? TROUBLESHOOTING

TROUBLESHOOTING SYSTEMATIC APPROACH ISOLATE A SOURCE OF A PROBLEM AND FIX IT THROUGH A PROCESS OF ELIMINATION FIX THE MOST OBVIOUS / EASIEST PROBLEM FIRST

QC QUESTIONABLE RESULT ANALYSER RELATED SAMPLE RELATED SAMPLE RELATED QC IS OUT OF RANGE ANALYSER RELATED QC ACCEPTABLE SAMPLE RELATED

ANALYSER RELATED ERRORS ELECTRONIC (HARDWARE / SOFTWARE) PRESSURE / HYDRAULIC REAGENT

3 PART DIFFERENTIAL CELL COUNTER FLAGS ARE FEW OPERATOR MUST BE ALERT

NORMOCYTIC NORMOCHROMIC Hb: 15.6 g/dL RBC: 5.19 x 106 / uL HCT: 45.9 % MCV: 88.6 fl MCH: 30.1 pg MCHC: 33.3 g/dL RDW: 12.9 % RULE OF 3 NORMOCYTIC NORMOCHROMIC

WBC : 7.0 x 103 / uL Lymph#: 1.8 x 103/uL Mid# : 0.3 x 103/uL Gran# : 4.9 x 103/uL Lymph%: 25.9 % Mid% : 4.3 % Gran%: 69.8 % PLT : 267 x 103 / uL MPV : 9.5 fL PDW : 14.2 PCT : 0.183 %

INTERFERENCES RBC FRAGMENTS, VERY SMALL RBC’s NUCLEATED RBC’s, LYSE RESISTANT RBC’s COLD AGGLUTININS HIGH LEUKOCYTE COUNTS (>50 x 103/uL) GIANT PLATELETS, PLATELETS CLUMPS LIPEMIC SAMPLE CRYOGLOBULINS CLOTTED SAMPLE AIR BUBBLES

CASE: 1 20 years / Female Breathlessness, easy fatiguability Lack of concentration, giddiness

MICROCYTIC HYPOCHROMIC ANEMIA Hb: L 4.2 g/dL RBC: L 3.09 x 106 / uL HCT: L 15.3 % MCV: L 55.0 fl MCH: L 13.5 pg MCHC: L 24.8 g/dL RDW: H 24.5 % WBC: 5.5 x 103 / uL PLT: H 561 x 103 /ul MICROCYTIC HYPOCHROMIC ANEMIA IDA THALASSEMIA ANEMIA OF CHRONIC DISORDERS

If M. I. < 13: Thalassemia MENTZER’S INDEX (M. I.) M. I . = MCV / RBC If M. I. > 13: IDA If M. I. < 13: Thalassemia

Suggestive of Iron deficiency Anemia Adv: Sr. Iron, TIBC, Ferritin = 17.8 Suggestive of Iron deficiency Anemia Adv: Sr. Iron, TIBC, Ferritin Iron: 12.30 37 - 145 ug/dL TIBC: 563 215 - 535 ug/dL % Transferrin: 3.23 13 - 45 % Ferritin : 1.80 13 - 150 ng/mL

Transaminases: Normal CASE: 2 23 years / Female Pregnant, 12 weeks Total Bil : 1.1 mg/dL Conjugated Bil: 0.3 mg/dL Transaminases: Normal

Hb: L 10.1 g/dL RBC: 4.89 x 106 / uL HCT: L 31.9 % MCV: L 65.0 fl MCH: L 22.8 pg MCHC: L 30.5 g/dL RDW: H 17.2 % WBC: 7.5 x 103 / uL PLT: 418 x 103 /ul

MICROCYTIC HYPOCHROMIC ANEMIA Adv: Sr. Iron, TIBC, Ferritin = 13.29 MICROCYTIC HYPOCHROMIC ANEMIA Adv: Sr. Iron, TIBC, Ferritin Hemoglobin electrophoresis

β Thalassemia trait with Iron deficiency Iron: 21.40 37 - 145 ug/dL TIBC: 405.10 215 - 535 ug/dL % Transferrin: 5.28 13 - 45 % Ferritin : 8.19 13 - 150 ng/ml Hemoglobin Electrophoresis HbA = 93.7 94.3 – 98.5 HbF = 0.9 0.2 – 2.0 HbA2 = 4.8 1.5 – 3.5 β Thalassemia trait with Iron deficiency

CASE: 3 46 years / Female Weakness, Paresthesia No organomegaly

MACROCYTIC ANEMIA Hb: L 7.1 g/dL RBC: L 1.80 x 106 / uL HCT: L 21.5 % MCV: H 119.9 fl MCH: H 39.4 pg MCHC: 34.0 g/dL RDW: H 20.6 % WBC: 3.8 x 103 / uL PLT: 127 x 103 /ul MACROCYTIC ANEMIA

MEGALOBLASTIC ANEMIA MACROCYTIC ANEMIA, FAVOUR MEGALOBLASTIC Adv: Sr. Vitamin B-12 and Folic acid Vitamin B-12 113.23 211 – 946 pg/ml FOLIC ACID 1.12 >5.38 ng/ml MEGALOBLASTIC ANEMIA

RETICULOCYTE RESPONSE THERAPEUTIC TRIAL RETICULOCYTE RESPONSE

MACROCYTIC ANEMIA Vitamin B 12 & Folic acid deficiency Myelodysplastic syndromes Hemolytic anemias Myelophthisic anemias Aplastic anemia Chronic Liver diseases

Adv: Sr. Vitamin B-12 and Folic acid MACROCYTIC ANEMIA Adv: Sr. Vitamin B-12 and Folic acid Vitamin B-12 874.89 211 – 946 pg/ml FOLIC ACID 27.46 >5.38 ng/ml ?

MACROCYTIC ANEMIA WITH FEATURES OF DYSPLASTIC HEMATOPOIESIS BONE MARROW & CYTOGENETIC STUDIES

CASE: 4 63 years / Male

M. I. = 72.3 / 3.86 = 18.73 DUAL PEAK Hb: L 9.0 g/dL RBC: L 3.86 x 106 / uL HCT: L 27.9 % MCV: L 72.3 fl MCH: L 23.3 pg MCHC: 32.2 g/dL RDW: H 29.2 % WBC: 8.3 x 103 / uL PLT: 258 x 103 /ul M. I. = 72.3 / 3.86 = 18.73 DUAL PEAK

POST BLOOD TRANSFUSION

CASE: 5 53 years / Female ? FEVER

Hb: L 9.4 g/dL PLT: H 689 x 103/uL WBC: H 310.6 x 103 / uL Lymph#: H 31.4 x 103/uL Mid# : H 43.8 x 103/uL Gran# : H 235.6 x 103/uL Lymph%: L 10.1 % Mid% : 14.1 % Gran%: H 75.8 %

Features compatible with a Myeloproliferative neoplasm: CHRONIC MYELOID LEUKEMIA IN CHRONIC PHASE Adv: Molecular / Cytogenetic studies for Philadelphia chromosome

HEMOGLOBIN PERFORMED BY MANUAL (CYANMETH) METHOD Hb: L 9.4 g/dL RBC: L 1.77 x 106 / uL HCT: L 16.6 % MCV: 94.1 fl MCH: H 53.1 pg MCHC:H 56.6 g/dL RDW: H 22.3 % PLT: H 689 x 103/uL HEMOGLOBIN PERFORMED BY MANUAL (CYANMETH) METHOD 8.3 g/dL 7.1 g/dL (after centrifugation)

Operated case of Ca-Ovary, on Chemotherapy, for follow up 63 years / Female Operated case of Ca-Ovary, on Chemotherapy, for follow up

Hb: 12.4 g/dL RBC: 4.57 x 106 / uL HCT: 35.9 % WBC : 6.7 x 103 / uL PLT : L 56 x 103 / uL MPV : H 14.5 fL PDW : H 19

SPURIOUS THROMBOCYTOPENIA EDTA induced platelet clumping Giant Platelets Platelet clumps EDTA induced platelet clumping

Central Clinical Laboratory Acknowledgement thank you Dr. P. R. Malur, Dr. Anita Borges Dr. C. N. Nair, Dr. Sumeet Gujral Dr. Mona Anand, Dr. Mani Drs: Archana, Anshuman, Aditi, Sunita, Kiran, Kanchan, Manu, Sanica. Entire Staff of Hematopathology Laboratory, TMH, Mumbai Ms. Salini Gopinathan Dr. Prasad P. Shenoy Central Clinical Laboratory Panjim – Goa