Validation procedures for cell analyzers

Dr Archana VazifdarDept. of Hemato-Pathology,

Super Religare Laboratories Limited, Mumbai

Principles of automation

• Impedance – count and size cells by change in resistance produced as they are suspended in an electrically conductive medium

• Optical scatter- measures scatter properties of cells by laser light– Single angle/ Multi-angle scatter

• RBC & Platelets measured in one channel

– RBC volume > 30-36 fl

– Platelet volume 2-20 fl

• Hb & WBC measured in second channel

• DLC in third channel

Interpretation of data

Normocytic Normochromic

RBC count

Spurious increase:• Giant PLT• High WBC counts (>50)

Spurious decrease:• Cold /warm agglutinins• Very small RBC• Cryoglobulins

ADVIA 120

CELL-DYN

COULTER

Platelet count

Spurious increase:• RBC/ WBC fragments• Cryoglobulins• Lipids

Spurious decrease:• Platelet clumps• Giant platelets

neutrolympho

Baso,mono, eos, blasts

WBC (FCM)

Normal WBC scatterplot

Normal WBC histogram

Impedance- VCS

Optical scatter: ADVIA120 DLC by Peroxidase method

Spurious increase

• PLT clumps & large platelets • Nucleated red cells• Resistant RBC’s

Spurious decrease:

• Clotted sample• Fragile cells- CLL• Lymphoid aggregates- UTI, B- cell NHL,

CMML• Storage associated degeneration

Flags

• A signal to the operator that the analyzed sample may have a significant abnormality/ does not meet acceptance criteria/ cannot be displayed

• Cause of errors:– Analyzer– Sample– Random run error

RBC flags

Suspect flags• N’rbc, R’rbc, Micro RBC, RBC fragments,

– interfere with WBC & platelet counts

• H & h errors• short sample, aged sample

Definitive flags• Anemia, anisocytosis, microcytosis, macrocytosis,

poikilocytosis• Erythrocytosis

FLAG:Anemia, Microcytosis, anisocytosis

Hb 8.5RBC 3.2

Left shift of curve:

MicrocytosisIron Deficiency Anemia

β thalassemia trait Anemia of chronic diseases

Conclusion:

s/o Iron Deficiency AnemiaAdvise Iron studies

ACTION:

RBC indicesMentzer’s index (MCV/RBC)=

18.3MI ≤ 13- BTT, ≥ 13- IDA

Flags:• N’rbc, Micro RBC/ RBC

fragments• Giant plt• Thrombocytopenia

Lt of curve not touching baseline:NoiseSchistocytes &/ extremely small rbcGiant platelets

PLT 140MPV 7.9PCT .148PDW 15

Hb 6.4

Conclusion:

RBC count falsely ↓Platelets falsely ↑ (mask t’penia)

Hemolytic anemia

Action:

• RBC Indices- MCV, RDW• PLT Histogram- MPV & PDW • Review PS- RBC morphology

-PLT count (100)

Bimodal peak: Dimorphic RBC population

Transfused cellsCombined deficiencyTherapeutic response in IDA

Hb- 8.6, MCH- 26.5, MCHC- 32.2

Flags:Dimorphic RBC population, anisocytosis

Action:

Review PS to identify cause

50/ F, Hb-8.9, MCV-73, MCH- 25.6, RDW-26.8

Blood transfusion

Dual/Combined deficiency

45/F, Severe pallorHb-5.1, MCV-96.7, MCH- 29.6, MCHC-31.4, RDW-24.5 TLC/Plt-Normal

S. Fe- 25TIBC- 144

S. Fe saturtn- 20.8S. B12- 158

Right portion of curve extended:RBC agglutinationN’rbcsLeukocytosis

Flags:H&H error, N’rbc, dimorphic redsAnemia, macrocytosis, anisocytosis

H&H

• Sample related problems- turbidity-↑ Hb– Lipemia/ TPN– Cryoglobulins

• Autoagglutination• Hemolysis (in-vitro/vivo)• Spurious ↓ Hct• Clotted sample

Spurious ↑MCHC:

corrected

Conclusion:False ↓ RBC, Hct, False ↑ MCV, MCH & MCHC

Cold agglutinin disease

After warming in H2O bath @ 37ºC for 15 mins

Action:Review PS: L/F agglutination vs n’rbc’s

Short sample (microtainer)Repeat collection

Causes of H&H mismatch:

• partial sample aspiration/ improper mixing

• Hb/ MCV measurement error/ very low• High WBC counts (interfere with Hb

measurment)• Cold agglutinins

PlateletsSmallest guys largest culprits!!

• As platelet counts fall, reliability of analyzer decreases.

• Conventional methods are unable to provide consistently accurate results in lower range

• Clinicians using thresholds of 5-10 X 109/l must be aware of the limitations in precision and accuracy of cell counters

Linearity : 10–1,000 X 109/l

Common platelets flags

• PLT Clumps – ↓Plt counts– Interferences with WBC Results (↑WBC

counts)• Giant platelets• Small platelets• PIC/POC delta- difference > 20,000• Thrombocytopenia- true/false

Increased small sized particles:

Noise, debris, lipids, bacteria, fungi ? Wiskott Aldrich syndrome

Conclusion:

Falsely elevated platelet counts

Flags:Small platelets

Debris/ noise

Action:

Review PS for platelet count

Conclusion:

Falsely ↑RBC countFalsely ↑WBC count

Falsely ↓ Plt count, ↑MPV

Giant platelets

Flags:Giant platelets, platelet clumpsCellular interference

Non fitted curve with increase in large cells:

Large platelets, clumps

PIC/POC delta

• Excessive noise included in impedance count

• Debris, bacteria, fungi• Plt clumps• Giant plt

45/M

IG, Band, BlastsAty ly, Variant lyMPO, non viable WBCN’RBC, rst RBCPlt clumpOutside Reportable RangeLeukocytosis, monocytosis, basophilia, eosinophiliaUnable to Find Clear Separation between WBC subpopulations

WBC Flags

Shoulder on the left of curve:

N’rbcLyse resistant RBCPlatelet clumps/ Giant platelets FibrinImpedance noise

Flags: IG, Blasts, eosinophilia,monocytosis, lymphopenia

CML

LeukocytosisThrombocytosisAnemia

Flags:Aty lymphocyte, Variant lymphocyteNon-viable wbcLeukocytosisT’penia

Acute Leukemia

38/F, k/c/o DM

Flag: leukocytosis, n’rbc, dimorphic reds

Conclusion:

21 nrbc’s/100 wbc- corr WBC= 17.35

DM in sepsis with liver abscess

Plt 100

VCS:• Quantitative • Operator independent• Routinely available• Inexpensive

INCREASE MEAN NEUTROPHIL VOLUME (MNV)DECREASE MEAN NEUTROPHIL SCATTER (MNS) – left shift

– Lacking leukocytosis or neutrophilia

Newer Aspects: VCS-Neutrophil population data

Suggestive of acute bacterial sepsis

Automated malaria detection

• “Gold standard” - thick & thin smear • Need for rapid, sensitive & cost-effective

screening technique

• Hemazoin pigment• Activation of neutrophils & monocytes• Increase volume heterogeneity (anisocytosis) of

monocytes & lymphocytes, detected by VCS

• ‘Positional parameters’, used as objective criteria for detecting presence of plasmodium

Clin. Lab. Haem., 26, 367–372 Automated detection of malaria

Normal Plasmodium falciparum

Monocytes

Reactive LY

Parasitized RBC

Vol SD lymphocyte X SD Monocyte / 100 > 3.7

Am J Clin Pathol 2006;126:691-698Briggs et al / MALARIA DETECTION USING VCS TECHNOLOGY

shoulder

• Specificity is 94% and sensitivity 98%

• PPV is 70% and NPV 99.7%.

• A flag indicating potential presence of malaria is a valuable diagnostic method for detection of malaria and may become a routine parameter in it’s diagnosis

Reticulocyte Indices

• most promising from a clinical viewpoint are the CHr and the MCVr.

• CHr: – directly reflects hemoglobin synthesis in marrow, & measures

iron availability.– ↓ IDA & BTT (independent of iron stores)

• MCVr: ↑rapidly following iron therapy – ↓ with the development of iron-deficiency– ↓ in macrocytosis after therapy with B12 &/or folic acid

• Available in very few analyzers, not standardized

Case 1 38/M, No history available

Result after treatment in H20 bath @ 37 C �

Cold agglutinin disease

27/M, Hb 7, MCV 94, MCH 32, MCHC 35.7, RDW 14.6, Plt 158

Flags: Blasts, IG, n’rbc, rbc fragments, giant platelets

Case 2

Conclusion:

Severe hemolysis following Primaquine ingestion in G6PD deficiency

50 nrbc’s/100 WBCSpherocytes +Giant platelets

Case 3 : 33/M, Thrombocytopenia X 6 mnths, no bleeding. All other parameters WNL, ? ITP

Flags: n’rbc, micro rbc/ rbc fragments

Action:

Change anticoagulant to Sodium Citrate

Platelet count- 243

Conclusion

EDTA dependant pseudothrombocytopenia(EDP)

EDP

EDTA dependant pseudothrombocytopenia (EDP):

• Hypothesis- antigen-binding site in the GPIIb/IIIa complex , normally hidden/cryptic, is modified by or exposed only in presence of EDTA

• In-vitro phenomena• Associated with autoimmune/ neoplastic pathology, but

also seen in healthy individuals• Abnormal plt from CMPD, more prone to clumping by

EDTA• Alternate anticoagulants; 10% trisodium citrate/ ACD

Case 4: 15/M, Fever

Conclusion:

Plasmodium falciparum , PI 15%Thrombocytopenia

Malaria discriminant factor= 6.3

THANK YOU

Archana Vazifdar, M.D.SRL RELIGARE LTD.