Validation Cell Analyzers

Validation procedures for cell analyzers

Dr Archana VazifdarDept. of Hemato-Pathology,

SRL Religare Ltd.

Validation

A documented act of demonstrating that a procedure, process, & activity will consistently lead to expected results

FDA 1987

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

• 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

Presenter

Presentation Notes

RBC-Plt- separated by Volume & refractive index based counting/sorting

Validation of automated CBC analyzers:

– interpretation of numeric, graphic & flag data

– Delta checks

– Review of peripheral smear

Interpretation of data

Presenter

Presentation Notes

3 steps for performing a CBC- interpretation of numeric, graphic & flag data, Delta checks, Review of peripheral smear

Normocytic Normochromic

RBC count

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

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

Presenter

Presentation Notes

RBC histo- X axis, volume, Y axis, No. of RBC, MCV- mean of distribution curve

ADVIA 120

CELL-DYN

COULTER

Platelet count

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

Spurious decrease:•Platelet clumps•Giant platelets

Presenter

Presentation Notes

neutrolympho

Baso,mono, eos, blasts

WBC (FCM)

Normal WBC scatterplot

Normal WBC histogram

Impedance- VCS

Presenter

Presentation Notes

The traditional microscopic method based on the count of 100 cells has 3 types of error: statistical error, distributional error owing to unequal distribution of cells in the smear, and error in identifying cells related to the subjective interpretation of the examiner. Newer analyzers analyze thousands of cells per sample and can produce morphologic and quantitative flags, which have significantly reduced error and allow reliable absolute counts at low and high concentrations. WBC analyzed by fcm, using impedance (VCS), Optical scatter (cytochem) VCS- Simultaneous measurement of volume, conductivity and scattered laser light, CELLS R ANALYZED IN NEAR- NATIVE STATE. x-axis represents laser light scatter, the y-axis volume and the z-axis conductivity

Optical scatter: ADVIA120DLC 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

Presenter

Presentation Notes

EDC include immature or atypical cells such as blasts, IGs, atypical lymphocytes, hematopoietic progenitor cells (HPCs), and NRBCs.70 The principal aims of the EDC are to further reduce the need for microscopic revision, to obtain more precise and accurate counts when elevated basophil/ monocyte counts are produced, they must be examined with caution because they can be artifacts due to the presence of abnormal cells such as blasts, plasma cells, and lymphoma cells.

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 flagsSuspect 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

Presenter

Presentation Notes

BTT & ACD- NORMAL RDW

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

PLT 140MPV 7.9PCT .148PDW 15

Hb 6.4

Presenter

Presentation Notes

FRBCs are identified only on the basis of size and hemoglobin content, independent of their shape

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

Presenter

Presentation Notes

NN anemias: Hypoplastic, malignancy, Sideroblastic (low retic) Acute bld loss, Sev hemolysis, AIHA, G6PD, Sickle,

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

Blood transfusion

Presenter

Presentation Notes

Volume/Hemoglobin Concentration (V/HC) cytogram hemoglobin concentration (chromia) is plotted along the x axis and cell volume/size is plotted along the y axis, Nine zone grid, central zone NN

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

Presenter

Presentation Notes

spuriously low RBC counts and to abnormally high MCV (each small RBC clump is considered as one single particle Haematocrit (RBC · MCV) is erroneous and spuriously low, contrasting with Hb that is measured after RBC lysis and is unaffected by agglutinins. As a rule, the MCHC is spurious, usually >36 g/dl.

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

Presenter

Presentation Notes

Thresholds for use of prophylactic plt tx has reduced frm 20 × 103/µL to 10 × 103/µL (20 × 109/L to 10 × 109/L). Other Authors have suggested that in patients without fever or bleeding, there may be even lower values. However, the utilization with confidence of these new thresholds requires knowledge of the limitations in precision and accuracy of the analyzers at these count levels.

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

Presenter

Presentation Notes

CAUSE- Plt clumps, giant plt, ….?

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 plateletsFibrinImpedance noise

Presenter

Presentation Notes

N’RBC: neonatal hemolytic disease, in premature neonates and neonates affected by hypoxia in the perinatal period. adults: thalassemic syndromes, myeloproliferative diseases (myelofibrosis), bone marrow metastases of solid tumors, extramedullary hematopoiesis, and all of the conditions of hematopoietic stress (eg, septicemia, massive hemorrhage, and severe hypoxia) Resistant RBC’s: physiological (neonates) pathological (abnormal hb, liver disease, uraemia, chemotherapy) extended lysis mode

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

Presenter

Presentation Notes

it is useful not only to identify the presence of NRBCs, but also to estimate the NRBC count: 1.to evaluate the efficacy of transfusion therapy, as with thalassemic syndromes in which it is advisable to maintain an NRBC concentration of less than 5/100 WBCs 2. persistence of NRBCs in the peripheral blood of subjects undergoing stem cell transplantation has been shown to be a poor prognostic factor 3. hospitalized patients after general or cardiothoracic surgery or with other nonhematologic disease, the mortality rate was 21.1% for patients with NRBCs, whereas it was 1.2% for patients without

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 P lasmodium 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

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)

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.

Validation Cell Analyzers

Documents

Transcript of Validation Cell Analyzers