Lab tests in HSCT

HedayatiHedayatiHedayatiHedayati AslAslAslAsl A.A.A.A.HedayatiHedayatiHedayatiHedayati AslAslAslAsl A.A.A.A.Pediatrics Hematologist & Pediatrics Hematologist & Pediatrics Hematologist & Pediatrics Hematologist & OncologistOncologistOncologistOncologistMAHAK Children’s Cancer Hospital MAHAK Children’s Cancer Hospital MAHAK Children’s Cancer Hospital MAHAK Children’s Cancer Hospital

Lab tests in HSCTHematology

Biochemistry

Virology

CoagulationCoagulation

Culture

Flow cytometry

HLA

Cytogenetic Study

Chimerism(

Other important additional information:

Name of Clinician/Consultant requesting the

testtest

2. Tests requested

3. Clinical details

4. Date of sample

Laboratory test required for donor selection shall be done by accredited laboratory and include at least the following:

• HLA-A, B, DR typing and other appropriate compatibility tests as

indicated by an accredited laboratory.

• ABO group and Rh type. Anti-A and Anti-B titre• ABO group and Rh type. Anti-A and Anti-B titrewhere appropriate.

• Infectious disease screening shall minimally include the following:

• HIV-1, HIV-2 HBV, HCV, CMV and syphilis. Where appropriate, additional test for HTLV-1, HTLV-2, EBV, HAV, VZV, HSV-I, HSVII , toxoplasmosis and cryptosporidium may be performed.

COLLECTION OF SPECIMENS

• VENIPUNCTURE PROCEDURE• Method: Percutaneous Venipuncture

• 1. Identify self to patient.• 2. Properly identify the patient using two unique identifiers. Acceptable

identifiers are patient first and last name and one of the following: date of birth, medical record number, social security number, or driver’s license number.number.

• 3. Verify diet restrictions.• 4. Position patient lying on back with face up or sitting with the appropriate

site exposed.• 5. Wash hands thoroughly and apply clean gloves.• 6. Select venipuncture site.• a. DO NOT USE an extremity with an A-V shunt or status/post mastectomy.• b. DO NOT USE a site with extensive scarring.• c. DO NOT USE a site with a hematoma.• d. DO NOT USE a site with an IV.

7. Prep overlying skin with alcohol using a circular motion. Chloraprep

may be used if patient is allergic to alcohol.

8. Before using, tap all tubes that contain additives to ensure that the

entire additive is dislodged from the stopper and the wall of the tube.

9. Make sure patient’s arm or other venipuncture site is in a downward

position to prevent reflux.

10. Apply tourniquet to extremity 2 inches proximal to desired site.10. Apply tourniquet to extremity 2 inches proximal to desired site.

11. Identify target vein with palpation and visualization.

12. Use thumb to apply tension downward distal to insertion site.

13. Verbally state to patient that the venipuncture is starting and insert

the needle at a 15-30˚ angle and ¼ to ½ inches below the intended

entry into the vein.

Venipuncture procedure when evacuated tubes are used:1. Insert blood collection tube into holder and onto needle up to the recessed

guideline on the Vacutainer® adapter.

2. Position the needle with the bevel up and the shaft parallel to the path of the vein.

3. Insert needle through skin at 15-30˚ angle and ¼ to ½ inches below intended entry

into vein.

4. Grasp the flange of the needle adapter and push the collecting tube forward until

the needle punctures the stopper. Observe for flow of blood into stopper. Maintain

tube below the needle.tube below the needle.

5. Remove tourniquet as soon as possible once blood flow is established.

6. Keep constant, slight forward pressure on the end of the tube to prevent release of

shut-off valve and stop of blood flow.

7. Fill the tube until the vacuum is exhausted and blood flow ceases.

8. When blood flow ceases, remove the tube from the holder. The shut-off valve

recovers the point, stopping blood flow until the next tube is inserted.

9. Tubes containing additives should be mixed immediately upon draw by inverting 5-

10 times. To avoid hemolysis, do not mix vigorously.

10. To obtain additional specimens, insert the next tube into the holder and repeat

steps 7-9.

Venipuncture procedure when using needle and syringe:1. Position the needle with the bevel up and the shaft parallel to the path of the vein.

2. Insert sterile needle or butterfly through the skin at a 15-30˚ angle ¼ to ½ inches below the

intended entry into the vein.

3. Pull back on plunger or syringe slowly until sufficient volume of sample is achieved.

4. Release tourniquet.

5. Withdraw needle and syringe.

6. Apply pressure to site with gauze pad.6. Apply pressure to site with gauze pad.

7. Pierce stopper of collection tube with needle; the evacuated tube will fill to the correct

amount of blood.

8. Immediately activate the safety feature according to manufacturer instructions and discard

without assembly into a sharps container.

9. Check patient’s arm to ensure bleeding has stopped.

10. Apply gauze pad secured lightly with tape to the puncture site.

11. Instruct patient to leave bandage in place for at least 15 minutes.

12. Label all blood tubes at patient’s time of draw.

13. Place labeled specimens in biohazard bag.

14. Discard gloves and wash hands.

15. Place specimen at courier pickup station for processing.

Blood culture collection procedure:

1. For each request for blood cultures on adults when a time or location is not specified by the

doctor, two sets of two bottles will be drawn. Each set of aerobic and anaerobic blood culture

bottles will be obtained from two different sites. Samples will NOT be taken from an arterial

line, heparin lock or a subclavian IV unless specifically ordered by the attending physician.

2. After selecting a good phlebotomy site, the tourniquet will be released and the site disinfected.

3. The site will be cleansed first with chloraprep® using a concentric spiral motion moving from

the site outward.the site outward.

4. Prep the site using an iodine prep using the same motion working from the site outward. Allow

the iodine to dry before drawing specimen.

NOTE: If the patient is allergic to iodine, another topical disinfectant may be used. Any deviation

from routine collection should be noted on the request form or sample bottles.

5. Perform the venipuncture and draw the sample according to procedure. Sample should flow

freely.

6. Carefully change syringe needle and blood transfer device and place 8-10mL of blood into each

vial using aseptic technique. Be sure not to contaminate bottle tops before entering bottle

with needle. Label and send to lab as soon as possible.

WHOLE BLOOD, SERUM or PLASMA COLLECTION

• The most common sample of laboratory testing is whole blood serum or plasma. The preferred collection method for adults is venipuncture using vacuum collection tubes. The method of collection is similar for whole blood, serum or plasma except for the anticoagulant used. The color of the stopper of the collection tube specifies the anticoagulant content.anticoagulant content.

• Blood should be obtained from a freely flowing venipuncture performed according to current nursing or laboratory venipuncture procedure. Tubes should be collected in the following stopper color order – red, blue, other. All tubes, except red top tubes, should be inverted gently several times in order to mix the anticoagulant. Adequate volume should be collected for the number and types of tests requested. Minimum blood volumes are noted in the collection manual for each test. If insufficient volume is collected, call the laboratory before sending.

LABORATORY TESTS

Lab evaluation

a) Hematology:

CBC with differential CBC with differential

PT/INR/PTT

ABO group and Rh type

antibody screen

A. Complete blood cell counts (CBC),

differential and platelet counts should be

• Measured at each office visit. Patients

receiving ganciclovir (or valganciclovir), daily

Trimethoprim/Sulfamethoxazole (TMP/SMX), Trimethoprim/Sulfamethoxazole (TMP/SMX),

Cellcept (mycophenolate mofetil), and

other myelosuppressive medication should

have a CBC at weekly intervals or more often

• when counts are low.

Blood Smear - Normal

b) Chemistry:

Sodium, Potassium, Chloride, Bicarbonate Glucose Calcium, Albumin Magnesium Phosphate Phosphate BUN, CreatinineTotal Bilirubin (direct and indirect if total bilirubin is elevated) Alkaline PhosphataseAST, ALT Total Protein LDH, Uric Acid

. Liver function tests (LFT's) (alkaline phosphatase, ALT, AST, LDH and total bilirubin)

• Should be measured at each office visit.

Patients receiving immunosuppressive

medications or other hepatotoxic drugs such medications or other hepatotoxic drugs such

as itraconazole, voriconazole, INH, should

have LFT's measured at two-week intervals or

more often when abnormalities are

• present.

Renal evaluation:

Urine analysis 24 hour urine for creatinine24 hour urine for creatinineclearance

Renal function tests (serum creatinine, BUN, and magnesium) should be measured at

• Each office visit. Patients receiving cyclosporine,

tacrolimus (formerly known as FK506),

amphotericin or other nephrotoxic drugs should amphotericin or other nephrotoxic drugs should

have renal function monitored at weekly intervals

or more often when abnormalities are present.

• Dose adjustment may be needed for medications

such as cyclosporine, tacrolimus, ganciclovir,

valciclovir, acyclovir, among others.

Drug levels:

Cyclosporine

tacrolimus (FK506)

Sirolimus (rapamycin)

Itraconazole blood levels should be monitoredItraconazole blood levels should be monitored

KETOCONAZOLE OR VORICONAZOLE SHOULD NOT BE

COADMINISTERED WITH SIROLIMUS.

Blood cultures should be drawn whenever clinically indicated.

• For high risk patients

(i.e., treatment with prednisone at a dose of

more than 1 mg/kg/day), weekly surveillance

blood cultures may be beneficial.blood cultures may be beneficial.

c) Serology: (must be documented within 30

days prior to first day of stem cell collection)

• Cytomegalovirus serology (*Anti-CMV IgG)

• Herpes simplex virus serology (HSV I & II)

• Varicella zoster virus (VZV) serology

• HIV serology (*Anti-HIV-1, *Anti-HIV-2)

• *HIV-1 Antigen

• *Anti-HTLV 1 and 2

• Serologic test for syphylis:

*RPR • *RPR

• Hepatitis A:

• Anti-HAV IgM and IgG

• Hepatitis B:

• *HbsAg - surface antigen

• *Anti-HBc

• Anti-HBs - surface antibody

• Hepatitis C:

• *Anti-HCV

CMV monitoring in blood should be:

CMV surveillance tests:

CMV monitoring can be performed using

CMV DNA by PCR or hybrid capture,

pp67 mRNA, or pp65 antigenemiapp67 mRNA, or pp65 antigenemia

(culture based assays are not

appropriate for monitoring.)

PCR is recommended over pp65

antigenemia

Bone marrow should be evaluated as clinically

indicated and according to specific protocol

Testing should include evaluation of morphology,

immunophenotyping, BCR/abl transcripts or other markers

of minimal residual disease, and cytogenetics as applicable.

Patients transplanted for chronic myeloid leukemia (CML) or Patients transplanted for chronic myeloid leukemia (CML) or

Philadelphia chromosomepositive acute lymphocytic

leukemia (Ph-positive ALL) should have blood tested for

BCR/abl transcripts at 6 month intervals for the first 2 years

after transplant and then at yearly intervals.

When BCR/abl transcripts are detected in the blood, a marrow

aspirate should be evaluated by cytogenetic testing,

morphology and molecular testing of blood samples should

be continued at 6-month intervals.

Stem Cell Laboratory

The Stem Cell Laboratory processes peripheral stem cell and bone marrow products for transplantation purposes. Services provided include:

• Cryopreservation, thawing, and infusion of bone • Cryopreservation, thawing, and infusion of bone marrow, stem cells, and donor lymphocytes

• Red cell depletion and volume reduction of bone marrow products

• Performance of specific cell isolation procedures on peripheral stem cell and bone marrow products

Flow Cytometry Laboratory

The Flow Cytometry laboratory performs flow cytometric analysis used by clinicians in diagnosing, monitoring, and treating patients with the following disorders:

acute and chronic leukemia, lymphoproliferative disease, multiple myeloma, immune deficiencies, multiple sclerosis, and paroxysmal nocturnal hemoglobinuria.

• Acute and chronic leukemia immunophenotyping• Acute and chronic leukemia immunophenotyping

• Lymphoproliferative disorder immunophenotying

• Multiple Myeloma Immunophenotyping

• Paroxysmal Nocturnal Hemoglobinuria testing

• T-cell and T-subset quantitation

• CD3 and CD20 quantitation

• CD34 (Stem Cell) quantitation

• T-cell, B-cell, NK-cell quantitation

MAJOR HISTOCOMPATIBILITY CLASSES

� Prior to discussing those aspects of T cell function

relevant to the pathogenesis of GVHD, it is helpful

to first briefly review the major Histocompatibility

Complex (MHC) or HLA (for Human Leukocyte Complex (MHC) or HLA (for Human Leukocyte

Antigens) in humans since these molecules

underlie the recognition of antigen by T cells.

� The MHC is highly polymorphic from individual

to individual, and segregates in families in a

Mendelian codominant fashion.

HLA Laboratory

The Histocompatibility (HLA) Laboratory provides HLA-related compatibility testing for solid organ transplants, bone marrow transplants, platelet transfusions, and HLA-associated diseases.

Services include:

• HLA low-resolution serological and low and high-resolution • HLA low-resolution serological and low and high-resolution PCR-based typing for solid organ and stem cell transplantation

• Compatibility testing services for deceased organ donor recovery and allocation

• HLA typing for diseases association

• HLA typing/matching and antibody screening for platelet transfusion and non-hemolytic transfusion Reactions

HLA

�The genes of the HLA locus encode two distinct classes of cell surface molecules, classes I and II.

�Class I molecules are expressed on the surfaces of virtually all nucleated cells at varying densities, while class II molecules are more restricted to cells of the immune system, primarily B lymphocytes and monocytes. system, primarily B lymphocytes and monocytes.

�There are three different class I (HLA-A, -B, -C) and class II (HLA-DQ, -DR, -DP) antigens. HLA-A, -B and -DR antigens appear to be the most important loci determining whether transplanted cells initiate a graft versus host reaction

Tissue typing

• HLA – Human Leucocyte Antigen

• Co-dominant expression of maternal and

paternal epitopes on all leucocytes

• HLA: class 1 ABC, class 2 DR(Q,P,T) class 3 • HLA: class 1 ABC, class 2 DR(Q,P,T) class 3

others

• Blood group can be different

• Sex does not have to be matched

HLA complex

HLA

Human MHC (chromosome 6)

Human MHC

GLO DN DM TAP LMP DO C4B C4A Bf C2 HSP TNF E J H G F

DP DQ DR B C A

Class II Class III Class I

Genes Ags

Family HLA Inheritance

Class I(HLA A, B, Cw)- Found on most nucleated cells & platelets-Present endogenous synthesized antigenic peptides- interact with CD8 on T cells

HLA class II molecules

Class II(DR)

-Found on B cells, monocytes, macrophage, dendritic cells and activated T cells-Present antigenic peptide derived from exogenous proteins - interact with CD4 on T cells

�For each full sibling, a patient has a one in four (25%) chance of a full match.a full match.

�The chance of having a donor is 1-(3/4)n ,where n is the number of siblings ..

Terazaki plate

Ab

Different Alleles

– Class IHLA A 451 allelesHLA B 782 allelesHLA C 238 allelesHLA C 238 alleles

– Class IIHLA DR 525 allelesHLA DQ 105 allelesHLA DP 147 alleles

PlasmaPlasma

PBLPBL

FicollFicoll

Ficoll Density=Ficoll Density=11..077077

RBCsRBCs

BuffyBuffy coatcoat

PMNPMN

The laboratory aims to meet the

following targets in reporting results.

• HLA Type 1-2 weeks

• (HLA-A,B,C,DR,DQ and DP, where applicable)

HLA Antibody Screening 1-2 weeks• HLA Antibody Screening 1-2 weeks

• Post transplant antibody monitoring 1-2 days

• HLA Type of local donor 4-5 hours

• Platelet testing (initial screen) 1-2 days

HLA TESTING FOR RECIPIENTS OF AUTOLOGOUSHAEMATOPOIETIC STEM CELL TRANSPLANTS

• The laboratory provides HLA class I typing and HLA antibody screening for patients requiring autologous haematopoietic stem cell transplant.

• This is to ensure that data is available should • This is to ensure that data is available should the patient become refractory to random donor platelet transfusions.

• Samples required are:10mls EDTA for PCR class I typing10mls Clotted Sample for HLA antibody testing

HLA TYPING FOR HAEMATOPOIETIC STEM CELL TRANSPLANT RECIPIENTS AND POTENTIAL DONORS

• Related family members who are being considered as potential donors are also HLA typed.

• On the request form for the potential donor it should be noted who the patient is.

• HLA typing is performed by both serological assays and • HLA typing is performed by both serological assays and PCR based assays depending on the level of resolution required.

• Samples required are:

• 10mls EDTA for PCR class I and II typing

• 10mls Clotted Sample for HLA antibody testing

• Potential Donor 10mls EDTA for PCR class I (and II typing if compatible for class I)

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MIXED CHIMERIC STATE

• In the field of HCT, mixed chimerism is defined as the concurrent presence of donor and recipient hematopoietic cells. Several methods have been used to assess mixed chimerism after hematopoietic cell hematopoietic cells. Several methods have been used to assess mixed chimerism after hematopoietic cell transplantation, including determination of the variable number of tandem repeats (VNTR) by restriction fragment length polymorphism (RFLP) analysis, polymerase chain reaction (PCR), and, if applicable, fluorescence in situ hybridization (FISH) for the Y chromosome .

• Engraftment status was routinely analyzed in

the bone marrow at one, two, six, and 12

months after HCT.

Transient mixed chimerism

• Transient chimerism is a dynamic condition

limited to the first two years after HCT. One limited to the first two years after HCT. One

risk factor is the cyclophosphamide dose used

in the conditioning regimen, with mixed

chimerism being less frequent at the full dose

of 200 mg/kg than at lower doses .

Persistent mixed chimerism

• After the second year of follow up, the persistently chimeric patients were submitted to yearly assessment of chimerism status for 2 to 11 years.

• Mixed chimerism was present in bone marrow at the same level as in the peripheral blood, with the exception of one patient in whom there were 25 percent donor cells in the bone marrow, but approximately 70 percent donor cells in the peripheral blood.