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mHam 2.0 – Complement-Dependent Cell Killing Assay

Justification

Complement-mediated diseases such as atypical hemolytic uremic syndrome (aHUS) and catastrophic antiphospholipid syndrome (CAPS) are difficult to diagnose. Accurate and rapid diagnosis is critical. The bioluminescent modified Ham (mHam) cell-based functional complement assay detects abnormal complement deposition using cell viability as the readout. Complement-mediated cell death in the mHam assay uses a human kidney cell line modified by genetic deletion of complement regulator CD46 and is blocked by addition of complement inhibitors. Positive mHam results will reflex to addition of C5 inhibitor to assess whether complement-mediated cell killing is blocked in vitro. Soluble C5b-9 levels are measured as part of this assay. This assay may help to diagnose and treat complement-mediated diseases. The '2.0' in the test name represents recent improvements in this assay (Cole, M. et al. Blood. 2024) from when the assay was first introduced (Gavriilaki E, et al. Blood. 2015). https://mham.machaondiagnostics.com/

STAT: < 24 hours (M-F)

Cell-based Assay

Draw Tube: Purple Top

Draw Tube: Red Top

Sample Type: Serum and EDTA Plasma

Specimen Requirements

Sample Type Volume Required Minimum Volume Stability
PREFERRED Serum and EDTA Plasma Two aliquots, 1mL each (Serum);One aliquot, 1mL (EDTA Plasma) Two aliquots, 0.5mL each (Serum);One aliquot, 0.5mL (EDTA Plasma) Frozen (-20C): 72 hours
Frozen (-80C): 6 months
ALTERNATIVE - - - -
REJECTION CRITERIA Thawed in transit, refrozen or clotted sample
SPECIAL INSTRUCTIONS Process and freeze samples within one hour of collection. Transport under dry ice.

General Information

METHODOLOGY Cell-based Assay
STAT TAT < 24 hours (M-F)
STAT TAT Performance > 90% of results released in 24 hours
ROUTINE TAT < 1 week
ALTERNATIVE NAMES Brodsky Assay, Modified Ham assay, functional complement activation assay, complement drug inhibition assay
DESCRIPTION Cell-based complement activation assay with a bioluminescence read-out. A 2024 publication (Cole, M. et al. Blood. 2024) showed that 5/5 (100%) acute complement-mediated thrombotic microangiopathy patients and 7/13 (54%) in remission had a positive mHam result. A 2020 publication showed that 6/7 (85.7%) catastrophic APS patients had a positive mHam result (Chaturvedi S, et al. Blood. 2020).
LIMITATIONS The mHam assay has a specificity of 85-93%. Patients treated with complement inhibitors will likely produce a negative result. Complement depletion or deficiency (e.g., low C4) in the sample may also produce false negatives. Incorrect or delayed sample processing or transport can be a major cause of complement depletion. Some conditions (e.g., pregnancy) known to increase complement may cause a positive result in the assay.
NORMAL RANGE >62.5% Cell Viability
ASSOCIATED TESTING soluble Complement 5b-9 (sC5b-9), aHUS 3.0 Genetic Panel, ADAMTS13 Activity, CFH Region Deletion/Duplication Analysis by MLPA, Anti-CFH Antibody
REFERENCES

1) Cole MA, Ranjan N, Gerber GF, Pan XZ, Flores-Guerrero D, McNamara G, Chaturvedi S, Sperati CJ, McCrae KR, Brodsky RA. Complement Biosensors Identify a Classical Pathway Stimulus in Complement-Mediated Thrombotic Microangiopathy. Blood. 2024 Oct 2:blood.2024025850. doi: 10.1182/blood.2024025850. Epub ahead of print. PMID: 39357054.
2) Chaturvedi S, Braunstein EM, Yuan X, Yu J, Alexander A, Chen H, Gavriilaki E, Alluri R, Streiff MB, Petri M, Crowther MA, McCrae KR, Brodsky RA. Complement activity and complement regulatory gene mutations are associated with thrombosis in APS and CAPS. Blood. 2020 Jan 23;135(4):239-251. doi: 10.1182/blood.2019003863. PMID: 31812994; PMCID: PMC6978159.
3) Gavriilaki E, Yuan X, Ye Z, Ambinder AJ, Shanbhag SP, Streiff MB, Kickler TS, Moliterno AR, Sperati CJ, Brodsky RA. Modified Ham test for atypical hemolytic uremic syndrome. Blood. 2015 Jun 4;125(23):3637-46. doi: 10.1182/blood-2015-02-629683. Epub 2015 Apr 10. PMID: 25862562; PMCID: PMC4784297.
SAMPLE REPORT Upon request
NEW YORK STATE APPROVED NY Restricted Laboratory Permit Required

Test Codes

ORDER CODE P3364
CPT CODE 86161, 86160
LOINC CODE 93244-2